AMINOPYRIDYLOXYPYRAZOLE COMPOUNDS
The present invention relates to novel aminopyridyloxypyrazole compounds that
inhibit activity of transforming growth factor beta receptor 1 (TGFpRl), pharmaceutical
compositions comprising the compounds, and methods of using the compounds to treat
cancer, preferably colon cancer, melanoma, hepatocellular carcinoma (HCC), renal
cancer, glioblastoma (GBM), pancreatic cancer, myelodysplastic syndrome (MDS), lung
cancer, and gastric cancer, and/or fibrosis, preferably liver fibrosis and chronic kidney
disease.
Transforming growth factor beta (TGF-beta or TGF ) is a multi-functional
cytokine which binds to the heteromeric complexes of TGF-beta type I and type II
serine/threonine kinase receptors and activates the TGF-beta receptor complex, which
phosphorylates and activates SMAD2 and SMAD3, which then associate with SMAD4
and migrate into the nucleus and regulate expression of different target genes. Key
players of TGF-beta receptor signal transduction pathway include TGF 1, TGFp2,
TGF 3, TGFpRl, TGFpR2, SMADs, SnoN, SARA, SKI, DAB, TRAP, TAK1, SMIF,
E2F4, E2F5, RBLl, RBL2, RBI, TFDPl, TFDP2, SMURFl, SMURF2, P300, CBP, and
UN. The SMAD mediated TGF-beta receptor pathway regulates various cellular and
physiological processes such as proliferation, differentiation, growth, migration,
myelination, cell cycle arrest, apoptosis and development.
Small molecule inhibitors of TGF Rl are already known in the art for the
treatment of cancer and/or fibrosis. See for example, WO2012/002680,
WO2009/022171, WO2004/048382, and WO2002/094833. Unfortunately, there is no
known curative treatments for many types of cancers or fibrosis. It would be desirable to
have additional small molecule inhibitors of TGF Rl for the treatment of cancer,
preferably colon cancer, melanoma, hepatocellular carcinoma (HCC), renal cancer,
glioblastoma (GBM), pancreatic cancer, myelodysplastic syndrome (MDS), lung cancer,
and gastric cancer, and/or fibrosis, preferably liver fibrosis and chronic kidney disease,
in particular compounds that are more selective for TGFpRl.
The present invention provides a compound of the formula:
wherein:
R1 is hydrogen, isopropyl, difluoromethyl, difluoroethyl, or cyclopropyl;
R2 is ethyl, tert-butyl, pyridin-2-yl, tetrahydropyran-4-yl, tetrahydrofuran-3-yl,
cyclopropyl, or cyclobutyl; and
R3 is carbamoylphenyl, pyridin-2-yl, ( 1-hydroxy- 1-methylethyl)pyridinyl, 1-
methyl-2-oxo-lH-pyridin-4-yl, 1-methylpyrazolyl, pyrazin-2-yl, 2-methoxypyrimidin-4-
yl, l-methyl-2-oxo-lH-pyrimidin-4-yl, pyridazin-3-yl, 6-chloropyridazin-3-yl, 6-
methylpyridazin-3-yl, or 6-methoxypyridazin-3-yl;
or a pharmaceutically acceptable salt thereof.
The present invention also provides 2-{4-[(4-{[l-cyclopropyl-3-(tetrahydro-2Hpyran-
4-yl)-lH-pyrazol-4-yl]oxy}pyridin-2-yl)amino]pyridin-2-yl}propan-2-ol or a
pharmaceutically acceptable salt thereof.
The present invention also provides 2-{4-[(4-{[l-cyclopropyl-3-(tetrahydro-2Hpyran-
4-yl)-lH-pyrazol-4-yl]oxy}pyridin-2-yl)amino]pyridin-2-yl}propan-2-ol -
4-methylbenzenesulfonate.
The present invention also provides crystalline 2-{4-[(4-{[l-cyclopropyl-3-
(te1rahydro-2H-pyran-4-yl)-lH-pyrazol-4-yl]oxy}pyridin-2-yl)amino]pyridin-2-
yl}propan-2-ol 4-methylbenzenesulfonate. The present invention further provides
crystalline 2-{4-[(4-{[l-cyclopropyl-3-(tetrahydro-2H-pyran-4-yl)-lH-pyrazol-4-
yl]oxy}pyridin-2-yl)amino]pyridin-2-yl}propan-2-ol 4-methylbenzenesulfonate
characterized by the X-ray powder diffraction pattern (Cu radiation, -1.54060 A)
comprising a peak at 17.8° with one or more peaks selected from the group consisting of
19.7°, 18.4°, and 22.0° (2±0.2°).
The present invention also provides a method of treating cancer, preferably colon
cancer, melanoma, hepatocellular carcinoma (HCC), renal cancer, glioblastoma (GBM),
pancreatic cancer, myelodysplastic syndrome (MDS), lung cancer, and gastric cancer, in a
patient in need of such treatment comprising administering the patient an effective
amount of a compound or salt of the present invention.
The present invention also provides a method of treating fibrosis, preferably liver
fibrosis and chronic kidney disease, in a patient in need of such treatment comprising
administering the patient an effective amount of a compound or salt of the present
invention.
The present invention also provides a pharmaceutical composition comprising a
compound or salt of the present invention, and one or more pharmaceutically acceptable
excipients, carriers, or diluents.
This invention also provides a compound or salt of the present invention for use in
therapy. Additionally, this invention provides a compound or salt of the present invention
for use in the treatment of cancer, preferably colon cancer, melanoma, hepatocellular
carcinoma (HCC), renal cancer, glioblastoma (GBM), pancreatic cancer, myelodysplastic
syndrome (MDS), lung cancer, and gastric cancer and/or fibrosis, preferably liver fibrosis
and chronic kidney disease. Furthermore, this invention provides the use of a compound
or a salt of the present invention in the manufacture of a medicament for treating cancer,
preferably colon cancer, melanoma, hepatocellular carcinoma (HCC), renal cancer,
glioblastoma (GBM), pancreatic cancer, myelodysplastic syndrome (MDS), lung cancer,
and gastric cancer and/or fibrosis, preferably liver fibrosis and chronic kidney disease
The following paragraphs describe preferred classes of the present invention:
a) R1 is difluoromethyl, difluoroethyl, or cyclopropyl;
b) R2 is pyridin-2-yl, tetrahydropyran-4-yl, or cyclopropyl;
c) R3 is carbamoylphenyl or ( 1-hydroxy-1-methylethyl)pyridinyl;
d) R1 is cyclopropyl and R2 is tetrahydropyran-4-yl;
e) R1 is cyclopropyl and R2 is cyclopropyl;
f) R1 is difluoroethyl and R2 is tetrahydropyran-4-yl;
g) R1 is difluoromethyl and R2 is pyrid-2-yl;
h) R1 is cyclopropyl, R2 is tetrahydropyran-4-yl, and R3 is (1-hydroxy-lmethylethyl)
pyridinyl;
i) R1 is cyclopropyl, R2 is cyclopropyl, and R3 is (1-hydroxy-lmethylethyl)
pyridinyl;
j ) R1 is difluoroethyl, R2 is tetrahydropyran-4-yl, and R3 is (1-hydroxy- 1-
methylethyl)pyridinyl; and
k) R1 is difluoromethyl, R2 is pyridin-2-yl, and R3 is carbamoylphenyl.
