Pyridoimidazolones as novel potent inhibitors of v-Raf murine sarcoma viral oncogene homologue B1 (BRAF)

Article abstract of DOI:10.1021/jm801509w

BRAF is a serine/threonine kinase that is mutated in a range of cancers, including 50-70% of melanomas, and has been validated as a therapeutic target. We have designed and synthesized mutant BRAF inhibitors containing pyridoimidazolone as a new hinge-binding scaffold. Compounds have been obtained which have low nanomolar potency for mutant BRAF (12 nM for compound 5i) and low micromolar cellular potency against a mutant BRAF melanoma cell line, WM266.4. The series benefits from very low metabolism, and pharmacokinetics (PK) that can be modulated by methylation of the NH groups of the imidazolone, resulting in compounds with fewer H-donors and a better PK profile. These compounds have great potential in the treatment of mutant BRAF melanomas.

Full text of DOI:10.1021/jm801509w

J. Med. Chem. 2009, 52, 2255–2264
2255
Pyridoimidazolones as Novel Potent Inhibitors of v-Raf Murine Sarcoma Viral Oncogene
Homologue B1 (BRAF)
Dan Niculescu-Duvaz,† Catherine Gaulon,† Harmen P. Dijkstra,† Ion Niculescu-Duvaz,† Alfonso Zambon,† Delphine Me´nard,†
Bart M. J. M. Suijkerbuijk,† Arnaud Nourry,† Lawrence Davies,† Helen Manne,† Frank Friedlos,† Lesley Ogilvie,†
Douglas Hedley,† Steven Whittaker,‡ Ruth Kirk,‡ Adrian Gill,§ Richard D. Taylor,§ Florence I. Raynaud,† Javier Moreno-Farre,†
Richard Marais,‡ and Caroline J. Springer*,†
Cancer Research UK Centre for Cancer Therapeutics, The Institute of Cancer Research, 15 Cotswold Road, Sutton, Surrey SM2 5NG, U.K.,
Cancer Research UK Centre for Cell and Molecular Biology, The Institute of Cancer Research, 237 Fulham Road, London SW3 6JB, U.K.,
and Astex Therapeutics Ltd., Registered in England at 436 Cambridge Science Park, Cambridge CB4 0QA, U.K.
ReceiVed December 1, 2008
BRAF is a serine/threonine kinase that is mutated in a range of cancers, including 50-70% of melanomas,
and has been validated as a therapeutic target. We have designed and synthesized mutant BRAF inhibitors
containing pyridoimidazolone as a new hinge-binding scaffold. Compounds have been obtained which have
low nanomolar potency for mutant BRAF (12 nM for compound 5i) and low micromolar cellular potency
against a mutant BRAF melanoma cell line, WM266.4. The series benefits from very low metabolism, and
pharmacokinetics (PK) that can be modulated by methylation of the NH groups of the imidazolone, resulting
in compounds with fewer H-donors and a better PK profile. These compounds have great potential in the
treatment of mutant BRAF melanomas.
Introduction
28113 for BRAF and sorafenib for CRAF (Figure 1). Sorafenib
(launched for renal cell carcinoma [RCC]14) failed to show
therapeutic activity in the treatment of malignant melanoma,15
presumably because of its poor cellular activity against
V600EBRAF. RAF265, PLX4032 and XL-281 are in phase 1
clinical trials, the first two for melanoma, but no results have
been reported yet. Therefore, V600EBRAF inhibitors are of great
topical interest currently.
We initiated a new series of BRAF inhibitors to improve upon
sorafenib in terms of both V600EBRAF potency and cell growth
inhibition. For this purpose, we have discovered a new hinge-
binding group, pyridoimidazolone, as a key structural element
of our new series of V600EBRAF inhibitors.
Most kinase inhibitors bind into the adenosine triphosphate
(ATP) pocket and form interactions with the hinge separating
the two lobes of the kinase domain. The hinge binds to the
adenine group of ATP, through generation of a network of
H-bonds. This motif has been targeted in the design of most
kinase inhibitors, which contain hydrogen bond accepting and
donating groups that can form noncovalent interactions with
the hinge essential for the potency of inhibition. Many hetero-
cyclic groups have been used as hinge-binding moieties to
generate interactions with the backbone of the hinge, for
example a 2-carboxamidopyridine group in sorafenib or a
4-aminoquinazoline group in gefitinib and erlotinib.16 We
implemented the bicyclic pyridoimidazolones as a novel hinge-
binding group in the design of new BRAF inhibitors. The
imidazolone moiety was envisaged as having two purposes:
affording two interactions with the hinge backbone, one due to
the pyridine nitrogen H-bond acceptor and the other Via the
3-NH donor of the imidazolone. In addition, projecting a
hydrophilic “edge” of the inhibitors toward the aqueous solvent
was envisaged as favorable. The imidazolone ring is also rigid,
and reduces the number of rotatable bonds compared to the
methylcarboxamide group of sorafenib. Reducing the number
of rotatable bonds can be desirable for improved PK properties.17
We combined our pyridoimidazolone hinge binding group
with a pharmacophore known to engender type II kinase
RAFa (so named from the v-raf oncogene that causes rapidly
growing fibrosarcomas in mice1) is a serine/threonine kinase
that is part of the mitogen-activated protein kinase (MAPK)
signal transduction cascade: RAF-MAPK/ERK kinase (MEK)-
extracellular regulated kinase (ERK) that regulates cell growth,
differentiation, and proliferation in response to external stimuli
such as growth factors.2 Following RAF activation, the cascade
is stimulated and MEK and ERK are sequentially phosphory-
lated and activated. ERK then regulates cell function by
phosphorylating more than 50 substrates in the cytosol and the
nucleus.3 There are three members of the RAF family (ARAF,
BRAF and CRAF). BRAF is mutated in approximately 2% of
human cancers, with particularly high frequencies in melanoma
(50-70%), ovarian (35%), thyroid (30%), and colorectal (10%)
cancers.4,5 The most common mutation (90%) is a valine for
glutamic acid substitution at position 600, termed V600EBRAF.
