Pyrazolopyridine inhibitors of B-RafV600E. Part 1: The development of selective, orally bioavailable, and efficacious inhibitors

Article abstract of DOI:10.1021/ml200025q

The V600E mutation of B-Raf kinase results in constitutive activation of the MAPK signaling pathway and is present in approximately 7% of all cancers. Using structure-based design, a novel series of pyrazolopyridine inhibitors of B-Raf^V600E was

Full text of DOI:10.1021/ml200025q

LETTER
pubs.acs.org/acsmedchemlett
Pyrazolopyridine Inhibitors of B-RafV600E. Part 1: The Development of
Selective, Orally Bioavailable, and Efficacious Inhibitors
Steve Wenglowsky,*,† Li Ren,† Kateri A. Ahrendt,† Ellen R. Laird,† Ignacio Aliagas,‡ Bruno Alicke,‡
Alex J. Buckmelter,† Edna F. Choo,‡ Victoria Dinkel,† Bainian Feng,‡ Susan L. Gloor,† Stephen E. Gould,‡
Stefan Gross,† Janet Gunzner-Toste,‡ Joshua D. Hansen,† Georgia Hatzivassiliou,‡ Bonnie Liu,‡
Kim Malesky,‡ Simon Mathieu,‡ Brad Newhouse,† Nicholas J. Raddatz,† Yingqing Ran,‡ Sumeet Rana,†
Nikole Randolph,† Tyler Risom,† Joachim Rudolph,‡ Scott Savage,‡ LeAnn T. Selby,† Michael Shrag,†
Kyung Song,‡ Hillary L. Sturgis,† Walter C. Voegtli,† Zhaoyang Wen,‡ Brandon S. Willis,†
Richard D. Woessner,† Wen-I Wu,† Wendy B. Young,‡ and Jonas Grina†
†Array BioPharma, 3200 Walnut Street, Boulder, Colorado 80301, United States
‡Genentech, Inc., 1 DNA Way, South San Francisco, California 94080-4990, United States
S Supporting Information
b
ABSTRACT: The V600E mutation of B-Raf kinase results in constitutive activation of the
MAPK signaling pathway and is present in approximately 7% of all cancers. Using structure-
based design, a novel series of pyrazolopyridine inhibitors of B-RafV600E was developed.
Optimization led to the identification of 3-methoxy pyrazolopyridines 17 and 19, potent,
selective, and orally bioavailable agents that inhibited tumor growth in a mouse xenograft
model driven by B-RafV600E with no effect on body weight. On the basis of their in vivo efficacy
and preliminary safety profiles, 17 and 19 were selected for further preclinical evaluation.
KEYWORDS: B-RafV600E, MAPK pathway, targeted therapy, pyrazolopyridine, amorphous spray-dried dispersion
he Ras/Raf/MEK/ERK (MAPK) signaling pathway trans-
duces signals from cell surface receptors to the nucleus
protein kinase inhibitors.14 Although there are no approved kinase
inhibitors that are selective for B-RafV600E, four are currently in
clinical trials including RAF265 (Chiron/Novartis),15 XL281/BMS-
908662 (Exelixis/BMS),16 GSK2118436 (GlaxoSmithKline),17 and
PLX4032 (1, Plexxikon/Roche),18 with others in preclinical develop-
ment.19-22 A detailed account of the discovery and clinical develop-
ment of 1, a selective B-RafV600E inhibitor that binds to the active
conformation of the kinase (DFG-in), has recently been published.23
The authors report that the propyl group of the sulfonamide effects
B-RafV600E selectivity by trapping a small lipophilic pocket enlarged by
an outward shift of the RC-helix.24 It was further reported that the
difluorophenyl ring of 1 occupies a hydrophobic pocket adjacent to
the gatekeeper residue, Thr529.
T
leading to cellular proliferation, differentiation, and survival.1
The Raf family consists of serine/threonine kinases A-Raf, B-Raf,
and C-Raf (Raf-1), which phosphorylate and activate MEK,
although with different biochemical potencies (B-Raf > C-Raf .
