Design and optimization of potent and orally bioavailable tetrahydronaphthalene raf inhibitors

Article abstract of DOI:10.1021/jm101479y

Inhibition of mutant B-Raf signaling, through either direct inhibition of the enzyme or inhibition of MEK, the direct substrate of Raf, has been demonstrated preclinically to inhibit tumor growth. Very recently, treatment of B-Raf mutant melanoma patients with a selective B-Raf inhibitor has resulted in promising preliminary evidence of antitumor activity. This article describes the design and optimization of tetrahydronaphthalene-derived compounds as potent inhibitors of the Raf pathway in vitro and in vivo. These compounds possess good pharmacokinetic properties in rodents and inhibit B-Raf mutant tumor growth in mouse xenograft models.

Full text of DOI:10.1021/jm101479y

ARTICLE
pubs.acs.org/jmc
Design and Optimization of Potent and Orally Bioavailable
Tetrahydronaphthalene Raf Inhibitors
,†
†
†
‡
†
Alexandra E. Gould,* Ruth Adams, Sharmila Adhikari, Kathleen Aertgeerts, Roushan Afroze,
†
†
†
†
†
Christopher Blackburn, Emily F. Calderwood, Ryan Chau, Jouhara Chouitar, Matthew O. Duffey,
†
†
†
†
†
†
†
Dylan B. England, Cheryl Farrer, Nancy Forsyth, Khristofer Garcia, Jeffery Gaulin, Paul D. Greenspan,
†
†
†
†
†
†
Ribo Guo, Sean J. Harrison, Shih-Chung Huang, Natalia Iartchouk, Dave Janowick, Mi-Sook Kim,
†
†
†
†
†
Bheemashankar Kulkarni, Steven P. Langston, Jane X. Liu, Li-Ting Ma, Saurabh Menon,
†
†
†
†
†
Hirotake Mizutani, Erin Paske, Christelle C. Renou, Mansoureh Rezaei, R. Scott Rowland,
†
†
†
†
†
†
Michael D. Sintchak, Michael D. Smith, Stephen G. Stroud, Ming Tregay, Yuan Tian, Ole P. Veiby,
†
†
†
†
†
‡
Tricia J. Vos, Stepan Vyskocil, Juliet Williams, Tianlin Xu, Johnny J. Yang, Jason Yano, Hongbo Zeng,
†
†
†
Dong Mei Zhang, Qin Zhang, and Katherine M. Galvin
†
Millennium Pharmaceuticals, Inc., 40 Landsdowne Street, Cambridge, Massachusetts 02139, United States
Takeda San Diego, 10410 Science Center Drive, San Diego, California 92130, United States
‡
S Supporting Information
b
ABSTRACT: Inhibition of mutant B-Raf signaling, through
either direct inhibition of the enzyme or inhibition of MEK, the
direct substrate of Raf, has been demonstrated preclinically to
inhibit tumor growth. Very recently, treatment of B-Raf mutant
melanoma patients with a selective B-Raf inhibitor has resulted
in promising preliminary evidence of antitumor activity. This
article describes the design and optimization of tetrahydro-
naphthalene-derived compounds as potent inhibitors of the Raf
pathway in vitro and in vivo. These compounds possess good
pharmacokinetic properties in rodents and inhibit B-Raf mutant
tumor growth in mouse xenograft models.
’
INTRODUCTION
At the time we began our work, sorafenib (1) was the only
compound with known Raf activity in the clinic. Sorafenib is
The mitogen-activated protein kinase (MAPK) signal trans-
V600E
reported to have a reasonable enzyme potency against
ΔB-
duction pathway regulates cellular growth, proliferation, and1
Raf and C-Raf (IC50 values of 38 and 6 nM, respectively) and
differentiation in response to many different external stimuli.
pathway inhibition in cells (IC values of 40-1200 nM depend-
50
This pathway is also frequently mutated in many types of cance2r
9
ing on the cell line). We took the sorafenib scaffold as a starting
point for our efforts and sought to identify compounds that
display robust Raf pathway inhibition in an in vitro and in vivo
setting. In this paper, we report the optimization of a series of
novel tetrahydronaphthalene-derived compounds with respect
to cellular activity and pharmacokinetic (PK) properties. In
addition, these compounds profoundly inhibit both tumor
growth and markers of Raf pathway activity in B-Raf mutant
melanoma xenografts.
and thus contains attractive targets for oncology drug discovery.
One component of this pathway, the Raf isoform B-Raf, has a
high rate of activating mutation in melanoma (50-70%) and
other cancers including papillary thyroid (49%), colorectal
3,4
(
∼15%), and ovarian (∼30%). The V600E activating muta-
tion is most common and significantly increases the basal level
5
activity of the enzyme. Inhibition of mutant B-Raf signaling,
6
through either direct inhibition of the enzyme or inhibition of
7
MEK, the direct substrate of Raf, has been demonstrated
preclinically to inhibit tumor growth. Very recently, treatment
of B-Raf mutant melanoma patients with a selective B-Raf
inhibitor has resulted in antitumor activity.
Received: November 17, 2010
Published: February 22, 2011
8
r 2011 American Chemical Society
1836
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|J. Med. Chem. 2011, 54, 1836–1846

Journal of Medicinal Chemistry
ARTICLE
a
Scheme 1. Synthesis of Amide-Based Raf Inhibitors
a
Reagents and conditions: (a) 4-Chloro-N-methylpicolinamide, KOtBu, DMPU, DMF, 100 °C. (b) (()-15 or (S)-15, Cs
2
CO
3
, DMF, heat and then
amide coupling. (c) Compound 17, Cs
2
CO
3
, DMF, heat and then amide coupling.
a
Scheme 2. Synthesis of 4-Fluoropyridine Lactam
a
Reagents and conditions: (a) t-Butyl carbamate, Pd(OAc)
6%. (c) Pd catalyst 29, 3,3-diethoxy-1-propene, DIPEA, H O, DMF, 140 °C, 47%.
2
, Xantphos, NaOH, H
2
2
2
O, dioxane, 80%. (b) n-BuLi, TMEDA, THF, -78 °C and then I ,
8
’
CHEMISTRY
We devised two modular approaches to the synthesis of the
lactam 11 is prepared in three steps from 2-chloro-4-fluoropyr-
idine (Scheme 2). Palladium-catalyzed amination provides the
Boc-protected aminopyridine (9). ortho-Lithiation and iodina-
tion set the stage for a one-pot palladium-catalyzed Heck coupling/
cyclization to provide lactam 11 (Scheme 2) in modest yield.
The synthesis of the (S)-tetrahydronaphthalene carboxylic
acid (S)-15 (Scheme 3) begins with the Evans' oxazolidinone
amide of phenylpropionic acid (12). Asymmetric alkylation of
this oxazolidinone with t-butyl bromoacetate proceeds with high
compounds of interest as shown in Scheme 1. The appropriately
substituted aniline is coupled with a phenylpropionic acid. The
resulting propanamide phenol and a substituted pyridine are heated
in the presence of a base to provide a biaryl ether (e.g., 2, Scheme 1).
Alternatively, the biaryl ether bond is formed first when the
appropriately substituted pyridine and phenol are heated in the
presence of cesium carbonate. These biaryl ethers are then further
reacted with either an aniline or a benzoic acid derivative to provide
the desired compounds (e.g., 4 and 8, Scheme 1).
12
diastereoselectivity to provide 13 in good yield. Deprotection
13
of the t-butyl ester, Friedel-Crafts cyclization, and removal of
The syntheses of several substituted pyridines such as 4-chloro-
the oxazolidinone gives tetralone acid 14. Subsequent reduction
10
N-methylpicolinamide,1 N-(4-nitropyridin-2-yl)acetamide, and
of the tetralone and deprotection of the methyl ether with BBr
affords the desired tetrahydronaphthalene carboxylic acid (S)-15
3
1
cyclopropylacetamide are described in the literature. Pyridine
1
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Journal of Medicinal Chemistry
ARTICLE
a
Scheme 3. Synthesis of S-Tetrahydronaphthalene Carboxylic Acid
a
Reagents and conditions: (a) THF, -78 °C, 89%. (b) NaHMDS, THF -78 °C and then t-butyl bromoacetate, 79%. (c) CF
3
SO
3
H, PhH, 80 °C, o/n.
(
d) H O , NaOH, THF 0 °C, 70% for two steps. (e) Pd/C, H , H SO , AcOH, 80 °C, 20 psi, 65%. (f) BBr , DCM, -78 to 0 °C, 74%.
2
2
2
2
4
3
a
Scheme 4. Synthesis of Amino Tetrahydronaphthalene
a
Reagents and conditions: (a) BnNH
2
, NaBH(OAc)
3
, AcOH, DCM, 100%. (b) Pd(OH)
O, reflux, 99%.
