The discovery of potent and selective pyridopyrimidin-7-one based inhibitors of B-RafV600E kinase

Article abstract of DOI:10.1016/j.bmcl.2012.04.015

Herein we describe the discovery of a novel series of ATP competitive B-Raf inhibitors via structure based drug design (SBDD). These pyridopyrimidin-7-one based inhibitors exhibit both excellent cellular potency and striking B-Raf selectivity. Optimization led to the identification of compound 17, a potent, selective and orally available agent with excellent pharmacokinetic properties and robust tumor growth inhibition in xenograft studies.

Full text of DOI:10.1016/j.bmcl.2012.04.015

Bioorganic & Medicinal Chemistry Letters 22 (2012) 3387–3391
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
The discovery of potent and selective pyridopyrimidin-7-one based inhibitors
of B-RafV600E kinase
Li Ren a, , Kateri A. Ahrendt a, Jonas Grina a, Ellen R. Laird a, Alex J. Buckmelter a, Joshua D. Hansen a,
⇑
Brad Newhouse a, David Moreno a, Steve Wenglowsky a, Victoria Dinkel a, Susan L. Gloor a,
Gregg Hastings a, Sumeet Rana a, Kevin Rasor a, Tyler Risom a, Hillary L. Sturgis a,
Walter C. Voegtli a, Simon Mathieu b
a Array BioPharma, Inc., 3200 Walnut Street, Boulder, CO 80301, United States
b Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080-4990, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Herein we describe the discovery of a novel series of ATP competitive B-Raf inhibitors via structure based
drug design (SBDD). These pyridopyrimidin-7-one based inhibitors exhibit both excellent cellular
potency and striking B-Raf selectivity. Optimization led to the identification of compound 17, a potent,
selective and orally available agent with excellent pharmacokinetic properties and robust tumor growth
inhibition in xenograft studies.
Received 22 February 2012
Revised 29 March 2012
Accepted 3 April 2012
Available online 10 April 2012
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
B-Raf
Kinase
Drug discovery
Pyridopyrimidin-7-one
Structure based drug design
F
The Raf family protein kinases, consisting of A-Raf, B-Raf, and
C-Raf, are central components of the MAPK pathway that regulates
cellular proliferation, differentiation, and survival.1 Raf kinases act
downstream of RAS and are responsible for MEK and ERK activa-
tion. BRAF gene mutations may lead to MAPK pathway amplifica-
tion via constitutive activation of B-Raf, and are present in ꢀ7%
of all cancers,2 with high frequency in malignant melanoma
(30–70%).3,4 The most common (>90%) mutation in B-Raf is a
glutamic acid for valine substitution at residue 600 (V600E),2
which leads to constitutive kinase activity 500-fold greater than
wild-type B-Raf,5 and correlates with increased malignancy and
decreased response to chemotherapy.6 A number of drug candi-
dates targeting the B-RafV600E mutation have entered clinical trials
in recent years. Several of these, such as vemurafenib7 and dabrafe-
nib8 have shown clinical efficacy, thus validating B-RafV600E as a
cancer target.
F
O
O
H
N
O
O
H
N
S
3
S
N
6
N
N
N
H
H
O
F
4
O
F
H2N
5
Br
1
2
B-RafV600E enzyme IC50 = 3900 nM
Malme-3M pERK IC50 = 6700 nM
B-RafV600E enzyme IC50 = 110 nM
Malme-3M pERK IC50 = 310 nM
Figure 1. B-RafV660E inhibitors 1 and 2.
keeper residue Thr529.9a The initial series of amide-linked B-
RafV600E inhibitors utilized a pyridine as a hydrogen bond acceptor,
which was postulated to bind to the –NH of hinge residue Cys532.
The micromolar enzymatic and cellular activity of pyridine amide 1
was improved 30-fold by introducing an amino group at the
6-position and a bromine atom at the 5-position.9a
Our lab recently reported the discovery of several novel series of
potent and selective inhibitors of B-RafV600E 9
. A common feature
An X-ray crystal structure9a of B-RafWT in complex with amino
pyridine amide 2 revealed that the newly installed 6-amino group
is hydrogen-bonded to the carbonyl of Cys532, the sulfonamide
moiety forms several hydrogen bonds with the backbone of the
DFG sequence, and the propyl chain occupies a small lipophilic
for these inhibitors (Fig. 1) is an amide linker connecting various
hinge-binding templates to an aryl sulfonamide, with the amide
–NH making a weak hydrogen bond to the hydroxyl of the gate-
pocket that is enlarged by an outward shift of the aC-helix (Fig. 2).
