BRAF inhibitors based on an imidazo[4,5]pyridin-2-one scaffold and a meta substituted middle ring

Article abstract of DOI:10.1021/jm901509a

We recently reported on the development of a novel series of BRAF inhibitors based on a tripartite A-B-C system characterized by a para-substituted central aromatic core connected to an imidazo[4,5]pyridin-2-one scaffold and a substituted urea linker. Here, we present a new series of BRAF inhibitors in which the central phenyl ring connects to the hinge binder and substrate pocket of BRAF with a meta-substitution pattern. The optimization of this new scaffold led to the development of lownanomolar inhibitors that permits the use of a wider range of linkers and terminal C rings while enhancing the selectivity for the BRAF enzyme in comparison to the para series.

Full text of DOI:10.1021/jm901509a

1964 J. Med. Chem. 2010, 53, 1964–1978
DOI: 10.1021/jm901509a
BRAF Inhibitors Based on an Imidazo[4,5]pyridin-2-one Scaffold and a Meta Substituted Middle Ring
†
Arnaud Nourry, Alfonso Zambon, Lawrence Davies, Ion Niculescu-Duvaz, Harmen P. Dijkstra, Delphine Menard,
†
†
†
†
†
ꢀ
Catherine Gaulon,† Dan Niculescu-Duvaz,† Bart M. J. M. Suijkerbuijk,† Frank Friedlos,† Helen A. Manne,† Ruth Kirk,‡
Steven Whittaker,‡ Richard Marais,‡ and Caroline J. Springer*,†
†Cancer Research UK Centre of Cancer Therapeutics, The Institute of Cancer Research, 15 Cotswold Road, Sutton, Surrey SM2 5NG, United
Kingdom, and ‡Cancer Research UK Centre for Cell and Molecular Biology, The Institute of Cancer Research, 237 Fulham Road, London SW3
6JB, United Kingdom
Received October 11, 2009
We recently reported on the development of a novel series of BRAF inhibitors based on a tripartite
A-B-C system characterized by a para-substituted central aromatic core connected to an imidazo-
[4,5]pyridin-2-one scaffold and a substituted urea linker. Here, we present a new series of BRAF
inhibitors in which the central phenyl ring connects to the hinge binder and substrate pocket of BRAF
with a meta-substitution pattern. The optimization of this new scaffold led to the development of low-
nanomolar inhibitors that permits the use of a wider range of linkers and terminal C rings while
enhancing the selectivity for the BRAF enzyme in comparison to the para series.
Introduction
lipophilic DFG-out pocket (BPII using the general kinase
nomenclature of the kinase binding pockets),16 a linker
(urea, amide, or sulfonamide) interacting with the salt bridge,
and a terminal aromatic group (phenyl or pyrazole) filling the
allosteric pockets created by the displacement of the DFG loop
(BPIII and BPIV).14,15 For clarity, we have named the hinge
binder, the middle ring, and the terminal aromatic group rings
A, B, and C, respectively, and the salt bridge binder BC linker.
This series of para-substituted compounds has shown excellent
activity against the V600EBRAF enzyme, with IC50 down to
1 nM and submicromolar cellular activities.15
All of the type II inhibitors that we reported on previously
feature a 1,4 (para) substituted aromatic B ring connecting the
hinge binding moiety with the salt-bridge linker. Docking
studies of our inhibitors into the BRAF/sorafenib cocrystal
structure (PDB code UWH)4 indicated that inhibitors with a
meta substitution pattern on this ring could indeed retain the
binding affinity observed for the para-substituted com-
pounds, while moving to a novel chemical area. We later
discovered that this hypothesis was supported by the fact that
the meta type I BRAF binder PLX403217 presents a 1,3 (meta)
substituted central aromatic ring.