2
, H
2
, EtOH, AcOH, 92%. (c) NaOH, EtOAc, 92% and then
S-(þ)-mandelic acid, iPrOH, MeOH, H
2
O, PhMe, 60%. (d) HBr, H
2
in good yield while maintaining enantiopurity. Careful control of
the methyl ether deprotection reaction conditions was crucial as
this substrate was prone to racemization, particularly on scale.
Table 1. Sorafenib and Initial Amide-Based Raf Inhibitors
14
To prepare the single enantiomer aminotetralin 17 (Scheme 4),
15
we modified a procedure described in the patent literature.
Reductive amination of 7-methoxy-2-tetralone and benzyl amine
followed by debenzylation provides 7-methoxy-2-aminotetralin
(
(
16). Crystallization of the free base of the aminotetralin 16 with
S)-mandelic acid using slightly modified solvents to those
16
previously reported, followed by HBr deprotection of the
methyl ether, provides the desired tetralin amine 17 in high ee
and good yield.
’
RESULTS AND DISCUSSION
V600E
a
a
a
ΔB-Raf IC50 (nM)
C-Raf IC50 (nM)
pERK IC50 (nM)
An examination of the crystal structure of sorafenib bound to
V600E
17
c
2
3
4
61 ( 11
47 ( 12
10 ( 2
9 ( 2
>25000
ΔB-Raf reveals several important features that contribute
c
to the potency of the molecule as it occupies the ATP binding site
of the enzyme in its DFG-out conformation. The pyridine ring
makes key contacts in the hinge region of the enzyme. The
trifluoromethyl group on the phenyl ring occupies a hydrophobic
pocket formed by regions of the DFG motif and the catalytic loop
of the enzyme, while the urea forms two hydrogen bonds with the
enzyme. We decided to explore the importance of these two
hydrogen bonds by replacing the urea with a variety of alternative
moieties and quickly identified 2 (Table 1) in which the urea of
sorafenib has been replaced with a propionamide. The enzyme
tolerates the increase in chain length as well as the loss of one of
the hydrogen bond donor groups of the urea. Both meta- and
>25000
b
b
b
32
28
2900
a
b
IC50 values are shown as mean values of three or more determinations.
IC50 values are shown as mean of two determinations. IC values are
shown as single determinations.
c
50
inhibition of extracellular signal-regulated kinase (ERK) kinase
phosphorylation (pERK) in A375 cells (a melanoma cell line that
V600E
contains
ΔB-Raf). Constraint of the flexible central core of
compound 3 (Table 1) through cyclization did not improve the
enzyme activity but did improve cellular activity, as tetralin 4
(Table 1), prepared as a racemic mixture, now has cellular activity
in the low micromolar range.
Having identified a series of novel compounds with modest
cellular activity, we next focused on improving the cellular
potency of the series while keeping a close eye on the
para-substituted molecules are acceptable (2 and 3, Table 1) and
have similar potency against both
Although these compounds were quite potent in our enzyme
assays, they did not show any activity in a cellular assay measuring
V600E
ΔB-Raf and C-Raf.
1
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ARTICLE
Table 2. Enzyme and Cellular Potencies of Raf Inhibitors
Amine 6 is a potent inhibitor of Raf in cells, but it has some
significant PK liabilities. While the compound has low in vivo
clearance (Table 3), it exhibits poor oral exposure and bioavail-
ability in rats. In an effort to improve the PK properties, we
focused on two areas of the molecule, the benzylic amine and the
pyridine amide moiety. Exploration of the amine moiety revealed
that various secondary and tertiary benzyl amines are all potent
inhibitors of Raf. Dimethylation of the primary benzyl amine of 6
provided tertiary amine 22, which maintained good potency and
demonstrated improved oral exposures (Table 3); the isopropyl
benzyl amine 23 also showed good cellular potency (Table 2)
and significantly improved oral exposures in a rat PK experiment
(
Table 3, discrete rat IV data were not obtained for this
molecule). Efforts to replace the amide substituent on the
pyridine with a variety of heterocycles did not lead to compounds
with an acceptable combination of cellular activity and in vivo
clearance. As mentioned above, more sterically demanding
amides were not well tolerated, but cyclization of the amide to
a lactam provided potent compounds with improved oral
exposures. A combination of the lactam and isopropyl benzyl
amine in one molecule yielded 7 as one of our better compounds
with respect to potency, in vivo clearance, and oral exposure
(
Table 3).
Continued optimization and exploration of the series led us to
examine the effect of reversing the internal amide in this series. As
exemplified by compound 8, this modification appeared to have
little impact on either the potency or the PK properties of the
molecule (Table 3). Interestingly, the more potent enantiomer
(
8, R) with this amide bond orientation has the opposite
stereochemistry when compared to the more potent enantiomer
in the opposite amide bond orientation (22, S).
Because of the straightforward nature of the synthesis of the
aminotetralin template required for this subseries, as well as the
lack of a risk of racemization (which was occasionally observed in
the synthesis of molecules related to compound 7), the focus of
our efforts now shifted to optimizing compound 8. We were able
to apply what we had learned from the optimization of the initial
tetralin scaffold to improve the PK properties of this subseries
a
IC50 values are shown as mean values of three or more determinations.
IC50 values are shown as mean of two determinations.
b
18
and quickly identified 26 (ML721) as a molecule with excellent
physicochemical and PK behavior of the molecules. Introduction
of a basic amine to the aryl amide portion of the molecule was
evaluated as a means of improving the aqueous solubility of the
series. Replacement of the 4-chloro substituent with a primary
benzyl amine at either the 4- or the 5-position of the phenyl ring
cellular potency and PK properties (Table 3).
Significant demethylation of 26 to the monomethyl benzyl
amine 28 was observed in vivo, and this metabolite was generally
found to be present at ∼30% of the circulating levels of 26 after
oral dosing, as measured by area under the curve (AUC). We
investigated 28 as a potential substitute for 26 and found that it
did not perform as well as desired in vivo either in rat PK studies
(
18 and 19, respectively, Table 2) was tolerated, although this
modification did not improve inhibitory activity in the enzyme or
cellular assays.
(
(
see Table 3) or in mouse PK/pharmacodynamic (PD) studies
data not shown). A variety of substituted benzyl amines (as well
Major improvements in enzyme and cellular activity were
observed with alteration of the substituent on the pyridine ring,
the hinge-binding element of the series. A simple reversal of the
pyridine amide moiety (which is expected to maintain this inter-
action) provided a 10-fold improvement in cellular potency
as other amine-containing moieties) were explored, and we
18
identified 27 (ML786) as a molecule that maintained good
cellular potency and acceptable PK properties but did not form
any known active metabolites in vivo.
(
cf. 19 and 5, Table 2). A cyclopropyl amide (compound 6)
was also well tolerated, but more sterically demanding amides
e.g., isobutyryl or pivaloyl) led to decreases in cellular and
Both of these compounds, 26 and 27, were found to have
activity in vivo. As can be seen from Table 4, both compounds
strongly inhibit the Raf pathway in vivo following a 75 or 100
mg/kg oral dose. As a class, these compounds have been found
to inhibit a significant number of kinases (Table 5) and are
inhibitors of wild-type B-Raf and C-Raf. Although 26 and 27
display significant receptor tyrosine kinase activity, we were
confident that this additional activity would not contribute
significantly to pERK inhibition in melanoma tumors with
constituitively activated B-Raf. This hypothesis was confirmed
(
enzyme activity (compounds and data not shown).
1
9
The enantiomers of 6 were separated by chiral HPLC, and
the absolute stereochemistry was subsequently confirmed by
synthesis of the individual enantiomers. While the enzyme
activity of these two antipodes is quite similar, (S)-enantiomer
2
0 (Table 2) appears to be somewhat more potent in a cellular
setting.
1
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ARTICLE
Table 3. Structures, Raf Potency, and Rrat PK Parameters for Key Raf Inhibitors
V600E
a
a
c
c
c
c
ΔB-Raf IC50 (nM)
pERK IC50 (nM)
Clp (L/h/kg)
Vss (L/kg)
po AUC1-24h (μM h)
po % F
b
d
d
6
2
2
7
2
8
2
2
2
2
8 ( 1
150
0.4
6.4
0.58
1.9
27
d
d
2
3
4 ( 1
130 ( 26
190 ( 42
110 ( 40
79 ( 37
70 ( 26
1.41
6.07
ND
3.87
ND
2.19
ND
0.75
3.93
2.87
e
f
5.0 ( 0.5
3.3 ( 0.3
ND
15.6
ND
20
0.32
ND
2.14
ND
0.23
0.44
0.73
20.7
ND
0.51
ND
98.6
35.9
12.4
b
4
2.9
ND
22
2.9 ( 0.5
3.2 ( 0.2
2.7 ( 0.5
2.1 ( 0.5
2.2 ( 0.5
b
5
6
7
8
1100
ND
100
85
50 ( 26
60 ( 30
45 ( 11
4.01
b
50
a
c
Unless otherwise noted, IC50 values are shown as mean values of three or more determinations. IC50 values are shown as means of two determinations.
