Development of novel, highly potent inhibitors of V-RAF murine sarcoma viral oncogene homologue B1 (BRAF): Increasing cellular potency through optimization of a distal heteroaromatic group

Article abstract of DOI:10.1021/jm900607f

We describe the design, synthesis, and optimization of a series of new inhibitors of V-RAF murine sarcoma viral oncogene homologue B1 (BRAF), a kinase whose mutant form (V600E) is implicated in several types of cancer, with a particularly high frequency in melanoma. Our previously described inhibitors with a tripartite A-B-C system (where A is a hinge binding pyrido[4,5-b] imidazolone system, B is an aryl spacer group, and C is a heteroaromatic group) were potent against purified ^V600EBRAF in vitro but were less potent in accompanying cellular assays. Substitution of different aromatic heterocycles for the phenyl based C-ring is evaluated herein as a potential means of improving the cellular potencies of these inhibitors. Substituted pyrazoles, particularly 3-tert-butyl-1-aryl-1H-pyrazoles, increase the cellular potencies without detrimental effects on the potency on isolated ^V600EBRAF. Thus, compounds have been synthesized that inhibit, with low nanomolar concentrations, ^V600EBRAF, its downstream signaling in cells [as measured by the reduction of the phosphorylation of extracellular regulated kinase (ERK)], and the proliferation of mutant BRAF-dependent cells. Concomitant benefits are good oral bioavailability and high plasma concentrations in vivo.

Full text of DOI:10.1021/jm900607f

J. Med. Chem. 2010, 53, 2741–2756 2741
DOI: 10.1021/jm900607f
Development of Novel, Highly Potent Inhibitors of V-RAF Murine Sarcoma Viral Oncogene
Homologue B1 (BRAF): Increasing Cellular Potency through Optimization of a Distal
Heteroaromatic Group
Bart M. J. M. Suijkerbuijk,†,§ Ion Niculescu-Duvaz,†,§ Catherine Gaulon,† Harmen P. Dijkstra,† Dan Niculescu-Duvaz,†
†
Delphine Menard, Alfonso Zambon, Arnaud Nourry, Lawrence Davies, Helen A. Manne, Frank Friedlos,
†
†
†
†
†
ꢀ
Lesley M. Ogilvie,† Douglas Hedley,† Filipa Lopes,† Natasha P. U. Preece,† Javier Moreno-Farre,† Florence I. Raynaud,†
Ruth Kirk,‡ Steven Whittaker,‡ Richard Marais,‡ and Caroline J. Springer*,†
†The Institute of Cancer Research, Cancer Research UK Centre for Cancer Therapeutics, 15 Cotswold Road, Sutton, Surrey SM2 5NG,
United Kingdom, and ‡The Institute of Cancer Research, Cancer Research UK Centre for Cell and Molecular Biology, 237 Fulham Road,
London SW3 6JB, United Kingdom. § These authors contributed equally to this work.
Received May 8, 2009
We describe the design, synthesis, and optimization of a series of new inhibitors of V-RAF murine
sarcoma viral oncogene homologue B1 (BRAF), a kinase whose mutant form (V600E) is implicated in
several types of cancer, with a particularly high frequency in melanoma. Our previously described
inhibitors with a tripartite A-B-C system (where A is a hinge binding pyrido[4,5-b]imidazolone
system, B is an aryl spacer group, and C is a heteroaromatic group) were potent against purified
V600EBRAF in vitro but were less potent in accompanying cellular assays. Substitution of different
aromatic heterocycles for the phenyl based C-ring is evaluated herein as a potential means of improving
the cellular potencies of these inhibitors. Substituted pyrazoles, particularly 3-tert-butyl-1-aryl-1H-
pyrazoles, increase the cellular potencies without detrimental effects on the potency on isolated
V600EBRAF. Thus, compounds have been synthesized that inhibit, with low nanomolar concentrations,
V600EBRAF, its downstream signaling in cells [as measured by the reduction of the phosphorylation of
extracellular regulated kinase (ERK)], and the proliferation of mutant BRAF-dependent cells. Con-
comitant benefits are good oral bioavailability and high plasma concentrations in vivo.
Introduction
ERK controls gene expression, cytoskeletal rearrangements,
and metabolism, causing cell transformation andcontributing
to tumor initiation and maintenance. Targeting this path-
way, for example, through inhibition of BRAF, is therefore a
main focus point of many current cancer therapeutics ap-
proaches.6-12
X-ray crystallographic studies of the kinase domain of
protein kinases together with structural probing by classical
medicinal chemistry have been important for the development
of specifically designed small molecule inhibitors that bind to
specific protein kinases with high association constants. The
application of these techniques in the development of new
anticancer drugs has led to thediscovery of a number of highly
successful kinase inhibitors, such as sorafenib,13,14 gefitinib,15
and imatinib,16-18 as well as erlotinib,19 sunitinib,20 and
dasatinib21 (see Figure 1).
V-RAF murine sarcoma viral oncogene homologue B1
(BRAFa) is a serine/threonine kinase that operates down-
stream from RAS and is responsible for activation of MAPK/
ERK kinase(MEK) and extracellularregulatedkinase (ERK)
activation in the mitogen-activated protein kinase (MAPK)
pathway, a conserved signaling cascade responsible for cell
proliferation and survival.1,2 Recent studies have shown that
mutations in BRAF are found in a number of cancers, such as
melanomas, ovarian, colorectal, and papillary thyroid can-
cers.3 Melanomas in particular show a high incidence of
BRAF mutations, the most common being a substitution of
glutamic acid for valine at position 600 (V600E).4 This point
mutation lies within the activation segment of the protein,
adjacent to the conserved DFG motif, and stimulates consti-
tutive kinase activity in BRAF.5 This, in turn, causes continu-
ous activation of MEK and ERK, the latter of which pho-
sphorylates several cytosolic enzymes and nuclear substrates.
Given the importance of BRAF as a therapeutic target,9,22
we initiated a program to develop a series of small molecule
BRAF inhibitors.23-27 The design and synthesis of novel
BRAF inhibitors based on a 7-substituted 1H-imidazo[4,5-b]-
pyridin-2(3H)-one hinge binding fragment (ring A in
Figure 2) connected to a substituted 1,4-phenylene or a 1,4-
naphthalene fragment (ring B in Figure 2) that in turn is
attached to a substituted aromatic group (ring C in Figure 2)
via a urea linker have been described.25 In addition, we have
shown that the overall potencies of these inhibitors could be
greatly enhanced by appropriate B-ring functionalization.26
*To whom correspondence should be addressed. Phone: þ44 20
87224214. Fax: þ44 20 8722 4046. E-mail: caroline.springer@icr.ac.uk.
a Abbreviations: RAF, rapidly growing fibrosarcoma; BRAF,
V-RAF murine sarcoma viral oncogene homologue B1; MAPK, mito-
gen-activated protein kinase; MEK, mitogen-activated protein kinase/
extracellular regulated kinase kinase; ERK, extracellular regulated
kinase; SRB, sulforhodamine B; PK, pharmacokinetics; THF, tetrahy-
drofuran; DMSO, dimethyl sulfoxide; SAR, structure-activity relation-
ship; F, percent oral bioavailability.
r
2010 American Chemical Society
Published on Web 03/03/2010
pubs.acs.org/jmc