BRAFa is a serine/threonine specific protein kinase and a
component of the ERK MAPK cell signaling pathway that
regulates essential cell responses to environmental changes.1,2
BRAF is also a human oncogene that is mutated in approxi-
mately 50% of malignant melanomas and at a lower fre-
quency in a variety of other human cancers (e.g., colorectal,
ovarian, andpapillarythyroidcancers).2-5 The most common
BRAF mutation in human cancer is the substitution of a
valine for glutamic acid at position 600 (termed V600EBRAF),
resulting in a protein that has 500-fold elevated protein kinase
activity compared to the wild type. V600EBRAF stimulates
sustained and constitutive activation of the ERK path-
way, inducing uncontrolled cell proliferation, increased
cell survival, and tumor progression.3,6 These data indicate
that V600EBRAF is an important therapeutic target in human
cancer,4,7,8 and some drug discovery and drug development
programs have been initiated.9 Important advances have been
made over the past decade in the field of the small molecule
kinase inhibitors, with some representative marketed exam-
ples of this class of inhibitors being gefitinib10 1, sorafenib11 2,
and imatinib12 3, shown in Figure 1.
According to the docking results, the binding mode of these
meta compounds in BRAF is similar to that of sorafenib.4 In
particular, the 2,3-dihydroimidazo[4,5]pyridin-2-one unit
forms a hydrogen bond with the backbone of Cys532 of the
hinge region, and 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. The chlorotri-
fluoromethyl phenyl group interacts with the BPIII and BPIV
hydrophobic pockets exposed by the displacement of the DFG
loop in the out conformation (Figure 3). No alternative modes
of binding to the target were evidenced by the docking analysis
of 12 and the other meta compounds presented in this paper.
The comparison between the docking poses of the meta
compound 12 and its para analogue 12a shown in Figure 3
Recently, wereported on the development of a series of type
II inhibitors13 (Figure 2) targeting the inactive conformation
of V600EBRAF for the treatment of mutant BRAF-driven
malignancies.14,15 These compounds are based on a 2,3-
dihydroimidazo[4,5]pyridin-2-one scaffold binding to the
hinge region of BRAF, an aromatic system occupying the
*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; ERK, extracellular regulated kinase; PK,
pharmacokinetics; Boc, tert-butoxycarbonyl; DMF, dimethylforma-
mide; TFA, trifluoroacetic acid; THF, tetrahydrofuran; DCM, dichlo-
romethane; SAR, structure-activity relationship.
r
pubs.acs.org/jmc
Published on Web 02/11/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5 1965
Figure 1. Structures of recent kinase inhibitors.
substitution between the 4-chloro-2-amino-3-nitropyridine
and various substituted aminophenols, in which the amino
group is either protected with a Boc group (4a-c) or unpro-
tected (4d-l). The protected intermediates 5a-c were used to
access the target compounds by route A. This route consists of
the formation of the AB ring system prior to the introduction
of the BC linker bearing a variety of C rings and allows ready
access to a panel of compounds with different BC linkers and
C rings. After reduction of the nitro group of compounds
5a-c by catalytic hydrogenation, the cyclic urea (compounds
7a-c) was formed using triphosgene in basic conditions.
VariousBClinkerswerethenintroducedfromthe deprotected
amino group of compounds 8a-c. The urea linker was
obtained by reaction of the amine with either an isocyanate
group or an activated carbamate, whereas the sulfonamide
linker was introduced by reaction with an aromatic sulfonyl
chloride. For the amide linker, acid chlorides or activated
carboxylic acids were used as C ring precursors depending on
the commercial availability of the starting material. The
reduced amide linker, with a methylene bridge instead of the
carbonyl group, was prepared using the corresponding sub-
stituted benzaldehyde as the C ring precursor under reductive
amination conditions. The alternative route B to the target
compounds was used when route A failed. In this case, the BC
linker was introduced on the amino intermediates 9d-l prior
to the formation of the A ring using the same methodologies
exemplified in route A.