Average of three rats dosed at 1 mg/kg iv or 10 mg/kg po. Vehicle, 10% HPbCD. Vehicle: po, 20% HPbCD; iv, PEG400/H O/EtOH/DMA
4:4:1:1). ND, not determined. Data from a single rat dosed at 10 mg/kg po. Vehicle, 20% HPbCD.
d
2
e
f
(
a
Table 4. Inhibition of pERK Formation In Vivo Following an
Oral Dose of Select Raf Inhibitors
Table 5. Selected Kinase Selectivity Data for 26 and 27
a
IC50 (nM)
26
27
d
e
hours postdose pERK (% inhibition) plasma concentration (μM)
V600E
ΔB-Raf
2.7
2.1
4.2
2.5
<0.5
7.0
190
11
b
2
6
7
4
8
2
4
8
2
93 ( 2
92 ( 1
72 ( 6
87 ( 4
90 ( 2
83 ( 7
28 ( 7
wt B-Raf
C-Raf
Abl-1
4.7
b
29 ( 5
2.2
b
c
4
c
0.4 ( 0.1
23 ( 4
27 ( 3
1.2
2
DDR2
EGFR
EPHA2
KDR
2.6
c
4900
18
4
3.0 ( 0.6
a
PK/PD studies were performed in nude mice bearing A375 M
59
6.2
170
1400
42
xenografts. Compounds were dosed as solutions in 10% HPbCD.
LCK
610
>10000
91
b
Xenografts were harvested for analysis at the time listed after a single
c
MEK
1
00 mg/kg dose. Xenografts were harvested for analysis at the time
d
p38R
listed after a single 75 mg/kg dose. Average of pERK levels determined
by Li-Cor quantitative Western blots (3 or 4 animals per group) as
PDGFR
RET
1100
1.6
47
e
compared to vehicle control set at 100%. Average of compound levels
0.8
in the plasma of mice determined by LC/MS/MS quantitation (3 or 4
animals per group).
a
IC50 values are shown as means of two determinations. ATP concen-
tration for each kinase = Kmapp. Single-concentration binding data for
20 kinases can be found in the Supporting Information, Table 1.
2
with the observation that neither sunitinib [a multi-RTK
(
receptor tyrosine kinase)] nor erlotinib (an EGFR inhibitor)
demonstrated significant pERK inhibition in A375 cells in a
cellular setting or in A375 M tumors upon in vivo dosing (data
not shown).
1
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ARTICLE
Figure 2. Crystal structure of 24 solved in complex with B-Raf (3.1 Å
resolution).
following procedure. Compounds were dissolved in DMSO, methanol,
or acetonitrile, and the solutions were analyzed using an Agilent 1100 LC
interfaced to a micromass Waters Micromass Zspray Mass Detector
(ZMD). One of two gradients was used to elute the compounds either a
formic acid (FA) gradient (acetonitrile containing 0-100% 0.1% formic
acid in water) or an ammonium acetate (AA) gradient (acetonitrile
containing 0-100% 10 mM ammonium acetate in water). All com-
pounds were determined to be >95% pure unless otherwise noted. High-
resolution mass spectra were recorded using a Thermo LTQ-FT Ultra
mass spectrometer equipped with an Eksigent nanoLC Ultra system.
About 500 pg of each compound was injected into an in-house packed
reverse phase nanobore column (75 μm ID) with Magic C18 resin
Figure 1. Inhibition of subcutaneous A375 M xenografts in immuno-
compromised mice by 26 and 27. The doses shown are the maximum
daily dose tolerated without body weight loss. Each compound was
administered po/qd for 21 days.
(Michrom BioResources, Auburn, CA) to a length of 12 cm. The pump
flow rate was 300 nL/min. Buffer A is the aqueous solvent and consisted
of 0.1% formic acid (Sigma, St. Louis, MO) in HPLC grade water (VWR
Scientific, West Chester, PA). Buffer B, the organic phase, consisted of
Furthermore, once daily dosing for 21 days at 100 mg/kg for
6 or 75 mg/kg for 27 induces partial regressions of the tumor
0
.1% formic acid in 90% acetonitrile (VWR Scientific) and 10% HPLC
grade water. The gradient was set as follows: 0 min, 2% buffer B; 32 min,
2% buffer B; 35 min, 90% buffer B; and 36 min, 2% buffer B. The
2
xenografts. These doses are well tolerated by the mice and show
no indication of toxicity or weight loss as compared to vehicle-
treated animals (Figure 1).
4
compounds were ionized using a New Objective PicoView Nanospray
Source with 2.2 kV voltage. The mass spectrometer was set to acquire
masses from 350 to 2000 D in positive mode. MS/MS spectra of each
compound were also acquired under data-dependent acquisition mode
to confirm the chemical structure of the compounds (data not shown).
A crystal structure of a representative of this series, compound
4, bound to the active site of wt B-Raf revealed the expected mode
2
of binding to the DFG-out conformation (Figure 2). Key hinge
contacts are achieved by the bicyclic pyridine lactam moiety
4
-[4-(3-{[4-Chloro-3-(trifluoromethyl)phenyl]amino}-
3-oxopropyl)phenoxy]-N-methylpyridine-2-carboxamide
2). A mixture of 3-(4-hydroxyphenyl)propanoic acid (4 ꢀ 250 mg,
nitrogens, the CF substituent occupies a key hydrophobic pocket,
3
20
and the central amide supplies a single hydrogen bond.
(
We began our work searching for novel compounds that
robustly inhibit the Raf pathway and demonstrate a strong
antitumor effect in models of B-Raf mutant melanoma at well-
tolerated doses. In identifying 26 and 27, we have fulfilled that
search. These molecules show potent inhibition of B-Raf in cells,
possess excellent physicochemical and PK properties, potently
inhibit the B-Raf pathway in mouse PK/PD studies, and show
good efficacy in B-Raf mutant xenograft studies.
2
4.1 mmol total), 4-chloro-3-(trifluoromethyl)aniline (4 ꢀ 190 mg, 15.5
mmol total), and HATU (4 ꢀ 0.58 g, 24.1 mmol total) were added to
four microwave-safe vials. To each vial, NMP (5 mL) and DIPEA (0.425
mL, 9.7 mmol) were added. The vials were then sealed and subjected to
microwave irradiation at 220 °C for 15 min. The reaction vessels were
unsealed, and the mixtures were combined and diluted with EtOAc. The
organic solution was extracted with 1 N HCl and brine, dried over
Na SO , filtered, and concentrated to give a brown oil. Purification by
2
4
2
column chromatography (SiO , elution with EtOAc in hexane) provided
N-[4-chloro-3-(trifluoromethyl)phenyl]-3-(4-hydroxyphenyl)propana-
’
EXPERIMENTAL SECTION
mide as a yellow oil that solidified upon exposure to DCM (960 mg, 47%
1
NMR spectra were recorded in the solvent reported on a Bruker 300
yield, ∼95% pure). H NMR (300 MHz, CDCl ): δ 7.74 (br d, J =
3
MHz Avance1 or 400 MHz Avance2 (5 mm QnProbe) using residual
solvent peaks as the reference. Compound purity was determined by
analysis of the diode array UV trace of an LC-MS spectrum using the
2.4 Hz, 1H), 7.65 (br dd, J = 8.5 Hz, 2.3 Hz, 1H), 7.42 (d, J = 8.7 Hz, 1H),
7.16 (br s, 1H), 7.10 (d, J = 8.5 Hz, 2H), 6.79 (d, J = 8.5 Hz, 2H), 2.99
(t, J = 7.4 Hz, 2H), 2.65 (t, J = 7.4 Hz, 2H).
1
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Journal of Medicinal Chemistry
ARTICLE
1
To a mixture of DMPU (0.7 mL) and DMF (2.8 mL) was added
to give the HCl salt of 4 as an off-white solid (175 mg, 42%). H NMR
N-[4-chloro-3-(trifluoromethyl)phenyl]-3-(4-hydroxyphenyl)propana-
(400 MHz, d -DMSO): δ 10.43 (s, 1H), 8.49 (d, J = 5.71 Hz, 1H), 8.21
(d, J = 2.48 Hz, 1H), 7.85 (dd, J = 8.81, 2.45 Hz, 1H), 7.61 (d, J = 8.83
6
1
0
mide (600 mg, 1.75 mmol) and 4-chloro-N-methylpicolinamide (328
t
mg, 1.92 mmol). To this solution was added KO Bu (590 mg, 5.25
Hz, 1H), 7.46 (d, J = 2.27 Hz, 1H), 7.23 (d, J = 8.30 Hz, 1H), 7.16 (dd,
J = 5.73, 2.57 Hz, 1H), 6.99 (d, J = 2.39 Hz, 1H), 6.95 (dd, J = 8.23, 2.51
Hz, 1H), 3.01-2.92 (m, 2H), 2.91-2.72 (m, 6H), 2.19-2.06 (m, 1H),
mmol). The reaction mixture was stirred at 100 °C for 18 h and then
cooled to room temperature, diluted with water, and extracted with
EtOAc and DCM. The combined organic solutions were washed with
þ
and 1.92-1.72 (m, 1H). LC-MS: m/z 504.3/506.3 [M þ H] , 502.4/
-
water and brine, dried over Na
2
SO
4
, filtered, and concentrated to give a
504.3 [M - H] (AA). HRMS: m/z calcd for C H N O ClF ([M þ
2
5
22
3
3
3
þ
brown oil. Purification by column chromatography provided 4-[4-(3-{[4-
chloro-3-(trifluoromethyl)phenyl]amino}-3-oxopropyl)phenoxy]-N-
H]) , 504.12963; found, 504.130179.