It will be understood by the skilled reader that free base forms of the compounds
of the present invention are capable of forming salts and such salts are contemplated to be
part of the present invention. The free base compounds of the present invention are
amines, and accordingly react with any of a number of inorganic and organic acids to
form pharmaceutically acceptable acid addition salts. Such pharmaceutically acceptable
acid addition salts and common methodology for preparing them are well known in the
art. See, e.g., P. Stahl, et al, HANDBOOK OF PHARMACEUTICAL SALTS:
PROPERTIES, SELECTION AND USE, (VCHA/Wiley-VCH, 2008); S.M. Berge, etal,
"Pharmaceutical Salts, " Journal of Pharmaceutical Sciences, Vol 66, No. 1, January
1977. It is understood by the skilled artisan that salt stoichiometry can be readily
determined. See for example, D. Risley, et al., Simultaneous Determination of
Positive and Negative Counterions Using a Hydrophilic Interaction Chromatography
Method, LCGC NORTHAMERICA, Vol 24, No. 8, August 2006 pages 776-785.
Certain of the compounds of the present invention are crystalline. It is well
known in the crystallography art that, for any given crystal form, the relative intensities of
the diffraction peaks may vary due to preferred orientation resulting from factors such as
crystal morphology and habit. Where the effects of preferred orientation are present,
peak intensities are altered, but the characteristic peak positions of the polymorph are
unchanged. See, e.g., The United States Pharmacopeia #23, National Formulary #18,
pages 1843-1844, 1995. Furthermore, it is also well known in the crystallography art that
for any given crystal form the angular peak positions may vary slightly. For example,
peak positions can shift due to a variation in the temperature or humidity at which a
sample is analyzed, sample displacement, or the presence or absence of an internal
standard. In the present e s s £ peak position variability of ± 0.2 in 2will take into
account these potential variations without hindering the unequivocal identification of the
indicated crystal form. Confirmation of a crystal form may be made based on any unique
combination of distinguishing peaks (in units of ° 2), typically the more prominent
peaks. The crystal form diffraction patterns, collected at ambient temperature and relative
humidity, are adjusted based on NIST 675 standard peaks at 8.853 and 26.774 degrees 2-
theta.
The compounds of the present invention can be prepared according to the
following synthetic schemes by methods well known and appreciated in the art. Suitable
reaction conditions for the steps of these schemes are well known in the art and
appropriate substitutions of solvents and co-reagents are within the skill of the art.
Likewise, it will be appreciated by those skilled in the art that synthetic intermediates
may be isolated and/or purified by various well known techniques as needed or desired,
and that frequently, it will be possible to use various intermediates directly in subsequent
synthetic steps with little or no purification. Furthermore, the skilled artisan will
appreciate that in some circumstances, the order in which moieties are introduced is not
critical. The particular order of steps required to produce the compounds of the present
invention is dependent upon the particular compound being synthesized, the starting
compound, and the relative liability of the substituted moieties, as is well appreciated by
the skilled chemist. All substituents, unless otherwise indicated, are as previously
defined, and all reagents are well known and appreciated in the art.
Some intermediates or compounds of the present invention may have one or more
chiral centers. The present invention contemplates all individual enantiomers or
diastereomers, as well as mixtures of the enantiomers and diastereomers of said
compounds including racemates. It is preferred that compounds of the present invention
containing at least one chiral center exist as single enantiomers or diastereomers. The
single enantiomers or diastereomers may be prepared beginning with chiral reagents or by
stereoselective or stereospecific synthetic techniques. Alternatively, the single
enantiomers or diastereomers may be isolated from mixtures by standard chiral
chromatographic or crystallization techniques. The skilled artisan will appreciate that in
some circumstances the elution order of enantiomers or diastereomers may be different
due to different chromatographic columns and mobile phases.
The designation of "isomer 1" in a compound name represents that the
corresponding intermediate or compound of the present invention is the first of two
eluting enantiomers when a mixture of a pair of enantiomers is separated by chiral
chromatography. The designation of "isomer 2" in a compound name represents that the
corresponding intermediate or compound of the present invention that is the second of
two eluting enantiomers when the mixture of a pair of enantiomers is separated by chiral
chromatography.
Compounds of the present invention may be synthesized as illustrated in the
following Schemes, where R1, R2, and R3 are as previously defined.
4 Formula I
Scheme 1: Synthesis of compounds of Formula I
Scheme 1 illustrates the general synthesis of compounds of Formula I. Compound
1 is reacted with 2-chloropyridin-4-ol in a suitable solvent such as dimethylformamide
(DMF) or acetone with a suitable base such as cesium carbonate or potassium carbonate
at room temperature or elevated temperature to afford Compound 2. Compound 2 is
reacted with l,l-dimethoxy-N,N-dimethyl-methanamine at elevated temperature to form
Compound 3. Compound 3 can be purified or used without further purification to react
with hydrazine in acetic acid to afford Compound 4. Compound 4 can react with a
suitable alkylation reagent such as potassium alkyltrifluoroborate or alkyboronic acid
under Chan-Lam coupling conditions to form Compound 5. More specifically, first heat
a suspension of 2,2'-bipyridine and copper(II)acetate in a suitable solvent such as 1,2-
dichloroethane to elevated temperature and purge with nitrogen, and then filter the
reaction mixture and add the filtrate to a mixture of Compound 4, a suitable boronate such
as potassium alkyltrifluoroborate or an alkylboronic acid, and a suitable base such as
sodium carbonate in a suitable solvent such as 1,2-dichloroethane. Heat the reaction
mixture to an elevated temperature to provide Compound 5. Compound 4 can also react
with a suitable alkyl halide such as alkyl iodide, alkyl bromide or alkyl chloride with a
suitable base such as sodium hydride in an appropriate solvent such as DMF or
tetrahydrofuran (THF) to afford Compound 5. Compound 5 is reacted with a suitable
amine under well-known Buchwald coupling conditions to provide a compound of
Formula I. More specifically, Compound 5 is reacted with a suitable amine at elevated
temperature in the presence of a suitable base such as cesium carbonate, a suitable ligand
reagent such as 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene, and a suitable catalyst
such as palladium(II)acetate in an appropriate solvent such as 1,4-dioxane to afford a
compound of Formula I.
6 Formula I
R is H
Scheme 2: Synthesis of compounds of Formula I when R is H
Scheme 2 illustrates the general synthesis of compounds of Formula I when R1 is
H. As illustrated in Step 4 of Scheme 1, when the alkylation reagent is 2-
(trimethylsilyl)ethoxymethyl chloride, Compound 6 can be obtained by alkylation
through Step 4 and Buchwald coupling reaction through Step 5. Compound 6 can react
with triethylsilane in trifuoroacetic acid to provide a compound of Formula I in which R1
is H. When R1 is H, it is known to skilled artisans that a compound of Formula I can exist
as a pair of tautomers in which the hydrogen can migrate between two nitrogens on the
pyrazolyl ring.
7 Formula I
R3 is (carbamoyl)phenyl
Scheme 3: Synthesis of compounds of Formula I when R3 is (carbamoyl)phenyl
Scheme 3 illustrates the general synthesis of compounds of Formula I when R3 is
a (carbamoyl)phenyl group. Compound 7 can be made by the method illustrated in Step 5
of Scheme 1 when R3 is a suitablely substituted benzonitrile. Compound 7 is reacted with
hydrogen peroxide and a suitable base such as potassium carbonate in dimethyl sulfoxide
(DMSO) to provide a compound of Formula I when R3 is a (carbamoyl)phenyl group.