Figure 2. General structure of BRAF inhibitor series with impor-
tant BRAF interactions.
suggests that the middle ring of compounds bearing a meta
substitution pattern lies partially in the lipophilic BPI pocket
situated next to the ATP binding site. We noticed previously15
that elaboration into the BPI pocket improved the selectivity
of BRAF inhibitors toward a panel of protein kinases; one
could therefore expect the meta-substituted inhibitors to
present a different and improved selectivity profile with
respect to the para-substituted compounds. The docking
poses of the A rings of 12 and 12a are very close, suggesting
that the SAR of this moiety reported for the para series14
could be extended to a meta series. Conversely, a difference
between the SAR behavior of the meta and para series could
be expected for the rest of the scaffold, as the poses of the B
ring-linker-C ring systems are less close (Figure 3). This
prompted us to focus our medicinal chemistry efforts toward
the modification of the B and C rings and of the linker, using
the 2,3-dihydroimidazo[4,5]pyridin-2-one moiety as the hinge
binder.
The change in the substitution pattern of the middle ring
from 1,4 to 1,3 should also modifythe overall conformation of
the scaffold so that changes in the physicochemical properties
of the compounds would be expected. The present work
reports the synthesis of a variety of such inhibitors and their
SAR analysis. The target scaffold for this study consists of the
2,3-dihydroimidazo[4,5]pyridin-2-one hinge binding moiety
and a central phenyl ring attached in the 3-position to the
terminal C ring via various linkers (Figure 4).
For the synthesis of the targets 82-89 bearing a reversed
amide linker (i.e., with the carbonyl group bound to the
middle ring), a slightly different strategy was applied
(Scheme 2), using methyl 3-hydroxybenzoate as starting
material. The AB ring system was then prepared using the
same methods described above, and the coupling of the linker
was carried out using substituted aromatic amine in the
presence of either trimethylaluminium (AlMe3)18 or sodium
bis(trimethylsilyl)amide (NaHMDS).19
Results and Discussion
The biological activities of our target compounds were
evaluated using three assays as previously reported.14,15
The first measures the potency against the V600EBRAF
mutant enzyme in vitro (IC50, BRAF). In order to assess
the inhibition of the target signaling pathway (RAF-ERK),
the phosphorylation of extracellular signal-regulated
kinase (ERK) was assessed in a cell-based assay (IC50,
pERK). Finally, the antiproliferative activity of the com-
pounds was assessed using a sulforhodamine B (SRB) cell
Chemistry
The general synthetic routes to the target compounds are
depicted in Scheme1. Thefirst step isanaromaticnucleophilic

1966 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5
Nourry et al.
Figure 3. Superposed docking poses of meta compound 12 (green) and para 12a (purple) in the BRAF active site.
linkers and C rings to improve in vitro potency. The bio-
logical results in the three assays (IC50 BRAF, IC50 pERK,
GI50 SRB) are shown in Table 2.
Among the compounds bearing a urea BC linker, 12 (with
a 4-chloro-3-trifluoromethylphenyl C-ring) and 23 (2-
fluoro-5-trifluoromethylphenyl C-ring) show good activity
against V600EBRAF (IC50=15 and 26 nM, respectively) but
poor cellular activities. In the sulfonamide series, com-
pounds 13-16 and 30 show poor activity in the BRAF and
pERK assays.
We previously reported how the introduction of large
lipophilic substituents on the middle B ring led to submicro-
molar cellular activities in the para series.15 Interestingly, in
the meta series we observe a similar level of cellular inhibition
with compounds 26-29, bearing an unsubstituted middle
ring in combination with 1-aryl-3-tert-butylpyrazoles based
C rings. Compound 26 is the most effective, with a pERK
IC50 of 830 nM and an SRB GI50 of 705 nM. The substitu-
tion of the tert-butyl group by the less bulky trifluoromethyl
group in position 3 of the pyrazole (29 versus 26) has a
negative impact on activity.
Figure 4. General structure of target series, presenting a meta
substitution pattern on B ring.
proliferation assay (GI50, SRB). All results are summarized
in Tables 1-3.
1,3-Substitution (Meta) versus 1,4-Substitution (Para). To
evaluate the effect on activity of the change in substi-
tution pattern, we compared selected 1,3-substituted inhi-
bitors with their corresponding 1,4- analogues, pre-
viously described.14,15 Table 1 shows the results obtained in
the V600EBRAF kinase assay for both series. In order to
confirm the validity of our model, we compared the para
compound 12a,14 bearing a urea BC linker and a phenyl
middle ring, with its meta counterpart 12: the two inhibitors
are essentially equipotent against V600EBRAF.