2742 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 7
Suijkerbuijk et al.
Figure 1. Structural formulas of recently developed kinase inhibitors.
Figure 2. General structural formulas of the BRAF inhibitors and one representative example.
A-B-C) but comprising a 1,3 (meta) substitution of the
middle ring B. These derivatives showed an improved selec-
tivity in a panel of kinases; however, the cellular activities
remained relatively weak (SRB > 700 nM), the most potent
inhibitor being 26 from reference 27 (see Figure 3). Never-
theless, we noticed that some aromatic C-rings (especially
substituted pyrazoles) could enhance significantly the cellular
potency of these inhibitors.
Therefore, in order to improve the cellular activity of our
previous 1,4 (para) substituted B-ring series,26 we set out to
optimize the C-ring fragment of the inhibitors focusing on
heteroaromatic C-rings. The results of the ensuing structure-
activity relationship (SAR) studies are described here.
Figure 3. Structural formula of 1,3-phenylene compound with
pyrazole ring C and biological data.
The inhibitors thus obtained exhibit BRAF IC50 values as
low as 1 nM, but their potencies on cells (pERK IC50 and
sulforhodamine B (SRB) GI50 on BRAF-dependent cells) are
on the order of several hundred nanomolar (for example,
compound 21a from ref 26; Figure 2).
Chemistry
In order to explore the SAR of the C-ring, a synthetic
approach was adopted in which the A-B portion was first
synthesized and functionalized. The amino group at the
B-ring para-position (with respect to the A-B linker)
was subsequently reacted with an appropriately activated
On the basis of docking studies, we developed a new series
of BRAF inhibitors with the same tripartite structure (rings

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 7 2743
Scheme 1. General Synthesis of the BRAF Inhibitors Described in the Current Paper
(see Scheme 2). Compounds 4g-m are commercially available.
Scheme 2. Synthesis of 1-Substituted 3-tert-Butyl-1H-pyrazol-
5-aminesa
The activated carbamates 5a-d and 5f-o were subsequently
obtained in excellent yields by reacting 4a-d and 4f-o with
phenyl chloroformate, or in the case of 5e, 4e was reacted with
isopropenyl chloroformate28 under basic conditions (Scheme 3).
In the final step, 5a-d and 5f-o were reacted with general
intermediate 7-(4-aminophenoxy)-1H-imidazo[4,5-b]pyridin-
2(3H)-one 6 in warm tetrahydrofuran (THF) or in dimethyl
sulfoxide (DMSO) to give the corresponding 1-substituted
3-(4-(2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-7-yloxy)phe-
nyl)ureas 1a-d and 1f-o (Scheme 4, Table 1). This series
of compounds, which contain both an unsubstituted 1H-
imidazo[4,5-b]pyridin-2(3H)-one ring A and an unsubstituted
phenyl ring B, served as a benchmark series to assess the
influence of the structural characteristics of ring C on the
relative activity of the compounds in the assays.
a Reagents and conditions: (i) 0.2 M ethanolic HCl or toluene, reflux.
Scheme 3. Synthesis of Activated Carbamatesa
The C-rings most beneficial for cellular potency found in
this study were subsequently connected to a variety of A-B
systems25-27 via either route A or route B. Route B utilizes
functionalized isocyanates for the coupling of the C-ring
fragment to an A-B intermediate. The C-ring fragments used
here are relatively electron-rich, and the derived isocyanates
tend to be less stable, which precludes their commercial
availability. Therefore, these isocyanates were prepared from
the amine via reaction with phosgene under basic conditions29
and usedimmediately in the nextreaction step. Thus, coupling
of a selection of A-B intermediates 7 to C-ring fragments,
either by route A or by route B, gave the corresponding
urea final compounds 2a-af in variable yields (see Scheme 5,
Table 2, and the Experimental Section).
a Reagents and conditions: (i) pyridine, THF, 0 °C f room tempera-
ture.
Selection of C-Ring Fragments
Three in vitro assays were used to determine the biological
activities of the compounds: (a) IC50 on purified V600EBRAF
(IC50, V600EBRAF); (b) IC50 for the inhibition of ERK phos-
phorylation in mutant BRAF expressing WM266.4 melanoma
cells (IC50, pERK); (c) IC50 for proliferation in WM266.4 cells
(GI50, SRB). These data are summarized in Tables 1 and 2.
The SAR presented is focused mainly on ring C. Some
structural diversity, however, has also been incorporated into
the A- and B-ring fragments of some compounds to probe
their effects on the activity of these compounds in the in vitro
assays, as well as their pharmacokinetic (PK) behavior. In
Table 1 an overview is presented of the assay results of
C-ring fragment to give the target A-B-C system. Thus, the
previously described general intermediates25-27 were reacted
with an aromatic heterocycle functionalized with either an
activated carbamate (route A) or an isocyanate group (route B)
to give the target compounds (see Scheme 1).
The carbamate functionalized heterocycles (5a-o) needed
for route A were synthesized in one or two steps, depending on
the commercial availability of the required heterocyclic amine.
For pyrazolyl amines 4a-f, the appropriately functionalized
hydrazine (3a-f) was condensed with 4,4-dimethyl-3-oxopen-
tanenitrile to give the corresponding 1,3-disubstituted-1H-
pyrazol-5-amines in yields ranging from 27% to quantitative