General Procedure B1, Amide Bond Formation. 7-({2-[-
(Methylamino)carbonyl]pyridine-4-yl}oxy)-1,2,3,4-tetrahydronaphth-
alene-2-carboxylic acid (400 mg, 1.22 mmol), 5-amino-2-cyanobenzo-
trifluoride (251 mg, 1.35 mmol), EDCI (258 mg, 1.35 mmol), and
DMAP (180 mg, 1.47 mmol) were combined in DCM (10 mL), and the
resulting mixture was stirred overnight at room temperature. The
reaction mixture was concentrated in vacuo, diluted with EtOAc, and
extracted with aqueous 1 N HCl solution. The organic phase was
methylpyridine-2-carboxamide as a colorless solid (100 mg, 12%
1
yield). H NMR (300 MHz, CD
3
OD): δ 10.11 (br s, 0.3H), 8.41
(d, J = 5.6 Hz, 1H), 8.07 (br d, J = 2.2 Hz, 1H), 7.74 (br dd, J = 8.7, 2.1
Hz, 1H), 7.48-7.51 (m, 2H), 7.36 (d, J = 8.5 Hz, 1H), 7.05 (d, J = 8.5
Hz, 2H), 6.98 (dd, J = 5.6, 2.5 Hz, 1H), 3.34 (s, 0.8H), 3.04 (t, J = 7.5
Hz, 2H), 2.92 (s, 3H), 2.71 (t, J = 7.5 Hz, 2H).
4
-[3-(3-{[4-Chloro-3-(trifluoromethyl)phenyl]amino}-3-
oxopropyl)phenoxy]-N-methylpyridine-2-carboxamide
3). A mixture of N-[4-chloro-3-(trifluoromethyl)phenyl]-3-(3-hydro-
washed with brine, dried over MgSO , filtered, and concentrated.
4
(
Purification by column chromatography (SiO , elution with 0-50%
2
xyphenyl)propanamide [585 mg, 1.70 mmol, prepared as described
above using 3-(3-hydroxyphenyl)propanoic acid and 4-chloro-
EtOAc in hexanes) provided 4-{[7-({[4-cyano-3-(trifluoromethyl)ph-
enyl]amino}carbonyl)-5,6,7,8-tetrahydronaphthalen-2-yl]oxy}-N-meth-
1
0
1
3-(trifluoromethyl)aniline], 4-chloro-N-methylpicolinamide
(318
ylpyridine-2-carboxamide as a white powder (480 mg, 79%). H NMR
mg, 1.87 mmol), and Cs CO (7.76 g, 8.5 mmol) in DMF (3.4 mL)
2
3
(400 MHz, CDCl
(m, 1H), 8.22 (d, J = 4.9 Hz, 1H), 8.11 (d, J = 1.8 Hz, 1H), 8.03 (dd, J =
.5, 1.9 Hz, 1H), 7.73 (d, J = 8.5 Hz, 1H), 7.52 (d, J = 2.5 Hz, 1H), 7.06
d, J = 8.3 Hz, 1H), 7.00 (dd, J = 5.6, 2.5 Hz, 1H), 6.81 (dd, J = 8.2, 2.3
3
): δ 9.30 (s, 1H), 8.33 (d, J = 5.6 Hz, 1H), 8.25-8.18
was heated at 100 °C overnight. The reaction mixture was concentrated
and diluted with EtOAc. The organic solution was washed with brine,
8
(
dried over MgSO
by column chromatography (SiO
as a white solid (10 mg, 1% yield). H NMR (400 MHz, CD OD): δ
4
, filtered, and concentrated. The residue was purified
2
, 0-30% EtOAc in hexanes) to give 3
Hz, 1H), 6.67 (d, J = 2.0 Hz, 1H), 3.05-2.97 (m, 4H), 2.88 (dd, J = 16.9,
3.4 Hz, 2H), 2.77-2.60 (m, 2H), 2.22-2.13 (m, 1H), and 2.01-1.89
1
3
8
7
7
2
.39 (d, J = 5.58 Hz, 1H), 8.03 (d, J = 2.43 Hz, 1H), 7.74-7.66 (m, 1H),
.50 (dd, J = 8.12, 5.65 Hz, 2H), 7.40 (d, J = 7.86 Hz, 1H), 7.22 (d, J =
.68 Hz, 1H), 7.06 (s, 1H), 7.03-6.96 (m, 2H), 3.05 (t, J = 7.48, 7.48 Hz,
(m, 1H).
4-{[7-({[4-(Aminomethyl)-3-(trifluoromethyl)phenyl]ami-
no}carbonyl)-5,6,7,8-tetrahydronaphthalen-2-yl]oxy}-N-
methylpyridine-2-carboxamide (18). 4-{[7-({[4-Cyano-3-(trifl-
uoromethyl)phenyl]amino}carbonyl)-5,6,7,8-tetrahydronaphthalen-2-
yl]oxy}-N-methylpyridine-2-carboxamide (200 mg, 0.4 mmol) was dis-
H), 2.93 (s, 3H), and 2.71 (t, J = 7.46, 7.46 Hz, 2H). LCMS: m/z 478.3
þ
[M þ H] (AA).
General Procedure A1, Biaryl Ether Bond Formation. A
mixture of 7-hydroxy-1,2,3,4-tetrahydronaphthalene-2-carboxylic acid
solved in 7.0 M NH in MeOH solution. To this solution was added
Raney 2800 nickel (50% v/v slurry in water, approx 2 mL), and the mix-
3
10
(8.00 g, 41.6 mmol), 4-chloro-N-methylpicolinamide (8.58 g, 50.3
mmol), and cesium carbonate (40.7 g, 125 mmol) in DMF (150 mL)
was heated at 100 °C overnight. The reaction was cooled to room temp-
erature and concentrated under vacuum. The resulting slurry was diluted
with water and acidified with 1 N HCl solution to pH 3. The mixture was
allowed to stand at room temperature, and a precipitate formed. This preci-
pitate was isolated by filtration, washed well with water, and thoroughly
dried to give 7-({2-[(methylamino)carbonyl]pyridin-4-yl}oxy)-1,2,3,4-
ture was stirred under an atmosphere of hydrogen gas for 5 h. The
reaction mixture was filtered through a pad of Celite, and the filtrate was
concentrated. Purification by column chromatography [SiO , elution
2
with 0-5% MeOH (with 1% NH OH) in DCM] provided 18 as a white
4
solid. This material was taken up in DCM and treated with 1 N HCl in
Et O. The solution was concentrated to give the HCl salt of 18 as a white
2
1
6
solid (110 mg, 52%). H NMR (300 MHz, d -DMSO): δ 10.73 (s, 1H),
tetrahydronaphthalene-2-carboxylic acid as a white solid (12.10 g, 89%).
8.86 (dd, J = 9.32, 4.51 Hz, 1H), 8.60 (bs, 2H), 8.51 (d, J = 5.69 Hz, 1H),
8.21 (d, J = 1.72 Hz, 1H), 7.94 (d, J = 8.54 Hz, 1H), 7.73 (d, J = 8.50 Hz,
1H), 7.44 (d, J = 2.40 Hz, 1H), 7.24 (d, J = 8.28 Hz, 1H), 7.18 (dd, J =
5.67, 2.52 Hz, 1H), 7.02 (d, J = 2.16 Hz, 1H), 6.97 (dd, J = 8.20, 2.37 Hz,
1H), 4.14-4.03 (m, 2H), 2.99-2.92 (m, 2H), 2.91-2.81 (m, 3H), 2.78
(d, J = 4.76 Hz, 3H), 2.18-2.06 (m, 1H), and 1.90-1.72 (m, 1H). LC-
1
H NMR (400 MHz, d
.8 Hz, 1H), 12.30 (s, 1H), 7.35 (d, J = 2.6 Hz, 1H), 7.20 (d, J = 8.3
Hz, 1H), 7.13 (dd, J = 5.6, 2.6 Hz, 1H), 6.99 (d, J = 2.4 Hz, 1H), 6.94
dd, J = 8.3, 2.6 Hz, 1H), 2.93 (d, J = 5.4 Hz, 1H), 2.83 (ddd, J = 9.4,
.8, 3.6 Hz, 1H), 2.72 (s, 1H), 2.16-2.06 (m, 1H), and 1.81-1.68
m, 1H).