We extended the comparison between the two series to
other BC linker/C ring combinations. For sulfonamide
linkers, all the compounds assessed (13-16) in the 1,3-series
show micromolar activity against V600EBRAF, whereas the
corresponding analogues bearing substituted C phenyl ring
in the 1,4-series (13a-15a) were inactive. Only unsubstituted
16a has shown activity against V600EBRAF (0.85 μM) and is
more active than its 1,3 equivalent. The same trend was
observed for the amide derivatives (17-20). For all the
compounds assessed, the amides of the 1,3-series show a
significant improvement in activity compared to their 1,4-
series counterparts. The example with the greatest differen-
tial is compound 20, which demonstrates a 1000-fold in-
crease in potency with respect to its analogue 20a.
We investigated the effect of different C-rings in combina-
tion with the amide linker. Among the panel of phenyl rings
explored, those bearing a fluorinated ether in the 3 position
(OCF3 or OCF2CF2H) are very potent against isolated
V600EBRAF but less active in the cellular pERK and
SRB assays (39, 43, 46). The 3-trifluoromethylthiophe-
nyl and 3-tert-butylphenyl groups also are favorable
(IC50 V600EBRAF for 47 and 48 is 5 and 17 nM, respectively).
Substitution on the meta position of C ring appears essential
to maintain activity (compare 39 versus 41), and the intro-
duction of a further methylene group between the amide
linker and the phenyl ring leads to inactive compounds
(compare 40 versus 39 and 35, 36 versus 19). The introduc-
tion of bulky groups (in 50-53, 20) or a fused ring (in 54-58)
on position 3 of the C ring does not lead to potency
improvement in the three assays. We also explored a variety
Variation of the BC Linker and the C Ring. We followed up
these positive results by examining a larger panel of BC

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5 1967
Scheme 1. Synthesis of 1,3-Substituted Inhibitors 12-78a
t
a Reagents and conditions: (i) BuOK, DMF, 80 °C; (ii) H2, Pd/C, EtOAc/EtOH; (iii) triphosgene, THF/pyridine; (iv) TFA, room temp. (v) For
X = CONH (urea): RC isocyanate, THF, room temp or RC carbamate, DMSO, 85 °C. For X = SO2 (sulfonamide): RC-sulfonyl chloride, pyridine,
room temp. For X = CO (amide): RC acid chloride, Et3N, THF, reflux or RC-carboxylic acid, DIC, HOBt, room temp. For X = CH2 (reduced amide)
RC-CHO, EtOH, Na(AcO)3BH, room temp.
Scheme 2. Synthesis of Inhibitors 82-89 with Reversed Amide BC Linkera
a Reagents and conditions: (i) NaH, DMSO, 100 °C; (ii) H2, Pd/C, EtOAc/EtOH; (iii) triphosgene, THF/pyridine, reflux; (iv) RC-NH2, AlMe3, THF,
reflux or RC-NH2, NaHMDS, THF, room temp.
of heterocycles as C ring. Substituted pyridines (59, 60) or
pyrazoles (61-66) show moderate or poor biological acti-
vities. Compounds 65 with 3-tert-butyl-1-phenyl-1H-pyra-
zole and 66 with 3-isopropyl-1-phenyl-1H-pyrazole as C
ring, analogues of the most effective pyrazole derivatives
with a urea linker, show nanomolar IC50 against puri-
fied V600EBRAF (65 and 66 show 274 and 87 nM, res-
pectively) but have micromolar activity in the cell-based
assays (13.7 μM for both compounds in the pERK assay
and 3.1 and 5.7 μM, respectively, in the SRB assay).

1968 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5
Nourry et al.
Table 1. V600EBRAF IC50 (μM) of Phenoxy-1H-imidazo[4,5-b]pyridin-2(3H)-ones Meta- and Para-Substituted on the Central Phenyl Ring or B Ringa
a (/) a denotes para counterparts taken from refs 14 and 15 for comparison.
