Dual binding site inhibitors of B-RAF kinase

Article abstract of DOI:10.1016/j.bmcl.2008.04.002

Computer aided modeling guided the design of a series of diarylimidazole compounds (11-22) intended to interact with both the ATP and adjacent allosteric binding domains of B-RAF kinase. Their ability to inhibit the function of B-RAF kinase and intracellular ERK1/2 phosphorylation were evaluated.

Full text of DOI:10.1016/j.bmcl.2008.04.002

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 2825–2829
Dual binding site inhibitors of B-RAF kinase
Ronald L. Wolin,* Scott D. Bembenek, Jianmei Wei, Shelby Crawford,
Katherine Lundeen, Anders Brunmark, Lars Karlsson,
James P. Edwards and Jonathan M. Blevitt
Johnson & Johnson Pharmaceutical Research & Development, L.L.C., 3210 Merryfield Row, San Diego, CA 92121, USA
Received 6 February 2008; revised 28 March 2008; accepted 1 April 2008
Available online 4 April 2008
Abstract—Computer aided modeling guided the design of a series of diarylimidazole compounds (11–22) intended to interact with
both the ATP and adjacent allosteric binding domains of B-RAF kinase. Their ability to inhibit the function of B-RAF kinase and
intracellular ERK1/2 phosphorylation were evaluated.
Ó 2008 Elsevier Ltd. All rights reserved.
The RAF (Ras Activating Factor) family of serine/thre-
onine kinases are members of the MAPK kinase signal-
ing cascade and participate in relaying extracellular
signals from the cell surface to the nucleus. The RAF
family comprises of three isoforms; A-RAF, B-RAF,
and C-RAF, which are involved in cell growth, differen-
tiation, and apoptosis. Clinically, it has been determined
that ꢀ66% of melanomas, ꢀ36% of thyroid tumors, and
ꢀ10% of colon cancers in humans can be correlated with
mutations that have occurred in the B-RAF kinase do-
main.1 While over 40 different mutations in the kinase
domain have been identified, the most prevalent muta-
tion arises from the replacement of a valine residue with
a glutamic acid residue located at position 600. This sub-
stitution, which lies within the kinase activation loop,
essentially precludes the need for phosphorylation and
results in constitutively active B-RAF that displays
approximately a 500-fold increase in kinase activity over
the wild-type isoform.2 As a result, the activation of the
MAPK signaling cascade through mutant B-RAF has
been linked to tumorigenesis in several types of tumors.
Based upon these observations, and the fact that B-RAF
is considered an important therapeutic target for cancer
management,3 we embarked upon a chemistry effort to-
wards identifying a small molecule that would inhibit
this signaling pathway.
High-throughput-screening of our compound collec-
tion identified several diarylimidazoles as low nM
inhibitors of B-RAF kinase of which 1 served as a
general representation of the type of hits identified.
Maintaining an ortho substituent (i.e., Cl) on the aryl
ring attached at the C-4 position of the imidazole ring
was important for retaining B-RAF kinase affinity and
selectivity. Substituents (i.e., Cl) at the meta- or para-
position of the C-4 aryl ring displayed less selectivity
for B-RAF. Compound 1 was chosen as an attractive
lead since related analogs suffered from poor physio-
chemical properties and/or microsomal instability.
Via slight SAR modifications we prepared a derivative
2 that inhibited B-RAF with improved potency and
possessed comparable solubility and metabolic stabil-
ity parameters.4 The 2,5-dichloro substituent was
mainly responsible for the boost in kinase potency,
and the conversion of the t-butylurea to the cyclic
urea was responsible for improving the solubility pro-
file (see Fig. 1).
Cl
Cl
4
3
N
N
Cl
N
H
N
H
1
5
N
N
N
N
O
O
NH
NH
N
N
H
N
H
HN
Keywords: B-RAF; Kinase inhibitors; Diarylimidazoles.
*
(2), IC50 (B-Raf) = 10 nM
(1), IC50 (B-Raf) = 83 nM
Corresponding author. Tel.: +1 858 784 3133; fax: +1 858 784
3116; e-mail: RWOLIN@PRDUS.JNJ.COM
Figure 1. SAR modifications.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.04.002

