The identification of potent, selective and CNS penetrant furan-based inhibitors of B-Raf kinase

Article abstract of DOI:10.1016/j.bmcl.2008.06.070

Modification of the potent imidazole-based B-Raf inhibitor SB-590885 resulted in the identification of a series of furan-based derivatives with enhanced CNS penetration. One such compound, SB-699393 (17), was examined in vivo to challenge the hypothesis that selective B-Raf inhibitors may be of value in the treatment of stroke.

Full text of DOI:10.1016/j.bmcl.2008.06.070

Bioorganic & Medicinal Chemistry Letters 18 (2008) 4373–4376
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Bioorganic & Medicinal Chemistry Letters
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The identification of potent, selective and CNS penetrant furan-based
inhibitors of B-Raf kinase
a
a
b
a
Andrew K. Takle a, , Mark J. Bamford , Susannah Davies , Robert P. Davis , David K. Dean ,
Alessandra Gaiba a, Elaine A. Irving b, Frank D. King a, Antoinette Naylor a, Christopher A. Parr a,
Alison M. Ray b, Alastair D. Reith d, Beverley B. Smith c, Penelope C. Staton b,
Jon G. A. Steadman a, Tania O. Stean b, David M. Wilson a
*
a Department 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
b Department of Biology, Neurology and GI Centre of Excellence for Drug Discovery, GlaxoSmithKline Pharmaceuticals,
New Frontiers Science Park, Third Avenue, Harlow, Essex CM19 5AW, UK
c Department of Drug Metabolism and Pharmacokinetics, Neurology and GI Centre of Excellence for Drug Discovery, GlaxoSmithKline Pharmaceuticals,
New Frontiers Science Park, Third Avenue, Harlow, Essex CM19 5AW, UK
d Discovery Technology Group, GlaxoSmithKline Pharmaceuticals, New Frontiers Science Park, Third Avenue, Harlow, Essex CM19 5AW, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
Modification of the potent imidazole-based B-Raf inhibitor SB-590885 resulted in the identification of a
series of furan-based derivatives with enhanced CNS penetration. One such compound, SB-699393 (17),
was examined in vivo to challenge the hypothesis that selective B-Raf inhibitors may be of value in the
treatment of stroke.
Received 30 April 2008
Revised 18 June 2008
Accepted 19 June 2008
Available online 24 June 2008
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
B-Raf
Kinase
Furan
Indanone-oxime
Stroke
Mitogen-activated protein (MAP) kinase signalling cascades are
responsible for controlling numerous intracellular processes.1 Typ-
ically such cascades consist of three tiers of protein kinases that are
sequentially activated in response to appropriate extra-cellular
stimuli. This leads to signal transduction and allows for amplifica-
tion to occur at each step of the cascade. The RAF-MEK-ERK MAP
kinase cascade is intimately involved in the regulation of cell cycle
progression and apoptosis, and has been implicated in both prolif-
erative and degenerative disease states. Activating mutations in
B-Raf, one of the Raf family members, are reported to be present
in 66% of malignant melanomas,2 whereas increased activation of
ERK1/2 has also been reported in a number of in vitro and in vivo
models of neuronal cell death.3,4 Furthermore, inhibition of the
cascade at the MEK level has led to a reduction in infarct volume
in rodent models of stroke.4
of the potent and extremely selective triaryl imidazole
SB-590885 (1). Here, we wish to report the result of further studies
to identify inhibitors with enhanced CNS penetration.
N
N
NMe2
H
N
H
N
O
Ph
N
N
Cl
N
OH
HO
SB-590885 (1)
B-Raf Kd 0.3nM
L-779,450 (2)
B-Raf Kd 2.4nM
We have previously reported our initial efforts to identify po-
tent inhibitors of B-Raf in order to assess their potential in the
treatment of stroke.5 These studies resulted in the identification
SB-590885 was initially assessed for its ability to inhibit B-Raf-
mediated ERK phosphorylation in rat pheochromocytoma (PC12)
cells following nerve growth-factor (NGF) stimulation, as a marker
of cellular pathway inhibition (Table 1). In this assay, SB-590885
showed a robust, dose-dependant inhibition of ERK phosphoryla-
* Corresponding author. Tel.: +44 (0)1279 627686; fax: +44 (0)1279 622260.
