The identification of potent and selective imidazole-based inhibitors of B-Raf kinase

Article abstract of DOI:10.1016/j.bmcl.2005.09.072

A novel triarylimidazole derivative, SB-590885 (33), bearing a 2,3-dihydro-1H-inden-1-one oxime substituent has been identified as a potent and extremely selective inhibitor of B-Raf kinase.

Full text of DOI:10.1016/j.bmcl.2005.09.072

Bioorganic & Medicinal Chemistry Letters 16 (2006) 378–381
The identification of potent and selective imidazole-based
inhibitors of B-Raf kinase
Andrew K. Takle,a,* Murray J. B. Brown,b Susannah Davies,a David K. Dean,a
Gerraint Francis,a Alessandra Gaiba,a Alex W. Hird,a Frank D. King,a Peter J. Lovell,a
Antoinette Naylor,a Alastair D. Reith,c Jon G. Steadmana and David M. Wilsona
aDepartment of Medicinal Chemistry, Neurology and GI Centre of Excellence for Drug Discovery, GlaxoSmithKline
Pharmaceuticals, New Frontiers Science Park, Third Avenue, Harlow, Essex CM19 5AW, UK
bDepartment of Screening and Compound Profiling, GlaxoSmithKline Pharmaceuticals, Medicines R&D Centre,
Gunnels Wood Road, Stevenage, Hertfordshire SG1 2NY, UK
cDepartment of High Throughput Biology, GlaxoSmithKline Pharmaceuticals, Medicines R&D Centre, Gunnels Wood Road,
Stevenage, Hertfordshire SG1 2NY, UK
Received 28 July 2005; revised 23 September 2005; accepted 28 September 2005
Available online 2 November 2005
Abstract—A novel triarylimidazole derivative, SB-590885 (33), bearing a 2,3-dihydro-1H-inden-1-one oxime substituent has been
identified as a potent and extremely selective inhibitor of B-Raf kinase.
Ó 2005 Elsevier Ltd. All rights reserved.
Mitogen-activated protein (MAP) kinases are a family
of serine/threonine protein kinases that participate in
signal transduction pathways controlling numerous
intra-cellular events.1 MAP kinases are regulated by
phosphorylation cascades whereby activation of an up-
stream kinase leads to phosphorylation of a downstream
substrate which itself has protein kinase activity. Typi-
cally in such MAP kinase cascades, three protein kinases
are sequentially activated in response to appropriate
extracellular stimuli and allow for signal amplification
at each step of the cascade.
focal cerebral ischemia in in vivo rodent models of
stroke.4 Furthermore, inhibition of the cascade at the
MEK level has proved to be neuroprotective leading
to a significant reduction in infarct volume in such
animal models.5
N
N
H
N
H
N
N
N
Cl
F
OH
The RAF-MEK-ERK MAP kinase cascade appears to
be intimately involved in the regulation of cell cycle pro-
gression and apoptosis. Indeed, activating mutations in
B-Raf, one of the Raf family members, are reported to
be present in 66% of malignant melanomas.2 Disruption
of this signaling cascade could thus offer a novel ap-
proach for cancer chemotherapy.3 Conversely, increased
activation of ERK1/2 has been reported in a number of
in vitro models of neuronal cell death and following
(2) B-Raf IC50 10nM
(1) B-Raf IC50 900nM
We sought to identify inhibitors of B-Raf, the likely ma-
jor Raf isoform in the central nervous system, to assess
their potential as neuroprotective agents in the treat-
ment of stroke.
Screening of the SmithKline Beecham compound bank
identified the tri-substituted imidazole (1) as a sub-
micromolar inhibitor of B-Raf. Concurrently we became
aware of a poster publication from Merck disclosing a
related tri-aryl imidazole 2 (L-779,450) as a highly
potent low nanomolar inhibitor of Raf.6 Compound (2)
Keywords: B-Raf; Imidazole.
*
Corresponding author. Tel.: +44 (0) 1279 627686; fax: +44 (0) 1279
622260; e-mail: Andy.K.Takle@gsk.com
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.09.072