As used herein, the following terms have the meanings indicated: "ACN" refers to
acetonitrile; "BSA" refers to bovine serum albumin; "DCM" refers to dichloromethane;
"DMF" represents N,N-dimethylformamide; "DMSO" refers to dimethyl sulfoxide;
"DTT" refers to dithiothreitol; "EDTA" refers to ethylenediaminetetraacetic acid;
"EGTA" refers to ethylene glycol tetraacetic acid; "ELISA" refers to enzyme-linked
immunosorbent assay; "EtOAc" refers to ethyl acetate; "EtOH" refers to ethanol; "FBS"
refers to fetal bovine serum ; "HEC" refers to hydroxyethylcellulose; "HPLC" refers to
high performance liquid chromatography; "" refers to in vivo target inhibition ;
"MS" refers to mass spectroscopy; "MeOH" refers to methanol; "NMR" refers to nuclear
magnetic resonance; "THF" refers to tetrahydrofuran; "TBS" refers to tris buffered saline;
"TED" refers to threshold effective dose; "UVW" refers to ultra-violet wavelength, and
"XRD" refers to X-ray diffraction.
Unless noted to the contrary, the compounds illustrated herein are named and
numbered using either ACDLABS or Accelrys Draw 4.1.
Preparation 1
2-(4-Bromo-2-pyridyl)propan-2-ol
Equip a three-liter, three-neck round bottom flask with an addition funnel, a reflux
condenser, a nitrogen inlet, and a temperature probe. Charge with methylmagnesium
bromide (3.2M in 2-methyltetrahydrofuran, 239.07 mL, 765.01 mmol) and cool in an ice
bath. To the addition funnel, add a solution of ethyl 4-bromopyridine-2-carboxylate (80.0
g, 347.73 mmol) in THF (800.0 mL). Add the solution dropwise to the methylmagnesium
bromide solution while keeping the internal temperature below 25°C. Remove the
cooling bath and allow stirring at 25°C for 30 minutes. Cool the reaction mixture to 5°C
and quench carefully with the dropwise addition of aqueous hydrochloric acid solution
(1M) while keeping the internal temperature below 30°C. Add additional aqueous
hydrochloric acid solution (1M) until the mixture reaches a pH of around 7. Remove the
cooling bath and dilute with ethyl acetate (EtOAc; 200 mL). Isolate the organic layer, dry
over anhydrous sodium sulfate, filter through a CELITE® plug and rinse with EtOAc.
Concentrate the filtrate to give an orange oil. Purify by using a silica gel plug eluting
with hexane/EtOAc (3/1) to give the title compound (63.15 g; 84.0% yield) as a colorless
oil. MS (m z): 216/218 (M+l/M+3).
Prepare the following compound essentially by the method of Preparation 1.
Table 1:
Preparation 3
2-(4-Amino-2-pyridyl)propan-2-ol
Charge a two-liter Parr reactor with a stirring bar, copper (powder mesh, 12.6 g,
198.6 mmol), 2-(4-bromo-2-pyridyl)propan-2-ol (63.1 g, 292.0 mmol) and ammonium
hydroxide (28 wt/wt% in water, 757.2 mL). Stir the reaction mixture under open air for
30 minutes until it is dark blue. Remove the stirring bar, attach a mechanical stirring top,
seal, and place on a stirrer. Heat the mixture to 100°C (inner, heating bath at 120°C) and
stir overnight. Cool the reaction mixture to room temperature and add 2-
methyltetrahydrofuran (600 mL). Filter through a CELITE® plug and rinse with 2-
methyltetrahydrofuran. Isolate the organic layer and extract the aqueous layer with 2-
methyltetrahydrofuran (200 mL). Combine the organic layers and dry over anhydrous
sodium sulfate. Filter, concentrate and dry under vacuum overnight to give the title
compound (31.3 g; 70.4% yield) as a yellow oil. MS (m/z): 153 (M+l).
Prepare the following compound essentially by the method of Preparation 3.
Table 2:
Preparation 5
2-Bromo- 1-tetrahydropyran-4-yl-ethanone
Method 1;
Add oxalyl chloride (28.69 mL, 330.73 mmol) dropwise to a mixture of
tetrahydropyran-4-carboxylic acid (39.13 g, 300.67 mmol) in DCM ( 250 mL) and DMF
(15 drops). Stir the mixture at room temperature for 2.5 hours under nitrogen.
Concentrate under reduced pressure and dissolve the residue in DCM (250 mL). Add the
resulting solution dropwise to (trimethylsilyl)diazomethane (2M in hexanes, 450 mL,
900.00 mmol) at -10°C and stir the mixture at room temperature overnight. Cool the
mixture to 0°C and add hydrobromic acid (48 wt wt% in water, 52 mL, 462.73 mmol)
dropwise. Stir the mixture at room temperature for two hours. Cool the mixture to 0°C
and add hydrobromic acid (48 wt/wt% in water, 26 mL, 231.36 mmol) dropwise. Stir the
mixture at room temperature for two hours. Add water (250 mL), DCM (250 mL) and
isolate the organic layer. Extract the aqueous layer with DCM (2 x 250 mL). Combine
the organic layers and wash with saturated aqueous sodium bicarbonate solution and
saturated aqueous sodium chloride. Dry over anhydrous sodium sulfate and concentrate
under reduced pressure to give the title compound (58.2 g; 93.48% yield) as a brown
solid. l NMR (300 MHz, CDC13) 4.00 (m, 2H), 3.95 (s, 2H), 3.45 (m, 2H), 2.98 (m,
1H), 1.78 (m, 4H).
Method 2;
Cool a solution of l-tetrahydropyran-4-ylethanone (10 g, 78.02 mmol) in
methanol (MeOH; 50 mL) to -10°C. Add bromine (4.01 mL, 78.02 mmol) dropwise. Stir
the mixture at 0°C for 45 minutes and then at 10°C for 45 minutes. Add an aqueous
solution of sulfuric acid ( 11M, 27.5 mL, 302.50 mmol) and stir the resulting mixture at
room temperature overnight. Add water and extract with diethyl ether three times.
Combine the organic layers. Wash with an aqueous solution of sodium bicarbonate and
water. Dry over anhydrous sodium sulfate and concentrate under reduced pressure to
give the title compound (12 g; 74.28% yield) as a white solid. 1H NMR (400.13 MHz,
CDCI3) 4.00 (m, 2H), 3.95 (s, 2H), 3.45 (m, 2H), 2.98 (m, 1H), 1.78 (m, 4H).
Preparation 6
2-[(2-Chloro-4-pyridyl)oxy] -1-tetrahydropyran-4-yl-ethanone
Method 1:
Add a solution of 2-bromo-l-tetrahydropyran-4-yl-ethanone (24.35 g, 117.60
mmol) in DMF (50 mL) dropwise to a stirring mixture of 2-chloropyridin-4-ol (13.85 g,
106.91 mmol) and cesium carbonate (69.67 g, 213.82 mmol) in DMF (380 mL) at room
temperature. Stir the resulting mixture at 90°C for 2.5 hours. Cool to room temperature
to give the crude mixture. Combine with a crude mixture of another 2.85 g (2-
chloropyridin-4-ol) scale reaction run as indicated above. Dilute the combined mixture
with water (200 mL) and EtOAc (300 mL). Isolate the organic layer and extract the
aqueous layer with EtOAc (3 x 250 mL). Combine the organic layers and wash with
water (100 mL) and saturate aqueous sodium chloride (100 mL). Dry over anhydrous
sodium sulfate, filter and concentrate the filtrate under reduced pressure to give the title
compound (29.32 g; 88.96% yield) as a brown oil. MS (m/z): 256 (M+l).