V600EBRAF shows a permanent 500-fold elevated kinase activ-
ity.6 In addition, V600EBRAF stimulates constitutive ERK signal-
ing in mammalian cells4,7,8 and it transforms established cells,
allowing them to grow as tumors in nude mice.4,7 In melanoma,
V600EBRAF stimulates proliferation and survival; inhibition of
BRAF signaling with siRNA inhibits proliferation and induces
apoptosis.9,10 These data validate BRAF as an important and
exciting therapeutic target in human cancer. Inhibitors of RAF
have been developed, such as PLX4032,11 RAF26512 and XL-
* To whom correspondence should be addressed. Phone: +44 20 8722
4214. Fax: +44 20 8722 4046. E-mail: caroline.springer@icr.ac.uk.
†
Cancer Research UK Centre for Cancer Therapeutics, The Institute of
Cancer Research.
‡
Cancer Research UK Centre for Cell and Molecular Biology, The
Institute of Cancer Research.
§
Astex Therapeutics Ltd.
a Abbreviations: RAF, rapidly growing fibrosarcoma; BRAF, V-RAF
murine sarcoma viral oncogene homologue B1; MAPK, mitogen-activated
protein kinase; MEK, MAPK/ERK kinase; ERK, extracellular regulated
kinase; PK, pharmacokinetics; Boc, tert-butoxycarbonyl; DMF, dimethyl-
formamide; TFA, trifluoroacetic acid; THF, tetrahydrofuran; DCM, dichlo-
romethane; iv, intravenous; SAR, structure-activity relationship.
10.1021/jm801509w CCC: $40.75
2009 American Chemical Society
Published on Web 03/26/2009

2256 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8
Niculescu-DuVaz et al.
Figure 1. Structures of RAF inhibitors sorafenib, PLX4032 and RAF265.
interacting with Glu501 Via both NH groups.19 We also explored
several alternatives, such as amides, sulfonamides, thioureas and
methyleneamides.
Two synthetic routes were developed. In route I, described
in Scheme 1, ring C is first coupled to 4-(4-aminophenoxy)-3-
nitropyridin-2-amine 2, whereafter the imidazolone is formed
in the last step. This route was used to obtain compound 5a. In
route II, the imidazolone is formed initially to generate the
complete ring A-ring B part of the inhibitors (the common
intermediate, for example compound 9 in Scheme 2) containing
an amino group that can be reacted in the last step with a variety
of electrophiles. This second route is more suitable to explore
quickly a range of different C-rings and B-C linkers in a
parallel fashion, since there is just one step from the common
intermediate to the final product. This method was adopted for
the synthesis of the rest of the BRAF inhibitors described here
(5b-x, 26, 27a-f, 28a-e, 29a-g).
Figure 2. General structure of the pyridoimidazolone-based BRAF
The starting material for both routes is 2-amino-4-chloro-3-
nitropyridine 1. The chlorine atom can be substituted selectively
with the 4-aminophenolate, whereby the phenolate is preferred
to the amine as nucleophile, to afford 4-(4-aminophenoxy)-3-
nitropyridin-2-amine 2.
Route 1. The amino group on the phenyl B-ring is more
nucleophilic than the one in position 2 of the pyridine (which
is deactivated both by the 3-nitro group and the pyridyl ring),
and can be reacted selectively with 4-chloro-3-trifluorometh-
ylphenyl isocyanate to afford urea 3. Reduction of the nitro
group and cyclization of the diamino compound 4 with
triphosgene afforded the target product 5a (Scheme 1).
Route 2. For the synthesis of the common intermediate 9,
4-(4-aminophenoxy)-3-nitro-2-aminopyridine 2 is protected
selectively with tert-butoxycarbonyl (Boc) at the 4-aminophe-
noxy group, the nitro group is subsequently reduced and the
resulting diamino compound 7 is reacted with phosgene to afford
compound 8. Intermediate 6 can also be obtained by direct
coupling of 2-amino-4-chloro-3-nitropyridine 1 with 4-N-Boc-
aminophenol. Subsequent deprotection with trifluoroacetic acid
(TFA) generates common intermediate 9 (Scheme 2). The Boc
protection-deprotection is required to avoid side reactions of
the 4-amino group on ring B with phosgene.
We wished to investigate the importance of the two NH
groups of the imidazolone, with N3 being expected to be
involved in hinge-binding. We hypothesized that substitution
of N1 or N3 or both nitrogen atoms of the imidazolone with a
methyl or ethyl group would be useful in answering this
question. Also, alkylation of one or both imidazolone NH groups
would remove one or two H-donors, yielding compounds that
would be more orally bioavailable. For this purpose, the
intermediates 16 (Scheme 3), 20, 20a (Scheme 4) and 21
(Scheme 5) were synthesized.
inhibitors described in this paper.
inhibitors. Type II kinase inhibitors occupy a hydrophobic
pocket adjacent to the ATP pocket and the gatekeeper residue,
created by the displacement of the DFG loop. Type II inhibitors
bind to a DFG-out or inactive conformation.18 Sorafenib is an
example of an inhibitor of the DFG-out conformation. Our
cocrystal structure of sorafenib with BRAF supports this binding
mode.19 The pharmacophore of type II kinase inhibitors consists
of a hinge-binding moiety linked to an aromatic/heteroaromatic
ring in the DFG-out pocket (BPII), a hydrogen bond donor/
acceptor and usually a lipophilic group in the pockets created
by the displacement of the DFG loop (BPIII and BPIV).18
The general structure of the inhibitors shown in Figure 2 fits
this model: the pyridoimidazolone hinge-binder (ring A) is
connected through an oxygen or a sulfur atom to the phenyl
ring B that is expected to occupy the DFG-out pocket (BPII).
Rings B and C are connected Via the H-donor/acceptor linker
B-C. Urea is a linker frequently used in type II inhibitors, for
example in sorafenib where it interacts with Glu501 and the
backbone of Asp594. Different H-donor/acceptor linkers
were also explored, e.g., thiourea, amide and sulfonamide. The
substituted phenyl ring C occupies the BPIV pocket in the
generalized kinase model, with the lipophilic substituent target-
ing the BPIII pocket.