A. K. Takle et al. / Bioorg. Med. Chem. Lett. 16 (2006) 378–381
379
Table 2. B-Raf activity of C5 modified imidazoles
was reported to demonstrate good selectivity over other
kinases tested, with the exception of p38 where the selec-
tivity was approximately 30-fold. Unfortunately 2 is
poorly soluble in aqueous systems (ꢀ0.005 mg/ml in
pH 5 buffer) thus precluding its use as an in vivo tool
where administration via iv infusion is desirable. We
sought to explore the SAR of the imidazole leads whilst
being mindful of the selectivity and developability issues
described above.
R2
H
5
N
N
R
Cl
OH
Compound
R
R2
B-Raf (FP)
Kd nM
2
13
14
15
16
17
H
H
4-Pyridyl
Phenyl
3-Pyridyl
2.4
>200
Our studies began with the synthesis of the hybrid mol-
ecule 3 which displayed comparable activity to 2.
Further investigation demonstrated that the imidazole
C2 position was tolerant of a variety of substituents
(Table 1). Heteroaryl derivatives such as 4 and 5 showed
comparable potency with 2, whereas the unsubstituted
derivative 6 only showed a modest reduction in potency.
Introduction of a basic amine substituent on either the
C2 aromatic or tert-butyl groups was well tolerated
(e.g., compounds 7 and 8) as was the carboxamide
linked derivative 9. Such derivatives lead to enhanced
aqueous solubility but also provide a handle for further
substitution (e.g., 10, 11) and modulation of the physi-
cochemical properties of the series.
H
>200
14
87
H
4-Pyrimidinyl
4-Pyridazinyl
2-Amino, 4-pyrimidinyl
H
CN
5.7
Our investigation of the imidazole C5 substituent
showed trends similar to those reported in the p38 ki-
nase area (Table 2).8 Replacement of the 4-pyridyl C5
substituent of 2 by phenyl- or 3-pyridyl groups (13 and
14, respectively) produced compounds that were essen-
tially devoid of activity in the FP assay, thus implying
that an appropriately positioned H-bond acceptor at
C5 is essential for activity. This is consistent with the
well-established binding mode of such imidazole-based
kinase inhibitors where the C5 substituent forms an H-
bond to the ÔhingeÕ region of the protein.8 As with p38,
B-Raf activity can be retained if the C5 pyridine group
is replaced by an alternative H-bond acceptor such as
in the pyrimidinyl- and pyridazinyl-derivatives 15 and
16, albeit with a 5- to 30-fold reduction in potency.
The potency drop can be mitigated through the intro-
duction of a 2-amino-4-pyrimidinyl substituent as in
17, which is postulated to pick up a second H-bonding
interaction with the hinge region of the protein.
Acylation of the C2 benzylamine 7 was also of value in
the generation of a fluorescent derivative 12 (SB-477790)
which proved to be suitable for the development of a
B-Raf fluorescent ligand binding assay (FP). The two
B-Raf assay formats showed a good correlation in rank
order potency but, due to increased throughput, the FP
format was used to generate all subsequent SAR.
O
O
H
N
N
N
O
H
NH2
N
Table 3. B-Raf activity of C4 modified imidazolesa,b
O
N
Cl
OH
H
N
H2N+
N
4
SB-477790 (12)
R3
Table 1. B-Raf activity of C2 modified imidazoles7
Compound
R3
B-Raf (FP)
Kd nM
N
H
N
R1
2
18
19
20
21
22
23
24
25
26
27
28
29
3-OH, 4-Cl
3-OH
2.4 (10)
8
N
316c (339)
Cl
4-OH
3-OMe
OH
>196
71
>196c (1585)
4-CH2OH
3-CH2OH
Compound R1
B-Raf B-Raf (FP)
IC50
nM
Kd nM
3-OH, 4-CH2OH
3-Cl, 4-CH2OH
4-CH@NOH
3-OH, 4-CH@NOH
3-Cl, 4-CH@NOH
4-C(Me)@NOH
4-C(NH2)@NOH
22
132
5
2
3
Ph
tBu
10
12
6
2.4
2
27
6
4
3-Pyridyl
2-Furyl
—
4.2
39
5.3
4.8
24
10
21
5
13
33
8
11
53
6
H
7
4-(H2NCH2)Ph
Me2CCH2NH2
CONH(CH2)3NMe2
Me2CCH2NHSO2Me
a Data from the B-Raf kinase assay is shown in parentheses (see
Ref. 7).
b Oxime derivatives isolated as mixtures of E and Z isomers.
c Data derived from a variant of the FP binding assay and hence is not
directly comparable.
8
6
9
40
14
10
11
Me2CCH2NHCONHPh(4-Cl) 13

