Selective inhibitors of the mutant B-Raf pathway: Discovery of a potent and orally bioavailable aminoisoquinoline

Article abstract of DOI:10.1021/jm901081g

The discovery and optimization of a novel series of aminoisoquinolines as potent, selective, and efficacious inhibitors of the mutant B-Raf pathway is presented. The N-linked pyridylpyrimidine benzamide 2 was identified as a potent, modestly selective inhibitor of the B-Raf enzyme. Replacement of the benzamide with an aminoisoquinoline core significantly improved kinase selectivity and imparted favorable pharmacokinetic properties, leading to the identification of 1 as a potent antitumor agent in xenograft models. 2009 American Chemical Society.

Full text of DOI:10.1021/jm901081g

J. Med. Chem. 2009, 52, 6189–6192 6189
DOI: 10.1021/jm901081g
Selective Inhibitors of the Mutant B-Raf
Pathway: Discovery of a Potent and Orally
Bioavailable Aminoisoquinoline
Adrian L. Smith,*,† Frenel F. DeMorin,† Nick A. Paras,†
Qi Huang,† Jeffrey K. Petkus,† Elizabeth M. Doherty,†
Thomas Nixey,† Joseph L. Kim,‡ Douglas A. Whittington,‡
)
Linda F. Epstein,‡ Matthew R. Lee,§ Mark J. Rose,
Figure 1. Structures of a potent, selective Raf inhibitor (1) and the
screening leads 2 and 3 from which it was derived.
Carol Babij,z Manory Fernando,z Kristen Hess,z
Quynh Le,z Pedro Beltran,z and Josette Carnahanz
related aminoquinazoline benzamides that potently inhibit
the enzyme and potently inhibit downstream phosphorylation
of ERK in the A375 human melanoma cell line bearing the
V600E mutation in vitro. Compounds 2 and 3 are representa-
tive of these leads (Figure 1).5 While these leads were strong
inhibitors of B-Raf,6 they also potently inhibited other kinases
with smaller gatekeeper residues and known DFG-out bind-
ing modes, including p38R, KDR, Lck, and Tie-2 (Table 1).5,7
In addition, rat pharmacokinetic properties were suboptimal
(short t1/2, moderate clearance, and generally low to modest
bioavailability; Table 1).
Departments of †Medicinal Chemistry, §Molecular Structure,
Pharmacokinetics and Drug Metabolism, and zOncology Research,
)
Amgen Inc., One Amgen Center Drive, Thousand Oaks, California
91320-1799, and ‡Department of Molecular Structure, Amgen Inc.,
360 Binney Street, Cambridge, Massachusetts 02142
Received July 21, 2009
Abstract: The discovery and optimization of a novel series of
aminoisoquinolines as potent, selective, and efficacious inhibitors
of the mutant B-Raf pathway is presented. The N-linked pyridyl-
pyrimidine benzamide 2 was identified as a potent, modestly
selective inhibitor of the B-Raf enzyme. Replacement of the benza-
mide with an aminoisoquinoline core significantly improved kinase
selectivity and imparted favorable pharmacokinetic properties,
leading to the identification of 1 as a potent antitumor agent in
xenograft models.