Chemistry
The structure of the inhibitors can be rationalized to 3
domains: the novel bicyclic hinge-binding pyridoimidazolone
(ring A), the phenyl ring in the DFG-out pocket (ring B) and
the aromatic group occupying the BPIV pocket (ring C) (Figure
2). Rings A and B are interconnected through either an O or an
S. The linker between rings B and C plays two critical roles, in
binding to Glu501 and in positioning the C ring in the DFG
pocket. We utilized the 1,3-disubstituted urea pharmacophore
as the B-C linker, since it was described as being effective in
The synthesis of common intermediate 16, methylated at the
N3 of the imidazolone, is shown in Scheme 3. The methylated
starting material 4-chloro-3-nitro-2-methylaminopyridine 12 is

Pyridoimidazolones as NoVel Potent Inhibitors of BRAF
Scheme 1. Synthetic Route I: Synthesis of Compound 5aa
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8 2257
a Reagents and conditions: (a) 4-amino-phenol, t-BuOK, DMF, 80 °C; (b) 4-chloro-3-trifluoromethylphenyl isocyanate, THF; (c) H2, Pd/C, EtOH; (d)
triphosgene, NEt3, THF.
Scheme 2. Synthesis of Unsubstituted Imidazolone Intermediate
MeI produced a complex mixture of products with a variable
degree of methylation. Therefore, a more selective method was
employed. Dimethylated common intermediate 20a was obtained
by a similar method to intermediate 20, starting from the
monomethylated intermediate 14 (Scheme 4).
9a
Interestingly, intermediate 21 with N1 and N3 dialkylated
with ethyl groups was obtained directly from intermediate 9 by
deprotonation with NaH and alkylation with ethyl iodide
(Scheme 5). In this case, alkylation proceeded selectively
following the deprotonation of the more acidic NH groups of
the imidazolone.
Common intermediate 25 with a sulfur linker instead of
oxygen between rings A and B can be synthesized in a similar
manner to 9, replacing the 4-N-Boc-aminophenol with 4-N-Boc-
aminothiophenol, as illustrated in Scheme 6.
a Reagents and conditions: (a) Boc2O, THF; (b) H2, Pd/C, EtOH; (c)
COCl2, pyridine, THF; (d) TFA.
Ureas with a range of aromatic rings C were obtained from
the reaction of common intermediates 9, 16, 20, 20a, 21 or 25
with aromatic isocyanates. For ureas 5d, 5h, 5i and 5j, the
respective isocyanates were unavailable commercially. These
compounds were obtained by reacting the common intermediates
with the phenyl carbamates of the respective amines instead.
Thiourea 26 was obtained from the common intermediate 9 and
the corresponding isothiocyanate. Aromatic amides (27a-f) and
methyleneamides (28a-e) were obtained from the common
intermediates and the respective acid chlorides. Sulfonamides
(29a-g) were also obtained from common intermediates and
sulfonyl chlorides. The general synthesis of all the final
compounds except 5a from common intermediates is presented
in Scheme 7.
obtained from 1 in a three-step procedure: protection of the
amino group with Boc, methylation with methyl iodide (MeI)
and sodium hydride (NaH) and Boc deprotection. Although
longer than the single step direct methylation of 4-chloro-3-
nitro-2-aminopyridine 1, this method has an improved yield
(>90% overall) and results in fewer impurities. Aromatic
nucleophilic substitution of the 4-chloro intermediate 12 with
4-N-Boc-aminophenolate affords intermediate 13, which is
converted to common intermediate 16 by a method similar to
that for intermediate 9 (Scheme 4).
Acylation of the diamino intermediate 7 with ethyl chloro-
formate occurs at the more nucleophilic 3-amino group on the
pyridyl ring (corresponding to N1 of the imidazolone). In order
to allow the selective methylation of N1, the Boc protecting
group was removed with TFA affording intermediate 18, which
contains three amino groups, with only the 3-pyridyl nitrogen
(the future N1 of the imidazolone) being modified as ethyl
carbamate. Reaction with 1 equiv of NaH removes the more
acidic proton of the carbamate, converting this nitrogen into a
stronger nucleophile than the other two amino groups. Addition
of MeI therefore selectively substitutes this position to afford
intermediate 19. Upon heating 19 in a microwave reactor with
sodium ethoxide, the carbamate cyclizes to imidazolone 20
(Scheme 4), methylated at the N1 of the imidazolone.
Results and Discussion
Our goal was to identify potent inhibitors of BRAF based
on our novel hinge-binding scaffold. The importance of the two
imidazolone NH groups for the binding affinity and the linker
between rings A and B was investigated, as was the structure-
activity relationship (SAR) around ring C and the linker between
rings B and C. Accordingly, the biological activities of
compounds 5a-x, 26, 27a-f, 28a-e, 29a-g were determined
in 2 separate assays: (1) inhibition of V600EBRAF kinase activity
(IC50 BRAF), and (2) cell growth inhibition of mutant BRAF
WM266.4 melanoma cells measured by sulforhodamine-B (GI50
SRB).
Attempts to obtain the imidazolone dimethylated at both N1
and N3 by direct methylation of intermediate 9 with NaH and

2258 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8
Scheme 3. Synthesis of N3-Methylated Intermediate 16a
Niculescu-DuVaz et al.
a Reagents and conditions: (a) Boc2O, NaH, THF; (b) MeI, NaH, THF; (c) TFA; (d) 4-(N-Boc-amino)-phenol, t-BuOK, DMF, 70 °C; (e) H2, Pd/C, EtOH;
(f) triphosgene, pyridine, THF; (g) TFA.
Scheme 4. Synthesis of N1-Methylated and N1,N3-Dimethylated
Intermediates 20 and 20aa
5t showing a less than 3-fold increase in BRAF IC50 and no
significant difference in its SRB GI50 compared to its nonm-
ethylated and monomethylated counterparts 5a, 5o and 5q. In
our model, the NH-3 of the imidazolone is facing the hinge
backbone. However, there is apparently enough space to fit a
methyl group and the NH-3 does not appear to be essential for
hinge-binding. Unsurprisingly, compound 5u, substituted with
two bulkier ethyl groups at both N1 and N3 of the imidazolone,
is 10-fold less active.