⇑
The bromine atom makes key lipophilic contacts to the side
chains of Ile463, Val471, Trp531, and Phe583.
Corresponding author.
E-mail address: li.ren@arraybiopharma.com (L. Ren).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.04.015

3388
L. Ren et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3387–3391
Table 1
B-Raf activity of compounds 3–9
F
O O
S
H
Ar
N
F
B-RafV600E IC50 (nM)
pERK IC50 (nM)
a
a
Compd
Ar
H
N
b
N
3
>10,000
—
N
N
b
4
5
6
340
—
N
b
3606
241
—
N
N
703
469
N
N
7
8
9
72
71
13
Br
Figure 2. X-ray crystal structure of 2 in complex with B-RafWT. The cleft surface is
rendered in violet, select residues are depicted in white, and the inhibitor is green.
Hydrogen-bonding interactions are illustrated with yellow dashed lines. Several
residues that are involved in hydrophobic interactions with 2 are omitted for clarity
and are described in the text. The propyl group resides in a pocket that is enlarged
N
N
N
445
O
N
by an outward shift of the
aC-helix. The DFG sequence (D594-G596) resides in its
active (DFG-in) conformation.
1163
O
(CH2)2Ph
The crystal structure also revealed two characteristic conforma-
tional features of the amide series: a near co-planar arrangement of
the amide group with the hinge-binding scaffold and a near 90° tor-
sion angle of the amide relative to the 2,6-di-halo substituted cen-
a
IC50 values reflect the average from at least two separate experiments.
Not determined.
b
tral phenyl ring. This resulted in
a near orthogonal spatial
arrangement between the propylsulfonamide bearing phenyl ring
and the hinge-binding pyridine ring, and positions them to interact
optimally with residues in the ATP pocket. Although the amide lin-
ker functioned efficiently as a spacer and a conformational control,
its chemical and metabolic stability were perceived as liabilities. In-
deed, potentially toxic aniline metabolites generated from in vivo
cleavage of the amide bond were observed. To address these con-
cerns, we initiated a program to identify alternative linkers.
was inactive. Modeling studies revealed that the 6,5-fused ring
system repositioned the central di-halo aryl ring and the propyl-
sulfonamide tail, rendering 3 unable to maintain key contacts with
the DFG motif. In contrast, molecular modeling suggested that the
propyl sulfonamide on the 6,6-fused template should occupy a
similar space when compared to the amide series. As predicted,
isoquinoline 4, showed ca. 10-fold improvement in enzyme inhibi-
tion over 1. The new core also made an improved hydrophobic con-
tact with Phe583 on the floor of the ATP pocket. Incorporating a
nitrogen atom into the second ring made a striking difference to
activity depending on the substitution pattern. Compound 5 was
less active most likely owing to a desolvation penalty upon burial
of a strong hydrogen bond acceptor. Binding activity was restored
for 2,6-naphthyridine 6, where N2 is more accessible to solvent,
with concomitant submicromolar cellular activity. Consistent with
the observation made in the amide series, 8-bromo naphthyridine
7 showed improved potency presumably due to added hydropho-
bic contacts with the protein. Further improvement over 6 could
also be achieved by introducing a carbonyl group into the second
ring. The resulting 2,6-naphthyridin-1-one 8 showed similar activ-
ity to 7. Finally, 1,6-naphthyridin-2-one 9, an inhibitor with re-
versed amide connectivity, showed the most promising enzyme
activity. Results from modeling of compound 9 suggested that
the potency gain could be attributed to improved hydrophobic
contact between the phenethyl substituent on the amide nitrogen
and residues of the P-loop (Fig. 4).
Our strategy was based on the observation that the amide linker
adopted a near co-planar conformation in relationship to the pyr-
idine ring. Wishing to retain the propyl sulfonamide moiety, a key
feature for the DFG-in/aC-helix shifted binding mode, we explored
the approach of connecting the amide to the 4-position of the pyr-
idine to form a fused heterocycle (Fig. 3). The resulting inhibitor
with the appropriate torsion angle between the newly introduced
bicycle and the propylsulfonamide bearing phenyl ring should
maintain all the critical interactions with the enzyme.