A-Raf).1 Mutations in the BRAF gene may lead to MAPK
pathway amplification via constitutive activation of B-Raf kinase
and are present in approximately 7% of all cancers.2 Mutated
B-Raf is most frequently associated with melanoma and has been
detected in up to 82% of cutaneous melanocyte nevi,3 66% of
primary melanomas,2 and 40-68% of metastatic melanomas.4,5
Additional cancers with aberrant activation of B-Raf due
to oncogenic mutations include papillary thryroid cancer
(29-83%),2,6,7 colorectal cancer (5-22%),2 cholangiocarcino-
ma (22%),8 and ovarian cancer (30%).2 Over 90% of the detected
mutations in B-Raf are a glutamic acid for valine substitution at
residue 600 (V600E),2 which leads to constitutive kinase activity
500-fold greater than B-RafWT and correlates with increased
malignancy and decreased response to chemotherapy.9-12 Thus,
cancers induced via constitutive activation of the MAPK signal-
ing pathway arising from B-RafV600E should be treatable by a
small molecule inhibitor targeted to disable this aberrant enzyme.13
Herein is described the discovery of selective, orally bioavailable, and
efficacious inhibitors of B-RafV600E that utilize a novel 3-methoxy
pyrazolopyridine hinge-binding template.
From structural evaluation and molecular modeling, it was
hypothesized that an amide linker could couple an aryl group in
this hydrophobic pocket to a hinge-binding heterocycle, while
Received: November 17, 2010
Accepted: February 26, 2011
Published: March 08, 2011
The targeted approach to cancer therapy development has
been validated by the growing number of marketed small-molecule
r
2011 American Chemical Society
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Scheme 1. Preparation of Pyridine Amides 3-5a
a Reagents and conditions: (a) Compound 2a, EDCI, HOBt, DMF, 22 °C, 5-36%.
Table 1. Enzymatic and Cellular Activities of B-RafV600E
Amide Series Inhibitors
IC50 (nM)
compd
enzymea
Malme-3Mb
A375b
3
4
3900
920
110
61
6700
2400
310
2400
94
>3000
>3000
1200
>3000
290
5
8
9
38
10
13
14
15
16
17
18
19
1
3.4
17
39
190
62
430
4.0
12
44
260
86
830
8.2
4.8
6.3
1.7
31c
40
650
19
150
56
560
Figure 1. X-ray crystal structure of 5 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 hydro-
phobic interactions with 5 are omitted for clarity and described in the
text. The propyl group resides in a pocket that is enlarged by an outward
shift of the RC-helix. The DFG sequence (D594-G596) resides in its
active (DFG-in) conformation.
20
240
61
190
a Biochemical assay (B-RafV600E). b Cellular phosphorylation assay
(pERK) using B-RafV600E mutant Malme-3M or A375 cell line. c Ref 23.
forming a hydrogen bond to the hydroxyl of Thr529. Linkage via
an amide bond would also allow for rapid and efficient screening
of several potential hinge-binding groups. The initial series of
amide-linked B-RafV600E inhibitors utilized pyridine as a hydrogen
bond acceptor, which was postulated to bind to the -NH of
hinge residue Cys532. Scheme 1 illustrates the synthesis of
pyridine-amides 3-5 and the structures of the two benzoic acid
coupling partners used in this report (2a and 2b).25
Inhibitor enzymatic activity was determined utilizing full-
length B-RafV600E. Inhibition of basal ERK phosphorylation in
Malme-3M cells was used as the mechanistic cellular assay and to
drive the structure-activity relationships for the amide series.26
Inhibition of ERK phosphorylation was also determined in the
A375 cell line and, while up to an order of magnitude less sensitive
than the Malme-3M cell line, generated an identical SAR.