2744 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 7
Suijkerbuijk et al.
Scheme 4. Synthesis of Inhibitors 1a-d and 1f-o via Activated Carbamate Route
compounds in which the A-ring (a 7-substituted 1H-imidazo-
[4,5-b]pyridin-2(3H)-one) and the B-ring (a 1,4-phenylene
linker) have been kept constant and in which only the C-ring
has been varied (1a-d,f-o). The C-ring fragments are mainly
based on a 1,3-disubstituted-1H-pyrazole core which is linked
through its 5-position to the B-ring via a urea bridge, which
proved essential for the preservation of activity.25
A striking observation from Table 1 is that all C-ring
modifications (with the notable exception of di-tert-butylpyr-
azole 1b and 4-tert-butylthiazole 1n) lead to inhibitors that, in
contrast to the previously reported phenyl C-ring compounds,25
exhibit submicromolar potencies on the isolated enzyme. The
lower potency of compound 1b may be explained by its bulky
three-dimensional 1-N-tert-butylpyrazole substituent. The ex-
ploration of the C-ring was initiated because of the difficulty of
achieving low nanomolar cellular activities (i.e., in the pERK
and SRB assays) with phenyl C-rings.25,26 Whereas a number of
C-ring modifications do not substantially ameliorate this beha-
vior, the use of a number of 1-aryl-3-tert-butylpyrazoles, such as
in 1h, 1i, 1k, and 1m, leads to a significant decrease of the pERK
IC50 and SRB GI50 with respect to the previously published
phenyl C-ring-containing compounds.25,26
The data in Table 1 indicate thatboth the3-tert-butyland1-
aryl substituents are essential for this improvement. Com-
pounds 1a, 1c, and 1g all possess a C-ring 3-tert-butyl
substituent, but the absence of an aryl group at the pyrazole
1-position renders them only moderately active on cells.
Conversely, the drop in cellular potency when going from a
3-tert-butyl to a 3-isopropyl substituent (cf. 1k and 1l) and
especially from a 3-tert-butyl to a 3-methyl substituent (cf. 1i
and 1j) supports the assumption that a tert-butyl substituent is
required at this position for effective cellular potency. A likely
explanation is that the change in lipophilicity, due to the
presence of the 3-tert-butyl group, drives the improvement in
cellular activity. In addition, the steric match between the tert-
butyl group and the kinase’s pocket beyond the gatekeeper
residue may be of importance, as discussed below.
1-Methylated compounds, 2a-g, generally exhibit good to
excellent potencies in all three in vitro assays. For 2a, 2b, 2e,
and 2g, 1-methylation can be directly compared to the
corresponding NH analogues 1g, 1h, 1k, and 1m. Whereas
it seems to influence the potency on BRAF only marginally,
1-methylation does have a substantially beneficial impact on
the pERK and SRB potencies of 2a, 2e, and 2g. In contrast,
3-methylation has a detrimental effect on the BRAF IC50,
which is consistent with the expected result of removing one
NH hydrogen bonding interaction with the hinge region of
the enzyme.31 Recent results, however, imply that these
compounds are more potent than suggested by the isolated
V600EBRAF enzyme assay.30 Cellular potencies benefit more
from 3-methylation than from 1-methylation of the scaffold,
whereas this order seems to be reversed for compounds with
a naphthyl B-ring. This is consistent with the greater reduc-
tion in H-bonding potential through removal of the peri-
pheral 3-NH H-bond donor and the overriding lipophilic
contribution to cell penetration provided by the naphthyl
group. Interestingly, for the fluoro B-ring series, a change
from unmethylated to 1,3-dimethylated does not seem to
affect the pERK IC50 values and SRB GI50 values (compare
2y and 2ab); it could be hypothesized that the effect on cell
permeability of removing one H-bond donor from the
imidazopyridinone scaffold is less significant in the case of
the fluorophenyl middle ring, which already locks into
position one strong H-bond donor of the urea group via
an intramolecular H-bond with the halogen atom.
A-B Linker. Substitution of the A-B oxygen linker of 1k
for a sulfur linker to give 2ae yields a compound that is
slightly less potent on the isolated enzyme, but whose cellular
activity was improved by a factor of ∼10 on pERK and ∼30
in the SRB assay, which is likely to be due to the higher
lipophilicity of this compound with a concomitant increase
in cell permeability. Because of its low solubility in aqueous
media, this line of investigation was discontinued.
Improvement of Cellular Potency
Variation of the A-B Fragment
Variations of the B-ring fragment of the inhibitors lead to
changes in the BRAF IC50, which have been discussed
previously.26 However, it is noteworthy that in the present
series similar trends are observed. Thus, all other groups being
equal, the potency of the inhibitors increases in the order
phenyl < naphthyl ∼ 3-F-phenyl < 3-MeS-phenyl.
A-Ring NH Methylation. After optimizing the C-ring
fragment of the inhibitors, we focused on connecting the
pyrazole C-ring to the optimized A-B ring fragments de-
scribed earlier (see Table 2 for synthesized compounds).
Methylation at the 1- and 3-positions of the A-ring was
performed with the aim of enhancing the PK profile of the
compounds by removal of one H-bond donor25 and of
investigating whether removal of the NH groups is tolerated.
The potencies of some of the compounds in this paper com-
pared to the corresponding 4-chloro-3-trifluoromethylphenyl
C-ring compounds discussed previously25,26 are presented in

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 7 2745
Table 1. Comparison of the Potencies of Inhibitors with Different C-Rings