SAR of A-B Linker. A replacement of the oxygen linker
between rings A and B with sulfur (compounds 5v-x) does
not have any effect on the BRAF IC50, but results in a better
cell growth inhibition. This is most probably due to an increased
cell membrane permeability because of its increased lipophilicity.
SAR of Ring C. The initial structure of the inhibitors contains
a urea linker between rings B and C, since 1,3-diarylureas have
been reported to be inhibitors of kinases (e.g., in sorafenib),
while the structure of the aryl ring C was varied. A relatively
large and lipophilic group in the 3-position (with respect to the
urea substituent) is an absolute requirement for activity. Such
a group can be CF3 (as in 5a, 5d,e, 5i-k, 5n,o, 5q, 5s-x),
tert-Bu (in 5p, 5r) or OCF3 (in 5h). The BRAF IC50 shows a
difference greater than 3 orders of magnitude between 5a and
5c, where the only difference is the meta CF3 group. In
compound 5m, the morpholine presumably plays the role of
the lipophilic group, since it is five times more potent than the
corresponding compound 5l, which lacks the morpholine group.
Compounds without the lipophilic meta group (5b, 5c) are
completely devoid of activity. According to modeling, the CF3
group fills the lipophilic pocket vacated when the DFG loop is
out, corresponding to the BPIII pocket in the generalized kinase
model.18,19 The 3-position is compulsory for the CF3 group in
order to maintain activity (5f with CF3 at position 2 and 5g
with CF3 at position 4 are inactive). The second substituent on
the aryl ring can contribute to an increased cellular activity.
Compound 5a has a GI50 (SRB) of 4.2 mM, whereas a similar
compound without the Cl (5e) has no measurable cellular
activity, despite its good potency in the BRAF assay. Similarly,
a fluorine atom or a methoxy group at the 6-position leads to
low micromolar cell growth inhibition (5i-k, 5s). A chlorine
atom at position 5, however, is not favorable for the activity
(5d is 5-fold less potent than 5a). The improved cellular activity
of these compounds bearing a second or third substituent on
ring C could be caused by improved cell permeability, due to
increased lipophilicity (for the Cl substituent) or to the removal
of an H-donor, the NH of the urea, by intramolecular H-bonding
with the fluoro or methoxy substituent at the 6-position.
a Reagents and conditions: (a) EtOCOCl, pyridine, THF; (b) TFA; (c)
MeI, NaH, THF; (d) EtONa, EtOH, microwave.
Scheme 5. Synthesis of Diethylated Intermediate 21a
a Reagents and conditions: (a) EtI, NaH, DMF.
The IC50 and GI50 results are presented in Tables 1, 2 and 3.
SAR of Ring A: Imidazolone. Compound 5a is a good
inhibitor of mutant BRAF and exhibits a 2-fold improvement
over sorafenib on the BRAF IC50 as well as a modest
improvement in the cellular assay GI50. Many of the synthesized
compounds (see Table 1) with this new scaffold show potent
BRAF inhibition and low micromolar cell growth inhibition,
attesting to the ability of the pyridoimidazolone to interact with
the hinge of mutant BRAF kinase.
Methylation of the N1 position of the imidazolone is tolerated,
with no difference in either the BRAF IC50 or the SRB GI50
(5o vs 5a). Since this nitrogen is likely to face the solvent, this
result is expected. Surprisingly, the methylation of the N3
position also shows little effect, with compounds 5q and 5s
being only slightly less potent or equipotent to their nonmethy-
lated counterparts 5a and 5k, respectively. Dimethylation at both
N1 and N3 of the imidazolone is also tolerated, with compound

Pyridoimidazolones as NoVel Potent Inhibitors of BRAF
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8 2259
Scheme 6. Synthesis of Intermediate 25 with a Sulfur A-B Linkera
a Reagents and conditions: (a) 4-(N-Boc-amino)-benzenethiol, NaH, DMSO; (b) H2, Raney-Ni, EtOH/AcOEt; (c) carbonyldiimidazole, THF; (d) TFA.
Scheme 7. Synthetic Route II: Synthesis of Final Compounds
from Common Intermediates
Metabolism and PK. Four compounds were assessed for
their metabolic liabilities in mouse liver microsomes (MLMs).
The compounds assessed and their percentage metabolism after
30 min incubation with MLMs are 5a (0%), 5o (11%), 5j (8%),
29b (5%). Thus the metabolism of all the compounds assessed
is low (<20%). PK assessments were performed on compounds
5a, 5o and 5q following administration (2 mg/kg intravenous
(iv)) to nude mice bearing human mutant BRAF melanoma
xenografts (WM266.4). The data are summarized in Table 4.
N-Methylation of the imidazolones greatly improves all the PK
parameters (AUC, Cmax and half-life are increased, and the
clearance is reduced to levels well below that of liver blood
flow).
Conclusion
A new series of potent BRAF inhibitors bearing a novel
pyridoimidazolone hinge-binding group has been designed.
Their synthesis, SAR and in ViVo PK are reported here. Five
compounds (5a, 5i, 5j, 5v and 5w) are more potent than the
licensed drug sorafenib (that was designed as a RAF inhibitor)
in two assays for mutant BRAF (BRAF IC50 and SRB GI50).
Up to a 4-fold improvement in BRAF IC50 is achieved, with a
potency as low as 12 nM for compound 5i. We hypothesize
that an improvement in cellular activity compared to sorafenib
of the compounds reported herein would provide beneficial
therapeutic efficacy in mutant BRAF- tumors including mela-
noma. Compound 5v is 4-fold more potent than sorafenib in
inhibiting WM266.4 melanoma cell growth. Overall, this novel
series is promising, with excellent in Vitro potency both against
the isolated mutant BRAF enzyme and in mutant BRAF driven
cells. The series has low metabolism and a good PK profile
that can be modulated by structural changes, warranting its
assessment in ViVo to determine whether these characteristics
translate into antitumor efficacy.