Method 2:
Add 2-bromo-l-tetrahydropyran-4-yl-ethanone (10.03 g, 48.42 mmol) and
potassium carbonate (10.14 g, 72.62 mmol) to a solution of 2-chloropyridin-4-ol (6.40 g,
48.42 mmol) in acetone (150 mL) and stir the resulting mixture at room temperature
overnight. Filter to remove the solid and wash the solid with DCM. Concentrate the
filtrate under reduced pressure to give the title compound quantitatively. MS (m/z): 256
(M+l).
Prepare the following compounds essentially by the Method 2 of Preparation 6.
Table 3:
Preparation 13
-Chloro-4-[(3 -tetrahydropyran-4-yl-1H-pyrazol-4-yl)oxy]pyridine
Stir a mixture of 2-[(2-chloro-4-pyridyl)oxy]-l-tetrahydropyran-4-yl-etlianone
(29.3 g, 114.59 mmol) and l,l-dimethoxy-N,N-dimethyl-methanamine (65 mL, 486.83
mmol) at 100°C for two hours. Cool to room temperature, concentrate under reduced
pressure and dissolve the residue in EtOAc (400 mL). Wash with water (100 mL) and
saturated aqueous sodium chloride (100 mL). Dry over anhydrous sodium sulfate and
concentrate under reduced pressure to give a brown solid. Dissolve in acetic acid (350
mL) and cool to 0°C. Add hydrazine monohydrate (16.8 mL, 345.66 mmol) and stir at
room temperature overnight under nitrogen. Pour the mixture into an ice/water mixture
(250 mL) and extract with EtOAc (4 x 200 mL). Combine the organic layers and wash
with water (200 mL), saturated aqueous sodium bicarbonate solution (100 mL) and
saturated aqueous sodium chloride (100 mL). Dry over anhydrous sodium sulfate, filter
and concentrate the filtrate under reduced pressure to give a brown oil. Purify the brown
oil by using a silica gel plug eluting with EtOAc. Combine the appropriate fractions and
concentrate under reduced pressure. Dry under vacuum to give the title compound (24.43
g; 76.22% yield) as a yellow solid. MS (m/z): 280 (M+1).
Prepare the following compounds essentially by the method of Preparation 13.
Table 4:
2-Chloro-4-[(3-
NH 15 tetrahydrofuran-3 -yl- 1H- 266 (M+l)
pyrazol-4-yl)oxy]pyridine
4-[(3-Tert-butyl-lH-
16 pyrazol-4-yl)oxy]-2- 252 (M+l)
chloro-pyridine
2-Chloro-4- [(3 -cyclobutyl- N H
17 lH-pyrazol-4- 250 (M+l)
yl)oxy]pyridine
2-Chloro-4-[(3- NH
18 cyclopropyl- 1H-pyrazol-4- 236 (M+l)
yl)oxy]pyridine
2-Chloro-4-[[3-(2-
19 pyridyl)- 1H-pyrazol-4- 273 (M+l)
yl]oxy]pyridine
Preparation 20
2-Chloro-4-(l-cyclopropyl-3-tetrahydropyran-4-yl-pyrazol-4-yl)oxy-pyridine
Method 1:
Reflux a mixture of 2,2'-bipyridine (13.73 g, 87.90 mmol) and copper(II)acetate
(15.97 g, 87.90 mmol) in 1,2-dichloroethane (244.3 mL) at 75°C for 25 minutes and then
cool to room temperature. Add a solution of 2-chloro-4- [(3 -tetany dropyran-4-yl-l Hpyrazol-
4-yl)oxy]pyridine (24.43 g, 79.91 mmol) in 1,2-dichloroethane (335.30 mL), then
add cyclopropylboronic acid (13.73 g, 159.82 mmol) and sodium carbonate (16.94 g,
159.82 mmol). Heat the reaction mixture at 75°C for two hours under an oxygen
atmosphere and cool to room temperature. Dilute with EtOAc (200 mL), filter through a
silica gel plug and rinse with EtOAc (250 mL). Wash the filtrate with water (200 mL)
and saturated aqueous sodium chloride (200 mL). Dry over anhydrous sodium sulfate,
filter and concentrate the filtrate under reduced pressure and dry the residue under
vacuum at room temperature overnight. Purify by silica gel column chromatography with
6-27% EtOAc in DCM to give the title compound (20.75 g; 81.2% yield) as a yellow
solid. MS (m/z): 320 (M+1).
Method 2:
Heat a suspension of 2,2'-bipyridine (28.8 g, 56.5 mmol) and copper(II)acetate
(8.2 g, 45.2 mmol) in 1,2-dichloroethane (50 mL) to 70°C and purge with nitrogen for 3
minutes. Filter and add the filtrate to a mixture of 2-chloro-4-[(3-tetrahydropyran-4-yllH-
pyrazol-4-yl)oxy]pyridine (8 g, 22.6 mmol), potassium cyclopropyl(trifluoro)borate
(6.7 g, 45.2 mmol) and sodium carbonate (4.8 g, 45.2 mmol) in 1,2-dichloroethane (50
mL). Heat the reaction mixture at 70°C for four days. Cool to room temperature. Filter
and rinse with DCM. Wash the filtrate with saturated aqueous ammonium chloride
solution and saturated aqueous sodium bicarbonate solution. Dry over anhydrous sodium
sulfate, filter and concentrate the filtrate under reduced pressure. Purify by silica gel
column chromatography with 1-10% MeOH in DCM to give the title compound (6.0 g;
82.2% yield). MS (m/z): 320 (M+1).
Prepare the following compounds essentially by Method 1 of Preparation 20.
Alteration in work up procedure is indicated.
Table 5:
Prepare the following compounds essentially by Method 2 of Preparation
Table 6:
Preparation 27
2-Chloro-4-(l -cyclopropyl-3-tetrahydrofuran-3-yl-pyrazol-4-yl)oxy-pyridine, isomer 1
Purify the racemic mixture of 2-chloro-4-(l-cyclopropyl-3-tetrahydrofuran-3-ylpyrazol-
4-yl)oxy-pyridine (Preparation 25) with chiral chromatography to afford the
first eluting enantiomer as the title compound. MS (m/z): 306 (M+l).
Purification condition: CHIRALPAK® IC; Mobile Phase: 20% ethanol (EtOH) in
carbon dioxide; Flow rate: 300 g/min; UVW: 240 nm; Retention time: 2.44 minutes.
Preparation 28
2-Chloro-4-(l -cyclopropyl-3-tetrahydrofuran-3-yl-pyrazol-4-yl)oxy-pyridine, isomer 2
Purify the racemic mixture of 2-chloro-4-(l-cyclopropyl-3-tetrahydrofuran-3-ylpyrazol-
4-yl)oxy-pyridine (Preparation 25) with chiral chromatography to afford the
second eluting enantiomer as the title compound. MS (m/z): 306 (M+l).
Purification condition: CHIRALPAK® IC; Mobile Phase: 20% EtOH in carbon
dioxide; Flow rate: 300 g/minute; UVW: 240 nm; Retention time: 2.93 minutes.