Modeling of 2 into the B-Raf crystal structure8 indicated
that it is well accommodated in the ATP-binding site of the
kinase domain of the protein, where the activation loop folds
out (DFG-out)9 to open up an extended hydrophobic pocket
in which the trifluoromethylaniline moiety forms favorable
hydrophobic interactions. An examination of the amino acid
residues surrounding the inhibitor 2 inB-Raf indicated a small
unoccupied cleft in the vicinity of the conserved X-DFG
motif at the start of the activation loop adjacent to the amide
linkage of the benzamide of 2 (Figure 2 ). A glycine residue
(Gly593) immediately precedes the DFG sequence in B-Raf
(X=G), but for many other kinases the corresponding residue
is larger (X=L, C, A, and A for p38R, KDR, Lck, and Tie-2,
respectively). The resulting side chain from the residue X is
directed toward this unoccupiedcleft, and modeling suggested
that filling this small unoccupied pocket would significantly
improve the kinase selectivity of 2. The aminoisoquinoline
12a was therefore designed and prepared (Schemes 1 and 2)
to achieve this while maintaining key hydrogen bonding
interactions through the endocyclic and exocyclic nitrogens
of the aminoisoquinoline to Asp594 and Glu501, respec-
tively.6
The aminoisoquinoline 12a exhibited a reduction in activity
against the recombinant B-Raf enzyme but was only slightly
less active in inhibiting phosphorylation of ERK in an A375
cellular assay (Table 1). It has been noted elsewhere that Raf
inhibitors can result in counterintuitive activation of Raf
kinase activity;10 therefore, structure-activity relationships
were principally derived from activity in the A375 cellular
assay.
Notably, activity of 12a against p38R and KDR was
markedly attenuated, as predicted from the initial modeling
studies with 2. Selectivity against Lck and Tie-2 was more
modest as might be predicted from the smaller side chain of
the alanine residue preceding the DFG motif in these kinases.
However, a comparison of the extended hydrophobic pockets
of the B-Raf, Lck, and Tie-2 structures suggested more room
in the vicinity of the 4-position of the aniline side chain for
B-Raf is a Ras-activated serine/threonine protein kinase
that plays a central role in transducing signals from cell
surface receptors to the nucleus through the mitogen-acti-
vated protein kinase (MAPKa) pathway (Ras/B-Raf/MEK/
ERK). This pathway has long been associated with human
cancers due to frequent oncogenic mutations identified in its
members.1 Ras mutations occur in ∼15% of cancers, and
B-Raf is also mutated at high frequency in certain cancers
including melanoma (∼66%), thyroid cancer (35-70%),
colorectal cancer (5-20%), and ovarian cancer (∼30%).2
Over 45 B-Raf mutations have been described, but by far
the most common mutation (∼90%) is Val600 f Glu in the
activation loop. This mutation renders the kinase constitu-
tively active. Given the high incidence of B-Raf mutations in
melanoma, together with few effective treatments currently
being available for metastatic melanoma, selectively targeting
the mutant B-Raf pathway offers clinicians an attractive new
approach to treating this deadly disease.3 There have been few
reports to date of selective inhibitors of the mutant B-Raf
pathway in an in vitro cellular setting that demonstrate robust
antitumor efficacy in vivo against tumors driven by the
mutant B-Raf pathway.4 We herein disclose work leading to
the discovery of 1 as such an agent.
A high-throughput screen against recombinant V600EB-Raf
led to the identification of a series of pyridylpyrimidine and
*To whom correspondence should be addressed. Phone: 805-447-
4662. Fax: 805-480-3016. E-mail: adrians@amgen.com.
aAbbreviations: MAPK, mitogen-activated protein kinase; ERK,
extracellular signal-regulated kinase; MEK, mitogen-activated protein
kinase/extracellular signal-regulated kinase kinase; SAR, structure-
activity relationship; PD, pharmacodynamic.
r
2009 American Chemical Society
Published on Web 09/18/2009
pubs.acs.org/jmc

6190 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20
Smith et al.