The micromolar enzymatic and cellular activity of pyridine
amide 3 revealed that additional interactions would be necessary
to achieve useful potency (Table 1). Molecular modeling in-
dicated that the addition of a 6-amino group to the pyridine of 3
could yield a hydrogen bond to the carbonyl of Cys532. The
4-fold improvement in enzymatic activity and nearly 3-fold
improvement in cellular activity of aminopyridine 4 substantiated
this approach. Addition of bromine at the 5-position of 4 led to
compound 5 and a further 8-fold increase in enzymatic and
cellular activity. The presumed binding model, with hydrogen
bonds to Cys532 and Thr529, was confirmed by an X-ray crystal
structure of amide 5 with B-Raf (Figure 1). The crystal structure
also revealed key lipophilic contacts made by the 5-bromo group
with the side chains of Ile463, Val471, Trp531, and Phe583,
which accounted for the potency increase over 4. However, despite
the >20-fold improvement in potency from 3to 5, the cellular activity
of the pyridine amides remained insufficient for in vivo efficacy.
To further improve the potency of the amide series, bicyclic
heterocycles were prepared as replacements for the pyridine
hinge-binding template. Cyclization of the hinge donor amine of
4 to the 5-position of the pyridine via a five-membered ring
suggests three potential bicyclic cores: imidazopyridine, pyrro-
lopyridine, and pyrazolopyridine. These bicycles were designed
to form a bidentate hydrogen-bonding interaction at the hinge,
enhance the interaction with the indole of Trp531, and provide a
point of diversity for analogue synthesis. Scheme 2 illustrates the
synthesis of imidazopyridine and pyrrolopyridine amide series
analogues 8-10. Although imidazopyridine 8 had encouraging
enzymatic activity, cellular activity was poor (Table 1). This result
was surprising given that a related imidazopyrimidine hinge binder
has demonstrated excellent cellular activity in an alternative series of
B-Raf inhibitors.22 While pyrrolopyridine 9 had similar enzymatic
activity to imidazopyridine 8, it was 20-fold more active in the
cellular assay. In addition, pyrrolopyridine 9 was significantly more
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Scheme 2. Preparation of Imidazopyridine and Pyrrolopyridine Amides 8-10a
a Reagents and conditions: (a) Compound 2a, EDCI, HOBt, DMF, 22 °C, 9% for 8, 53% for 9. (b) Compound 9, n-bromosuccinimide, CHCl3, 82%.
Scheme 3. Preparation of Pyrazolopyridines 13-19a
a Reagents and conditions: (a) Sodium nitromalonaldehyde monohydrate, AcOH or water, 90 °C, 38-94%. (b) Br2, NaOH, dioxane, 0-22 °C, 69%.
(c) Forty percent HNMe2/water, microwave, 160 °C, 54%. (d) H2, 10% Pd/C, EtOAc/MeOH, 22 °C, or SnCl2, EtOAc, reflux, 82-99%. (e) Benzoic
acid 2a or 2b, EDCI, HOBt, DMF, 22 °C, 35-82%.
potent than aminopyridines 4 and 5, demonstrating the advantage
of a bicyclic hinge binder. Bromination at the 3-position of
pyrrolopyridine 9 afforded compound 10 and a 2-fold improvement
in cellular activity. This result indicated that potency could be
increased by an appropriate substitution at the 3-position of the
bicyclic hinge-binding template.
The pyrazolopyridine hinge-binding template was prepared
concurrently and ultimately provided the most potent B-RafV600E
inhibitors from the amide series. A key advantage of this template
included the facility with which substituents of varying electro-
nics could be synthetically incorporated at the 3-position of the
core (Scheme 3). Nitromalonaldehyde condensation27 with
3-substituted-5-aminopyrazoles 11a-c provided pyrazolopyri-
dines 12a-c. Bromination of 12a introduced a synthetic pre-
cursor (12d) from which a 3-amino pyrazolopyridine (12e) was
obtained. Nitro reduction of 12a-e followed by coupling to
benzoic acids 2a or 2b provided amides 13-19.