Due to synthetic accessibility, initial hinge-binding template
evaluation focused on fused heterocycles with a single hinge con-
tact to the main chain –NH of Cys532. It was anticipated that once
an optimal scaffold was identified, activity could be further im-
proved 4- to 5-fold by installing an amino group at the appropriate
position to interact with the carbonyl of Cys532.9a Selected exam-
ples are shown in Table 1.10
Pyrrolopyridine 3 was prepared in an attempt to retain the
amide –NH interaction with the gatekeeper residue Thr529, but
F
F
O
O
S
O O
H
N
W
Z
S
N
N
N
X
Y
N
H
H
O
F
F
H2N
H2N
Br
2
Figure 3. Schematic illustration of the amide linker replacement strategy.

L. Ren et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3387–3391
3389
Table 3
B-Raf activity of compounds 11, 13–15
R2
6
O O
S
H
N
N
2
R1
H2N
R1, R2
N
a
a
Compd
B-RafV600E IC50 (nM)
pERK IC50 (nM)
682
11
13
14
15
F, F
H, H
H, F
140
b
>10,000
>10,000
128
—
—
b
H, Me
>10,000
a
IC50 values reflect the average from at least two separate experiments.
Not determined.
b
Figure 4. Model of 9 illustrating the possibility for hydrophobic contact between
residues of the P-loop and the phenethyl group.
Subsequent optimization focused on 6,6-fused bicyclic systems
represented by compounds 4, 6, 8, and 9 due to their initial encour-
aging activities. As pointed out earlier, targeting the interaction
with the carbonyl of Cys532 via an amino group became the next
priority. However, the considerable synthetic challenges associated
with 6 and 8 precluded further investigation of these scaffolds. Se-
lected examples from expansion of templates 4 and 9 are shown in
Table 2.
Amino isoquinoline 10, with an appropriately placed NH2 moi-
ety, showed no improvement in enzyme potency over 4. Increased
activity was observed for 11, by converting the hinge-binding mo-
tif in 10 from an isoquinoline to a quinazoline. The potency of the
aminoquinazoline scaffold could be further enhanced via the strat-
egy that was employed previously (9 vs 4). The resulting pyrido-
pyrimidin-7-one analog 12 showed encouraging activity in both
enzyme and cellular assays, and served as a starting point for fur-
ther optimization.
In parallel to the on-going efforts to identify an optimal bi-aryl
template to replace the amide linker, the effect of varying the sub-
stitutions at the 2 and 6-position on the central phenyl ring was
briefly evaluated with the aminoquinazoline series (Table 3).
Removing both fluorines (13) completely abolished any B-Raf
activity and a mono-fluoro at the 6-position (14) was also inactive.
Figure 5. X-ray crystal structure of 19 in complex with B-RafWT. The color scheme
is as described in Figure 2, and the observed interactions are consistent with those
depicted for compound 2. The hydroxypropyl group is involved in a hydrogen bond
with the sidechain of Ser536.
Table 2
B-Raf activity of compounds 10–12
F
O O
S
Ar
N
H
F
a
a
Compd
Ar
B-RafV600E IC50 (nM)
pERK IC50 (nM)
N
N
N
b
10
549
—
H2N
H2N
H2N
11
13
140
10
682
93
N
N
N
O
Figure 6. Edge view of the X-ray crystal structure of 19 illustrating the added
contacts at the exit of the ATP cleft. Several of these residues are expected to be
involved in the potency enhancements for compounds described in Table 4.
a
IC50 values reflect the average from at least two separate experiments.
Not determined.
b

3390
L. Ren et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3387–3391
Table 4
fold. We reasoned that further improvement in potency could be
B-Raf activity of compounds 12, 16–23
achieved by exploring contacts to the outer edge of the ATP cleft
(defined by residues Gly534-Ser536, and the sidechains of Ile463,
Trp531); illustrated in Figure 6 (vide infra); selected examples
are shown in Table 4.
Alkyl substitutions in general gave a 2- to 3-fold improvement in
cell potency over 12. The most potent among these was compound
17, a cyclopropylmethyl derivative with a pERK IC50 of 27 nM.