Table 1. Activity of Compounds in B-Raf Enzyme and Cellular Assays, Selected Kinase Counterscreening Data, and Rat Pharmacokinetic Parameters
enzyme inhibition, IC50 (nM)
rat pharmacokineticsc
compd
A375 pERK inh, IC50 (nM)a
B-Rafb
p38R
12
KDR
Lck
Tie-2
t1/2 (h)
CL ((L/h)/kg)
Vss (L/kg)
F (%)
2
15
27
53
34
35
44
26
29
270
22
27
1.8
0.46
1.7
17
4.2
1.0
3.5
0.27
81
3.6
1.8
1.3
2.3
4.5
3.5
4.7
2.7
2.8
2.9
2.1
1.6
1.9
3.6
3.7
2.2
1.7
0.37
2.2
0.72
16
24
3
3.5
12a
12b
13
14
15
16
18
19
20
1
>1600
>1600
>1600
>1600
450
760
>25000
300
47
1.2
22
83
3.4
56
280
12
520
24
0.67
0.29
0.11
0.73
0.33
100
100
76
39
18
>1000
420
170
150
210
23
400
8.1
110
5.4
70
350
5.8
100
>1600
110
51
>25000
230
240
110
21
>1000
200
>1000
120
3.9
4.8
3.5
1.8
5.5
4.3
1.9
24
55
80
21
1.6
78
83
0.65
0.40
>1600
1000
a In-cell Western assay performed using LiCOR Odyssey scanner; inh=inhibition. b Recombinant V600EB-Raf kinase domain. c Compounds were
dosed at 2 mg/kg iv and 5 mg/kg po in male Sprague-Dawley rats. t1/2 is the plasma elimination-phase half-life (iv).
Scheme 2. Preparation of Aminoisoquinolinesa
Figure 2. Model of 2 docked into the DFG-out structure of B-Raf.
The isoquinoline core is overlaid onto the structure of 2, where it
occupies a small hydrophobic cleft unavailable in other kinases
containing any residue larger than a glycine at the position corre-
sponding to Gly593.
a Reagents and conditions: (a) LiHMDS, Pd2(dba)3, 2-dicyclohexylpho-
sphino-20-(N,N-dimethylamino)biphenyl, THF, 70 °C, 16 h, 29-56%;
(b) LiHMDS, THF, 45 °C, 1 h, 72%.
Scheme 1. Preparation of Aminoisoquinoline Intermediatesa
prepared (Scheme 2). Not only did 12b demonstrate an
improvement in activity against B-Raf while maintaining
excellent selectivity against p38R and KDR, selectivity against
Lck and Tie-2 was also markedly improved.6 A signific-
ant improvement in rat pharmacokinetics was noted in going
from the benzamide 2 to the aminoisoquinoline 12a (Table 1).
The aniline substituent was identified as a major source of
oxidative metabolism/clearance, and changes to the aniline
substitution pattern (12a f 12b) resulted in very favorable
pharmacokinetic profiles (Table 1).
To improve potency, attention was focused on the pyrimi-
dine portion of the molecule. This forms a bidentate hydrogen
bonding interaction in the hinge region of the B-Raf protein
(i.e., between the pyrimidine N1 and Cys532 (NH) and
between the pyrimidine C2-H and Gln530 (CdO)).11 It
was reasoned that an additional hydrogen bond to Cys532
(CdO) would be accessible with the aminomethyl group of 13
(Scheme 2). This turned out to be detrimental to activity
a Reagents and conditions: (a) CHCl3, reflux; (b) EtOCOCl, P(OEt)3,
against the B-Raf enzyme but interestingly maintained
CHCl3, 0 f 22 °C, 23 h, then TiCl4, CHCl3, reflux, 12 h, 78% (2 steps);
potency in the A375 pERK cellular assay. However, this
(c) KNO3, H2SO4, 0 °C, 1.5 h, 95%; (d) mCPBA, CH2Cl2, 0 °C, 4 h;
change was detrimental to selectivity, particularly against
Lck and Tie-2. Further SAR exploration of the aniline sub-
stituent of 13 using analogous chemistry demonstrated the
i
(e) POCl3, CH2Cl2, 70 °C, 3 h, 86% (2 steps); (f) PrOH, microwave,
170 °C, 16 min, 79-84%; (g) SnCl2, EtOH, 75 °C, 16 h, 66-72%.