Varying the substituent at the 3-position of the pyrazolopyr-
idine resulted in improvements in the enzymatic and cellular
Figure 2. X-ray crystal structure of 17 in complex with B-RafWT
.
activity of the inhibitors while significantly modulating their
pharmacokinetic and physicochemical properties. Unsubstituted
pyrazolopyridine 13 was similarly active to the corresponding
pyrrolopyridine 9 (Table 1). Most substitutions at the 3-position
[Br- (14), Me- (15), and Me2N- (16)] produced inhibitors
within a narrow range of activity and with no significant improve-
ment in potency over 13. One exception to the flat SAR observed
at the 3-position of the pyrazolopyridine template was obtained
by the introduction of a methoxy group.28 This led to 3-methoxy
pyrazolopyridine 17, the most cell potent inhibitor in the amide
series. Compounds with halogen substitutions on the benzamide
moiety were also examined. While substitution of chloro
for fluoro at the 6-position of the benzamide resulted in a 3-fold
improvement in the enzymatic activity within two pairs of
compounds, there was no change in cellular activity (18 vs 13
Notation is as described for Figure 1. In addition to the hydrogen-
bonding interactions described in Figure 1, the sulfonamide NH of 17 is
in close contact to the main chain NH of Asp594, indicating that the
sulfonamide is deprotonated.23
and 19 vs 17). Compounds 17 and 19 both had similar potency
to PLX4032 in the A375 cell line but were 3-fold more potent in
Malme-3M cells. The X-ray crystal structure of 17 in complex
with B-Raf further confirmed the amide series binding model
(Figure 2). Importantly, the proposed interaction of the bicyclic
hinge-binding core with Trp531 was validated: A π-stacking
interaction between the pyrazole portion of the bicycle and the
side chain indole of Trp531 was revealed and likely contributes to
the improvement in potency over the pyridine amides 3-5.22
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Table 2. Mouse Pharmacokinetics and Physicochemical Properties of Pyrrolo- and Pyrazolopyridine Amides
compd
30 mg/kg po AUCa (% F)
100 mg/kg po AUCa (% F)
CLb
Vdc
mouse PPBd
mp (°C)
sol. at pH 6.5, 7.4e
9
10
13
14
15
16
17
18
19
200 (76)
49 (71)
40 (100)
52.5
1.9
7.2
15
0.20
0.24
0.68
97.9
99.4
87.1
98.4
89.9
90.8
97.4
97.1
99.1
243
227
173
220
181
181
229
236
261
1, 4
<1, <1
80, 150
1, 3
18.3
380, 740
290, 450
4, 9
1.09
190 (48)
147 (67)
1080 (53)
664 (51)
50.7 (1.2)
1.3
2.3
1
0.11
0.17
0.11
15, 48
<1, 2
a μg h/mL. b mL/min/kg. c L/kg. d Plasma protein binding (%). e μg/mL.
Table 3. Dose-Ranging Tumor Growth Inhibition Studies in
Colo205 Mouse Xenografts
Both the nature of the bicyclic template and its substituents
were found to have significant effects on the pharmacokinetic
and physicochemical properties of the amide series inhibitors
(Table 2). Pyrrolo- and pyrazolopyridine amides were dosed
orally in mice at 30 mg/kg as a solution in 40% PEG400/10%
ethanol/50% water, while select compounds were also dosed
intravenously. These compounds typically exhibited high oral
bioavailability (% F) and oral exposure (AUC). The total
clearance (CL) and volume of distribution (Vd) were low and,
with the exception of 10, correlated with plasma protein binding
(PPB). Aqueous solubility varied across the series and correlated
with melting point (mp), which broadly ranged from 173 to
261 °C; these data suggest that subtle crystal packing effects exist
within this series, which could be modulated by varying the
substituents around the molecule. Unfortunately, the com-
pounds with the highest aqueous solubilities also exhibited the
lowest oral exposures (13, 15, and 16). The substitution of
chloro for fluoro at the 6-position of the benzamide ring had
pronounced effects on pharmacokinetic and physicochemical
properties: attenuated aqueous solubility, increased PPB, lower
IV clearance, and significantly higher oral AUC (18 vs 13 and
19 vs 17). Overall, compounds 17 and 19 possessed the best
combined potency and exposure from this series, which illus-
trates the advantage of the 3-methoxy pyrazolopyridine hinge-
binding template. However, when these two compounds were
dosed orally in mice at 100 mg/kg using the same solution
formulation, compound 17 exhibited a dose proportional in-
crease in exposure, while the oral bioavailability of 19 dropped to
1%. The low aqueous solubility and high melting point of
this compound likely led to crystallization in the gut preventing
absorption.