Installation of aryl and heteroaryl groups resulted in the identifica-
tion of several highly active B-Raf inhibitors, several of which exhib-
ited sub-nanomolar enzyme activity with single digit nanomolar
cellular potency. For instance, compound 23 showed an impressive
F
O O
S
H
N
N
F
RHN
N
N
O
a
a
Compd
R
B-RafV600E IC50 (nM) pERK IC50 (nM)
12
16
H
Me
10
9
93
45
17
3
27
pERK IC50 of 1 nM.
WT
18
19
CH2CH2OH
CH2CH2CH2OH
6
7
31
41
An X-ray crystal structure of B-Raf
in complex with 19 con-
firmed our anticipated binding mode and the hydroxypropyl group
is involved in a hydrogen bond with the sidechain of Ser536 (Fig. 5).11
The pyridopyrimidin-7-ones were prepared according to Scheme 1.
Condensation of 4-(methylamino)-2-(methylthio)pyrimidine-5-carb-
aldehyde 24 with methyl 2-(3-amino-2,6-difluorophenyl)acetate 25
in the presence of K2CO3 afforded 6-(3-amino-2,6-difluorophenyl)-8-
methyl-2-(methylthio)pyrido[2,3-d]pyrimidin-7(8H)-one 26. Mono-
sulfonylation of aniline 26 was accomplished in a two-step fashion
to give 27. Activation of the methylthio moiety via oxidation, followed
by displacement with amines furnished compounds 16–23.
O
20
15
73
21
22
23
Ph
0.5
0.5
5
3
1
3-Pyridyl
1-Methyl-pyrazole-4-yl 0.4
a
IC50 values reflect the average from at least two separate experiments.
These results indicate that the 2-substituent is critical for potency
and are consistent with SAR from the amide series.9b Although the
enzyme potency of 15 is equivalent to 11, the 6-methyl derivative
lacks any cellular activity. While the exact reason favoring the 2,6-
difluoro substitution pattern is unclear, possible explanations in-
clude effects of the fluorines on hydrophobic contacts, torsion an-
gle, and/or the pKa of the sulfonamide motif.
The similar SAR trends from two distinct series on the central
phenyl ring convinced us to use the 2,6-difluoro phenyl sulfon-
amide tail for the optimization of the pyridopyrimidin-7-one scaf-
Due to their potent cellular activity, 21–23 were screened in a
panel of 65 protein kinases at 1 lM. Unfortunately, these analogs
showed strong inhibition (>90%) of multiple kinases such as ABL,
FGR, KDR, LCK and SRC. These findings were consistent with liter-
ature reports on PD166285,12 a related compound without the pro-
pylsulfonamide, which is a multi-kinase inhibitor. On the contrary,
alkyl derivatives 17, 18 and 19 exhibited excellent kinase selectiv-
ity toward non-Raf kinases. For example, the inhibitory activity of
F
F
CHO
NH
N
N
NH2
a
+
MeOOC
F
NH2
MeS
N
MeS
N
N
O
F
25
26
24
F
F
O
O
O O
S
S
b, c
N
N
H
d, e
N
N
H
F
F
MeS
N
N
O
RHN
N
N
O
27
16 23
-
Scheme 1. Preparation of pyridopyrimidin-7-ones: Reagents and conditions: (a) K2CO3, DMF, 80 °C; (b) n-PrSO2Cl, NEt3, DCM; (c) NaOH, MeOH/THF; (d) m-CPBA, DCM; (e)
NH2R, 90 °C.
Table 5
In vitro ADME and pharmacokinetic properties of 17–19
Caco-2b
Hepatocyte clearancea
Observed clearancec
Vd
AUCf
%F
d
Compd
17
18
19
High
High
High
17
75
46
3
74
—
0.46
1
20
133
1
20
33
18
—
e
e
a
b
c
d
e
f
Mouse hepatocyte clearance (ml/min/kg).
Caco-2 permeability classification: low (<2 Â 10À6 cm/s), medium (2–8 Â 10À6 cm/s), high (>8 Â 10À6 cm/s).
Mouse IV PK at 2.5 mg/kg (ml/min/kg).
l/kg.
Not determined.
Mouse PO PK at 30 mg/kg (lM h).