380
A. K. Takle et al. / Bioorg. Med. Chem. Lett. 16 (2006) 378–381
Early investigation of the imidazole C4 position SAR
demonstrated that the 3-hydroxy phenyl substituent
was key for potent B-Raf activity (Table 3). The
mono-substituted 3-hydroxy derivative 18 showed
comparable affinity to 2, whereas the 4-hydroxy isomer
19 and 3-methoxy derivative 20 showed significantly
reduced B-Raf affinity. These data suggested that the
3-hydroxy group was acting as an H-bond donor
and so we embarked upon a search to identify alterna-
tive groups to perform this role. We were encouraged
to find that the 4-hydroxymethyl derivative 21 showed
modest affinity in the FP assay (Kd 71 nM), which was
in contrast to the regioisomeric 3-hydroxymethyl deriv-
ative 22. Further substitution of the C4 phenyl ring
was tolerated as exemplified by the 3-hydroxy- and
3-chloro-4-hydroxymethyl derivatives 23 and 24 but
this had only a modest effect on potency. Transforma-
tion of 21 into the oxime derivative 25 however pro-
duced a 15-fold increase in potency (Kd 5 nM). Once
again, substitution with hydroxyl- or chloro-substitu-
ents in the 3-position was tolerated (26 and 27)
although the hydroxyl derivative 26 showed a 5-fold
drop in potency compared to 25. Further exploration
of the oxime unit demonstrated that substitution on
the oxime carbon atom was also tolerated in the form
of the ketoxime 28 (Kd 11 nM), although the amidox-
ime derivative 29 showed a 10-fold drop in potency
relative to 25.
Cyclization between the 3 and 4 positions of the C4 substi-
tuent to form the 2,3-dihydroindene and 3,4-dihydro-
naphthalenone derivatives (30–32) also provided some
interesting SAR findings (Table 4). Whilst the racemic
1-hydroxy 2,3-dihydroindene 30 showed weak affinity,
the oxime 31 (Kd 1.3 nM) was extremely potent with activ-
ity at least equal to that of the early lead 2. The ring
expanded 3,4-dihydro-1(2H)-naphthalenone oxime 32
(Kd 17 nM) also showed significant activity, although this
was 10-fold lower than that of 31. SB-590885 (33, Kd
0.3 nM), the C2 (4-dimethylaminoethoxy) phenyl ana-
logue of 31, was prepared to improve the aqueous solubil-
ity (>1 mg/ml in pH 5 buffer) but also showed enhanced
potency, being some 8-fold more potent than the early
lead 2.
N
H
NMe2
N
O
N
N
HO
SB-590885 (33)
B-Raf Kd 0.3nM
Table 4. B-Raf activity of cyclic oximes
The selectivity profiles of SB-590885 (33) and 2 were as-
sessed against a panel of 21 protein kinases (Table 5).
The data confirmed the generally good selectivity profile
N
H
N
N
X
Table 6. Selectivity of SB-590885 (33) and (2) versus p38a, GSK3b
and lck
(CH2)n
Compound
Fold selectivity versus B-Raf
Compound
X
n
B-Raf (FP) Kd nM
p38a
GSK3b
lck
70
>1000
30
31
32
H, OH (R/S)
HON
HON
1
1
2
>200
1.3
17
2
33
7
>1000
30
>1000
Table 5. Selectivity profiling of SB-590885 (33) and (2)a
Com AMPK Chk1 CK2 GSK JNK1 lck MAPK2 MAPK MEK1 MSK1 p70 Phos. PKA PKB PKC PRAK ROCK- p38 p38 p38 p38d SGK
pound
3b
À3 83
25
AP-K2
S6K
K
a
a
II
a
b
c
2
12
14
1
9
0
88 À3
À1
5
7
26
13
17
12
44
2
19
18
3
95 81
46
5
À15
2
33
8
19
39 11
9
14
38
3
À2
8
10
26
9
10
10 17
a Values are %inhibition at 10 lM drug concentration in kinase activity assays in the presence of 100 lM ATP (see Ref. 9 for details).
N
N
N
N
OTBDMS
H
N
OH
OH
O
O
NMe2
iii, iv
v
vi
O
(37)
N
R
MeON
MeON
X
(40) X = NOMe
(41) X = O
X
vii
viii
(33) X = NOH
(38)
(39)
(34) X = O; R = Br
(35) X = NOMe; R = Br
(36) X = NOMe; R = CHO
i
ii
Scheme 1. Reagents and conditions: (i) MeONH2 HCl, pyridine, ethanol (97%); (ii) nBuLi, DMF, THF À60 °C (65%); (iii) LDA, THF À60 °C, add
36; (iv) TBAF, THF (64% over two steps); (v) DMSO, oxalyl chloride, triethylamine dichloromethane À60 °C to room temperature (94%); (vi)
4-(2-dimethylaminoethoxy)benzaldehyde, ammonium acetate, acetic acid 100 °C (30%); (vii) 5 M HCl, acetone, dioxane 100 °C (56%); (viii) 50% aqs
NH2OH, ethanol, reflux (100%).