Experimental Section
Materials and Methods. All starting materials, reagents and
solvents for reactions were reagent grade and used as purchased.
Chromatography solvents were HPLC grade and were used without
further purification. Reactions were monitored 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 a temperature of 22 °C using the following solvent systems:
solvent A, methanol; solvent B, 0.1% formic acid in water at a
flow rate of 1 mL/min. Gradient starting with 10% A/90% B from
0 to 0.5 min, 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
SAR of Linker B-C. Replacement of the urea group with
an amide or sulfonamide group results in poor activity (27a-f,
Table 2, and 29a-g, Table 3). The only compound with
submicromolar activity is sulfonamide 29b with a naked phenyl
group; any attempt to substitute on the phenyl ring leads to
compounds with lower activity. Compound 26 is still active,
but less so than the equivalent urea 5a, in both mutant BRAF
assays, therefore the thiourea provides no advantage over the
urea linker. The better activity of the urea group compared to
the other linkers reported could be due to the interactions of
one or both of its NH-donors with Glu501,19 or because it is
orienting ring C and its 3-substituent on the correct vector
toward the DFG pocket, or both. Interestingly, the methylenea-
mides 28d and 28e, which are the urea isostere equivalents of
5h and 5e respectively, and would present the same conforma-
tion for ring C, have no activity on BRAF. This supports the
suggestion that at least the urea NH vicinal to ring C is crucial
for activity.

2260 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8
Table 1. BRAF Inhibitors with Urea or Thiourea Linker B-C
Niculescu-DuVaz et al.

Pyridoimidazolones as NoVel Potent Inhibitors of BRAF
Table 1. Continued
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8 2261
nm, and ionization was positive or negative ion electrospray.
Samples were supplied as 1 mg/mL in DMSO or methanol with 3
µL injected on a partial loop fill. The purity of the final compounds
was determined by HPLC as described above and is 95% or higher
unless specified otherwise. NMR spectra were recorded in DMSO-
d6 on a Bruker DPX 250 MHz or a Bruker Advance 500 MHz
spectrometer.
1-(4-(2,3-Dihydro-2-oxo-1H-imidazo[4,5-b]pyridin-7-yl-oxy)-
phenyl)-3-phenyl-urea (5b). 7-(4-Amino-phenyloxy)-2,3-dihydro-
2-oxo-1H-imidazo[4,5-b]pyridine (9) (65 mg, 0.27 mmol) was
suspended in dry pyridine (3 mL) and heated at 50 °C. Phenyl
isocyanate (30 µL, 0.28 mmol) was added; the reaction mixture
was heated at reflux for 2 h. After cooling at room temperature,
DCM (20 mL) was added, and the precipitate formed was filtered
and washed with more DCM, to afford the title compound (67 mg,
1-(4-(2,3-Dihydro-2-oxo-1H-imidazo[4,5-b]pyridin-7-yl-oxy)-
phenyl)-3-(4-chloro-3-(trifluoromethyl)phenyl)urea (5a). Triph-
osgene (10 mg, 0.03 mmol) was added to a solution of 1-(4-(2,3-
d i a m i n o p y r i d i n - 4 - y l - o x y ) p h e n y l ) - 3 - ( 4 - c h l o r o - 3 -
(trifluoromethyl)phenyl)urea (4) (44 mg, 0.1 mmol) and triethylamine
(30 µL, 0.21 mmol) in dry THF (1 mL), cooled at 0 °C. The mixture
was stirred at 0 °C for 15 min, and at room temperature for 1 h.
The precipitate formed was filtered off, and the filtrate evaporated.
The residue was purified by column chromatography (eluent
gradient AcOEt to THF:AcOEt 1:1), to afford the title compound
(11 mg, 24%). 1H NMR (δ, ppm, DMSO-d6): 6.35 (d, 1H, HPy,5, J
) 6.7 Hz), 7.14 (d, 2H, Harom,Ph,3+5, J ) 10.0 Hz), 7.54 (d, 2H,
1
69%). H NMR (δ, ppm, DMSO-d6): 6.34 (d, 1H, HPy,5, J ) 7.5
Hz), 6.98 (t, 1H, Harom,Ph′,4, J ) 7.5 Hz), 7.13 (d, 2H, Harom,Ph,3+5, J
) 8.8 Hz), 7.29 (t, 2H, Harom,Ph′,3+5), 7.46 (d, 2H, Harom,Ph′,2+6), 7.53
(d, 2H, Harom,Ph,2+6), 7.76 (d, 1H, HPy,6), 8.67 (s, 1H, NHurea,1), 8.76
(s, 1H, NHurea,3), 11.19 (s, 1H, NHPy3), 11.35 (s, 1H, NHPy2).
LC-MS: m/z 362 (M+ + H, 100). HRMS: m/z calcd for
C19H15N5O3 [M + H]+, 362.1248; found, 362.1241.
1-(4-Chlorophenyl)-3-(4-(2-oxo-2,3-dihydro-1H-imidazo[4,5-
b]pyridin-7-yl-oxy)phenyl)urea (5c). A mixture of p-chlorophe-
nylisocyanate (24.5 mg, 0.16 mmol) and 7-(4-aminophenoxy)-1H-
imidazo[4,5-b]pyridin-2(3H)-one (9) (30 mg, 0.12 mmol) in
anhydrous THF (1.5 mL) was stirred at room temperature for 14 h.
The solvent was evaporated, and the solid residue was washed with
Et2O to afford the title compound (44 mg, 91%) as an off-white
Harom,Ph,2+6), 7.64 (bs, 2H, Harom′), 7.77 (d, 1H, HPy,6), 8.12 (s, 1H,
Harom′), 8.94 (s, 1H, NHurea,1), 9.18 (s, 1H, NHurea,3), 11.19 (s, 1H,
NHPy3), 11.36 (s, 1H, NHPy2). LC-MS: m/z 463 (M, 100). HRMS:
m/z [M + H]+ calcd for C20H14ClF3N5O3, 464.0737; found,
464.0739.