Preparation 29
2-Chloro-4-[l-(difluoromethyl)-3-(2-pyridyl)pyrazol-4-yl]oxy-pyridine
Cool a solution of 2-chloro-4-[[3-(2-pyridyl)-lH-pyrazol-4-yl]oxy]pyridine (2.0 g,
7.33 mmol) in DMF (73.34 mL) in an ice bath and add sodium hydride (60% in mineral
oil, 880.02 mg, 22.00 mmol) portionwise. Stir the mixture at 0°C for 10 minutes, allow it
to warm to room temperature and stir for 10 minutes. Add difluoroiodomethane (10 wt%
in THF, 27.19 mL, 36.67 mmol) and stir the reaction mixture at 45°C overnight. Cool to
room temperature and dilute with EtOAc. Wash with 5% aqueous lithium chloride
solution first and then wash with saturated aqueous sodium chloride. Dry over anhydrous
sodium sulfate, filter and concentrate the filtrate under reduced pressure. Purify the
residue by silica gel column chromatography with 0-50% EtOAc in DCM. Combine the
appropriate fractions and concentrate under reduced pressure. Purify the residue by silica
gel column chromatography with 0-10% EtOAc in DCM to give the title compound (1.56
g; 65.9% yield). MS (m/z): 323 (M+l).
Prepare the following compounds essentially by the method of Preparation 29.
Alterations in solvent, base, and/or reaction temperature are indicated.
Table 7:

2-Chloro-4-(3-
MS (m/z): 278 room
cyclopropyl-1-isopropyl- (M+l) temperature
pyrazol-4-yl)oxy-pyridine
2-Chloro-4-(l -isopropyl-
MS (m/z): 322 room
3-tetrahydropyran-4-yl-
(M+l) temperature
pyrazol-4-yl)oxy-pyridine
2-Chloro-4-(3 -cyclobutyl-
MS (m/z): 292 room
1-isopropyl-pyrazol-4-
(M+l) temperature
yl)oxy-pyridine
THF,
2-Chloro-4-[l-(2,2-
potassium
difluoroethyl)-3-(2- MS (m/z): 337
tert- pyridyl)pyrazol-4-yl]oxy- (M+l)
butoxide,
pyridine
50°C
2-Chloro-4-[3- Cesium
cyclopropyl- 1-(2,2- MS (m/z): 300
difluoroethyl)pyrazol-4- (M+l) carbonate,
50°C
yl]oxy-pyridine
2-Chloro-4-[l-(2,2-
Cesium
difluoroethyl)-3- MS (m/z): 344
tetrahydropyran-4-yl- (M+l) carbonate,
50°C
pyrazol-4-yl]oxy-pyridine
Preparation 41
-Chloro-4- {[3-(pyridin-2-yl)-1-{[2-(trimethylsilyl)ethoxy]methyl} -1H-pyrazol-4-
yl]oxy}pyridine
Add sodium hydride (60% suspension in mineral oil, 484 mg, 12.10 mmol) to a
solution of 2-chloro-4-[[3-(2-pyridyl)-lH-pyrazol-4-yl]oxy]pyridine (3.0 g, 11.00 mmol)
in THF (110 mL) at 0°C. Stir for 1 minutes at 0°C and add 2-
(trimethylsilyl)ethoxymethyl chloride (2.02 g, 12.10 mmol). Stir the reaction mixture at
room temperature overnight. Concentrate the mixture. Partition the residue between
DCM and water. Isolate the organic layer and dry over sodium sulfate. Filter the mixture
and concentrate the filtrate under reduced pressure. Purify the residue by silica gel
column chromatography with 0-30% EtOAc in hexane to give the title compound (3.64 g;
82.1% yield). MS (m/z): 403 (M+l).
Preparation 42
4-[[4-(1-Cyclopropyl-3 -tetrahydropyran-4-yl-pyrazol-4-yl)oxy-2-
pyridyl]amino]benzonitrile
Purge a solution of 2-chloro-4-(l -cyclopropyl-3-tetrahydropyran-4-yl-pyrazol-4-
yl)oxy-pyridine (400 mg, 1.2 mmol), p-aminobenzonitrile (219.9 mg, 1.9 mmol), cesium
carbonate (568.5 mg, 1.7 mmol), 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene
(134.6 mg, 0.23 mmol) in 1,4-dioxane (15 mL) with nitrogen for five minutes. Treat the
resulting mixture with palladium(II)acetate (26.1 mg, 0.12 mmol) and purge with nitrogen
for 5 minutes. Close the vial and stir at 100°C for two hours then 80°C over the weekend.
Cool to room temperature, filter through a CELITE® plug and wash with 5% MeOH in
DCM. Concentrate the filtrate to give the title compound (467 mg; 100% yield). MS
(m/z): 402 (M+l).
Prepare the following compounds essentially by the method of Preparation 42.
Alterations in catalyst, and/or solvent are indicated.
Table 8:
Example 1
-{4-[(4-{[l-Cyclopropyl-3-(tetrahydro-2H-pyran-4-yl)-lH-pyrazol-4-yl]oxy}pyridin-2-
yl)amino]pyridin-2-yl}propan-2-ol
Method 1:
Purge a solution of 2-chloro-4-(l-cyclopropyl-3-tetrahydropyran-4-yl-pyrazol-4-
yl)oxy-pyridine (45.6 g, 142.6 mmol), 2-(4-amino-2-pyridyl)propan-2-ol (26.0 g, 171.1
mmol) and sodium phenate (26.5 g, 228.2 mmol) in 1,4-dioxane (456 mL) with nitrogen
for 20 minutes. Treat the resulting mixture with 4,5-bis(diphenylphosphino)-9,9-
dimethylxanthene (8.25 g, 14.3 mmol) and bis(dibenzylideneacetone)palladium (4.10 g,
7.13 mmol). Reflux for 2 1 hours. Cool the reaction to room temperature and stir
overnight. Filter through a CELITE® plug and wash with DCM (500 mL). Concentrate
the filtrate onto silica gel. Purify by silica gel column chromatography with 0-10%
MeOH in EtOAc. Concentrate appropriate fractions and dry under vacuum overnight to
give the title compound (58.7 g; 91.7% yield). MS (m/z): 436 (M+1). Several batches of
the product are produced using the above method. Dissolve the combined batches of the
title compounds (92.4 g) in EtOH ( 1 L). Treat the solution with QUADRASIL® MP (100
g, 1.0-1.5 mmol/g) and agitate at 60°C for one hour. Cool to room temperature and filter
to remove the solids. Concentrate to remove the solvent. Dissolve the residue in EtOH
(500 mL) while heating at 100°C. Then cool the mixture slowly to room temperature and
add water (500 mL) slowly. Cool the mixture to 5°C while stirring. Collect the solid by
filtration and dry under vacuum at 45°C overnight to give the title compound (81.8 g).
MS (m/z): 436 (M+1).
Method 2;
Dissolve 2-chloro-4-( 1-cyclopropyl-3 -tetrahydropyran-4-yl-pyrazol-4-yl)oxypyridine
(400 mg, 1.2 mmol) in 1,4-dioxane (15 mL) in a vial. Add 2-(4-amino-2-
pyridyl)propan-2-ol (266.5 mg, 1.6 mmol), cesium carbonate (568.5 mg, 1.7 mmol), 4,5-
bis(diphenylphosphino)-9,9-dimethylxanthene (134.6 mg, 0.23 mmol) and purge with
nitrogen for 5 minutes. Add palladium(II)acetate (26.1 mg, 0.12 mmol) and purge with
nitrogen for 5 minutes. Seal the vial and stir at 100°C overnight. Cool the reaction to
room temperature, filter through a CELITE® plug and wash with 5% MeOH in DCM.
Concentrate and purify by reverse phase chromatography (Redisep Rf Gold High
Performance CI 8 Reverse Phase Column, 0-100% formic acid/acetonitrile (ACN) in
formic acid/water). Concentrate appropriate fractions and dry under vacuum to give the
title compound (341 mg; 67.3% yield). MS (m/z): 436 (M+1).