2746 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 7
Table 1. Continued
Suijkerbuijk et al.
a Single determination.
well-known to enhance passive transport of compounds
across the cell membrane. Alternatively, the improvement
might be due to the kinetics of binding. In all but one case,
the p-tolyl-functionalized pyrazole increases the potency more
than the phenyl-functionalized pyrazole.
Scheme 5. Synthesis of Inhibitors 2a-ag via Either Activated
Carbamate Route A or Isocyanate Route B
Correlation between pERK IC50 and SRB GI50
A linear regression analysis of the correlation between the
pERK IC50 and SRB GI50 data for 39 inhibitors was per-
formed. The analysis yielded a linear relationship between the
two variables, expressed as
SRB ¼ 1:47ðpERKÞ þ 1:09
ð1Þ
Table 3. In general, these phenylpyrazoles are, according to
the CS Chemoffice LogP software, more lipophilic than the
analogous compounds with the phenyl C-ring. A striking
observation is that C-ring substitution of a 4-chloro-3-tri-
fluoromethylphenyl group by a 3-tert-butyl-1-aryl-1H-pyra-
zole (aryl = phenyl or p-tolyl) group leads to an increase of
potency in the cell-based pERK and the SRB cell proliferation
assays. Higherlipophilicity of the lattercompounds compared
to the former may be a rationale for this behavior, as this is
n ¼ 39
s ¼ 2:25 ðstandard error of the regression modelÞ
F ¼ 4:2 ꢀ 10-21 ðF probability of the modelÞ
with a regression coefficient r of 0.953 (r2 = 0.91) and a cross-
validation index (q2) of 0.76 (see Figure 4). These data indicate
that SRB GI50 and pERK IC50 correlate significantly with a
ratio close to unity. This finding provides further evidence for

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 7 2747
Table 2. Comparison of the Potencies of Inhibitors with Different Combinations of A-, B-, and C-Ringsa

2748 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 7
Table 2. Continued
Suijkerbuijk et al.
the hypothesis that direct targeting of V600EBRAF inhibits
proliferation of melanoma cells. These findings confirm that
this series inhibits cell growth through inhibition of the ERK
pathway, which makes the influence of off-pathway effects on
proliferation unlikely.
Cellular Selectivity
The SRB GI50 for the BRAF wild-type WM1361 mela-
noma cell line that does not express mutant BRAF was
determined for selected compounds (1k, 2r, 2y, 2z) in order
to provide evidence for the specificity of the compounds for

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 7 2749
Table 2. Continued
BRAF-driven cell proliferation. The results are shown in
Table 4, compared to the SRB GI50 on mutant BRAF
melanoma cell line WM266.4. Unlike sorafenib, for which
the activity is independent of BRAF status, these inhibitors

2750 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 7
Table 2. Continued
Suijkerbuijk et al.
are all selective up to 49-fold for the mutant BRAF WM266.4
cell line. This selectivity strongly supports the proposed
mechanism of action of this series through the inhibition of
oncogenic BRAF.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 7 2751
Table 2. Continued
a For a number of compounds (2j, 2l, 2o-r, 2u, 2w, 2x, 2ab, 2af), the BRAF IC50 values are 1-2 orders of magnitude higher than their pERK IC50 and
SRB GI50 values. This is due to the short incubation time of the BRAF assay, which is 45 min, compared to the much longer incubation times of the
pERK and SRB assays (6 and 96 h, respectively) and not because of the intrinsically lower inhibitory activities of those compounds. b Citric acid salt of 2j.
c The other two replicates were >10 μM.
PK Properties
vivo in BALB/cAnNCrl mice. The results are presented in
Table 5. Most of the phenylpyrazole compounds show a
significant improvement in F compared to the 4-chloro-3-
trifluoromethylphenyl C-ring compounds, which generally do
The oral bioavailability (F), half-life (t1/2), and maximum
concentrations of some of the inhibitors were determined in