B-Raf compared with Lck and Tie-2 and therefore offered a
potential way of further improving the kinase selectivity of
12a. The corresponding 4-chloroaniline 12b was therefore
ability to modulate kinase selectivity with 14-16 while retain-
ing equivalent B-Raf/A375 cellular activity and favorable
pharmacokinetic properties (Table 1).

Letter
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20 6191
Scheme 3. Preparation of Aminoquinazoline Derivativesa
a Reagents and conditions: (a) THF/5 N HCl (1:1), reflux, 14 h, 87%;
(b) H2, Pd/C, EtOH, 94%; (c) NaNO2, conc HCl, then KI, 88%;
(d) N-methyl-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)quinazolin-2-
amine, Pd(PPh3)4 (cat.), Na2CO3, 1,4-dioxane/water, microwave, 160 °C,
15 min, 52%; (e) POCl3, neat, 100 °C, 2 h, 82%; (f) Ar-NH2, Pd2(dba)3
(cat.), DavePhos, LiHMDS, 1,4-dioxane, microwave, 150 °C, 10 min,
40-59%.
Figure 3. Crystal structure of 1 solved in complex with B-Raf at
˚
2.7 A resolution.
Scheme 4. Preparation of Pyridylpurine Aminoisoquinoline 1a
Figure 4. Strong correlation observed between in vivo V600EB-Raf
MAPK pathway signaling inhibition 6 h postdose orally and A375
tumor growth inhibition in A375 SQ2 tumor-bearing mice dosed
orally for 14 days (same doses).
A pharmacodynamic (PD) assay in mouse measuring tumor
biochemical target coverage (in a dose and time dependent
manner) in A375 tumor lysate was performed to help in
designing dosing schedule for compounds in efficacy studies.13
The 6 h time point following oral dosing across multiple doses
and compounds was found to be highly predictive of in vivo
efficacy, as illustrated by the significant correlation (R2=0.86)
observed between in vivo pERK inhibition and A375 tumor
growth inhibition across all Raf inhibitors tested (Figure 4).
Compound 1was profiled in the A375 SQ2 xenograft model13
in mouse based on its in vitro potency, kinase selectivity, and
favorable rat pharmacokinetic profile. Pharmacokinetics in
a Reagents and conditions: (a) 3,4-dihydro-2H-pyran, TsOH (cat.),
EtOAc, reflux, 1 h, 99%; (b) 2-fluoropyridin-3-ylboronic acid, A-Phos
(cat.), KOAc, EtOH, water, 80 °C, 2 h, 96%; (c) 9b, LiHMDS, THF,
0 °C, 1 h; (d) HCl, water, reflux, 1 h, 73% (two steps).
Replacement of the aminopyridylpyrimidine portion of 13
with the aminoquinazoline fragment from the screening lead3
gave the corresponding analogue 18 (Scheme 3). While 18
retained good activity against the B-Raf enzyme accompanied
with good kinase selectivity, a notable drop in A375 cellular
activity was observed. Cellular activity could be restored by
varying the aniline substituent, for example with 19 and 20,
but at the expense of kinase selectivity (Table 1).
Rationalizing that rotational freedom of the aminomethyl
group of 13-16 may have introduced an entropic penalty for
binding to B-Raf, it was conformationally locked to form a
purine. The corresponding pyridylpurine 1 was therefore
prepared (Scheme 4). This resulted in a significant improve-
ment in potency against the B-Raf enzyme and in the A375
pERK cellular assay (Table 1). A cocrystal structure of 1 with
B-Raf revealed that the purine not only forms an additional
hydrogen bonding interaction with Cys532 but also allows a
favorable π-stacking interaction with the side chain of Trp531
which is probably responsible for the bulk of the improvement
in potency (Figure 3).12 Additionally, kinase selectivity rela-
tive to 13 was greatly increased and 1 displayed a very
favorable rat pharmacokinetic profile.