Prepare the following compounds essentially by the Method 2 of Example 1.
Alterations in base, catalyst, ligand, and/or solvent are indicated.
Table 9:

Example 62
4-[(4-{[l-Cyclopropyl-3-(tetrahydro-2H-pyran-4-yl)-lH-pyrazol-4-yl]oxy}pyridin-2-
yl)amino]benzamide
Add potassium carbonate (80.4 mg, 0.58 mmol) to a solution of 4-[[4-(lcyclopropyl-
3-te1rahydropyran-4-yl-pyrazol-4-yl)oxy-2-pyridyl]amino]benzonitrile (467
mg, 1.16 mmol) in DMSO (5 mL). Add 30% hydrogen peroxide (1.77 mL, 17.45 mmol)
and stir the reaction mixture at ambient temperature overnight. Dilute with water and
extract with DCM four times. Combine the organic layers and wash with saturated
aqueous sodium chloride. Dry over anhydrous sodium sulfate. Filter the mixture and
concentrate the filtrate under reduced pressure. Purify the residue by reverse phase
chromatography (Redisep Rf Gold High Performance C18 Reverse Phase Column, 0-
100% formic acid/ACN in formic acid/water) to give the title compound (220 mg; 45.9%
yield). MS (m z): 420 (M+1).
Prepare the following compounds essentially by the method of Example 62.
Table 10:
Example 70
2-{4-[(4-{[3-Cyclopropyl-l-(propan-2-yl)-lH-pyrazol-4-yl]oxy}pyridin-2-
yl)amino]pyridin-2-yl}propan-2-ol
Purge a solution of methyl 4-[[4-(3-cyclopropyl-l-isopropyl-pyrazol-4-yl)oxy-2-
pyridyl]amino]pyridine-2-carboxylate (298 mg, 0.76 mmol) in THF (6 mL) in a sealed
vial with nitrogen. Add methylmagnesium bromide (3M in diethyl ether, 1.01 mL, 3.03
mmol) dropwise and stir the mixture at room temperature for two hours. Concentrate the
mixture under reduced pressure and dilute the residue with DCM and saturated aqueous
sodium bicarbonate. Isolate the organic layer and extract the aqueous layer with DCM.
Combine the organic layers and wash with saturated aqueous sodium chloride. Dry over
sodium sulfate, filter and concentrate the filtrate. Purify by silica gel column
chromatography with 5-10% MeOH in DCM to give the title compound (160 mg; 53.69%
yield). MS (m/z): 394 (M+l).
Example 71
2- {5 -[(4- {[3-(Pyridin-2-yl)- 1H-pyrazol-4-yl]oxy }pyridin-2-yl)amino]pyridin-2-
l}pro an-2-ol
Cool a solution of 2-[5-[[4-[3-(2-Pyridyl)-l-(2-trimethylsilylethoxymethyl)
pyrazol-4-yl]oxy-2-pyridyl]amino]-2-pyridyl]propan-2-ol (500 mg, 0.96 mmol) in
trifluoroacetic acid (3 mL) to 0°C in an ice bath. Add triethylsilane ( 1 mL, 6.24 mmol).
Stir the reaction mixture at room temperature overnight. Concentrate and purify the
residue by reverse phase chromatography (Redisep Rf Gold High Performance CI8
Reverse Phase Column, 0-100% 10 mM ammonium bicarbonate in ACN). Concentrate
the appropriate fractions to remove ACN. Extract the remaining aqueous mixture with
DCM, isolate organic layer, and dry over sodium sulfate. Filter and concentrate the
filtrate under reduced pressure to give the title compound (168 mg; 44.9% yield). MS
(m/z): 389 (M+l).
Example 72
N-[4-( {1-(Difluoromethyl)-3-(tetrahydrofuran-3-yl)- lH-pyrazol-4-yl} oxy)pyridin-2-
yl] ridazin-3-amine, isomer 1
Purify the racemic mixture of N-[4-[l-(difluoromethyl)-3-tetrahydrofuran-3-ylpyrazol-
4-yl]oxy-2-pyridyl]pyridazin-3-amine (Preparation 49) with chiral
chromatography to afford the first eluting enantiomer as the title compound. MS (m/z):
375 (M+1).
Purification conditions: CHIRALPAK® IC; Mobile Phase: 30% isopropanol
containing 0.2% isopropyl amine in carbon dioxide; Flow rate: 70 g/minute; UVW: 280
nm; Retention time: 3.93 minutes.
Prepare the following compound essentially by the method of Example 72.
Alternate purification conditions are indicated.
Table 11:
X-Rav Powder Diffraction Collection Procedure for Examples 77-79
Obtain the XRD patterns of crystalline solids on a Bruker D4 Endeavor X-ray
powder diffractometer, equipped with a CuKa source (= 1.54060 A) and a Vantec
detector, operating at 35 kV and 50 mA. Scan the sample between 4 and 40° in 2, with a
step size of 0.009° in 2and a scan rate of 0.5 seconds/step, and with 0.6 mm divergence,
5.28 fixed anti-scatter, and 9.5 mm detector slits. Pack the dry powder on a quartz sample
holder and obtain a smooth surface using a glass slide. Collect the crystal form
diffraction patterns at ambient temperature and relative humidity.
Example 77
2-{4-[(4-{[l-Cyclopropyl-3-(tetrahydro-2H-pyran-4-yl)-lH-pyrazol-4-yl]oxy}pyridin-2-
yl)amino]pyridin-2-yl}propan-2-ol, (2Z)-but-2-enedioate (1:1)
Add 2- {4- [(4- {[1-cyclopropyl-3-(tetrahydro-2H-pyran-4-yl)- 1H-pyrazol-4-
yl]oxy}pyridin-2-yl)amino]pyridin-2-yl}propan-2-ol (142 mg) in ACN (2 mL). The solid
dissolves completely while stirring at 80°C/1000 rpm. Add maleic acid (48 mg, 1.20
equivalents, in 1mL of ACN at 80°C) to the resulting solution. The mixture is cloudy
initially but quickly becomes a clear solution. Stop heating and stirring. Cool the
solution to room temperature. Add another 2 mL of ACN to suspend the solid. Isolate
the white solid by vacuum filtration and dry the solid in place on the filter for 15 minutes
under an air stream. Dry the resulting solid in a 65°C vacuum oven overnight to afford
the title compound (132 mg, 73.4% yield). The theoretical percentage of maleic acid ion
in the formed salt for a mono salt is 2 1.0 %. Counterion analysis by HPLC determines
that the actual percentage of maleic acid ion in the formed salt is 17.2 %. The counterion
analysis indicates a mono salt.
X-Rav Powder Diffraction of Example 77
A prepared sample of Example 77 is characterized by an XRD pattern using
CuKa radiation as having diffraction peaks (2-theta values) as described in Table 13
below, and in particular having peaks at 9.6° in combination with one or more of the
peaks selected from the group consisting of 12.5°, 17.5°, and 16.9°; with a tolerance for
the diffraction angles of 0.2 degrees.