1
solid. H NMR (δ, ppm, DMSO-d6): 6.33 (d, 1H, HPy,5, J ) 6.0
Hz), 7.12 (d, 2H, Harom,Ph,3+5, J ) 8.9 Hz), 7.32 (d, 2H, Harom,Ph,2+6
,

2262 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8
Table 2. BRAF Inhibitors with Amide Linker B-C
Niculescu-DuVaz et al.
Table 3. BRAF Inhibitors with Sulfonamide Linker B-C
Table 4. Pharmacokinetics Data for Selected Inhibitors
compound tissue Cmax (nmol/L) AUC (h ·nmol/L) T1/2 (h) CL (L/h)
5a
5o
5q
plasma
tumor
plasma
tumor
1057
207
2454
1152
3230
8078
718
468
3256
4504
16382
9461
0.80
1.24
2.58
9.72
4.72
4.35
0.117
0.160
0.017
0.006
0.050
0.009
plasma
musclea
a Muscle was used as a surrogate for tumor.
1
85%). H NMR (δ, ppm, DMSO-d6): 6.32 (d, 1H, HPy,5, J ) 6.0
Hz), 7.12 (d, 2H, Harom,Ph,3+5, J ) 9.0 Hz), 7.39 (bs, 1H, Harom′,4),
7.55 (d, 2H, Harom,Ph,2+6, J ) 9.0 Hz), 7.75 (d, 1H, HPy,6, J ) 6.0
Hz), 7.84 (m, 2H, Harom′,2+6), 9.27 (bs, 1H, NHurea), 9.51 (bs, 1H,
NHurea), 11.21 (bs, 1H, NHPy3), 11.38 (bs, 1H, NHPy2). LC-MS:
m/z 464 (M + H, 100). HRMS: m/z [M + H]+ calcd for
C20H14ClF3N5O3, 464.0737; found, 464.0745.
J ) 8.9 Hz), 7.50 (∼t, 4H, Harom′), 7.75 (d, 1H, HPy,6, J ) 6.0 Hz),
8.82 (bs, 1H, NHurea), 8.85 (bs, 1H, NHurea), 11.17 (bs, 1H, NHPy3),
11.34 (bs, 1H, NHPy2). LC-MS: m/z 396 (M+ + H, 100). HRMS:
m/z [M + H]+ calcd for C19H15ClN5O3, 396.0863; found, 396.0866.
Using the same procedure the following compounds were
synthesized: 5e-g, 5k-x.
1-(3-Chloro-5-(trifluoromethyl)phenyl)-3-(4-(2-oxo-2,3-dihy-
dro-1H-imidazo[4,5-b] pyridin-7-yl-oxy)phenyl)urea (5d). A
mixture of phenyl 3-chloro-5-(trifluoromethyl)phenyl-carbamate (50
mg, 0.15 mmol) and 7-(4-aminophenoxy)-1H-imidazo[4,5-b]pyri-
din-2(3H)-one (9) (30 mg, 0.12 mmol) in anhydrous DMSO (1 mL)
was stirred at 85 °C for 2 h. After cooling, the solution was diluted
with AcOEt (10 mL), washed with H2O (2 × 10 mL) and brine
(10 mL). The solvent was evaporated and the residue was washed
with Et2O to afford the title compound as a white solid (48 mg,
Using the same procedure the following compounds were
synthesized: 5h, 5i,j.
N-(4-(2-Oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-7-yl-oxy)-
phenyl)benzamide (27a). 7-(4-Aminophenoxy)-2,3-dihydro-2-oxo-
1H-imidazo[4,5-b]pyridine (9) (30 mg, 0.13 mmol) and triethy-
lamine (22.3 µL, 0.16 mmol) were mixed in dry THF (3 mL), and
benzoyl chloride (19.0 µL, 0.16 mmol) was added. This mixture
was heated to reflux for 20 h, and subsequently the solvent was
evaporated under vacuum. The obtained residue was dissolved in
acetone (2 mL), and upon addition of H2O a solid precipitated.
This solid was collected, washed with water (2 × 2 mL) and Et2O
(2 × 2 mL) and dried. The title compound was obtained as a light
brown solid (44 mg, 80%). 1H NMR (δ, ppm, DMSO-d6): 6.39 (d,
1H, HPy,5, J ) 5.0 Hz), 7.19 (d, 2H, Harom,Ph,3+5, J ) 7.5 Hz),
7.51-7.63 (m, 3H, Harom,Ph′,3+4+5), 7.78 (d, 1H, HPy,6), 7.86 (d, 2H,

Pyridoimidazolones as NoVel Potent Inhibitors of BRAF
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8 2263
Harom,Ph,2+6), 7.97 (d, 2H, Harom,Ph′,2+6), 10.36 (s, 1H, NHamide), 11.22
(s, 1H, NHPy3), 11.39 (s, 1H, NHPy2). LC-MS: m/z 347 (M + H,
100). HRMS: m/z [M + Na]+ calcd for C19H14N4O3, 369.0958;
found, 369.0954.
Using the same methodology the following compounds were
synthesized: 27b-d, 27f, 28a-c.
Muscle, tumor and plasma samples were snap frozen in liquid
nitrogen and then stored at -70 °C prior to analysis. All procedures
involving animals were performed in accordance with national
Home Office regulations under the Animals (Scientific Procedures)
Act 1986 and within guidelines set out by the Institute’s Animal
Ethics Committee and the United Kingdom Coordinating Com-
mittee for Cancer Research’s ad hoc Committee on the Welfare of
Animals in Experimental Neoplasia.23 Plasma and tissue extractions
were performed as previously described.24 Analysis was also
performed by multiple reaction monitoring of the compounds and
the internal standard roscovitine on the triple quadrupole with an
external calibration and 3 quality controls at levels between 10 and
10000 nM. PK parameters were derived from WinNonLin Non-
Compartmental analysis (Pharsight, version 3.2).