2752 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 7
Suijkerbuijk et al.
Table 3. Comparison of 4-Chloro-3-trifluoromethylphenyl C-Rings
with 3-tert-Butyl-1-aryl-1H-pyrazole C-Rings
Table 4. Selectivity of Inhibitors on Wild Type and Mutant BRAF Cell
Lines
pERK IC50
improvementb
SRB GI50
improvementb
SRB GI50
(μM), WM1361
SRB GI50
(μM), WM266.4
compd
log P a
compd
fold difference
1k
1m
2e
2g
2i
4.2
4.7
4.4
4.9
4.4
4.9
5.2
5.7
5.4
5.9
5.4
5.9
4.3
4.8
5.1
3.9
5.7
62
11
14
37
80
61
137
14
29
43
65
14
2.1
39
33
29
sorafenib
4.5
3.2
5.2
0.39
1
1k
2r
2y
2z
8
1.2
0.024
0.039
0.046
49
17
17
113
116
280
16
0.66
0.79
2j
2o
2p
2r
Table 5. Pharmacokinetic Data for Selected Compounds, Based on
Plasma Levels after a 10 mg/kg Single Dose po
compda
40
40
2u
2w
2x
2y
2z
2ad
48
F (%)
t1/2 (h)b
max concn (nM)c
116
45
1md
2g
6.5
34
30
33
5
4.0 (15)
3.4 (24)
3.4 (24)
1.91 (20.5)
0.8 (5)
3674 (0.5, 12)
39594 (1, 600)
22675 (1, 840)
35492 (1.5, 1480)
4207 (0.5, 60)
16
54
2j
2rd
2s
11
a Calculated using the ChemDraw LogP function. b The reference
pERK IC50 or GI50 values are those of analogous 4-chloro-3-trifluoro-
methylphenyl C-ring compounds with the same A-ring and B-ring as the
analogues 3-tert-butyl-1-aryl-1H-pyrazol-5-yl compounds. The im-
provement values calculated in this table are the ratio of the pERK
IC50 or GI50 values of the 4-chloro-3-trifluoromethylphenyl C-ring
compounds to those of the corresponding 3-tert-butyl-1-aryl-1H-pyr-
azol-5-yl analogues.
2u
2y
43
24
7
8.4 (24)
2.8 (19)
5.3 (24)
12.6 (>6)
2.1 (18)
9.4 (>18)
43320 (1, 2700)
15020 (0.25, 390)
7425 (1.5, 160)
18024 (0.25, 1060)
4444 (0.5, 135)
8120 (0.5, 226)
2z
2aa
2ab
2ac
17
59
31
a 10 mg/kg single dose po unless otherwise noted. b In parentheses the
number of hours the concentration of a compound is above its GI50 after
oral administration. c In parentheses the tmax (h) and the ratio between
the plasma concentration of compound and its GI50 at tmax after oral
administration. d 20 mg/kg single dose po.
conformation of the BRAF and p38R proteins, respectively,
and are thus classified as type II kinase inhibitors.33 On the
basis of this rationale, we propose a tentative binding
mode of the inhibitors described here, which is depicted in
Figure 5. Within this model, the A-ring fragment of the
inhibitor (as exemplified here by 1m) forms a hydrogen
bond with the hinge region of the kinase through its
pyridine nitrogen atom, possibly in conjunction with its
ortho imidazolone proton. This fragment is connected
through the 1,4-phenylene or -naphthalene bridge to the
urea part, which forms a network of hydrogen bonds with
the enzyme. As in the structure of doramapimod with p38
MAP kinase, the p-tolyl part of the pyrazolyl fragment
probably protrudes through the BP-IV pocket into the E2
binding pocket (nomenclature adopted from Liao).32 In
contrast, its tert-butyl substituent fills the hydrophobic BP-
III pocket in a similar way as the C-ring CF3 group of
sorafenib does in BRAF. The docking of compound 2y on
the sorafenib/BRAF cocrystal structure (PDB code UWH)5
confirms this model. The docking pose showing selected
amino acids and key ligand-protein interactions is pre-
sented in Figure 6. As expected, the binding mode is
reminiscent of that of sorafenib, with the pyridoimidazo-
lone unit forming a hydrogen bond with the backbone
of Cys532 of the hinge region. Three more H bonds
are formed by the urea moiety of the inhibitor, two between
the NH groups and the Glu501 side chain and one between
the carbonyl moiety and the backbone of Asp594 of
the DFG motif. Notably, the fluoro atom on the B ring
of 2y elaborates into the BPI cavity of BRAF, which is
not occupied by sorafenib, while the tert-butylpyrazole
of the C ring of 2y forms extensive interactions with
the kinase pocket beyond the gatekeeper residue, in
particular between the tert-butyl group and the BPIII
pocket.
Figure 4. Graph showing the correlation between the pERK IC50
and SRB GI50.
not exhibit F > 5%. N1-Methylation seems to be beneficial
for the F of the compounds (cf. 2g vs 1m, respectively).
Furthermore, a lipophilic C-ring appears to increase F relative
to a more polar C-ring (2r and 2u vs 2s). Metabolism of the
compounds by human and mouse liver microsomes is gene-
rally low (<30% at 30 min, data not shown). All compounds
reach concentrations well above their GI50 values following
oral administration, some greater than 1000-fold. This sug-
gests that effective in vivo concentrations can be easily
achieved by oral dosing.
Rationale of Binding
Liao recently published a comprehensive overview of the
binding modes of kinase inhibitors to their targets.32 In that
context, the structural elements of the inhibitors described
here are quite similar to those found in sorafenib and in dora-
mapimod (Figure 1), which bind to the inactive, DFG-out