We investigated the effect on activity of reversing the
amide moiety. The compounds substituted by a fluorinated
ether group (OCF3 or OCF2CF2H) or a fluorinated thioether
group (SCF3) in position 3 of the C ring show generally
similar or slightly weaker biological activities (82, 85, 86).
Conversely, compound 87 with 3-tert-butyl-1-phenyl-1H-
pyrazole as C ring shows a significant improvement in the
cell-based assays while maintaining its activity against pur-
ified V600EBRAF (87 and 65: 356 and 274 nM, respectively,
for V600EBRAF assay; 1.09 and 13.7 μM for pERK assay;
and 0.989 and 3.1 μM for SRB assay). We also prepared an
analogue of 39 in which the carbonyl group of the amide
linker was removed (67). Unsurprisingly, removal of one of
the salt-bridge binders is detrimental to V600EBRAF and
pERK activity.
The choice of the most effective BC linker for the 1,3-series
appears to be dependent on the C-ring used. For example,
when the C-ring is the 4-chloro-3-(trifluoromethyl)phenyl

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5 1969
Table 2. Exploration of Various C Ring and B-C Linker Combinations

1970 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5
Table 2. Continued
Nourry et al.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5 1971
Table 2. Continued

1972 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5
Table 2. Continued
Nourry et al.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5 1973
Table 2. Continued
group, the compound bearing an urea linker is more potent
than its parent or reversed amide analogues (12 versus 17 and
83), whereas when the same ring is substituted by a tert-butyl
group, compounds with an amide linker are more active than
the corresponding urea-linked compounds (e.g., 48 versus
24). With a pyrazole derivative C-ring, the reversed amide or
urea linkers are almost equipotent (27 versus 87).
It is clear that the meta substituted middle ring allows a
wider range of linkers compared to its para counterpart.
Among the linkers explored, amides, sulfonamides, and
ureas are active for the meta substitution, while for the
para-substituted compounds only the urea group is tole-
rated.14
kinases. The compounds were chosen in order to provide the
widest possible structural diversity, with 27 presenting a urea
BC linker and a pyrazole C ring, 78 an amide linker and a
phenyl C ring, and 87 a reversed amide linker and a pyrazole
C ring. We show here that the three compounds display an
improvement in the selectivity profile with respect to the
previously reported para series,15 with no significant inhibi-
tion reported against most of the kinases of the panel (see
Figure 5). SRC, LCK, and p38R and p38β (MAP) kinases are
common hits with all the compounds, with the exception of
87 (see Figure 5). This is unsurprising, as the same three
proteins have been shown to be sensitive to type II ligands
that, like 78, elaborate into the BPI pocket.15,21-23
Substitutions on the B Ring. The SAR of the B ring was also
examined to optimize potency, and the results are summar-
ized in Table 3. Rings A (2,3-dihydroimidazo[4,5]pyridin-2-
one) and C (3-trifluoromethoxyphenyl) and the BC amide
linker were kept constant. The compound of reference for
this study is 39, with no substituent on B ring. The insertion
of a methyl group in position 2, 4, or 6 (compounds 70, 68, or
69, respectively) has a detrimental effect on activity, leading
to inactive compounds. Chlorine has been introduced in
various positions of the B ring (compounds 71-73). The
2,4-substitution is tolerated (72) but does not improve the
biological activity with respect to the parent compound. In
contrast, the insertion of a methoxy group in position 5
improves the SRB activity but is detrimental to pERK
activity. The incorporation of one or several atoms of
fluorine on the B ring (76-78) leads to compounds with
good activities against V600EBRAF and slightly improved
activities in the cell-based assays. Overall, these results are
consistent with the meta substituents buttressing the BPI
pocket of the BRAF protein, as depicted in Figure 2.
Kinase Selectivity. We anticipated that the change in the
geometry of the central middle ring would influence the
selectivity profile of the meta-substituted inhibitors. To
evaluate this change in selectivity, we submitted compounds
27, 78, and 87 (with a V600EBRAF IC50 of 89, 7, and 356 nM,
respectively) to the MRC National Centre for Protein
Kinase Profiling20 for the assessment of their activity at a
single concentration of 1 μM in a panel of 69-77 protein
Interestingly, the selectivity of the meta compounds is
further enhanced when the urea linker is replaced by an
amide. Compound 87, bearing a phenylpyrazole C-ring and
a reverse amide linker, appears remarkably selective within
the kinase panel, inhibiting only p38R and, to a lesser extent,
p38β and JNK. In addition, compound 78 shows remarkable
selectivity in view of its potency (V600EBRAF IC50 of 7 nM),
as it inhibits only SRC, LCK, and p38R and p38β.