L. Ren et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3387–3391
3391
2500
2000
1500
1000
500
References and notes
Vehicle, 10 ml/kg
17, 30 mg/kg QD
1. Peyssonnaux, C.; Eychene, A. Biol. Cell. 2001, 93, 53.
2. Davies, H.; Bignell, G. R.; Cox, C.; Stephens, P.; Edkins, S.; Clegg, S.; Teague, J.;
Woffendin, H.; Garnett, M. J.; Bottomley, W.; Davis, N.; Dicks, E.; Ewing, R.;
Floyd, Y.; Gray, K.; Hall, S.; Hawes, R.; Hughes, J.; Kosmidou, V.; Menzies, A.;
Mould, C.; Parker, A.; Stevens, C.; Watt, S.; Hooper, S.; Wilson, R.; Jayatilake, H.;
Gusterson, B. A.; Cooper, C.; Shipley, J.; Hargrave, D.; Pritchard-Jones, K.;
Maitland, N.; Chenevix-Trench, G.; Riggins, G. J.; Bigner, D. D.; Palmieri, G.;
Cossu, A.; Flanagan, A.; Nicholson, A.; Ho, J. W. C.; Leung, S. Y.; Yuen, S. T.;
Weber, B. L.; Seigler, H. F.; Darrow, T. L.; Paterson, H.; Marais, R.; Marshall, C. J.;
Wooster, R.; Stratton, M. R.; Futreal, P. A. Nature 2002, 417, 949.
3. Pollock, P. M.; Harper, U. L.; Hansen, K. S.; Yudt, L. M.; Stark, M.; Robbins, C. M.;
Moses, T. Y.; Hostetter, G.; Wagner, U.; Kakereka, J.; Salem, G.; Pohida, T.;
Heenean, P.; Duray, P.; Kallioniemi, O.; Hayward, N. K.; Trent, J. M.; Meltzer, P.
S. Nat. Genet. 2003, 33, 19.
4. (a) Gorden, A.; Osman, I.; Gai, W.; He, D.; Huang, W.; Davidson, A.; Houghton, A.
N.; Busam, K.; Polsky, D. Cancer Res. 2003, 63, 3955; (b) Kuman, R.; Angelini, S.;
Czene, K.; Sauroja, I.; Hahka-Kemppinen, M.; Pyrhonen, S.; Hemminki, K. Clin.
Cancer Res. 2003, 9, 3362.
5. Wan, P. T.; Garnett, M. J.; Roe, S. M.; Lee, S.; Niculescu-Duvaz, D.; Good, V. M.;
Jones, C. M.; Marshall, C. J.; Springer, C. J.; Barford, D.; Marais, R. Cell 2004, 116,
855.
6. (a) Samowitz, W. S.; Sweeney, C.; Herrick, J.; Albertsen, H.; Levin, T. R.;
Murtaugh, M. A.; Wolff, R. K.; Slattery, M. L. Cancer Res. 2005, 65, 6063; (b)
Riesco-Eizaguirre, G.; Gutiérrez-Martínez, P.; García-Cabezas, M. A.; Nistal, M.;
Santisteban, P. Endocr-Relat. Cancer 2006, 13, 257; (c) Houben, R.; Becker, J. C.;
Kappel, A.; Terheyden, P.; Bröcker, E. B.; Goetz, R.; Rapp, U. R. J. Carcinog. 2004,
3, 6.
7. Puzanov, I.; Flaherty, K. T.; Sosman, J. A.; Grippo, J. F.; Su, F.; Nolop, K.; Lee, R. J.;
Bollag, G. Drugs Fut. 2011, 36, 191.
8. Kefford, F. F.; Arkenau, H.; Brown, M. P.; Millward, J. R.; Infante, J. R.; Long, G. V.;
Ouellet, D.; Curtis, M.; Lebowitz, P. F.; Falchook, G. S. J. Clin. Oncol. 2010, 28,
8503. 15s Abstr.
97%
11
74%
96%
8
35%
3
0
1
5
Day of Study
Figure 7. Tumor growth inhibition of 17 in LOX xenograft.
17 was assessed at 1
the human kinome at [ATP] ꢀKm, ATP. Only one kinase (LIMK1)
showed >80% inhibition at 1 M besides B-Raf and C-Raf.
Compounds 17–19 were highly permeable in a Caco-2 perme-
ability assay and showed variable stability in mouse hepatocytes
(Table 5). Importantly, the most stable analog 17 exhibited low
clearance in mouse pharmacokinetic studies and afforded the high-
est oral exposure level. On the contrary, the least stable derivative
18 showed high clearance with very low AUC.