mouse were similar to those observed in rat (CD1 mouse: t1/2
=
3.3 h; CL=0.19 (L/h)/kg; Vss=1.1 L/kg; F=79%). The ability
of 1 to modulate ERK phosphorylation levels in the tumor was
demonstrated in the A375 PD assay, where a 2.5 mg/kg dose
administered orally resulted in 69% inhibition of ERK phos-
phorylation 6 h following a single dose, and a 5.0 mg/kg dose
resulted in a corresponding 77% inhibition. Repeat oral dosing
in an established A375 xenograft model in mouse13,14 resulted in
tumor growth inhibition with an ED50 of 1.3 mg/kg QD
(AUC0-24h = 2.7 μg h/mL) and 85% tumor regression at
3
5 mg/kg QD (AUC0-24h=14 μg h/mL) after dosing for 14 days.
3
In summary, a novel series of selective inhibitors of the
mutant B-Raf pathway is disclosed. Identification of the lead
compounds 2 and 3 as multikinase inhibitors that potently
inhibited B-Raf kinase, combined with an understanding of
how they bind in the target and off-target kinases, allowed a
structure-based approach to guide the medicinal chemistry
team. A relatively uncommon G-DFG motif in Raf provided

6192 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20
Smith et al.
an opportunity for kinase selectivity that was exploited suc-
cessfully with an aminoisoquinoline core. Additional selectiv-
ity was exploited through subtle differences in the extended
hydrophobic pockets of the kinases, and potency was opti-
mized through changes in the hinge-binding region through
the introduction of a purine moiety. This led to the identifica-
tion of 1 as a potent, highly selective inhibitor of Raf6 that
demonstrates potent antitumor activity in an in vivo melano-
ma model driven by the mutant B-Raf pathway. Although 1
was apparently well tolerated, this group will present else-
where data that suggest potent Raf inhibitors can lead to
compensatory activation of the MAPK pathway with unde-
sirable functional consequences observed in normal tissues
and certain other non-B-Raf mutant tumor models in vivo.
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Acknowledgment. We thank our colleagues Stephanie
Geuns-Meyer for the synthesis of 2, Rob Wahl for performing
the high-throughput screen that identified 2, Christina Boucher,
Christie Binder, and Shaun Flynn for running kinase counter-
screening assays, Xiao Ding, Michelle Kelly, and Tom Menges
for performing pharmacokinetic studies, and Andrew Tasker,
Rick Kendall, and Terri Burgess for helpful discussions.
Note Added after ASAP Publication. There was an error in
the Abbreviations in the version of this paper published on
September 18, 2009. The revised version was published on
September 22, 2009.
(6) No selectivity was observed for these compounds between V600EB-
Raf and wild type B-Raf or C-Raf in enzyme assays in vitro.
(7) Pargellis, C.; Tong, L.; Churchill, L.; Cirillo, P. F.; Gilmore, T.;
Graham, A. G.; Grob, P. M.; Hickey, E. R.; Moss, N.; Pav, S.;
Regan, J. Inhibition of p38 MAP kinase by utilizing a novel
allosteric binding site. Nat. Struct. Biol. 2002, 9, 268–272.
Supporting Information Available: Synthesis details for com-
pounds described herein, analytical data, X-ray crystallographic
data, kinase counterscreening data for 1, and PD-efficacy data
for the compounds shown in Figure 4. This material is available
free of charge via the Internet at http://pubs.acs.org.
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C. J.; Springer, C. J.; Barford, D.; Marais, R. Mechanism of
activation of the RAF-ERK signaling pathway by oncogenic
mutations of B-RAF. Cell 2004, 116, 855–867.
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(12) The cocrystal structure of B-Raf with 1 has been deposited in the
Protein Data Bank with accession code 3IDP.
(13) Human A375 cells were selected in vivo by passaging twice
subcutaneously in CD1 nu/nu mice to optimize for tumor take
and growth. This cell line was designated A375 SQ2.
(14) Compound was dosed QD in a randomized, blinded experiment for
14 days starting 17 days postimplantation.