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 7 2753
procedures. Chromatography solvents were HPLC grade and
were used without further purification. Reactions were mon-
itored by thin layer chromatography (TLC) analysis using
Merck silica gel 60 F-254 thin layer plates. Flash column
chromatography was carried out on Merck silica gel 60
(0.015-0.040 mm) or in disposable Isolute Flash Si and Si II
silica gel columns. Preparative TLC was performed on either
Macherey-Nagel [809 023] precoated TLC plates SIL G-25
UV254 or Analtech [2015] precoated preparative TLC plates,
2000 μm with UV254. LCMS analyses were performed on a
Micromass LCT/Water’s Alliance 2795 HPLC system with a
Discovery 5 μm, C18, 50 mm ꢀ 4.6 mm i.d. column from
Supelco at 22 °C using the following solvent systems: solvent
A (methanol) and solvent B (0.1% formic acid in water) at a flow
rate of 1 mL/min. The gradient started with 10% A/90% B from
0 to 0.5 min and then 10% A/90% B to 90% A/10% B from 0.5
to 6.5 min and continuing at 90% A/10% B up to 10 min. From
10 to 10.5 min the gradient reverted back to 10% A/90% B
where the concentrations remained until 12 min. UV detection
was at 254 nm, and ionization was positive or negative ion
electrospray. The molecular weight scan range was 50-1000.
Samples were supplied as 1 mg/mL in DMSO or methanol with 3
μL injected on a partial loop fill. NMR spectra were recorded in
DMSO-d6 on a Bruker DPX 250 MHz or a Bruker Advance 500
MHz spectrometer. Chemical shifts (δ) are given in ppm and are
referenced to residual, not fully deuterated, solvent signal (i.e.,
DMSO-d5). Coupling constants (J) are given in Hz. HRMS
analyses were performed on an Agilent 6210 time of flight mass
spectrometer with an Agilent 1200 series HPLC system. The
internal references were caffeine ([M þ H]þ 195.087 652), re-
serpine ([M þ H]þ 609.280 657) and (1H,1H,3H-tetrafluoro-
pentoxy)phosphazene ([M þ H]þ 922.009 798). The mobile
phase consisted of 0.1% formic acid in doubly distilled de-
ionized water (solvent A) and HPLC-grade methanol (solvent
B) with all runs executed at 1.0 mL/min flow rate. For condition
A, the column Restek Ultra Aqueous 3 μm C18 (30 mm ꢀ 4.6 mm
i.d.) was used with a gradient starting at 90:10 (A/B) from 0.0 to
0.2 min, then 90:10 to 10:90 from 0.2 to 4.0 min, and continuing
at 10:90 from 4.0 to 9.25 min before reverting back to 90:10 at
10 min. The purities of the final compounds described here were
determined by HPLC as described above and are 95% or higher
unless stated otherwise.
Figure 5. Proposed binding mode of 1m to BRAF.
Figure 6. Docking pose of compound 2y into the sorafenib-
BRAF cocrystal structure.
Conclusions
Building on the knowledge and insights gained from pre-
vious studies, a series has been synthesized. These are based on
a 7-aryloxy-1H-imidazo[4,5-b]pyridin-2(3H)-one scaffold that
is linked to an aromatic heterocyclic fragment (ring C) via
a urea bridge. The use of 3-tert-butyl-1-aryl-1H-pyrazoles
instead of the previously used substituted phenyl groups as
C-rings led to a marked improvement in cellular potency in
vitro, while modifications on the rest of the scaffold enhanced
PK properties such as oral bioavailability and half-life in vivo.
Seven compounds have oral bioavailabilities of g30% up to
59% for 2ab. This illustrates that these inhibitors can reach
effective in vivo concentrations following oral administration,
and their further investigation in vivo in xenograft models is
warranted.
Linear regression analysis was carried out using TSAR
3.3 (Accelrys Inc., San Diego, CA). Docking studies were
performed using GOLD,34 version 3.1. Partial charges of the
ligand were derived using the Charge-2 CORINA 3D package in
TSAR 3.3, and its geometry was optimized using the COSMIC
module of TSAR. The calculations were terminated if the energy
difference or the energy gradient were smaller than 1 ꢀ 10-5 kJ.
Ten docking solutions were generated in the docking run, and
the best three were stored for analysis.
General Methods and Examples for the Synthesis of Inhibitors
1a-o and 2a-af. Route A1. A mixture of the appropriate
phenyl carbamate and substituted aniline in anhydrous THF
˚
with or without 4 A molecular sieves was heated for the stated
Experimental section
amount of time. After dilution with EtOAc and filtration, the
solution was washed with 0.5 M citric acid(aq), saturated
NaHCO3(aq), and brine. The organic layer was dried over
MgSO4 and filtered, and the solvent was evaporated under
reduced pressure. The residue was finally washed with Et2O to
afford the title compound as a solid.
Materials and Methods. Starting materials, reagents, and
solvents for reactions were reagent grade and used as purchased.
7-(4-Aminophenoxy)-1H-imidazo[4,5-b]pyridin-2(3H)-one 6,25
7-(4-aminophenoxy)-1-methyl-1H-imidazo[4,5-b]pyridin-2(3H)-one,
7-(4-aminophenoxy)-3-methyl-1H-imidazo[4,5-b]pyridin-2(3H)-one,25
7-(4-aminophenoxy)-1,3-dimethyl-1H-imidazo[4,5-b]pyridin-2(3H)-
one,25 7-(4-aminonaphthalen-1-yloxy)-1H-imidazo[4,5-b]pyr-
idin-2(3H)-one,26 7-(4-aminonaphthalen-1-yloxy)-1-methyl-1
H-imidazo[4,5-b]pyridin-2(3H)-one,26 7-(4-aminonaphthalen-1-yl-
oxy)-3-methyl-1H-imidazo[4,5-b]pyridin-2(3H)-one,26 7-(4-ami-
no-3-fluorophenoxy)-1H-imidazo[4,5-b]pyridin-2(3H)-one,26
7-(4-amino-3-(methylthio)phenoxy)-1H-imidazo[4,5-b]pyridin-
2(3H)-one,26 and 7-(4-aminophenylthio)-1H-imidazo[4,5-b]-
pyridin-2(3H)-one25 were synthesized according to described
1-(3-tert-Butyl-1-phenyl-1H-pyrazol-5-yl)-3-(4-(2-oxo-2,3-di-
hydro-1H-imidazo[4,5-b]pyridin-7-yloxy)phenyl)urea (1k). Route A1
was employed, using 5k (97 mg, 0.29 mmol) and 6 (50 mg,
˚
0.20 mmol) with 4 A molecular sieves at 50 °C for 17 h. The title
compound was obtained as a slightly orange solid (43 mg, 45%). 1H
NMR, δ: 1.28 (s, 9H, tert-Bu), 6.32 (d, 1H, J = 6.0, HPy,5), 6.37 (s,
1H, HPyz,4), 7.10 (d, 2H, J = 9.0, Harom,Ph,3þ5), 7.37-7.54 (m, 5H,
Harom,Ph-Pyz), 7.47 (d, 2H, J = 9.0, Harom,Ph,2þ6), 7.75 (d, 1H, J =
6.0, HPy,6), 8.40 (bs, 1H, NHurea), 9.11 (bs, 1H, NHurea), 11.16