To assess further the selectivity profile of the series, the
IC50 of the same three compounds was assessed by Invitro-
gen against a focused panel of 16 protein kinases. The results
are summarized in Table 4. It is notable that all the com-
pounds assessed are more active on V600EBRAF than on
WTBRAF, ranging from a 3-fold selectivity for 87 to 30-fold
selectivity for 27. Among the kinases tested in this panel, the
compounds show activity only toward the BRAF isoform
CRAF and to a partial extent toward KDR and RET.
Cellular Selectivity. In order to provide evidence for the
specificity of the compounds for BRAF-driven cell pro-
liferation, the SRB GI50 for the BRAF wild-type WM1361
melanoma cell line was determined for compounds 27 and
87 and compared to the SRB GI50 on the mutant BRAF
melanoma cell line WM266.4. The results are shown in
Figure 6, along with the SRB GI50 of the multikinase
inhibitor sorafenib on the same cell lines. While for sorafenib
the activity is independent of BRAF status, our inhibitors
are up to 53-fold selective for the mutant BRAF WM266.4
cell line. This selectivity strongly supports our proposed

1974 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5
Table 3. Optimization of B Ring
Nourry et al.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5 1975
Table 4. IC50 (μM) of Selected Compounds 27, 78, and 87 toward a
Panel of Protein Kinases
kinase
27
78
87
V600E-BRAF
WT-BRAF
FGFR1
0.089
3.140
>10
0.007
0.042
>10
4.840
0.200
0.216
5.020
>10
>10
0.608
>10
2.170
2.390
0.003
0.237
0.356
1.090
4.250
4.160
1.370
0.883
>10
>10
>10
1.500
>10
9.710
7.490
0.126
0.202
FLT1 (VEGFR1)
KDR (VEGFR2)
KIT
1.530
0.196
1.340
0.584
>10
LCK
COT
p38γ
p38R
MET
1.810
0.152
>10
PDGFRR
PDGFRβ
CRAF (DD)
RET
1.840
1.550
0.198
0.698
mechanism of action of this series through the inhibition of
oncogenic BRAF.
In Vitro Potency and Cellular Activity. It is noted that in
the case of some compounds, e.g., 78, there is a large
differential between the potency for V600EBRAF in vitro
kinase inhibition and the cellular assays. This is likely to be
due to a number of factors such as greater ATP concentra-
tion in cells, the extent of compound uptake by the cells, and
lipophilicity.
Importantly, our selected compounds show a close corre-
lation between the inhibition of both ERK phosphorylation
and cell proliferation in a BRAF mutant cell line. These cells
are absolutely dependent upon mutant BRAF-mediated
activation of the ERK MAPK pathway, and therefore, loss
of ERK phosphorylation is tightly associated with loss of
BRAF kinase activity and decreased proliferation.
Conclusion
We have synthesized a novel series of type II BRAF
inhibitors in which the aromatic middle ring connects to
the hinge binder and the allosteric pocket binder in a 1,3-
substitution pattern. This new scaffold shows potent activity
in a V600EBRAF assay and allows the use of a wider range of
linkers and terminal C rings as salt bridge and allosteric
pocket binders, respectively. The introduction of one or more
atoms of fluorine on the central B ring achieves low-nanomolar
potencies on the V600EBRAF enzyme assay and submicromolar
potencies for several compounds. An important advantage
of this series of BRAF inhibitors is the improved selectivity
against other kinases and improved cellular selectivity com-
pared to the parent para series, as shown by the assessment of
selected inhibitors from this series on two panels of protein
kinases.