Based on the optimal combination of activity and pharmacoki-
netic properties, 17 was advanced to a tumor growth inhibition
(TGI) study in nude mice with established LOX (B-RafV600E) xeno-
grafts. Compound 17 was administered at a daily dose of 30 mg/
kg QD from day 1 to day 4, and tumor volume was measured on
day 1, 3, 5, 8, and 11 (Fig. 7). After 4 days of dosing, a 74% TGI re-
sponse was registered on day 5. More importantly, prolonged inhi-
bition was observed even after dosing was stopped. For example,
96% & 97% TGI were reported on day 8 and 11. These findings sug-
gest that compound 17 is a highly efficacious B-Raf inhibitor.
In summary, using structure based drug design (SBDD), we have
discovered a series of pyridopyrimidin-7-one-based B-Raf inhibi-
tors with excellent potency and selectivity profiles. Optimization
led to the identification of compound 17, a potent, selective and or-
ally available B-Raf inhibitor with excellent pharmacokinetic prop-
erties and robust tumor growth inhibition in xenograft studies.
lM against a panel of 228 kinases from across
l
9. (a) Wenglowsky, S.; Ren, L.; Ahrendt, K. A.; Laird, E. R.; Aliagas, I.; Alicke, B.;
Buckmelter, A. J.; Choo, E. F.; Dinkel, V.; Feng, B.; Gloor, S. L.; Gould, S. E.; Gross,
S.; Gunzner-Toste, J.; Hansen, J. D.; Hatzivassiliou, G.; Liu, B.; Malesky, K.;
Mathieu, S.; Newhouse, B.; Raddatz, N. J.; Ran, Y.; Rana, S.; Randolph, N.; Risom,
T.; Rudolph, J.; Savage, S.; Selby, L. T.; Shrag, M.; Song, K.; Sturgis, H. L.; Voegtli,
W. C.; Wen, Z.; Willis, B. S.; Woessner, R. D.; Wu, W.-I.; Young, W. B.; Grina, J.
ACS Med. Chem. Lett. 2011, 2, 342; (b) Wenglowsky, S.; Ahrendt, K. A.;
Buckmelter, A. J.; Feng, B.; Gloor, S. L.; Gradl, S.; Grina, J.; Hansen, J. D.; Laird, E.
R.; Lunghofer, P.; Mathieu, S.; Moreno, D.; Newhouse, B.; Ren, L.; Risom, T.;
Rudolph, J.; Seo, J.; Sturgis, H. L.; Voegtli, W. C.; Wen, Z. Biorg. Med. Chem. Lett.
2011, 21, 5533; (c) Ren, L.; Laird, E.; Buckmelter, A. J.; Dinkel, V.; Gloor, S. L.;
Grina, J.; Newhouse, B.; Rasor, K.; Hastings, G.; Gradl, S. N.; Rudolph, J. Bioorg.
Med. Chem. Lett. 2012, 22, 1165; (d) Wenglowsky, S.; Moreno, D.; Rudolph, J.;
Ran, Y.; Ahrendt, K. A.; Arrigo, A.; Colsen, B.; Gloor, S. L.; Hasting, G. Bioorg. Med.
Chem. Lett. 2012, 22, 912.
10. Inhibitor enzyme activity was determined utilizing full-length B-RafV600E
.
Inhibition of basal ERK phosphorylation in Malme-3M cells was used as the
mechanistic cellular assay. For assay description see: Laird, E.; Lyssikatos, J.;
Welch, M.; Grina, J.; Hansen, J.; Newhouse, B.; Olivero, A.; Topolav, G. WO
2006/084015 A2, 2006.
Acknowledgments
11. Coordinates for the B-Raf crystal structure have been deposited in the PDB:
accession code 4E4X.
12. Panek, R. L.; Lu, G. H.; Klutchko, S. R.; Batley, B. L.; Dahring, T. K.; Hamby, J. M.;
Hallak, H.; Doherty, A. M.; Keiser, J. A. J. Pharmacol. Exp. Ther. 1997, 1433.
The authors thank Susan Rhodes and Jennifer Otten for Caco-2
determinations. The authors also thank Dr. Joachim Rudolph for
critical review of the manuscript and helpful suggestions.