2826
R. L. Wolin et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2825–2829
The report from Wan and co-workers2 detailing the first
x-ray crystal structure for a small molecule ligand (3,
BAY-43-9006, Sorafenib)5 bound to B-RAF kinase
prompted us to compare our chemical series to the crys-
tal structure coordinates of Sorfenib to determine what
binding elements of the kinase may be shared by the
two molecules. The crystallographic data revealed the
pyridyl amide portion of (3) occupies the relatively con-
served ATP pocket while the aryl urea portion (motif E,
Fig. 2) of the molecule resides in an uncharacteristically
deep hydrophobic pocket and serves as the allosteric
binding motif. Consequently, the unique hydrophobic
pocket of B-RAF appeared to offer an opportunity to
achieve a less promiscuous kinase inhibitor. From our
docking model studies using the coordinates deposited
by Wan et. al. (PDB ID 1UWH)2 on a series of diarylim-
idazoles related to 1, it appeared that a meta-anilino
intermediate (8, Scheme 1) would provide a handle for
directing a branching group into the hydrophobic pock-
et as desired. Additionally, one could envision that the A
and B rings of 2 approximate the conformation occu-
pied by rings C and D of (3). The main difference effect-
ing the spatial orientation of the aryl rings is the scaffold
linking the two aryl rings together; an imidazole ring
versus an ether linkage (Figs. 2 and 3).
NHCO2Et
NHCO2Et
Me
O
a
c
b
N
N
SMe
4
N
N
O
N
N
O
5
SMe
6
SMe
R
NH2
NHCO2Et
HN
e-f
N
N
N
N
d
Y
Y
Y
N
H
N
H
H
N
N
N
N
N
N
8
9
7
SMe
SMe
Men(O)S
R
Y = CF3, Cyclopropane
n = 1, 2
R = See Table 1 for substituents
HN
g
N
Y
O
H
N
N
H
R1
=
NH
N
N
N
NH
10
R1
CONH2
NHCH3
Scheme 1. Synthetic route to the diarylimidazole analogs. Reagents
and conditions: (a) LiHMDS, THF, 0 °C, 30 min, then the Weinreb
Amide, 0 °C ! rt, 24 h (b) Br2, CH2Cl2, 0 °C ! rt, 1 h, then, DMSO,
Et3N, 65 °C, 1.5 h. (c) Y-CHO, NH4OAc, HOAc, 65 °C, 16 h (d)
KOH, H2O, EtOH, 80 °C, 24 h (e) 4-chlorophenyl acetylchloride, or
3,4-dichlorophenyl sulfonylchloride, or 4-chloro-3-trifluoromethyl-
phenyl isocyanate Et3N, CH2Cl2, (f) UHP, TFAA, CH3CN, 0 °C,
45 min, or m-CPBA, CH2Cl2, 0 °C, 30 min (g) R1-NH2, dioxane, 80–
120 °C, 4–16 h.
To test the dual-site inhibitor hypothesis, we constructed
a number of meta-substituted compounds. The synthetic
route used to assemble these inhibitors is outlined in
Scheme 1. The anion of pyrimidine 4 was condensed
with the Weinreb amide (derived from 3-aminobenzoic
acid)6 to afford the aryl ketone 5. Kornblum7 oxidation
of 5 provided the a-diketone 6, which was treated with
cyclopropane carboxaldehyde or trifluoroacetaldehyde
hydrate in the presence of ammonium acetate and acetic
acid to afford the C2-substituted imidazoles 7. Hydroly-
sis of the ethyl carbamate under basic conditions
afforded the amine 8, which was subsequently treated
with the appropriate electrophile to provide the urea,
sulfonamide or amide intermediates. Oxidation of the
thiomethyl moiety employing m-CPBA afforded the
corresponding sulfoxide or sulfone 9 which was subse-
quently displaced with the requisite amine in refluxing
dioxane to provide adducts 10 in yields ranging from
40% to 70%. Displacement with 3-aminobenzamide
required the addition of TFA to facilitate this
transformation.
Cl
Cl
CF3
Figure 3. Crystal structure of Sorafenib overlaid with the docking
model of our desired template in B-RAF. The arrow emphasizes the
predicted position (meta) for attaching substituents to our template to
allow for access to the hydrophobic pocket.
CF3
HN
HN
E
HN
HN
O
O
Cl
A
N
C
N
Cl
B
C
NH
NH
O
N
For consistency, the same urea component present in
Sorafenib (3) was installed onto the aniline intermediate
10 and three different groups were appended to the
pyrimidine ring for evaluation (R1 = methylamine, 3-
aminobenzamide, and 1-(2-amino-ethyl)-imidazolidin-
2-one) to afford the proof-of-concept analogs 11–13
(Table 1). As the data in Table 1 demonstrate, all three
N
N
D
N
D
HN
O
N
HN
O
O
N
NH
HN
N
NH
(2)
(11)
(3) BAY 43-9006
Figure 2. Rationale for the hybrid strategy.