N-(4-(2-Oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-7-yl-oxy)-
phenyl)-3-(trifluoromethoxy)benzamide (27e). To 7-(4-aminophe-
noxy)-1H-imidazo [4,5-b]pyridin-2(3H)-one (9) (50 mg, 0.21 mmol)
in anhydrous dioxane (7 mL) pyridine (38 µL, 1.3 equiv) and
3-trifluoromethyloxy-benzoyl chloride (56 µL, 0.25 mmol) were
added under stirring and nitrogen atmosphere and heated for 20 h
at 80 °C (block). The solvent was evaporated under vacuum and
the solid residue dissolved in 10 mL of AcOEt and washed with
brine (2 × 10 mL), solution of citric acid (10 mL) and brine (10
mL) again. The organic solution was evaporated under vacuum and
the solid residue recrystallized from AcOEt when the title compound
(18 mg, 20%, purity 91%) was obtained. 1H NMR (δ, ppm, DMSO-
d6): 6.39 (d, 1H, HPy,5, J ) 5.9 Hz), 7.19 (d, 2H, Harom3+5, J ) 9.0
Hz), 7.61-7.63 (m, 1H, Harom), 7.70 (t, 1H, Harom, J ) 8.0 Hz),
7.78 (d, 1H, HPy,6, J ) 5.9 Hz), 7.84 (d, 2H, Harom,Ph,2+6, J ) 9.0
Hz), 7.91 (s, 1H, Harom), 8.02 (d, 1H, Harom, J ) 7.9 Hz), 10.45 (s,
1H, NHamide), 11.17 (s, 1H, NHPy2), 11.43 (s, 1H, NHPy3). LC-MS:
m/z 431.2 (M+, 100). HRMS: (M+) calcd for C21H26N5O3, 431.0967;
found, 431.0967.
Acknowledgment. This work is supported by Cancer
Research UK (refs: C309/A2187 and C107/A10433), The
Wellcome Trust, The Institute of Cancer Research and The Isle
of Man Anti-Cancer Association. We acknowledge NHS funding
to the NIHR Biomedical Research Centre. We thank Professors
Paul Workman and Julian Blagg for helpful discussions and
support.
Supporting Information Available: Experimental details of the
synthesis and analytical characterization of compounds 5e-x, 26,
27b-d, 27f, 28a-e, 29b-g, and intermediates 2-4, 6-25
described in this paper and purity data for the final compounds.
This material is available free of charge via the Internet at http://
pubs.acs.org.
Using the same procedure the following compounds were
synthesized: 28d,e.
N-(4-(2-Oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-7-yl-oxy)-
phenyl)-3-(trifluoromethyl)-4-chloro-benzenesulfonamide (29a).
7-(4-Aminophenoxy)-2,3-dihydro-2-oxo-1H-imidazo[4,5-b]pyri-
dine (9) (30 mg, 0.13 mmol) was suspended in dry pyridine (3
mL), and 4-chloro-3-(trifluoromethyl)benzene sulfonyl chloride
(44.4 mg, 0.16 mmol) was added. The resulting solution was stirred
at room temperature for 20 h and subsequently the solvent was
evaporated under vacuum. The obtained residue was dissolved in
acetone (4 mL) and upon addition of H2O a solid precipitated. The
solid was collected, washed with H2O (2 × 2 mL) and Et2O (2 ×
2 mL) and dried to give the title compound as an off-white solid
(44 mg, 57%). 1H NMR (δ, ppm, DMSO-d6): 6.28 (d, 1H, HPy,5, J
) 5.8 Hz), 7.12 (s, 4H, Harom,Ph), 7.75 (d, 1H, HPy,6), 7.98 (s, 2H,
Harom,Ph′), 8.05 (s, 1H, Harom,Ph′), 10.47 (s, 1H, NHSO2), 11.17 (s,
1H, NHPy3), 11.40 (s, 1H, NHPy2). LC-MS: m/z 485 [(M + H)+,
100]. HRMS: m/z [M + H]+ calcd for C19H12ClF3N4O4S, 485.0299;
found, 485.0298.
References
(1) Wellbrock, C.; Karasarides, M.; Marais, R. The RAF proteins take
center stage. Nat. ReV. Mol. Cell Biol. 2004, 5, 875–885.
(2) Chiloeches, A.; Marais, R. Mitogen-activated protein kinase cascades
as therapeutic targets in cancer. In Targets for Cancer Chemotherapy:
Transcription Factors and Other Nuclear Proteins; Humana Press Inc.:
Totowa, NJ, 2002; pp 179-206.
(3) Kolch, W. Meaningful relationships: the regulation of the Ras/Raf/
MEK/ERK pathway by protein interactions. Biochem. J. 2000, 351,
289–305.
(4) Davies, H.; Bignell, G. R.; Cox, C.; Stephen, P.; et al. Mutation of
the BRAF gene in human cancers. Nature 2002, 417, 949–954.
(5) Tuveson, D. A.; Weber, B. L.; Herlyn, M. BRAF as a potential
therapeutic target in melanoma and other malignancies. Cancer Cell
2003, 4, 95–98.
(6) Garnett, M. J.; Marais, R. Guilty as charged: B-RAF is a human
oncogene. Cancer Cell 2004, 6, 313–319.
(7) Wellbrock, C.; Ogilvie, L.; Hedley, D.; Karasarides, M.; Martin, J.;
Niculescu-Duvaz, D.; Springer, C. J.; Marais, R. V599EB-RAF is an
oncogene in melanocytes. Cancer Res. 2004, 64, 2338–2342.
(8) Ikenoue, T.; Hikiba, Y.; Kanai, F.; Tanaka, Y.; Imamura, J.; Imamura,
T.; Ohta, M.; Ijichi, H.; Tateishi, K.; Kawakami, T.; Aragaki, J.;
Matsumura, M.; Kawabe, T.; Omata, M. Functional analysis of
mutations within the kinase activation segment of B-Raf in human
colorectal tumors. Cancer Res. 2003, 63, 8132–8137.
(9) Karasarides, M.; Chiloeches, A.; Hayward, R.; Niculescu-Duvaz, D.;
Scanlon, I.; Friedlos, F.; Ogilvie, L.; Hedley, D.; Martin, J.; Marshall,
C. J.; Springer, C. J.; Marais, R. B-RAF is a therapeutic target in
melanoma. Oncogene 2004, 23, 6292–6298.