A. K. Takle et al. / Bioorg. Med. Chem. Lett. 16 (2006) 378–381
381
of 2 but also identified some activity against GSK3b and
References and notes
lck. SB-590885, on the other hand, appeared to be de-
void of significant activity against this panel of enzymes.
More in-depth profiling of both compounds against
p38a, GSK3b, and lck in the fluorescent binding assay
format confirmed the enhanced selectivity profile for
SB-590885 (Table 6).
1. Pearson, G.; Robinson, F.; Beers, T.; Xu, B.; Karandikar,
M.; Berman, K.; Cobb, M. H. Endocr. Rev. 2001, 22, 153.
2. Davies, H. et al. Nature 2002, 417, 949.
3. (a) Chang, F.; Steelman, L. S.; Shelton, J. G.; Lee, J. T.;
Navolanic, P. M.; Blalock, W. L.; Franklin, R.; McCu-
brey, J. A. Int. J. Oncol. 2003, 22, 469; (b) Hilger, R. A.;
Scheulen, M. E.; Strumberg, D. Onkologie 2002, 25, 511.
4. see: Irving, E. A.; Barone, F. C.; Reith, A. D.; Hading-
ham, S. J.; Parsons, A. A. Mol. Brain Res. 2000, 77, 65,
and references therein.
5. (a) Namura, S.; Iihara, K.; Takami, S.; Nagata, I.; Kicku-
chi, H.; Matsushita, K.; Moskowitz, M. A.; Bonventre, J.
V.; Alessandrini, A. Proc. Natl. Acad. Sci. U.S.A 2001, 98,
11569; (b) Wang, X.; Wang, H.; Xu, L.; Rozanski, D. J.;
Sugawara, T.; Chan, P. H.; Trzaskos, J. M.; Feuerstein, G.
Z. J. Pharmacol. Exp. Ther. 2003, 304, 172.
6. Heimbrook, D. C.; Huber, H. E.; Stirdivant, S. M.; Patrick,
D. R.; Claremon, D.; Liverton, N.; Selnick, H.; Ahern, J.;
Conroy, R.; Drakas, R.; Falconi, N.; Hancock, P.; Robin-
son, R.; Smith, G.; Olif, A. American Association for Cancer
Research, New Orleans, April 1998 poster #3793.
7. Compound activity is determined as either the ability of
test compounds to inhibit B-Raf mediated phosphoryla-
tion of kinase dead MEK (data expressed as IC50) or as a
B-Raf-binding affinity derived from the fluorescent ligand
displacement assay (FP, data expressed as Kd). For assay
details, see Dean, D. K.; Takle, A. K.; Wilson, D. M. PCT
Int. Appl. WO 02/24680.
Triaryl imidazoles, such as SB-590885 (33), were pre-
pared as outlined in Scheme 1.10,11 Thus, commercially
available 5-bromo-2,3-dihydro-1H-inden-1-one 34 was
converted to the O-methyl oxime 35 and then formylat-
ed by treatment with n-butyl lithium followed by the
addition of DMF, to afford 36. Reaction of 36 with
the anion derived from the 4-hydroxymethylpyridine
derivative 37,12 followed by desilylation, afforded the ra-
cemic 1,2-diol 38. This was then oxidized to the dione 39
under Swern conditions and converted to the imidazole
40 in modest yield, by treatment with 4-(2-dimethylami-
noethoxy) benzaldehyde and ammonium acetate in ace-
tic acid at 100 °C. Hydrolysis of the O-methyl oxime, by
treatment with 5 M hydrochloric acid and acetone in
dioxane, formed the ketone 41 which was converted to
33 by treatment with aqueous hydroxylamine.
In summary, SB-590885 (33), a potent and extremely
selective inhibitor of B-Raf kinase, was identified follow-
ing an evaluation of the SAR of a series of imidazole
based leads. SB-590885 represents an excellent molecule
with which to investigate the role of B-Raf in neurode-
generative and other disease states.
8. Adams, J. L.; Badger, A. M.; Kumar, S.; Lee, J. C. Prog.
Med. Chem. 2001, 38, 1.
9. Davies, S. P.; Reddy, H.; Caivano, M.; Cohen, P.
Biochem. J. 2000, 351, 95.
10. All novel compounds gave satisfactory 1H NMR and LC/
MS data in full agreement with their proposed structures.
11. SB-590885 was isolated as a 4 to 11:1 mixture of E and Z
isomers. The configuration of the major (E) isomer was
confirmed by NMR.
Acknowledgments
The authors thank colleagues from the departments of
Gene Expression and Protein Biochemistry for the pro-
vision of human recombinant reagents and Screening
and Compound Profiling for kinase activity data. We
also acknowledge the Division of Signal Transduction
Therapy, University of Dundee, for the generation of
kinase selectivity data.
12. Gallagher, T. F.; Siebel, G. L.; Kassis, S.; Laydon, J. T.;
Blumenthal, M. J.; Lee, J. C.; Lee, D.; Boehm, J. C.; Frier-
Thompson, S. M.; Abt, J. W.; Soreson, M. E.; Smietana,
J. M.; Hall, R. F.; Garigipati, R. S.; Bender, P. E.; Erhard,
K. F.; Krog, A. J.; Hofmann, G. A.; Sheldrake, P. L.;
McDonnell, P. C.; Kumar, S.; Young, P. R.; Adams, J. L.
Bioorg. Med. Chem. 1997, 5, 49.