-{[7-({[4-Chloro-3-(trifluoromethyl)phenyl]amino}car-
6
-DMSO): δ 8.48 (d, J = 5.6 Hz, 1H), 8.76 (d, J =
4
(
7
(
þ
-
MS: m/z 499.4 [M þ H] , 497.4 [M - H] (FA).
4
General Procedure B2, Amide Bond Formation. 7-(2-Acet-
amidopyridin-4-yloxy)-1,2,3,4-tetrahydronaphthalene-2-carboxylic acid
(1.00 mg, 3.06 mmol), DIPEA (1.60 mL, 9.19 mmol), and HATU (1.75
g, 4.60 mmol) were combined in DMF (30 mL), and the solution was
stirred for 30 min. tert-Butyl [4-amino-2-(trifluoromethyl)benzyl]-
carbamate (889 mg, 3.06 mmol) was added, and the resulting mixture
was stirred overnight at room temperature. Water was added to the
reaction mixture, and the resulting precipitate was isolated by filtration,
washed well with water and hexane, and dried under vacuum. Purifica-
bonyl)-5,6,7,8-tetrahydronaphthalen-2-yl]oxy}-N-methyl-
pyridine-2-carboxamide (4). 4-Chloro-3-(trifluoromethyl)aniline
(160 mg, 0.817 mmol), 7-({2-[(methylamino)carbonyl]pyridine-4-
yl}oxy)-1,2,3,4-tetrahydronaphthalene-2-carboxylic acid (254 mg,
0
0
.778 mmol), EDCI (149 mg, 0.778 mmol), and DMAP (114 mg,
.943 mmol) were suspended in DCM (2.74 mL) in a microwave vial.
The vial was sealed, and the reaction mixture was subjected to micro-
wave irradiation at 60 °C for 5 min. Water and DCM were added to the
reaction mixture, and the phases were separated. The organic phase was
tion by column chromatography (SiO , elution with 20-80% EtOAc in
2
dried over MgSO , filtered, and concentrated. Purification by column
hexane) provided tert-butyl [3-{[((2S)-7-{[2-(acetylamino)pyridin-4-
4
chromatography (SiO , elution with 10-50% EtOAc in hexanes)
provided 210 mg of desired product. This material was taken up in
yl]oxy}-1,2,3,4-tetrahydronaphthalen-2-yl)carbonyl]amino}-5-(trifluo-
romethyl)benzyl]carbamate as an off white solid (1.21 g, 66%) H NMR
2
1
DCM and treated with 1 N HCl in Et O. The solution was concentrated
(300 MHz, CDCl
3
): δ 8.20 (s, 1H), 8.05 (d, J = 6.1 Hz, 1H), 7.83
2
1
842
dx.doi.org/10.1021/jm101479y |J. Med. Chem. 2011, 54, 1836–1846

Journal of Medicinal Chemistry
ARTICLE
(
(
1
s, 1H), 7.75-7.70 (m, 1H), 7.12 (d, J = 8.3 Hz, 1H), 6.90-6.79
m, 2H), 6.71 (dd, J = 6.1, 2.3 Hz, 1H), 5.01 (s, 1H), 4.32 (d, J = 6.0 Hz,
H), 3.14-2.85 (m, 4H), 2.84-2.60 (m, 2H), 2.21 (s, 3H), 2.17-2.12
(400 MHz, d
6
-DMSO): δ 9.47 (s, 1H), 8.32 (dd, J = 8.9, 5.5 Hz, 1H),
7.18 (dd, J = 7.2, 5.5 Hz, 1H), and 1.44 (s, 9H). LC-MS: m/z 339 [M þ
þ
H] (FA).
(m, 1H), 2.02-1.92 (m, 1H), and 1.45 (s, 9H).
General Procedure C, tert-Butyl Carbamate Deprotection.
tert-Butyl [3-{[((2S)-7-{[2-(acetylamino)pyridin-4-yl]oxy}-1,2,3,4-tet-
rahydronaphthalen-2-yl)carbonyl]amino}-5-(trifluoromethyl)benzyl]-
carbamate (1.21 g, 2.02 mmol) was dissolved in a 4.0 M HCl in dioxane
solution (10 mL), and the solution was stirred for 2 h. The mixture was
concentrated in vacuo. Purification of the resulting residue by column
chromatography (SiO
2
, elution with 4-10% MeOH (with 2%
NH OH) in DCM) provided a white solid. This solid was dissolved
4
A round-bottomed flask was charged with tert-butyl (4-fluoro-3-
iodopyridin-2-yl)carbamate (20.0 g, 59.2 mmol), 3,3-diethoxy-1-pro-
pene (13.5 mL, 88.7 mmol), DMF (150 mL), water (50 mL), DIPEA
in DCM (10 mL), and 2.0 M HCl in Et
2
O was added (2 mL). This
mixture was stirred for 5 min and then concentrated in vacuo to give
2
1
(
(
2S)-7-{[2-(acetylamino)pyridin-4-yl]oxy}-N-[3-(aminomethyl)-5-
(15.4 mL, 88.7 mmol), and Pd catalyst 29 (480 mg, 0.827 mmol), and
trifluoromethyl)phenyl]-1,2,3,4-tetrahydronaphthalene-2-carboxami-
the reaction mixture was warmed to 140 °C. After 5 h, the reaction
mixture was cooled in a refrigerator for 2 days. The precipitate was
isolated by filtration, washed with diethyl ether, and dried to give 3.25 g
of pink needles. The filtrate was concentrated to give a reddish semisolid.
This material was redissolved in DCM, and the solution was passed
1
de 2HCl (5) as a white solid (807 mg, 70%). H NMR (300 MHz,
d -DMSO, HCl salt): δ 11.23 (br s, 1H), 10.74 (s, 1H), 8.43 (s, 2H),
3
6
8
(
2
.23 (d, 1H), 8.13-7.99 (m, 2H), 7.58 (s, 1H), 7.35 (s, 1H), 7.28-7.22
m, 1H), 7.05-6.94 (m, 2H), 6.87-6.80 (m, 1H), 4.13-4.02 (m,
H), 2.98-2.79 (m, 5H), 2.10 (s, 4H), and 1.79 (br s, 1H). LC-MS:
through 200 g of SiO . Concentration of the resulting solution provided
2
þ
-
m/z 499.7 [M þ H] ; 497.4 [M - H] (FA). HRMS: m/z calcd for
a red/orange residue, which was recrystallized from 2-propanol (150
mL) to give 9.4 g of a pink solid. Purification of this pink solid by column
þ
C
26
H
25
F
3
N
-Fluoro-3,4-dihydro-1,8-naphthyridin-2(1H)-one (11).
4
O
3
([M þ H]) , 499.19515; found, 499.195700.
5
chromatography (SiO , elution with 0-75% EtOAc/DCM) provided
2
Palladium acetate (341 mg, 1.52 mmol) and 4,5-bis(diphenyl-
phosphino)-9,9-dimethylxanthene (1.76 g, 3.04 mmol) were added
to a three-necked, round-bottomed flask, and the flask was purged
three times with argon. Degassed 1,4-dioxane (240 mL) was added,
and the mixture was stirred and degassed again with argon. To this
solution was added a solution of 2-chloro-4-fluoropyridine (20 g,
1.41 g of a white powder. Overall, 4.66 g of 5-fluoro-3,4-dihydro-1,8-
naphthyridin-2(1H)-one (11) was isolated (47% yield). H NMR (400
1
MHz, d -DMSO): δ 10.7 (s, 1H), 8.11 (dd, J = 8.5, 5.7 Hz, 1H), 6.91
6
(dd, J = 8.8, 5.7 Hz, 1H), 2.91-2.85 (m, 2H), and 2.55-2.50 (m, 2H).
þ
LC-MS: m/z 167 [M þ H] (FA).
(2S)-N-[3-[(Isopropylamino)methyl]-5-(trifluoromethyl)-
phenyl]-7-[(7-oxo-5,6,7,8-tetrahydro-1,8-naphthyridin-4-
yl)oxy]-1,2,3,4-tetrahydronaphthalene-2-carboxamide
(7). A mixture of 5-fluoro-3,4-dihydro-1,8-naphthyridin-2(1H)-one
(11, 50.0 mg, 0.301 mmol), (2S)-7-hydroxy-1,2,3,4-tetrahydro-naphtha-
lene-2-carboxylic acid [(S)-15, 63.6 mg, 0.331 mmol], cesium carbonate
(294 mg, 0.903 mmol), and N,N-dimethylacetamide (1.40 mL) was
combined. The mixture was sealed in a microwave vial and heated in the
microwave at 150°C for 1 h. Water (5 mL) was added, and the mixture was
allowed to stir for a few minutes. The clear solution was then neutralized by
the addition of 1 N HCl solution, and the resulting suspension was filtered
through a Celite pad. The solid residue was washed with MeOH, and the
filtrate was concentrated in vacuo. The residue was purified by column
1
52 mmol) in degassed 1,4-dioxane (120 mL), tert-butyl carbamate
(
(
19.6 g, 167 mmol), NaOH (8.88 g, 222 mmol), and degassed water
4.0 mL, 222 mmol). The resulting mixture was stirred at 100 °C.