(10) Hingorani, S. R.; Jacobetz, M. A.; Robertson, G. P.; Herlyn, M.;
Tuveson, D. A. Suppression of BRAF(V599E) in human melanoma
abrogates transformation. Cancer Res. 2003, 63, 5198–5202.
(11) www.clinicaltrials.gov. NCT00405587: Safety Study of PLX4032 in
Patients With Solid Tumors.
(12) www.clinicaltrials.gov. NCT00304525: A Study to Evaluate RAF265,
an Oral Drug Administered to Subjects With Locally Advanced or
Metastatic Melanoma.
Using the same method the following compounds were synthe-
sized: 29b-g.
V600EBRAF Kinase Assay and SRB IC50 for BRAF Inhibi-
tors. These assays have been described by Niculescu-Duvaz et al.20
Compound Metabolism. Microsomal incubations evaluating
phase I metabolism were performed as previously described.21
Compounds were incubated at 10 µM concentrations 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 chro-
matography mass spectrometry with electrospray in positive ioniza-
tion mode on 2 instruments as previously described.22 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 conditions similar to
those described in Nutley et al.3
Pharmacokinetics. Forty-eight female Crl:CD1-Foxn1nu mice
bearing mutant BRAF WM266.4 tumor xenografts were utilized
for the PK analysis of compounds 5a and 5p. Twenty-four female
BALB/cAnNCrl mice at least 6 weeks of age were used for the
PK analysis of compound 5q. The mice were dosed iv (2 mg/kg,
10 mL/kg, in DMSO:Tween 20:water 10:1:89 v:v). At intervals of
5, 15, 30 min, 1, 2, 4, 6 and 24 h postdosing, 3 mice were placed
under isoflurane anesthesia and blood for plasma preparation was
taken by terminal cardiac puncture into heparinized syringes.
Femoral muscle was also sampled from the BALB/cAnNCrl mice.
Tumor samples were taken from the Crl:CD1-Foxn1nu mice.
(13) www.clinicaltrials.gov. NCT00451880: Study of XL281 in Adults With
Solid Tumors.
(14) Ahmad, T.; Eisen, T. Kinase inhibition with BAY 43-9006 in renal
cell carcinoma. Clin. Cancer Res. 2004, 10, 6388S–6392S.
(15) Eisen, T.; Ahmad, T.; Flaherty, K. T.; Gore, M.; Kaye, S.; Marais,
R.; Gibbens, I.; Hackett, S.; James, M.; Schuchter, L. M.; Nathanson,
K. L.; Xia, C.; Simantov, R.; Schwartz, B.; Poulin-Costello, M.;
O’Dwyer, P. J.; Ratain, M. J. Sorafenib in advanced melanoma: a
Phase II randomised discontinuation trial analysis. Br. J. Cancer 2006,
95, 581–586.

2264 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8
Niculescu-DuVaz et al.
(16) Collins, I.; Workman, P. Design and development of signal transduction
inhibitors for cancer treatment: Experience and challenges with kinase
targets. Curr. Signal Transduction Ther. 2006, 1, 13–23.
(17) Veber, D. F.; Johnson, S. R.; Cheng, H.-Y.; Smith, B. R.; Ward, K. W.;
Kopple, K. D. Molecular Properties That Influence the Oral Bioavail-
ability of Drug Candidates. J. Med. Chem. 2002, 45, 2615–2623.
(18) Liao, J. J.-L. Molecular Recognition of Protein Kinase Binding Pockets
for Design of Potent and Selective Kinase Inhibitors. J. Med. Chem.
2007, 50, 409–424.
(19) Wan, P. T. C.; Garnett, M. J.; Roe, S. M.; Lee, S.; Niculescu-Duvaz,
D.; Good, V. M.; Project, C. G.; Jones, C. M.; Marshall, C. J.; Springer,
C. J.; Barford, D.; Marais, R. Mechanism of activation of the RAF-
ERK signaling pathway by oncogenic mutations of B-RAF. Cell 2004,
116, 855–867.
cokinetics of geldanamycin analogues in mice. Cancer Chemother.
Pharmacol. 2004, 54, 475–486.
(22) Nutley, B. P.; Raynaud, F. I.; Wilson, S. C.; Fischer, P. M.; Hayes,
A.; Goddard, P. M.; McClue, S. J.; Jarman, M.; Lane, D. P.; Workman,
P. Metabolism and pharmacokinetics of the cyclin-dependent kinase
inhibitor R-roscovitine in the mouse. Mol. Cancer Ther. 2005, 4, 125–
139.
(23) Workman, P.; Twentyman, P.; Balkwill, F. United Kingdom Co-
ordinating Committee on Cancer Research guidelines for the welfare
of animals in experimental neoplasia, 2nd Ed. Br. J. Cancer 1998,
77, 1–10.
(24) Raynaud, F. I.; Whittaker, S. R.; Fischer, P. M.; McClue, S.; Walton,
M. I.; Barrie, S. E.; Garrett, M. D.; Rogers, P.; Clarke, S. J.; Kelland,
L. R.; Valenti, M.; Brunton, L.; Eccles, S.; Lane, D. P.; Workman, P.
In vitro and in vivo pharmacokinetic-pharmacodynamic relationships
for the trisubstituted aminopurine cyclin-dependent kinase inhibitors
olomoucine,bohemine and CYC202. Clin. Cancer Res. 2005, 11,
4875–4887.
(20) Niculescu-Duvaz, I.; Roman, E.; Whittaker, S. R.; Friedlos, F.; Kirk,
R.; Scanlon, I. J.; Davies, L. C.; Niculescu-Duvaz, D.; Marais, R.;
Springer, C. J. Novel Inhibitors of B-RAF Based on a Disubstituted
Pyrazine Scaffold. Generation of a Nanomolar Lead. J. Med. Chem.
2006, 49, 407–416.
(21) Smith, N. F.; Hayes, A.; Nutley, B. P.; Raynaud, F. I.; Workman, P.
Evaluation of the cassette dosing approach for assessing the pharma-
JM801509W