E-mail address: Andy.K.Takle@gsk.com (A.K. Takle).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.06.070

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A. K. Takle et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4373–4376
Table 1
carboxamido-C2 substituents bearing solubilising basic amino
groups, the cellular activity of these derivatives was variable (Table
2). More disappointing was the finding that the CNS penetration of
this series of compounds was routinely poor.
Cell-based pathway inhibition and rat in vivo pharmacokinetic parameters for
SB-590885 (1) and L-779,450 (2)a7
Compound
Pathway inhibition (% inhibition)
Blood clearance
(ml/min/kg)
Brain:blood
ratio (BB)
In an effort to overcome the problem of inadequate CNS pene-
tration of this series of molecules, we turned our attention to mod-
ifying the core heterocycle and in so doing reducing the number of
H-bond donors (HBD) and the polar surface area (PSA) of the mol-
ecules (Table 3).9 We were pleased to find that replacement of the
central imidazole core by either isomeric furan (10 and 12) or pyr-
role (11 and 13) groups afforded molecules with potent B-Raf affin-
ity, demonstrable cellular activity and enhanced CNS penetration.
This was most notable for the furan derivatives, which benefited
from both reduced PSA and HBD count, and especially so for the
C2-(4-pyridyl)furan isomer (10).
Having identified this series of furan-based inhibitors with
intrinsically superior CNS penetration, we sought to tune the over-
all properties of the molecules through a re-investigation of the
SAR of the C5 position (equivalent to the C2 position of the imidaz-
ole core) of the C2-(4-pyridyl)furans (Table 4). The C5 position was
once again tolerant of a variety of diverse aryl (not shown), alkyl,
and carboxamido-substituents that retained potent B-Raf affinity
and good cellular activity. It was notable, however, that whilst im-
1
l
M
3 lM
1
2
99
—
101
100
45
77
0.14:1
<0.1:1
a
The brain:blood (BB) ratio was calculated as the ratio of brain:blood concen-
trations following a constant rate (3.5–5 ml/kg/h) iv infusion to steady-state (6–
16 h) and at a target dose level of between 1 and 3 mg free base/kg/h. Blood
clearance was calculated as dose-rate/steady-state blood concentration.
tion similar to that seen with the early lead molecule L-779,450
(2).6 However, due to the inter assay variability, all novel com-
pounds were assessed relative to the activity of a standard 3 lM
(micromolar) concentration of L-779,450 as an internal control
(i.e., 100% refers to equivalent inhibition to that produced by
3 l
M of L-779,450 in that particular assay).7
Having demonstrated the ability of our tool compounds to inhi-
bit B-Raf-mediated signalling in a cellular context, we next exam-
ined their in vitro neuroprotective activity. Compounds were
tested for their ability to protect rat hippocampal slice cultures
from death induced by oxygen and glucose deprivation (OGD).8
We were encouraged to find that both compounds displayed po-
tent neuroprotective activity in this assay; L-779,450 showing
Table 3
In vitro and in vivo activities of core modified B-Raf inhibitors
approximately 80% protection at 10
lM and SB-590885 showing
a comparable degree of protection at 0.1
lM.
N
Unfortunately, despite its encouraging in vitro profile,
SB-590885 showed moderate blood clearance and poor CNS pene-
tration in the rat (Table 1). Whilst the blood clearance rate was
acceptable, since any therapy would likely be administered by con-
tinuous intra venous (iv) infusion, the poor penetration of the CNS
(brain:blood ratio (BB) 0.14:1) was sub-optimal for this indication.