R. L. Wolin et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2825–2829
Table 1. Substituent effects on B-RAF activity
2827
R
HN
N
Y
N
H
N
N
R1
Compound
R
R1
Y
B-RAF
IC50 (lM)
V600E C-RAF
IC50 (lM)
RLMa HLMa pERk
Solubilityb
IC50 (lM) (lM)
O
11
12
13
CONHPh-3–CF3-4–Cl
C3H5 0.066
C3H5 0.021
C3H5 0.035
0.022
0.016
0.061
0.014
44
57
nd
50
80
nd
2.3
1.4
2.1
6.2
0.3
0.4
HN
N
NH
NH
NH
NH
CONHPh-3–CF3-4–Cl NHCH3
NH
0.005
0.031
CONHPh-3–CF3-4–Cl
NH2
NH2
NH2
NH2
O
O
14
15
16
SO2CH2Ph-3,4-Cl
C3H5 0.343
C3H5 0.469
C3H5 0.887
0.141
0.138
0.274
0.041
0.026
0.049
52
87
44
85
60
76
na
na
na
na
na
na
HN
N
SO2CH2Ph-3,4-Cl
SO2CH2Ph-3,4-Cl
NHCH3
NH
O
O
17
18
19
COCH2Ph-4-Cl
COCH2Ph-4-Cl
COCH2Ph-4-Cl
C3H5 0.007
C3H5 0.009
C3H5 0.004
0.002
0.003
0.003
0.001
0.001
0.0005
60
nd
58
38
nd
53
3
35
HN
N
NHCH3
NH
5.1
2.4
0.4
0.5
O
O
20
21
22
COCH2Ph-4-Cl
COCH2Ph-4-Cl
COCH2Ph-4-Cl
CF3
CF3
CF3
0.054
0.03
0.04
0.02
34
81
47
73
74
71
30
7.5
HN
N
NHCH3
NH
0.018
0.085
0.002
0.017
12.6
30
0.48
0.35
0.108
O
3
BAY-43-9006/Sorafenibc
0.015
0.014
0.003
na
80
8.3
6.3
a Microsomal values are given as per cent of parent compound remaining after 30 min of incubation. The reported values are within a 10% range of
error.
b Solubility was determined by the method reported in Ref. 9.
c Values were determined in-house.
analogs possessed good inhibitory activity against B-
RAF kinase with IC50 values ranging from 21 to 66
nM, and slightly improved inhibition against the
V600E mutant and C-RAF isoforms. Additionally,
compounds 11–13 were slightly more potent than (3)
at inhibiting ERK phosphorylation in a cellular assay.
For example, compound 11, which exhibited a better
solubility profile by ꢀ15-fold than the corresponding
derivatives 12 and 13, retained similar cellular activity.
To improve compound solubility we replaced the urea
moiety with similarly substituted sulfonamide and
amide appendages. Table 1 shows that the sulfonamide
analogs 14–16 displayed a 4- to 28-fold loss of kinase
activity relative to ureas 11–13 , and lacked the neces-
sary potency to be analyzed in the pERK cellular assay.8
In contrast, the amide derivatives 17–19 provided a 2-
to 10-fold increase in kinase potency relative to 11–13
; however, a corresponding improvement in inhibiting
ERK phosphorylation was not observed. Interestingly,
while the solubility profile for compound 17 was en-
hanced 5-fold relative to compound 11, this parameter
did not translate to an increase in potency in the ERK
phosphorylation cellular assay. One of the factors that
appears to hamper the lack of correlation between en-
zyme potency and functional activity that we have ob-
served in this series (Table 1) seems to be tied to the
compounds solubility profile. Generally, it has been
our observation that analogs exhibiting higher solubility