After 1.5 h, the reaction mixture was cooled to room temperature and
filtered through a pad of Celite. The pad was washed well with
dioxane, and the filtrate was concentrated under reduced pressure to
dryness. The resulting solid was recrystallized from 2-propanol
(
∼250 mL) to give tert-butyl (4-fluoropyridin-2-yl)carbamate (9)
1
as a pale yellow crystalline solid (25.65 g, 79.5% yield). H NMR (400
MHz, d -DMSO): δ 10.10 (s, 1H), 8.26 (dd, J = 9.5, 5.6 Hz, 1H), 7.60
dd, J = 12.3, 2.3 Hz, 1H), 6.95 (ddd, J = 8.3, 5.6, 2.3 Hz, 1H), and
6
(
1
þ
.47 (s, 9H). LC-MS: m/z 213 [M þ H] (FA).
An oven-dried, three-neck, round-bottomed flask equipped with an
chromatography (SiO , eluting with 2-10% MeOH in DCM) to give
2
overhead stirrer, temperature probe, and addition funnel was charged
with tert-butyl (4-fluoropyridin-2-yl)carbamate (31.8 g, 150 mmol),
TMEDA (56.6 mL, 375 mmol), and THF (200 mL). The solution was
cooled to -78 °C, and a solution of n-BuLi (2.50 M in hexane, 150 mL,
(2S)-7-[(7-oxo-5,6,7,8-tetrahydro-1,8-naphthyridin-4-yl)oxy]-1,2,3,4-
tetrahydronaphthalene-2-carboxylic acid (42 mg, 41% yield), ee
93.2% (ee determined by chiral HPLC, Column: IA 4.6 mm ꢀ 250 mm,
1
eluting with 100/0.1 EtOH/TFA, at 0.5 mL/min for 50 min). H NMR
375 mmol) was added dropwise so that the reaction mixture tempera-
6
(400 MHz, d -DMSO): δ 11.94 (s, 1H), 10.10 (s, 1H), 7.57 (d, J = 5.8 Hz,
ture remained below -70 °C. The reaction mixture was stirred at -
1H), 6.76 (d, J = 8.3 Hz, 1H), 6.54-6.44 (m, 2H), 5.89 (d, J = 5.8 Hz, 1H),
2.64-2.34 (m, 6H), 2.33-2.23 (m, 1H), 2.16-2.12 (m, 2H), 1.79-1.65
(m, 1H), and 1.42-1.28 (m, 1H).
7
8 °C for 1 h, and a solution of I (95.2 g, 375 mmol) in THF (160 mL)
2
was added via addition funnel. The addition was controlled to keep the
reaction mixture temperature below -70 °C, and the resulting mixture
(2S)-7-[(7-Oxo-5,6,7,8-tetrahydro-1,8-naphthyridin-4-yl)oxy]-1,2,3,
4-tetrahydronaphthalene-2-carboxylic acid and tert-butyl [3-amino-
5-(trifluoromethyl)benzyl]isopropylcarbamate were combined as de-
scribed in General Procedure B2, Amide Bond Formation, and the Boc
group was removed as described in General Procedure C, tert-Butyl
was stirred at -78 °C for 1 h. A solution of NaHSO (61 g, 580 mmol) in
4
water (200 mL) was added to the reaction mixture as it warmed to room
temperature. Ethyl acetate was added, and the two-phase mixture was
stirred at room temperature for 1 h. Water (500 mL) was added, and the
phases were separated. The aqueous phase was extracted with EtOAc (3
Carbamate Deprotection, to give 7 as the HCl salt (67% yield over both
1
ꢀ
400 mL), and the organic phases were combined, dried over MgSO
4
,
steps). H NMR (400 MHz, d
6
-DMSO): δ 10.71 (s, 1H), 10.61 (s, 1H),
filtered, and concentrated to give an off white solid. This solid was
suspended in DCM (50 mL), and the solid was isolated by filtration and
washed with a minimum of DCM. The filtrate was concentrated and
filtered to give a second crop of product. The solids were combined and
dried under vacuum to give tert-butyl (4-fluoro-3-iodopyridin-2-
9.07 (s, 2H), 8.11 (s, 1H), 8.05 (s, 1H), 7.99 (d, J = 5.9 Hz, 1H), 7.68
(s, 1H), 7.19 (d, J = 8.3 Hz, 1H), 6.95-6.88 (m, 2H), 6.32 (d, J = 5.9 Hz,
1H), 4.22-4.17 (m, 2H), 3.38-3.28 (m, 1H), 3.01-2.75 (m, 7H), 2.54
(t, J = 7.7, 7.7 Hz, 2H), 2.16-2.07 (m, 1H), 1.88-1.72 (m, 1H), 1.30
(s, 3H), and 1.28 (s, 3H). HRMS: m/z calcd for C30
H]) , 553.24210; found, 553.24265.
H
31
F
3
N
4
O
3
([M þ
1
þ
yl)carbamate (10) as a white solid (45.63 g, 86% yield). H NMR
1
843
dx.doi.org/10.1021/jm101479y |J. Med. Chem. 2011, 54, 1836–1846

Journal of Medicinal Chemistry
ARTICLE
General Procedure B3, Amide Bond Formation. A solution
of N-(4-{[(7R)-7-amino-5,6,7,8-tetrahydronaphthalen-2-yl]oxy}pyri-
din-2-yl)cyclopropanecarboxamide (10.2 g, 31.5 mmol) and HCl (2.0
3.24-3.16 (m, 3H), 3.06-2.94 (m, 3H), 2.90 (s, 6H), 2.81-2.77
(m, 2H), 2.26-2.19 (m, 1H), 2.01-1.89 (m, 1H). HRMS: m/z calcd for
þ
C
29
H
29
F
3-(1-Amino-1-methylethyl)-N-{(2R)-7-[(7-oxo-5,6,7,8-tet-
3
N
4
O
3
([M þ H]) , 539.22645; found, 539.22700.
M solution in Et
2
O, 32 mL, 63 mmol) in pyridine (170 mL) was treated
with 3-[(dimethylamino)methyl]-5-(trifluoromethyl)benzoic acid Li
salt (9.18 g, 36.3 mmol) and EDCI (9.67 g, 50.5 mmol). This solution
was stirred overnight and then partitioned between EtOAc (500 mL)
rahydro-1,8-naphthyridin-4-yl)oxy]-1,2,3,4-tetrahydronap-
hthalen-2-yl}-5-(trifluoromethyl)benzamide (27). 3-{1-
[(tert-Butoxycarbonyl)amino]-1-methylethyl}-5-(trifluoromethyl)benzoic
acid and 5-{[(7R)-7-amino-5,6,7,8-tetrahydronaphthalen-2-yl]-
oxy}-3,4-dihydro-1,8-naphthyridin-2(1H)-one were combined as
described in General Procedure B3, Amide Bond Formation, with-
out the addition of HCl and General Procedure C to provide 27
3
and half-saturated NaHCO
extracted with DCM (2 ꢀ 100 mL), and the combined organic phases
were washed with brine, dried over Na SO , filtered, and concentrated.
Purification by column chromatography (SiO , elution with 100% DCM
to 15% EtOH, 15% MeCN, and 75% DCM) provided N-[(2R)-7-
{2-[(cyclopropylcarbonyl)amino]pyridin-4-yl}oxy)-1,2,3,4-tetrahydro-
3
solution (500 mL). The aqueous phase was
2
4
2
1
(44.1 g, 61%). H NMR (400 MHz, CD OD): δ 8.06 (s, 1H), 7.87
3
(
(s, 1H), 7.75 (d, J = 6.88 Hz, 1H), 7.67 (s, 1H), 6.95 (d, J = 8.40 Hz,
naphthalen-2-yl]-3-[(dimethylamino)methyl]-5-(trifluoromethyl)ben-
zamide (8) as a beige powder (14.3 g, 82%).
General Procedure D, HCl Salt Formation. Compound 8(14.3
1
1
H), 6.68-6.63 (m, 2H), 6.35 (d, J = 6.93 Hz, 1H), 4.05-3.95 (m,
H), 2.88-2.81 (m, 3H), 2.70-2.58 (m, 3H), 2.45 (t, J = 7.46, 7.46
Hz, 2H), 1.91-1.83 (m, 1H), 1.67-1.55 (m, 1H), and 1.47
þ
g, 25.9 mmol) was dissolved in Et
to 0 °C. HCl (2.0 M in Et O, 39 mL, 78 mmol) was added dropwise over
0 min, and a precipitate appeared immediately. The mixture was warmed to
2
O (200 mL), and the solution was cooled
(
5
s, 6H). HRMS: m/z calcd for C H F N O ([M þ H]) ,
2
9
29
3
4
3
2
39.22645; found, 539.22700.