Experimental Section
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. 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 and then 10% A/90% B to
Figure 5. Kinase selectivity of compounds 27, 78, and 87 tested
against a panel of 69 kinases at the MRC National Centre for
Protein Kinase Profiling. The color bar represents the percent
activity relative to the control.

1976 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5
Nourry et al.
Figure 6. Growth inhibition of WM266.4 (BRAF mutant) and WM1361 (NRAS mutant) melanoma cell lines following a 5-day exposure to 27
(A) or 87 (B) and analysis by SRB assay.
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% where the concentrations remained until 12 min. UV
detection was at 254 nm, and ionization was positive or negative
ion electrospray. Molecular weight scan range is 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 ona Bruker DPX 250operatingat 250.13 MHz or on a
Bruker advance 500 operating at 500.26 MHz. The signal of the
deuterated solvent was used as internal reference; the chemical
shifts are expressed in ppm (δ). Coupling constants (J) are given
in Hz. HRMS analyses were performed on 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), reserpine ([M þ H]þ 609.280 657), and (1H,1H,3H-tetra-
fluoropentoxy)phosphazene ([M þ H]þ 922.009798). Mobile
phase consisted of 0.1% formic acid in doubly distilled deionized
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 purity of the final compoundswas determined byHPLC
as described above and is 95% or higher unless specified other-
wise.
geometries 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. Ten docking
solutions were generated per docking run with GOLD, and
the best three were stored for analysis.
1-(4-Chloro-3-(trifluoromethyl)phenyl)-3-(3-(2-oxo-2,3-dihydro-
1H-imidazo[4,5-b]pyridin-7-yloxy)phenyl)urea (12). Method A.
A mixture of 4-chloro-3-(trifluoromethyl)phenyl isocyanate
(33 mg, 0.16 mmol) and 7-(3-aminophenoxy)-1H-imidazo-
[4,5-b]pyridin-2(3H)-one 8a (30 mg, 0.13 mmol) in anhydrous
THF (1.5 mL) was stirred at room temperature for 14 h. Next,
the solvent was evaporated and the solid residue was washed
with Et2O to afford 12 as an off-white solid (33 mg, 55%). 1H
NMR δ: 6.45 (d, 1H, HPy,5, J=5.8), 6.78 (d, 1H, Harom, J=8.2),
7.25 (d, 1H, Harom, J = 7.9), 7.34-7.39 (m, 2H, Harom),
7.55-7.67 (m, 2H, Harom), 7.79 (d, 1H, HPy,6, J=5.8), 8.06 (s,
1H, Harom), 9.14 (s, 1H, NHurea), 9.29 (s, 1H, NHurea), 11.23 (s,
1H, NH), 11.42 (s, 1H, NH). LC-MS: m/z 464 (M þ H, 100).
HRMS: m/z calcd for C20H14ClF3N5O3 ([M þ H]þ), 464.0737;
found, 464.0727.
4-Chloro-N-(3-(2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-
7-yloxy)phenyl)-3-(trifluoromethyl)benzenesulfonamide (13).
Method B. 7-(3-Aminophenoxy)-1H-imidazo[4,5-b]pyridine-
2(3H)-one 8a (30 mg, 0.13 mmol) was suspended in dry pyridine
(3 mL), and 4-chloro-3-(trifluoromethyl)benzene-1-sulfonyl
chloride (44.4 mg, 0.16 mmol) in pyridine (2 mL) was added.
The resulting solution was stirred at room temperature for
20 h, and subsequently the solvent was removed in vacuo.
The obtained residue was dissolved in acetone (4 mL), and
upon addition of water a solid precipitated. This solid was
collected, washed with water (2 ꢀ 2 mL) and Et2O (2 ꢀ 2 mL),
and dried under vacuum. 13 was obtained as an off-white
Biological Assays. V600EBRAF Kinase Assay and SRB IC50
for BRAF Inhibitors. These assays have been described by
Niculescu-Duvaz et al.14
pERK Kinase Assay. This assay has been described by
ꢀ
15
Menard et al.