Table 12: X-ray powder diffraction peaks of Example 77
Example 78
2-{4-[(4-{[l-Cyclopropyl-3-(tetrahydro-2H-pyran-4-yl)-lH-pyrazol-4-yl]oxy}pyridin-2-
yl)amino]pyridin-2-yl}propan-2-ol, methanesulfonate (1:1)
Add 2- {4- [(4- {[1-cyclopropyl-3 -(tetrahydro-2H-pyran-4-yl)- 1H-pyrazol-4-
yl]oxy}pyridin-2-yl)amino]pyridin-2-yl}propan-2-ol ( 113 mg) in acetone (2 mL). The
solid dissolves completely while stirring at 60°C/1000 rpm. Add methanesulfonic acid
(21 x , 1.24 equivalents) to the resulting solution. Stop heating and stirring. Cool the
solution to room temperature. Add another 3 mL of acetone to suspend the solid. Isolate
the white solid by vacuum filtration and dry the solid in place on the filter for 15 minutes
under air stream. Dry the resulting solid in a 65°C vacuum oven overnight to afford the
title compound (87 mg, 63.08% yield). The theoretical percentage of methanesulfonic
acid ion in the formed salt for a mono salt is 18.1%. Counterion analysis by HPLC
determines that the actual percentage of methanesulfonic acid ion in the formed salt is
16.2 %. The counterion analysis indicates a mono salt.
X-Rav Powder Diffraction of Example 78
A prepared sample of Example 78 is characterized by an XRD pattern using
CuKa radiation as having diffraction peaks (2-theta values) as described in Table 14
below, and in particular having peaks at 7.0° in combination with one or more of the
peaks selected from the group consisting of 14. 1°, 10.8°, and 18.6°; with a tolerance for
the diffraction angles of 0.2 degrees.
Table 13: X-ray powder diffraction peaks of Example 78
8 7.9 49.1
9 4.5 48.1
10 17.8 47.6
Example 79
2-{4-[(4-{[l-Cyclopropyl-3-(tetrahydro-2H-pyran-4-yl)-lH-pyrazol-4-yl]oxy}pyridin-2-
yl)amino]pyridin-2-yl}propan-2-ol 4-methylbenzenesulfonate ( 1:1)
Add 2- {4- [(4- {[1-cyclopropyl-3 -(tetrahydro-2H-pyran-4-yl)- 1H-pyrazol-4-
yl]oxy}pyridin-2-yl)amino]pyridin-2-yl}propan-2-ol (122 mg) in EtOAc (2 mL).
Dissolve the solid completely while stirring at 80°C/1000 rpm. Add p-toluenesulfonic
acid monohydrate (1.23 equivalents, in 1mL of EtOAc at 80°C) to the resulting solution.
Slurry the mixture at 80°C/1000 rpm for 30 minutes. Turn off the heat and keep stirring
the mixture at 1000 rpm as it cools to room temperature. Isolate the resulting white solid
by vacuum filtration and dry the solid in place on the filter for 15 minutes under air
stream. Dry the resulting solid in a 65°C vacuum oven overnight to afford the title
compound (159 mg, 93.40% yield). The theoretical percentage of p-toluenesulfonic acid
ion in the formed salt for a mono salt is 29.3%. Counterion analysis by HPLC determines
that the actual percentage of p-toluenesulfonic acid ion in the formed salt is 28.3%. The
counterion analysis indicates a mono salt.
X-Rav Powder Diffraction of Example 79
A prepared sample of Example 79 is characterized by an XRD pattern using
CuKa radiation as having diffraction peaks (2-theta values) as described in Table 15
below, and in particular having peaks at 17.8° in combination with one or more of the
peaks selected from the group consisting of 19.7°, 18.4°, and 22.0°; with a tolerance for
the diffraction angles of 0.2 degrees.
Table 14: X-ray powder diffraction peaks of Example 79
Signaling via the TGF pathway has been associated with cancer and tumor
progression in several indications (Elliott et. al. (2005) J Clin Oncol 23:2078; Levy et. al.
(2006) Cytokine & Growth Factor Rev 17:41-58). There are several types of cancer
where TGFP ligands produced by the tumor or by the stroma in the tumor
microenvironment may participate in tumor progression. MATLyLu rat prostate cancer
cells (Steiner and Barrack ( 1992) Mol. Endocrinol 6 :15-25) and MCF-7 human breast
cancer cells (Arteaga, et al. (1993) Cell Growth and Differ. 4:193-201) became more
tumorigenic and metastatic after transfection with a vector expressing the mouse TGFpi.
TGF-has been associated with angiogenesis, metastasis and poor prognosis in human
prostate and advanced gastric cancer (Wikstrom, P., et al. (1998) Prostate 37: 19-29;
Saito, H. et al. (1999) Cancer 86: 1455-1462). In breast cancer, poor prognosis is
associated with elevated TGF-(Dickson, et al. (1987) Proc. Natl. Acad. Sci. USA
84:837-841; Kasid, et al. (1987) Cancer Res. 47:5733-5738; Daly, et al. (1990) J. Cell
Biochem. 43:199-211; Barrett-Lee, et al. (1990) Br. J Cancer 61:612-617; King, et al.
(1989) J. Steroid Biochem. 34:133-138; Welch, et al. (1990) Proc. Natl. Acad. Sci. USA
87:7678-7682; Walker, et al. (1992) Eur. J. Cancer 238:641-644) and induction of TGF-
ΐ by tamoxifen treatment (Butta, et al. (1992) Cancer Res. 52:4261- 4264) has been
associated with failure of tamoxifen treatment for breast cancer (Thompson, et al. (1991)
Br. J. Cancer 63:609-614). Anti TGFpi antibodies inhibit the growth of MDA-231
human breast cancer cells in athymic mice (Arteaga, et al. (1993) J. Clin. Invest. 92:2569-
2576), a treatment which is correlated with an increase in spleen natural killer cell
activity. CHO cells transfected with latent TGFpi also showed decreased NK activity
and increased tumor growth in nude mice (Wallick, et al. (1990) J. Exp. Med. 172:1777-
1784). Thus, TGF-secreted by breast tumors may cause an endocrine immune
suppression. High plasma concentrations of TGFpi have been shown to indicate poor
prognosis for advanced breast cancer patients (Anscher, et al. (1993) N. Engl. J. Med.
328:1592-1598). Patients with high circulating TGFP before high dose chemotherapy
and autologous bone marrow transplantation are at high risk for hepatic veno-occlusive
disease (15-50% of all patients with a mortality rate up to 50%) and idiopathic interstitial
pneumonitis (40-60% of all patients). The implication of these findings is 1) that elevated
plasma levels of TGFp can be used to identify at risk patients and 2) that reduction of
TGFP signaling could decrease the morbidity and mortality of these common treatments
for breast cancer patients.
Recent publications have also suggested that TGF signaling may be important in
driving resistance of tumors to standard of care therapies, including chemotherapies and
receptor tyrosine kinases (WO2012138783). Specifically, in colon cancer, a specific gene
expression signature has been shown to isolate a group of patients who are resistant to
common first line treatments. These tumor cells regain sensitivity to therapy when the
TGF pathway is blocked with a TGF RI specific small molecule inhibitor (Huang, et. al.
(2012) Cell 151:937-950; Sadanandam et. al. (2013) Nat Med 19:619-625; Vermeulen et.
al. (2013) Nat Ned 19:614-618; Roepman et. al. (2014) 134:552-562).
Myleodysplastic syndromes (MDS) are disorders of the hematopoietic system in
the myeloid compartment and are characterized by ineffective production of myeloid
cells. MDS is linked to alterations of the TGFp pathway represented by reduced SMAD7
levels. SMAD7 is an inhibitory SMAD which functions to inhibit TGFp mediated
SMAD signaling and is downstream of ligand activated signaling through TGFpRI and
TGFpRII. Overexpression of SMAD7 is thus thought to lead to over-activation of TGFp
signaling in MDS, and this phenotype can be reversed by treating with a TGFpRI small
molecule inhibitor (Zhou et. al. (201 1) Cancer Res. 7 1:955-963). Similarly, in
glioblastoma (GBM), TGFp ligand levels are elevated and related to disease progression.