V600E and C-Raf Enzyme Assays. Raf enzyme activity was
1
room temperature and stirred for 15 min. The hygroscopic precipitate was
isolated by filtration, redissolved in EtOH and MeOH, concentrated in
determined using a Flash Plate Assay format by adding 15 μL of a
solution containing 50 mM HEPES, pH 7.5, 0.025% Brij 35, 10 mM
DTT, and 10 nM Raf (
plate containing compound and was incubated for 20 min. A substrate
solution (15 μL) containing 50 mM HEPES, pH 7.5, 0.025% Brij 35,
5
DRGFPRARYRARTTNYNSSRSRFYSGFNSRPRGRVYRGRARATS-
V600E
vacuo, and dried in a vacuum oven at 37 °C overnight. Compound 8 2HCl
3
ΔB-Raf or C-Raf) to the wells of an assay
1
was provided as an off white solid (14.2 g, 88%). H NMR (400 MHz, d
DMSO, HCl salt): δ 11.62-11.77 (m, 1H), 10.77 (s, 1H), 8.43 (br s, 2H),
.22 (d, 1H), 8.06 (s, 1H), 8.03 (s, 1H), 7.59 (s, 1H), 7.33 (s, 1H), 7.24 (d,
H), 7.04-6.95 (m, 2H), 6.88-6.82 (m, 1H), 4.05-4.11 (m, 2H), 2.99-
.75 (m, 5H), 2.17-1.72 (m, 3H), and 0.91-0.80 (m, 4H). LC-MS: m/z
6
-
8
1
2
5
2
mM β-glycerol phosphate, 10 mM MnCl , 2 μM peptide (Biotin-
WYSPY-NH , New England Peptide), 1 μM ATP, 0.1 mg/mL BSA, and
2
þ
-
3
3
25.6 [M þ H] , 523.3 [M - H] (FA). HRMS: m/z calcd for
P ATP 0.5 μCi/reaction was then added. The reaction mixture was
þ
C H F N O ([M þ H]) , 553.24210; found, 553.242651.
30
31
3
4
3
incubated for 3 h and then stopped by the addition of 50 μL of 100 mM
EDTA. The stopped reaction mixture (65 μL) was transferred to a Flash
Plate (Perkin-Elmer) and incubated for 1 (C-Raf) or 2 (
The wells were washed three times with 0.02% Tween-20. Plates were
read on a TopCount analyzer.
General Procedure A2, Biaryl Ether Bond Formation. A
mixture of (7R)-7-amino-5,6,7,8-tetrahydronaphthalen-2-ol hydrobro-
mide (17, 16.7 g, 68.2 mmol), 5-fluoro-3,4-dihydro-1,8-naphthyridin-
V600E
ΔB-Raf) h.
2
1
(1H)-one (11, 11.36 g, 64.95 mmol), and cesium carbonate (63.49 g,
94.9 mmol) in DMF (216 mL) was stirred at 140 °C for 2 h. The
pERK Cell-Based Assay. Inhibition of Raf kinase activity in whole
cells was assessed by determining the decrease in phosphorylation of the
pERK, a kinase downstream of Raf, phosphorylated by MEK. A375 cells
were seeded in a 96-well cell culture plate (12 ꢀ 103 cells/75 μL/well)
and incubated overnight at 37 °C. Compounds were added, and cells
were incubated with Raf kinase inhibitors for 3 h at 37 °C. Medium was
removed, and cells were fixed with 4% paraformaldehyde for 15 min at
room temperature. Four percent paraformaldehyde was replaced with
methanol for a 15 min treatment and then blocked with 10% sheep
serum and 1% BSA in PBS for 1 h. Cells were then incubated with anti-
pERK antibody (1:100, Cell Signaling Technologies, #9101 L) (20 μL/
well) overnight at 4 °C. After they were washed with PBS three times,
the cells were stained with antirabbit horseradish peroxidase-linked
antibody from donkey (1:100, Amersham Bioscience #NA9340) for
1 h at room temperature. Cells were washed three times with 0.5%
Tween-20 in PBS and then three more times with PBS. 3,3 ,5,5 -
Tetramethylbenzidine liquid substrate system (50 μL/well) was added,
and cells were incubated for 20-30 min at room temperature. The
optical density was read at 650 nm. The cells were then washed 3-5
times with PBS to remove color solution. Results were normalized for
the protein content in each well using a BCA protein assay kit (Pierce).
Xenograft Studies. The A375 M cell line (a derivative of A375 )
was licensed from MD Anderson Cancer Center. Nude NCR (ν/ν)
mice were purchased from Taconic. All animals were housed and
handled in accordance with the Guide for the Care and Use of
Laboratory Animals and Millennium Institutional Animal Care and
Use Committee guidelines. Six to eight week old mice were inoculated
reaction was not complete, so additional (7R)-7-amino-5,6,7,8-tetrahy-
dronaphthalen-2-ol hydrobromide (17, 1.70 g, 6.82 mmol) was added.
After an additional 1 h at 140 °C, the reaction mixture was cooled to
room temperature and carefully treated with an aqueous 1 M HCl
solution. The reaction mixture was then diluted with DCM and filtered
through Celite. The phases were separated, and the aqueous phase was
brought to pH 7 by the addition of a solution of NaOH (1.0 M). The
brown precipitate was then removed by filtration through Celite, and the
filtrate was washed with DCM. The aqueous phase was then brought to
pH 14 by addition of a solution of NaOH (1.0 M), and an off-white
precipitate formed. This suspension was treated with solid NaCl and
stirred for 1 h. The solid was isolated by filtration and dried under
vacuum to give 5-{[(7R)-7-amino-5,6,7,8-tetrahydronaphthalen-2-
yl]oxy}-3,4-dihydro-1,8-naphthyridin-2(1H)-one as an off-white solid
1
(
17.7 g, 88% yield). H NMR (400 MHz, d -DMSO): δ 10.6-10.4 (br s,
6
0
0
1
2
2
1
H), 7.94 (d, J = 5.8 Hz, 1H), 7.13 (d, J = 7.9 Hz, 1H), 6.87-6.81 (m,
H), 6.27 (d, J = 5.8 Hz, 1H), 3.05-2.96 (m, 1H), 2.93-2.79 (m, 4H),
.77-2.66 (m, 1H), 2.55-2.50 (m, 2H), 2.46-2.38 (m, 1H), 1.92-
.84 (m, 1H), 1.82-1.60 (br s, 2H), and 1.51-1.40 (m, 1H). LC-MS:
þ
m/z 310 [M þ H] (AA).
3-[(Dimethylamino)methyl]-N-{(2R)-7-[(7-oxo-5,6,7,8-tet-
rahydro-1,8-naphthyridin-4-yl)oxy]-1,2,3,4-tetrahydronap-
22
hthalen-2-yl}-5-(trifluoromethyl)benzamide (26). 3-[(Dime-
thylamino)methyl]-5-(trifluoromethyl)benzoic acid Li salt and 5-{[-
3
(
7R)-7-amino-5,6,7,8-tetrahydronaphthalen-2-yl]oxy}-3,4-dihydro-1,8-
naphthyridin-2(1H)-one were combined as described in General Pro-
cedures B3, Amide Bond Formation, and D, HCl Salt Formation, to
1
6
provide 26 (20.9 g, 67%). H NMR (400 MHz, CD
3
OD): δ 8.36 (s, 1
subcutaneously with A375 M cells (5 ꢀ 10 ) in the right flank. Mice
H), 8.30 (s, 1H), 8.10-8.06 (m, 2H), 7.33-7.26 (m, 1H), 7.04-6.96
m, 2H), 6.68 (d, J = 7.0 Hz, 1H), 4.51 (s, 2H), 4.40-4.31 (m, 1H),
were dosed orally with 0.2 mL of 26 or 27 or vehicle only as a control.
The vehicle was 10% (2-hydroxypropyl)-β-cyclodextrin (HPbCD) in
(
1
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dx.doi.org/10.1021/jm101479y |J. Med. Chem. 2011, 54, 1836–1846

Journal of Medicinal Chemistry
ARTICLE
water, and the pH was adjusted to between 4 and 6 by the addition of 0.1
N NaOH.
For efficacy studies, mice were randomized to generate groups of
(2) Roberts, P. J.; Der, C. J. Targeting the Raf-MEK-ERK mitogen-
activated protein kinase cascade for the treatment of cancer. Oncogene
2007, 26, 3291–3310.
(3) Davies, H.; Bignell, G. R.; Cox, C.; Stephens, P.; Edkins, S.;
1
2
0 per treatment with equivalent average initial tumor volume (150-
00 mm ) and dosed daily for 21 days. Tumor volumes were measured
3
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.; Busterson, 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. Mutations of
the BRAF Gene in Human Cancer. Nature 2002, 417, 949–954.
twice a week using vernier calipers, and volumes were calculated using
2
the formula L ꢀ W ꢀ 0.5.