Docking. Inhibitors 12 and 12a were docked on BRAF (PDB
code UWH) using GOLD, version 3.1.124 In order to prepare
the receptor for docking, the crystal structure was protonated
using the Protonate3D tool of MOE, and the ligand and water
molecules were then removed. The active site was defined using a
1
solid (38 mg, 60%). H NMR δ: 6.23 (d, 1H, HPy,5, J=5.7),
6.76 - 6.98 (m, 3H, Harom), 7.35 (m, 1H, Harom), 7.73 (m, 1H,
H
arom), 7.94-7.96 (m, 2H, Harom), 8.05 (d, 1H, HPy,6,
J = 5.7) 10.62 (s, 1H, NHSO2), 11.13 (s, 1H, NH), 11.40 (s,
1H, NH). LC-MS: m/z 485 (M þ H, 100). HRMS: m/z
calcd for C19H13ClF3N4O4S ([M þ H]þ), 485.0298; found,
485.0297.
˚
radius of 10 A from the backbone oxygen atom of Asp594 of the
ATP binding pocket. Partial charges of the ligands were derived
using the Charge-2 CORINA 3D package in TSAR 3.3 and their

Article
N-(2,3,4-Trifluoro-5-(2-oxo-2,3-dihydro-1H-imidazo[4,5-b]-
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5 1977
strongsupportandhelpfulreading of the manuscript. Meirion
Richards, Dr. Maggie Liu, and Dr. Amin Mirza are thanked
for providing technical assistance.
pyridin-7-yloxy)phenyl)-3-(trifluoromethoxy)benzamide (78).
Method C. N-(5-(2,3-Diaminopyridin-4-yloxy)-2,3,4-trifluoro-
phenyl)-3-(trifluoromethoxy)benzamide 11l (90 mg, 0.20 mmol)
and triethylamine (35 μL, 0.25 mmol) were mixed in dry THF
(7.7 mL), and 3-(trifluoromethoxy)benzoyl chloride (57 mg,
0.25 mmol) was added. This mixture was heated to reflux for
20 h, and subsequently the solvent was removed in vacuo. The
obtained residue was dissolved in acetone (3 mL), and upon
addition of water a solid precipitated. This solid was collected,
washed with water (2 ꢀ 3 mL) and Et2O (2 ꢀ 3 mL), and dried to
afford the title compound 78 (39 mg, 40%). 1H NMR δ: 6.65 (d,
1H, HPy,5, J=6.0), 7.32 (t, 1H, Harom, J=6.5), 7.63 (d, 1H,
Supporting Information Available: Experimental details of the
synthesis of compounds 14-30, 32-35, 37-66, 68-77, 79-82,
and 84-89 and intermediates; analytical characterization of all
compounds. This material is available free of charge via the
Internet at http://pubs.acs.org.
References
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arom, J=8.0), 7.68 (t, 1H, Harom, J=8.0), 7.84 (d, 1H, HPy,6,
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phenyl)-2-(3-trifluoromethylphenyl)acetamide (31). 31 was syn-
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6.27 - 6.31 (m, 3H, Harom), 6.48 (dd, 1H, Harom, J=8.2, J=1.4),
6.58 (t, 1H, NHamine, J=6.1), 7.10 (t, 1H, Harom, J=8.1), 7.21 (d,
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11.08 (s, 1H, NHIm), 11.29 (s, 1H, NHIm). LC-MS: m/z 417
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1H-imidazo[4,5-b]pyridin-7-yloxy)benzamide (83). Method D. A
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mixture was heated under reflux for 19 h. The mixture was
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aqueous HCl (1.5 mL). After evaporation of solvent, the residue
was retaken in CH2Cl2, washed with saturated solution of
NaHCO3 and then with brine, dried over MgSO4, and evapo-
rated under vacuum. The obtained residue was chromatogra-
phied (eluent EtOAc), and 83 was obtained as a slightly yellow
solid (48 mg, 30%).
Acknowledgment. This work is supported by Cancer
Research UK (grants C309/A2187 and C107/A10433), the
Wellcome Trust, the Institute of Cancer Research, and the Isle
of Mann Anti-Cancer Association. We acknowledge NHS
funding to the NIHR Biomedical Research Centre. Professors
Paul Workman and Julian Blagg are acknowledged for their

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