2754 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 7
Suijkerbuijk et al.
(bs, 1H, NHPy3), 11.34 (bs, 1H, NHPy2). 13C NMR, δ: 30.1, 32.0,
95.6, 105.2, 112.8, 119.7, 120.4, 124.2, 127.2, 129.2, 136.4, 137.0,
138.5, 141.2, 145.8, 146.8, 148.5, 151.5, 154.1, 160.7. LC-MS: m/z
484([Mþ H]þ, 100).HRMS(EI):m/zcalcd for C26H26N7O3 ([Mþ
H]þ), 484.2097; found, 484.2101.
1-(1-N-p-Tolyl-3-tert-butylpyrazol-5-yl)-3-(4-(2-oxo-2,3-dihydro-
1H-imidazo[4,5-b]pyridin-7-yl-oxy)naphthalen-1-yl)urea (2p). Route
B was employed, using 4m (40 mg, 0.17 mmol) and 7-(4-amino-
naphthalen-1-yloxy)-1H-imidazo[4,5-b]pyridin-2(3H)-one (40 mg,
0.13 mmol) for 14 h. The title compound was obtained as an off-
white solid (66 mg, 93%). Purity: 96%. 1HNMR,δ:1.28(s,9H,tert-
Bu), 2.40 (s, 3H, CH3), 6.21 (d, 1H, J = 5.9, HPy,5), 6.39 (s, 1H,
1-(3-tert-Butyl-1-(4-fluorophenyl)-1H-pyrazol-5-yl)-3-(4-(2-
oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-7-yl-oxy)naphthalen-
1-yl)urea (2m). Route A1 was employed, using 5h (50 mg,
0.15 mmol) and 7-(4-aminonaphthalen-1-yloxy)-1H-imidazo-
Hpyr), 7.27(d,1H, J=8.3, Harom),7.37(d,2H, J=8.1, Harom),7.45
(d, 2H, J=8.3, Harom), 7.59(t, 1H, J=7.6, Harom), 7.64(t, 1H, J=
7.0, Harom), 7.69 (d, 1H, J=5.9,HPy,6),7.89(d,1H,J=8.3,Harom),
7.95 (d, 1H, J = 8.3, Harom), 8.07 (d, 1H, J = 8.5, Harom), 8.72 (s,
1H, NHurea), 9.06 (s, 1H, NHurea), 11.32 (s, 1H, NHPy2), 11.38 (s,
1H, NHPy3). LC-MS: Rf = 5.05 min; m/z 547.2 (Mþ, 100). HRMS
(EI): m/z calcd for C31H30N7O3 ([M þ H]þ), 548.2405; found,
548.2410.
1-(1-N-p-Tolyl-3-tert-butylpyrazol-5-yl)-3-(4-(2-oxo-2,3-di-
hydro-1-N-methyl-1H-imidazo[4,5-b]pyridin-7-yl-oxy)naphthalen-
1-yl)urea (2u). Route B was employed, using 4m (35 mg, 0.15 mmol)
and 7-(4-aminonaphthalen-1-yloxy)-1-N-methyl-1H-imidazo[4,5-b]-
pyridin-2(3H)-one (40 mg, 0.13 mmol) for 14 h. The title compound
was obtained as an off-white solid (52 mg, 71%). Purity: 84%. 1H
NMR, δ: 1.28 (s, 9H, tert-Bu), 2.40 (s, 3H, CH3), 3.53 (s, 3H, CH3),
6.29 (d, 1H, J = 5.9, HPy,5), 6.39 (s, 1H, Hpyr), 7.24 (d, 1H, J = 8.4,
˚
[4,5-b]pyridin-2(3H)-one (29 mg, 0.10 mmol) with 4 A molecular
sieves at 50 °C for 17 h. The title compound was obtained as a
1
brown solid (12 mg, 22%). H NMR, δ: 1.35 (s, 9H, tert-Bu),
6.20 (d, 1H, J = 6.0, HPy,5), 6.40 (s, 1H, HPyz,4), 7.29 (d, 2H, J =
8.5, Harom,Naph), 7.41 (t, 2H, J = 8.5, Harom,4-F-Ph,3þ5), 7.58-
7.70 (m, 5H, Harom,Naphþ4-F-Ph), 7.87 (d, 1H, J = 8.5, Harom,Naph),
7.94 (d, 1H, J = 8.5, Harom,Naph), 8.05 (d, 1H, J = 6.0 HPy,6),
8.76 (s, 1H, NHurea), 9.06 (s, 1H, NHurea), 11.37 (bs, 1H, NHPy3),
11.43 (bs, 1H, NHPy2). LC-MS: Rf = 7.62 min; m/z 552.2 ([M þ
H]þ, 100). HRMS (EI): m/z calcd for C25H26N7O3 ([M þ H]þ),
552.2159; found, 552.2164.
Route A2. A solution of the appropriate phenyl carbamate
˚
and substituted aniline in anhydrous DMSO with or without 4 A
molecular sieves was heated for the stated amount of time. After
cooling to room temperature, the solution was diluted in EtOAc,
washed twice with H2O, and washed once with brine. The
organic layer was dried over MgSO4 and filtered, and the solvent
was evaporated under reduced pressure. The solid residue was
washed with Et2O to afford the title compound.
1-(3-tert-Butyl-1-p-tolyl-1H-pyrazol-5-yl)-3-(4-(2-oxo-2,3-di-
hydro-1H-imidazo[4,5-b]pyridin-7-yloxy)phenyl)urea (1m). Route
A2 was employed, using 5m (53 mg, 0.15 mmol) and 6 (30 mg,
0.12 mmol) at55°C for 3.5h. Thetitle compound was obtained as
an off-white solid (40 mg, 67%). 1H NMR, δ: 1.27 (s, 9H, tert-
Bu), 2.37 (s, 3H, CH3Ph), 6.31 (d, 1H, J = 6.0, HPy,5), 6.34 (s, 1H,
HPyz,4), 7.09 (d, 2H, J = 8.9, Harom,Ph,3þ5), 7.33 (d, 2H, J = 8.4,
Harom,p-tol), 7.40 (d, 2H, J = 8.4, Harom,p-tol), 7.47 (d, 2H, J = 8.9,
Harom), 7.36 (d, 2H, J = 8.2, Harom), 7.45 (d, 2H, J = 8.2, Harom),
7.60 (t, 1H, J=7.5, Harom), 7.67(t,1H, J=7.6, Harom), 7.74 (d, 1H,
J = 5.9, HPy,6), 7.87 (d, 1H, J = 8.3, Harom), 8.07 (d, 2H, J = 8.3,
H
arom), 8.71 (s, 1H, NHurea), 9.06 (s, 1H, NHurea), 11.65 (s, 1H,
NHPy3). LC-MS: Rf = 5. min; m/z 562.2 ([M þ H]þ, 100). HRMS
(EI): m/z calcd for C32H32N7O3 ([M þ H]þ), 562.2561; found,
562.2566.
Biological Assays. V600EBRAF Kinase Assay and SRB IC50
for BRAF Inhibitors. These assays have been described by
Niculescu-Duvaz et al.23 BRAF assays were performed in
triplicate for each compound except for compound 1n for which
a single measurement was performed. The SRB assays were
performed in quadruplicate.
pERK Kinase Assay. This assay has been described by
ꢀ
compounds 1b, 1g, 1o, which are single measurements.
H
arom,Ph,2þ6), 7.74 (d, 1H, J = 6.0, HPy,6), 8.38 (bs, 1H, NHurea),
26
Menard et al. and was performed in triplicate except for
9.14 (bs, 1H, NHurea), 11.16 (bs, 1H, NHPy3), 11.34 (bs, 1H,