In an effort to rescue the poor pharmacokinetics of compound
19 at higher doses and because of concerns with daily dosing of
high concentrations of PEG400 in potential preclinical toxicol-
ogy and xenograft studies, 17 and 19 were reformulated as a 25%
amorphous spray-dried dispersion (SDD) on hydroxypropyl
methylcellulose acetate succinate (HPMCAS) polymer.29 Brid-
ging pharmacokinetic studies in mice with 17 dosed orally at 2.5,
10, 40, and 80 mg/kg revealed a linear dose response, albeit with
oral bioavailabilities lower than with the soluble PEG400-based
solution formulation (F = 19-25 vs 48-51%). Nonetheless,
exposure exceeded the levels projected to achieve efficacy in
mouse models of cancer driven by B-RafV600E. Compound 19
was also dosed as the SDD formulation in mice at 10, 30, 60, and
100 mg/kg and demonstrated decreasing oral bioavailability with
increasing dose (F = 53, 28, 19, and 13%). Although a linear
dose response was not achieved, this formulation provided a
a
a
compd
no. of days dosed
ED50
ED90
max TGI (%)b
17
19
20
17
20
11
80
50
76
95
a mg/kg. b Tumor growth inhibition as a percent of vehicle control.
significant improvement in exposure at 100 mg/kg for 19 (13 vs 1%),
higher bioavailability than 17 at 10 mg/kg (53 vs 25%), and
likewise exceeded the levels projected to achieve efficacy.
To further characterize in vitro potency and in preparation for
mouse xenograft studies, inhibition of cellular viability by com-
pound 17 was measured against a panel of 13 melanoma and 10
colon cancer cell lines driven by the B-RafV600E mutation, and
potencies were compared to PLX4032 (1). In melanoma, 17 was
2-3-fold more potent than 1 in 8 of the 13 cell lines tested and
equivalent in the remaining 5, while in colon cancer, 17 was
2-14-fold more potent than 1 in 9 of 10 cell lines examined.
Compound 19 was screened against four of the colon cancer cell
lines and was similarly more potent than 1. For Colo205, in
which mouse xenograft studies were run, the EC50 values for 17
and 19 were 80 and 70 nM as compared to 240 nM for 1.
The activity of 17 and 19 was also determined against a diverse
panel of kinases and demonstrated similar selectivity profiles with
>100-fold selectivity for 223/228 kinases examined. Compounds
17 and 19 displayed no inhibition of cytochrome P450 enzymes
at 25 μM, no hERG channel inhibition at 100 μM, and were
negative in the Ames test for genotoxicity. On the basis of their in
vitro potency and favorable pharmacokinetic properties, 3-meth-
oxy pyrazolopyridines 17 and 19 were selected for dose-ranging
tumor growth inhibition studies in the Colo205 mouse xeno-
graft model.
The SDD of 17 and 19 were administered at ascending doses
once daily for 17 or 20 days in mice with established Colo205
xenografts (Table 3). Tumor growth inhibition was observed for
both compounds with a maximum tumor regression of 76% at
125 mg/kg for 17 and 95% at 100 mg/kg for 19. Analysis of
tumor samples indicated that in vivo efficacy requires sustained
inhibition of pERK levels of at least 50% for 6 h.30 Given that
compound 19 was similarly active to 17 in the biochemical assay
and in several cellular assays, the superior efficacy of 19 across the
entire dose range of the xenograft study was presumably due to
its higher exposure. The low volumes of distribution for both
compounds (Table 2) did not abate the efficacy of the com-
pounds. Significantly, over the course of the dosing period, no
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LETTER
toxicity and no significant alterations in body weight were
observed at any dose for either 17 or 19.