2828
R. L. Wolin et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2825–2829
Figure 4. Docking models for the amide and sulfonamide analogs.
values displayed inferior functional activity. We attrib-
uted this to their difficulty in penetrating the lipophilic
cellular membrane and hence, would be hindered from
reaching the intracellular target.9 Again, both the sul-
fonamide and amide derivatives displayed slightly better
IC50 values against the V600E mutant and C-RAF iso-
forms than the wild-type isoform. It is worth noting that
truncating the size of the substituent appended to the
pyrimidine ring from the 1-(2-aminoethyl)-imidazoli-
din-2-one and 3-aminobenzamide moieties to the smal-
ler methylamine group produced equivalent or better
B-RAF kinase inhibition than the other two pyrimidine
substituents.10 This result was encouraging given that we
were interested in reducing the molecular weight of the
hybrid analogs. Moreover, modeling suggested that this
group was directed to a solvent exposed region of the
protein that was not expected to hinder the ATP pocket
and allosteric binding interactions. Analogs containing
the methylamine moiety also provided slightly improved
rat and human microsomal stability data, although we
observed a significant decrease in solubility relative to
the 1-(2-aminoethyl)-imidazolidin-2-one moiety.
tional conformational strain results in weaker B-RAF
inhibitory activity.
Data from a related series of diarylimidazoles related to 1
and 2 demonstrated that replacing the cyclopropyl ring at
the C2-position of the imidazole ring with a trifluoro-
methyl group tended to improve the solubility and meta-
bolic stability for this class of compounds. These findings
encouraged us to incorporate a trifluoromethyl group in
this current hybrid-series as well. As the data in Table 1
illustrate relative to the amide analogs 17–19 , the trifluo-
romethyl derivatives 20–22 displayed a 3- to 26-fold de-
crease in kinase activity. Also, a measurable decrease in
the potency of 2- to 12-fold was observed in the pERK
cellular assay. Only the metabolic and solubility charac-
teristics remained similar to those observed for the amide
derivatives 17–19 . We postulate that the decrease in
pERK cellular activityis due tothe lower pKa values asso-
ciated with the C2-trifluoromethyl substituent, which
causes the NH of the imidazole to become ionized under
physiological pH.11 Hence, the resulting charged species
is less able to penetrate the lipid by-layer of the cell effi-
ciently, and thus weaker functional activity is observed.
In an effort to correlate the observed B-RAF kinase
inhibitory data to the interactions with the protein, we
performed manual docking simulations using the data
from the crystal structure.2 The proposed binding mode
for the amide moiety is shown in the left panel of Figure
4, in which the amide proton of the ligand forms a
hydrogen bond with glutamic acid E500 of the kinase.
The binding mode for the ureas is similar to the one
shown here for the amides. Therefore, the difference in
activity between the amides and the ureas (of up to
10-fold) may in part be due to a desolvation penalty that
is incurred by the urea because of the presence of the ex-
tra nitrogen. Moreover, the glutamic acid E500 residue
forms a salt bridge with the nearby lysine K482. From
our modeling studies this suggests that E500 should
not form two hydrogen bonds with the urea but rather
only one, like the amide, with the proximal amide pro-
ton. In the right panel of Figure 4, the proposed binding
mode for the sulfonamides shows that like the amides
and the ureas the nitrogen atom makes a hydrogen bond
interaction with E500. However, the most notable differ-
ence is the effect the ‘extra’ oxygen (shown by the arrow)
has on the fit in the binding site. According to our dock-
ing models the sulfonamide analogs clash with the pro-
tein surface that leads to a binding destabilization
relative to the amides or urea analogs. Hence, this addi-
In summary, potent inhibitors of B-RAF kinase were
identified that appear to bind to both the ATP binding
pocket as well as an adjacent allosteric region. The cur-
rent physiochemical and pharmacokinetic properties
associated with these molecules, however, limit their
evaluation in lengthy xenograph studies.12 Compounds
11, 14, 17 and 20 were evaluated for kinase selectivity
against a panel of 50 representative kinases (data not
shown)13 and only compounds 11 and 17 displayed
>50% inhibition at 10 lM concentration against 4 ki-
nases within the panel suggesting that identifying a
selective B-RAF inhibitor is achievable.14
Acknowledgment
We express our thanks to Michele Rizzolio, Casey Lee
and Michelle Herrmann for conducting the solubility
and microsomal stability experiments for this study.
References and notes
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8. Compounds possessing IC50 values >100 nM in the B-
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9. The aqueous solubility of compounds was determined
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diluted in DMSO to a concentration of 10 mM. Subse-
quently, these were diluted 1:100 in 1 mL of 70 mM
NaH2PO4, pH 7.4, and mixed for 24 h at room temper-
ature. The samples were then centrifuged at 14,000 rpm in
a bench top microfuge (Model 5420, Eppendorf, Ham-
burg, Germany) for 15 min. After transferring 800 lL to a
fresh tube, samples were re-centrifuged as above and
600 lL was then transferred to an HPLC vial. The samples
were analyzed by reversed phase chromatography (Zorbax
C18, Agilent HPLC). Standards of 100, 10, and 1 lM were
prepared in DMSO to generate a standard curve. Com-
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peak areas from the standard curve generated from a
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Scheme 1.
for compounds 17–19
respectively.
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