We thus set out to improve the CNS penetration of the series
(target BB P 0.5:1) whilst being mindful of the need to retain
solubility enhancing groups to meet the likely requirements of a
putative iv agent. Our initial approach was to modify the imidazole
C2 position since we had previously shown that B-Raf was tolerant
of a wide variety of substitutions at this position.5 Whilst potent
B-Raf binding was achieved with a number of aryl-, alkyl-, and
NMe2
X
Y
O
N
HO
Compound
X
Y
B-Raf (FP)
Kd (nM)
Pathway inhibition
(% inhibition)
PSA (HBD) BB ratio
1
l
M
3 lM
1
NH
O
N
0.3
99
75
66
60
17
101
87
74
85
66
87 (2)
71 (1)
74 (2)
71 (1)
74 (2)
0.14:1
1.33:1
0.27:1
0.56:1
0.33:1
10
11
12
13
CH 0.5
NH CH 0.7
CH 0.9
CH NH 2.4
O
Table 2
In vitro and in vivo activities of C2 modified imidazolesa
N
Table 4
H
N
In vitro and in vivo activities of C5 substituted furans
R1
N
N
O
R
N
HO
Compound
R1
B-Raf (FP)
Kd (nM)
Pathway
inhibition
(% inhibition)
BB
ratio
N
HO
Compound
R
B-Raf (FP)
Kd (nM)
Pathway
inhibition
Blood
clearance
BB
ratio
1
l
M
3 lM
3
4-[2-(2-Dimethylamino
propyloxy)]pyrimidine
C(Me2)CH2NH2
0.5
88
100
0.1:1
(% inhibition) (ml/min/kg)
1
l
M
3 lM
4
5
6
7
8
9
4.9
1.7
6.3
15.8
1.4
1.3
61
36
41
96
59
33
À8
59
98
4-Piperidine
14
15
4-Piperidine
4-Piperidine-
1-(CH2)2OMe
CONH(CH2)2-NMe2 0.5
4-Morpholino-
methyl
2.4
2.7
93
83
100
96
78
100
0.24:1
1.39:1
4-Piperidine-1-(CH2)2OMe
4-Morpholino-methyl
CONH(CH2)2-morpholine
CONHPh(3-CH2NMe2)
0.09:1
0.09:1
À10
61
16
17
93
91
NT
95
91
54
0.1:1
0.8:1
77
7.2
a
Compound potency is expressed as a B-Raf binding affinity (Kd) derived from a
fluorescent ligand binding assay (FP). See Ref. 8 for details.
NT, not tested.

A. K. Takle et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4373–4376
4375
Table 5
Selectivity profiling of SB-590885 (1) and SB-699393 (17)a
Compound AMPK Chk1 CK2 GSK3 b JNK1 Lck MAPK2 MAPKAP-K2 MEK1 MSK1 p70 S6K Phos.K PKA PKB
a
PKCa
PRAK ROCK-II p38
a
p38b p38
c
p38d SGK
1
17
14
6
9
7
8
9
25
À9
19
À2
39 11
9
4
14
12
38
7
13
À3
3
À8
À2
À4
8
5
10
3
18
11
26
11
46
12
9
12
10
8
10
0
17
0
3
7
a
Values are % inhibition at 10 lM drug concentration in kinase activity assays in the presence of 100 lM ATP (see Ref. 10 for details).
proved CNS penetration was a general feature of the furan deriva-
tives, the incorporation substituents bearing additional HBDs, such
as the unsubtituted piperidine (14) or carboxamide derivative (16),
once again resulted in a reduction in the brain:blood ratio. It was
also notable that blood clearance rates could be modulated by
modification of the furan C5 position, although in this case SAR
was difficult to interpret. As a result of our efforts, the C5 morpho-
lino-methyl derivative 17 (SB-699393) was identified as a mole-
cule possessing the optimal balance of potency, cellular activity
and pharmacokinetic properties. When assessed against a panel
of 21 protein kinases, SB-699393 (17) also showed an excellent
selectivity profile comparable to that of the imidazole lead SB-
590885 (1) (Table 5).
measure of pharmacodynamic activity in the brain, we chose to
assess the compounds ability to inhibit hippocampal ERK phos-
phorylation in rats subjected to cold water stress.11 We were
gratified to find that pretreatment with a 3 or 10 mg/kg iv bolus
of SB-699393, 5 min prior to cold water stress, was successful in
reducing the induced hippocampal ERK phosphorylation (Fig. 1).