Salem, G.; Pohida, T.; Heenean, P.; Duray, P.; Kallioniemi, O.; Hayward,
N. K.; Trent, J. M.; Meltzer, P. S. High frequency of B-Raf mutations in
nevi. Nat. Genet. 2003, 33, 19–20.
In summary, selective, orally bioavailable, and efficacious
inhibitors of B-RafV600E were developed. These inhibitors were
based on a novel 3-methoxy pyrazolopyridine hinge-binding
template, which evolved through structure-based design from a
simple pyridine lead (3). While the activity of 3 was micromolar
in both enzymatic and cellular assays, the introduction of a
hydrogen bond donor, utilization of a bicyclic hinge-binding
template, and addition of lipophilic contacts provided potent
pyrrolo- and pyrazolopyridine inhibitors of B-RafV600E. Optimi-
zation at the 3-position ultimately led to 3-methoxy pyrazolopyr-
idines 17 and 19, which demonstrated a 300-fold improvement
in potency over the lead pyridine 3. Compounds 17 and 19 were
highly potent against a broad panel of cancer cell lines driven by
B-RafV600E, particularly those derived from colon cancer, and
displayed significant antitumor activity in an in vivo colon cancer
model driven by B-RafV600E. Although 17 and 19 were well
tolerated in mice during the xenograft studies, RAF inhibitors
have been reported to activate the MAPK pathway in B-RafWT
cells.19,20,22,31-33 This paradoxical activation has been linked to
enhanced proliferation and stimulated tumor growth in vivo, and
hyperplasia of normal epithelial cells was observed in mice.34
Pyrazolopyridine Raf inhibitors likewise activate the MAPK
pathway, and the results from these studies will be presented
elsewhere.
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’ ASSOCIATED CONTENT
S
Supporting Information. Experimental procedures for
b
the synthesis of 3-5, 8-10, 13-19, kinase selectivity, and cell
viability data for 17 and 19 and details of in vitro and in vivo
assays. This material is available free of charge via the Internet at
http://pubs.acs.org.
’ AUTHOR INFORMATION
(14) Johnson, L. N. Protein kinase inhibitors: Contributions from
structure to clinical compounds. Q. Rev. Biophys. 2009, 42, 1–40.
(15) ClinicalTrials.gov web site. http://www.clinicaltrials.gov
(accessed Feb 22, 2011). A study to evaluate RAF265, an oral drug
administered to subjects with locally advanced or metastatic melanoma.
(16) Schwartz, G. L.; Robertson, S.; Shen, A.; Wang, E.; Pace, L.;
Dials, H.; Mendelson, D.; Shannon, P.; Gordon, M. A phase I study of
XL281, a selective oral RAF kinase inhibitor, in patients (Pts) with
advanced solid tumors. J. Clin. Oncol. 2009, 27, 15sAbstr. 3513.
(17) Kefford, R.; Arkenau, H.; Brown, M. P.; Millward, M.; Infante,
J. R.; Long, G. V.; Ouellet, D.; Curtis, M.; Lebowitz, P. F.; Falchook, G. S.
Phase I/II study of GSK2118436, a selective inhibitor of oncogenic
mutant BRAF kinase, in patients with metastatic melanoma and other
solid tumors. J. Clin Oncol. 2010, 28, 15sAbstr. 8503.
Corresponding Author
*Tel: 303-641-1076. E-mail: steve.wenglowsky@arraybiopharma.
com.
’ ACKNOWLEDGMENT
We thank Josh Ballard for HRMS data and Ben Colson for
solubility determinations. We also thank Jim Blake for helpful
discussions.
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