NHPy2). 13C NMR, δ: 20.4, 30.0, 31.8, 95.2, 105.2, 112.8, 119.6,
120.3, 124.1, 129.5, 136.0, 136.4, 136.5, 136.9, 141.1, 145.7, 146.8,
148.5, 151.5, 154.0, 160.3. LC-MS: m/z 498 ([M þ H]þ, 100).
HRMS (EI): m/z calcd for C27H28N7O3 ([M þ H]þ), 498.2254;
found, 498.2234.
1-[1-N-(4-Fluorophenyl)-3-tert-butylpyrazol-5-yl]-3-(4-(2-oxo-
2,3-dihydro-1-N-methyl-1H-imidazo[4,5-b]pyridin-7-yl-oxy)nap-
htalen-1-yl)urea (2q). Route A2 was employed, using 5h (177 mg,
0.5 mmol) and 7-(4-aminonaphthalen-1-yloxy)-1-N-methyl-1H-
imidazo[4,5-b]pyridin-2(3H)-one (100 mg, 0.33 mmol) at 42 °C
for 18 h. The title compound was obtained as an off-white solid
(130 mg, 70%). Purity: 76%. 1H NMR, δ: 1.28 (s, 9H, tert-Bu),
3.53 (s, 3H, CH3), 6.29 (d, 1H, J = 6.0, HPy,5), 6.39 (s, 1H, Hpyr),
7.24 (d, 1H, J = 8.3, Harom), 7.32 (t, 1H, J = 8.8, Harom), 7.39 (t,
2H, J = 8.8, Harom), 7.60-7.66 (m, 3H, Harom), 7.73 (d, 1H, J =
6.0, HPy,6), 7.84 (d, 1H, J = 8.3, Harom), 8.06 (d, 1H, J = 8.9,
Compound Metabolism. Microsomal incubations evaluating
phase I metabolism were performed as previously described.35
Compounds were incubated at 10 μM for 30 min. The reaction
was stopped by the addition of methanol (3 volumes), containing
roscovitine (500 nM) used as an internal standard. The parent
compound and metabolites were measured by liquid chromato-
graphy-mass spectrometry with electrospray in positive ioniza-
tion mode on two instruments as previously described.36 The
chromatographic separation was achieved on a Synergi polar
RP column (5 cm, 4 μm particle, 4.6 mm i.d.; Phenomenex,
Cheshire, U.K.) with gradient and mass spectrometry condi-
tions similar to those described by Nutley et al.36
Pharmacokinetics. Female BALB/cAnNCrl mice at least 6
weeks of age were used for the PK analyses except for the
intravenous administration of 1m, which was carried out with
female Crl:CD1-Foxn1nu mice bearing V600E mutant BRAF
WM266.4 tumor xenografts. The mice were dosed intra-
venously (2 mg/kg, equivalent to ∼4 μmol/kg, 10 mL/kg, in
DMSO/Tween-20/water, 10:1:89 v/v/v) or orally by gavage (10
mg/kg, equivalent to ∼20 μmol/kg except for 2r whose dosage
was 20 mg/kg, 10 mL/kg, in DMSO/water, 1:19 v/v). Samples
were taken at seven or eight time points between 5 min and 18
or 24 h for the intravenous route and at six or eight time points
between 15 min and 18 or 24 h for the oral route. Three mice
were used per time point per route. They were placed under
halothane or isoflurane anesthesia, and blood for plasma
preparation was taken by terminal cardiac puncture into
heparinized syringes. Plasma samples were snap frozen in
liquid nitrogen and then stored at -70 °C prior to analysis.
All procedures involving animals were performed in accor-
dance with national Home Office regulations under the
H
arom), 8.08 (d, 1H, J = 8.8, Harom), 8.72 (s, 1H, NHurea), 9.02 (s,
1H, NHurea), 11.65 (s, 1H, NHPy3). LC-MS: Rf = 7.94 min; m/z
566.2 ([M þ H]þ, 100). HRMS (EI): m/z calcd for C31H27FN7O3
([M þ H]þ), 566.2316; found, 566.2314.
Route B. The appropriate pyrazolyl-5-amine was dissolved in
a volume of CH2Cl2 (∼50 mM), and an equal volume of
saturated NaHCO3(aq) was added. The biphasic mixture was
stirred and cooled to 0 °C with an ice/water bath. After 10 min, 2
equiv of phosgene (1.9 M solution in toluene) were added. The
mixture was vigorously stirred for 10 min, the organic layer was
isolated, washed with H2O, dried (MgSO4), and concentrated to
about 100 mM. This solution (1-2 equiv) was added to a
solution of the appropriate amine in THF. The solution was
stirred for a given amount of time at room temperature, the
solvents were evaporated, and the solid residue was washed with
Et2O to afford the title compound.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 7 2755
Animals (Scientific Procedures) Act 1986 and within guidelines
set out by the Institute’s Animal Ethics Committee and
the United Kingdom Coordinating Committee for Cancer
Research’s Ad Hoc Committee on the Welfare of Animals in
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Acknowledgment. Wethank Cancer ResearchUK(Grants
C309/A8274 and C107/A10433), the Wellcome Trust, the
Instituteof CancerResearch, andthe Isle ofMan Anti-Cancer
Association for funding. We are grateful to Neus Cantarino,
Meirion Richards, and Dr. Amin Mirza for providing tech-
nical assistance and to Professors Julian Blagg and Paul
Workman for suggestions, support, and helpful comments.
Supporting Information Available: Synthetic details and
characterization data for 4a-f, 5a-o, 1a-j,l,n,o, and 2a-l,n,
o,r-t,v-z,aa-af. This material is available free of charge via the
Internet at http://pubs.acs.org.
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