In order to assess its neuroprotective activity, SB-699393 was
administered as a 5 h continuous iv infusion at doses ranging
from 0.3 to 10 mg/kg/h, starting 1 h post-insult, in a permanent
middle cerebral artery occlusion model of stroke in normotensive
rats.12 In this model, efficacy would be determined by an
improvement in neurological deficit and/or a reduction in infarct
lesion volume relative to vehicle. Unfortunately, SB-699393 failed
to demonstrate a significant improvement in either parameter
when assessed 24 h post-injury. These results, whilst apparently
at odds to earlier reports that link prolonged ERK activation to
neuronal cell death,4 suggest that selective inhibition of B-Raf
alone is not sufficient to elicit significant neuroprotection in ro-
dent models of stroke.
With a potent, selective and CNS penetrant molecule in hand
(SB-699393), we next set out to assess its activity in vivo. As a
Saline/
no swim
Saline/
swim
Swim/SB-699393-B
(3mg/kg iv)
KDa
47.5
•
•
•
•
•
•
•
•
• • •
•
•
•
•
•
•
Treatment group
SB-699393 (17) was prepared as shown in Scheme 1.13 Thus,
commercially available 5-bromo-2,3-dihydro-1H-inden-1-one 18
was converted to the O-methyl oxime 19 then to the boronic acid
20 by treatment with n-butyl lithium followed by the addition of
trimethyl borate. Meanwhile, 2,3-dibromo furan was reacted with
4-pyridyl boronic acid under Suzuki cross-coupling conditions to
regioselectively generate the 2-(4-pyridyl)furan 22 in 54% yield.14
Suzuki coupling between the furan 22 and the boronic acid 20 fur-
nished the disubstituted furan 23 (74%) which was then formylat-
ed, by treatment with LDA followed by the addition of DMF, to
afford 24 in 56% yield. Reductive amination with morpholine and
polymer-bound trimethylammonium cyanoborohydride afforded
the trisubstituted precursor 25. Hydrolysis of the O-methyl oxime
with 5 M hydrochloric acid and acetone in dioxane formed the ke-
32.5
Saline/
no swim
Saline/
swim
Swim/SB-699393-B
(10mg/kg iv)
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
Treatment group
KDa
47.5
32.5
Figure 1. Inhibition of cold water stress-induced hippocampal ERK phosphoryla-
tion by SB-699393 (17). An equivalent loading of tissue samples is used in each
lane.
N
R
Br
O
O
iii
iv
X
Br
Br
(18) X = O; R = Br
(19) X = NOMe; R = Br
(20) X = NOMe; R = B(OH)2
(21)
(22)
i
ii
O
N
N
N
O
O
N
O
v
vi
CHO
MeON
MeON
X
(25) X = NOMe
(26) X = O
(17) X = NOH
vii
viii
(23)
(24)
Scheme 1. Preparation of SB-699393 (17).13 Reagents and conditions: (i) MeONH2 HCl, pyridine, ethanol (96%); (ii) nBuLi, trimethyl borate, THF À78 °C to room temperature
(58%); (iii) 4-pyridyl boronic acid, Pd(OAc)2, PPh3, K2CO3, 2:1 DME–water, reflux (54%); (iv) (20), Pd(OAc)2, PPh3, K2CO3, 2:1 DME–water, reflux (74%); (v) LDA, DMF, THF
À78 °C (56%); (vi) morpholine, polymer-bound trimethylammonium cyanoborohydride, methanol, acetic acid (61%); (vii) 5 M HCl, acetone, dioxane 100 °C; (viii) 50% aqueous
NH2OH, ethanol, reflux (64% for two steps).