For PK/PD studies, mice were euthanized, and blood was collected
by cardiac puncture and processed to plasma. Compound levels were
measured in plasma by LC/MS/MS. Xenograft tumors were excised and
trimmed of necrotic areas and flash-frozen for PD analysis. pERK levels
were measured in tumor lysates by Li-Cor quantitative Western blot
using a rabbit monoclonal antibody (pERK1/2-Thr202/Tyr204, Cell
Signaling Catalog #4377).
(4) Lee, J. H.; Lee, E.-S.; Kim, Y.-S. Clinicopathologic Significance of
BRAF V600E Mutations in Papillary Carcinomas of the Thyroid. Cancer
2007, 110, 38–46.
’
ASSOCIATED CONTENT
(
5) Gray-Schopfer, V.; Wellbrock, C.; Marais, R. Melanoma biology
and new targeted therapy. Nature 2007, 445, 851–857.
6) King, A. J.; Patrick, D. R.; Batorsky, R. S.; Ho, M. L.; Do, H. T.;
S
Supporting Information. Synthetic details and charac-
b
(
terization data for 7-(2-acetamidopyridin-4-yloxy)-1,2,3,4-tetra-
Zhang, S. Y.; Kumar, R.; Rusnak, D. W.; Takle, A. K.; Wilson, D. M.;
Hugger, E.; Wnag, L; Karreth, F.; Lougheed, J. C.; Lee, J.; Chau, D.;
Stout, T. J.; May, E. W.; Rominger, Ch. M.; Schaber, M. D.; Lou., L.;
Lakdawala, A. S.; Adams, J. L.; Contractor, R. G; Smalley, K. S. M.;
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tion of a genetic therapeutic index for tumors expressing oncogenic
BRAF by the kinase inhibitor SB-590885. Cancer Res. 2006,
66, 11100–11105. Tsai, J.; Lee, J. T.; Wang, W.; Zhang, J.; Cho, H.;
Mamo, S.; Bremer, R.; Gillette, S.; Kong, J.; Haass, N. K.; Sproesser, K.;
Li, L.; Smalley, K. S. M.; Fong, D.; Zhu, Y.-L.; Marimuthu, A.; Nguyen,
H.; Lam, B.; Liu, J.; Cheung, I.; Rice, J.; Suzuki, Y.; Luu, C.; Settachatgul,
C.; Shellooe, R.; Cantwell, J.; Kim, S.-H.; Schlessinger, J.; Zhang, K. Y. J.;
West, B. L.; Powell, B.; Habets, G.; Zhang, C.; Ibrahim, P. N.; Hirth, P.;
Artis, D. R.; Herlyn, M.; Bollag, G. Discovery of a selective inhibitor of
oncogenic B-Raf kinase with potent antimelanoma activity. Proc. Natl.
Acad. Sci. U.S.A. 2008, 105, 3041–3046. Yang, H.; Higgins, B.; Kolinsky,
K.; Packman, K.; Go, Z.; Iyer, R.; Kolis, S.; Zhao, S.; Lee, R.; Grippo, J. F.;
Schostack, K.; Simcox, M. E.; Heimbrook, D.; Bollag, G.; Su, F. RG7204
hydronaphthalene-2-carboxylic acid, N-(4-{[(7R)-7-amino-
5
,6,7,8-tetrahydronaphthalen-2-yl]oxy}pyridin-2-yl)cyclopropa-
necarboxamide, N-(4-nitropyridin-2-yl)-acetamide, N-(4-nitropyri-
din-2-yl)-cyclopropane-carboxamide, tert-butyl [3-amino-5-(trifluo-
romethyl)benzyl]isopropylcarbamate, 3-[(dimethylamino)methyl]-
5
-(trifluoromethyl)benzoic acid Li salt, 3-{1-[(tert-butoxycarbo-
3
nyl)amino]-1-methylethyl}-5-(trifluoromethyl)benzoic acid, and
compounds 6, (()-15, (S)-15, 17, and 19-25; and kinase binding
data for 26 and 27. This material is available free of charge via the
Internet at http://pubs.acs.org.
’
AUTHOR INFORMATION
Corresponding Author
*Tel: 617-761-6959. Fax: 617-551-8907. E-mail: sandy.
gould@mpi.com.
(
PLX4032), a Selective BRAFV600E Inhibitor, Displays Potent Anti-
tumor Activity in Preclinical Melanoma Models. Cancer Res. 2010,
0, 5518–5527. Joseph, E. W.; Pratilas, C. A.; Poulikakos, P. I.; Tadi,
’
ACKNOWLEDGMENT
7
We thank Ashok Patil and Nanda Gulavita for assistance in
M.; Wang, W.; Taylor, B. S.; Halilovic, E.; Persaud, Y.; Xing, F.; Viale, A.;
Tsai, J.; Chapman, P. B.; Bollag, G.; Solit, D. B.; Rosen, N. The RAF
inhibitor PLX4032 inhibits ERK signaling and tumor cell proliferation in
a V600E BRAF-selective manner. Proc. Natl Acad. Sci. U.S.A. 2010,
purification; Xiaofeng Yang for determining the exact masses;
David Lok and Nina Molchanova for determining the HPLC
purity of our compounds; Bi-Ching Sang, Garret Textor, Matt
Kroeger, and Gyorgy Snell from Takeda San Diego for their
assistance in determining the crystal structure of compound 24;
and Matt Jones, Ling Xu, David Lok, Susan Chen, and Shaoxia Yu
for determining plasma levels of the compounds described in this
paper. We also thank ALS, which is supported by the Director,
Office of Science, Office of Basic Energy Sciences, Materials
Sciences Division of the U.S. Department of Energy, under contract
DE-AC03-76SF00098 at Lawrence Berkeley National Laboratory.
107, 14903–14908.
(7) Solit, D. B.; Garraway, L. A.; Pratilas, C. A.; Sawai, A.; Getz, G.;
Basso, A.; Ye, Q.; Lobo, J. M.; She, Y.; Osman, I.; Golub, T. R.; Sebolt-
Leopold, J.; Sellers, W. R.; Rosen, N. BRAF mutation predicts sensitivity
to MEK inhibition. Nature 2006, 439, 358–362. Davies, B. R.; Logie, A.;
McKay, J. S.; Martin, P.; Stelle, S.; Jenkins, R.; Cockerill, M.; Cartlidge,
S.; Smith, P. D. AZD6244 (ARRY-142886), a potent inhibitor of
mitogen-activated protein kinase/extracellular signal-regulated kinase
kinase 1/2 kinases: mechanism of action in vivo, pharmacokinetic/
pharmacodynamic relationship, and potential for combination in pre-
clinical models. Mol. Cancer Ther. 2007, 6 (8), 2209–2219.
’
ABBREVIATIONS USED
(8) Bollag, G.; Hirth, P.; Tsai, J.; Zhang, J.; Ibrahim, P. N.; Cho, H.;
MAPK, mitogen-activated protein kinase; ERK, extracellular signal-
regulated kinase; MEK, mitogen-activated protein kinase; PK,
pharmacokinetic; Clp, plasma clearance; Vss, steady state volume
of distribution; AUC, area under the curve; HPbCD, (2-hydro-
xypropyl)-β-cyclodextrin; PK/PD, pharmacokinetic/pharmacody-
namic;RTK, receptor tyrosine kinase
Spevak, W.; Zhang, C.; Zhang, Y.; Habets, G.; Burton, E. A.; Wong, B.;
Tsang, G.; West, B. L.; Powell, B.; Shellooe, R.; Marimuthu, A.; Nguyen,
H.; Zhang, K. Y. J.; Artis, D. R.; Schlessinger, J.; Su, F.; Higgins, B.; Iyer,
R.; D'Andrea, K.; Koehler, A.; Stumm, M.; Lin, P. S.; Lee, R. J.; Grippo,
J.; Puzanov, I.; Kim, K. B.; Ribas, A.; McArthur, G. A.; Sosman, J. A.;
Chapman, P. B.; Flaherty, K. T.; Xu, X.; Nathanson, K. L.; Nolop, K.
Clinical efficacy of a RAF inhibitor needs broad target blockade in
BRAF-mutant melanoma. Nature 2010, 467, 596–599. Flaherty, K.;
Puzanov, I.; Sosman, J.; Kim, K.; Ribas, A.; McArthur, G.; Lee, R. J.;
Grippo, J. F.; Nolop, K.; Chapman, P. Phase I study of PLX4032: Proof
of concept for V600EBRAF mutation as a therapeutic target in human
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19) Additional kinase binding data for 26 and 27 can be found in
the Supporting Information, Table 1.
20) Crystallization work was done with wt B-Raf protein (residues
2
(
(
445-726). The crystallization conditions were as follows: 14.025% PEG
8000, 0.8 M NP Lithium Cl, 0.06 M Tris base, and 0.04 M Tris Cl. A
50 mM stock concentration of compound was made in 100% DMSO.
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concentration of 1 mM compound at room temperature. The precipitate
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clear omit electron density for the compound was observed. The data
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(
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1
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