4376
A. K. Takle et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4373–4376
5. Takle, A. K.; Brown, M. J. B.; Davies, S.; Dean, D. K.; Francis, G.; Gaiba, A.; Hird, A.
W.; King, F. D.; Lovell, P. K.; Naylor, A.; Reith, A. D.; Steadman, J. G.; Wilson, D.
M. Bioorg. Med. Chem. Lett. 2006, 16, 378.
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.; Robinson, R.; Smith, G.; Olif, A. American Association for Cancer Research, New
Orleans, April 1998, Poster #3793.
7. PC12 cells were pretreated with compounds for 60 min prior to stimulation
with NGF. After 10 min cells were lysed and the lysates separated by SDS–
PAGE. Following transfer to nitrocellulose membranes, levels of
phosphorylated-ERK1/2 were determined following antibody probing.
tone 26, which was converted to 17 by treatment with aqueous
hydroxylamine in 64% yield.
In conclusion, we have extended our understanding of the SAR
associated with this series of extremely potent and selective 2,3-
dihydro-1H-inden-1-one oxime substituted heterocyclic B-Raf
inhibitors. In so doing, we have identified the furan derivative
SB-699393 (17) which possesses enhanced CNS penetration; how-
ever, this molecule failed to show significant neuroprotective ef-
fects when evaluated in a rodent model of stroke.
Pretreatment with
3 lM L-779,540 (2) routinely caused approx. 90%
inhibition of NGF-stimulated ERK1/2 phosphorylation.
8. For details of the B-Raf fluorescent ligand binding assay (FP) and the OGD in
vitro neuroprotection assay, see Dean, D. K.; Takle, A. K.; Wilson, D. M. PCT Int.
Appl. WO 02/24680.
Acknowledgments
The authors thank colleagues from the departments of Gene
Expression and Protein Biochemistry for the provision 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.
9. Molecular polar surface area (PSA) has long been recognised as a contributing
factor to the brain penetration of drug-like molecules. See Kelder, J.;
Grootenhuis, P. D. J.; Bayada, D. M.; Delbressine, L. P. C.; Plemen, J.-P. Pharm.
Res. 1999, 16, 1514; PSA values quoted in this paper are calculated using ‘2D’
methods. See Ertl, P.; Rohde, B.; Selzer, P. J. Med. Chem. 2000, 43, 3714.
10. Davies, S. P.; Reddy, H.; Caivano, M.; Cohen, P. Biochem. J. 2000, 351, 95.
11. Rats were subjected to a cold water swim (18 °C) for 10 min. The test animals
were then sacrificed, the brains removed, dissected and hippocampal phospho-
ERK levels assessed by Western blotting. Test compounds were administered
as an iv bolus 5 min prior to the cold water swim.
References and notes
12. See Ref. 3, and references therein, for details of the rat, permanent middle
cerebral artery occlusion model of stroke.
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. See Irving, E. A.; Barone, F. C.; Reith, A. D.; Hadingham, S. J.; Parsons, A. A. Mol.
Brain Res. 2000, 77, 65. and references therein.
4. (a) Namura, S.; Iihara, K.; Takami, S.; Nagata, I.; Kickuchi, 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.
13. All novel compounds gave satisfactory 1H NMR and LC/MS data in full
agreement with their proposed structures. Oximes were isolated as mixtures of
E and Z isomers in which the E isomer predominated. For experimental details
of the synthesis of SB-699393 (17) see Bamford, M. J.; Dean, D. K.; Naylor, A.;
Takle, A. K.; Wilson, D. M. PCT Int. Appl. WO 03/022840.
14. Precedence exists for the regioselective coupling of 2,3-dihalo-substituted
furans, see, for example, Bach, T.; Kruger, L. Eur. J. Org. Chem. 1999, 2045.
Nonetheless, the structure of 22 was also confirmed by HMBC NMR
spectroscopy.