An antisense oligonucleotide therapeutic, API 002, has been shown to be potentially
active in a subset of GBM patients (Bogdahn et. al. (201 1). Curr Pharm Biotechnol). In
melanoma, TGFp pathway signaling activation has also been linked to resistance to
BRAF and MEK inhibitors (Sun et. al. (2014) Nature. 508:1 18-122).
Many malignant cells secrete transforming growth factor-P (TGF-P), a potent
immunosuppressant, suggesting that TGF production may represent a significant tumor
escape mechanism from host immunosurveillance (Flavell et. al. (2010) Nat Rev
Immunol 10:554-567; Kast et. al. (1999) Leukemia 13:1188-1199). Establishment of a
leukocyte sub-population with disrupted TGFP signaling in the tumor-bearing host offers
a potential means for immunotherapy of cancer alone or in combination with one or more
other immunotherapies, for example in combination with one or more PD-1 inhibitor such
as nivolumab, pembrolizumab, PD-L1 inhibitors, cancer vaccines, and bispecific immune
engaging molecules such as IMCgplOO. TGFp ligand produced by lymphocytes has been
shown preclinically to antagonize tumor immune surveillance (Donkor et. al. (2012)
Development. Oncoimmunology 1:162-171, Donkor et. al. (2011) Cytokine Immunity
35:123-134); disrupting this axis preclinically has been shown to provide anti-tumor
benefit in murine models and in vitro (Zhong et. al. (2010) Cancer Res 16:1191-1 205;
Petrausch et. al. (2009) J Immunol 183:3682-3689); Wakefield et. al. (2013) Nat. Rev
Cancer 13:328-341). A transgenic animal model with disrupted TGFP signaling in T
cells is capable of eradicating a normally lethal TGFP over expressing lymphoma tumor,
EL4 (Gorelik and Flavell, (2001) Nature Medicine 7(10): 1118-1 122). Down regulation
of TGFP secretion in tumor cells results in restoration of immunogenicity in the host,
while T-cell insensitivity to TGFP results in accelerated differentiation and
autoimmunity, elements of which may be required in order to combat self-antigenexpressing
tumors in a tolerized host. The immunosuppressive effects of TGFp have also
been implicated in a subpopulation of HIV patients with lower than predicted immune
response based on their CD4/CD8 T cell counts (Garba, et al. J. Immunology (2002) 168:
2247-2254). A TGFP neutralizing antibody was capable of reversing the effect in culture,
indicating that TGFP signaling inhibitors may have utility in reversing the immune
suppression present in this subset of HIV patients.
During the earliest stages of carcinogenesis, TGFpl can act as a potent tumor
suppressor and may mediate the actions of some chemopreventive agents. However, at
some point during the development and progression of malignant neoplasms, tumor cells
appear to escape from TGFp-dependent growth inhibition in parallel with the appearance
of bioactive TGFP in the microenvironment. The dual tumor suppression/tumor
promotion roles of TGFP have been most clearly elucidated in a transgenic system over
expressing TGFP in keratinocytes. While the transgenics were more resistant to
formation of benign skin lesions, the rate of metastatic conversion in the transgenics was
dramatically increased (Cui, et al (1996) Cell 86(4):531-42). The production of TGFpi
by malignant cells in primary tumors appears to increase with advancing stages of tumor
progression. Studies in many of the major epithelial cancers suggest that the increased
production of TGFP by human cancers occurs as a relatively late event during tumor
progression. Further, this tumor-associated TGFP provides the tumor cells with a
selective advantage and promotes tumor progression. The effects of TGFp on cell/cell
and cell/stroma interactions result in a greater propensity for invasion and metastasis.
Tumor-associated TGFP may allow tumor cells to escape from immune surveillance since
it is a potent inhibitor of the clonal expansion of activated lymphocytes. TGFP has also
been shown to inhibit the production of angiostatin. Cancer therapeutic modalities such
as radiation therapy and chemotherapy induce the production of activated TGFp in the
tumor, thereby selecting outgrowth of malignant cells that are resistant to TGFP growth
inhibitory effects. Thus, these anticancer treatments increase the risk and hasten the
development of tumors with enhanced growth and invasiveness. In this situation, agents
targeting TGFP-mediated signal transduction might be a very effective therapeutic
strategy. The resistance of tumor cells to TGFp has been shown to negate much of the
cytotoxic effects of radiation therapy and chemotherapy and the treatment-dependent
activation of TGFP in the stroma may even be detrimental as it can make the
microenvironment more conducive to tumor progression and contributes to tissue damage
leading to fibrosis. The development of TGFP signal transduction inhibitors is likely to
benefit the treatment of progressed cancer alone and in combination with other therapies.
Additionally, it is known in the art that TGF signaling is involved in fibrotic
conditions such as liver fibrosis and chronic kidney disease. See for example, Ueha S, et.
al. 2012. Front Immunol. 3:71. Cellular and molecular mechanisms of chronic
inflammation-associated organ fibrosis; Bottinger et al. 2002. J Amer Soc Nephrol.
13:2600. TGF-Signaling in Renal Disease; Trachtman H., et al. 2011. Kidney
International 79:1236. A phase 1, single-dose study of fresolimumab, an anti-TGF-
antibody, in treatment-resistant primary focal segmental glomerulosclerosis; and
Rosenbloom J, et. al. 2010. Narrative review: fibrotic diseases: cellular and molecular
mechanisms and novel therapies. Ann Intern Med 152: 159-166.
The following assays demonstrate that the exemplified compounds inhibit
TGF Rl in a biochemical assay, at the cellular level, and in an animal model.
Biochemical Assay for TGFBRl Activity
The purpose of this in vitro assay is to identify compounds that inhibit TGFpRl.
Protein Expression and Purification
Insert the nucleotide sequence encoding amino acids 200-503 of human TGFpRl
(NM 004612.2) with amino acid Thr at position 204 changed to Asp into
PFASTBAC™1 (Invitrogen, Cat# 10360-014) vector with N-terminal HIS tag. Generate
baculovirus according to the protocol of the BAC-TO-BAC® Baculovirus Expression
System (Invitrogen, Cat# 10359-016). Infect Sf9 cells at 1.5 x 106 cells/mL using 15 mL
PI virus per liter of culture and incubate at 28°C for 48 hours. Harvest the cells and store
at -80°C for subsequent protein purification. Conduct protein purification at 4°C.
Suspend pellets from 2L culture in 100 mL buffer A (50 mM Tris-HCl, pH8, 200 mM
NaCl, 1mM DTT, 5mM imidazole, 10% glycerol) containing 0.2% Triton X-100 and
Roche complete EDTA-free protease inhibitor cocktail and homogenize. Clarify the cell
lysates by centrifugation in a Bechman JA-18 rotor for 45 minutes at 16,500 rpm.
Incubate the supernatant with 5 mL of Ni-NTA metal affinity resin (Qiagen) for three
hours. Pack the resin onto a column and wash with buffer A. Elute the HISTGFPR1(
200-503)(T204D) protein with 0-400 mM imidazole gradient in buffer A. Pool
and concentrate the HIS-TGFPR1(200-503)(T204D) containing fractions and load onto a
HiLoad 16.600 Superdex 200 column (GE Healthcare Bioscience). Elute the column
with storage buffer (50 mM Tris-HCl, pH7.5, 150 mM NaCl, ImM DTT). Pool and
concentrate the HIS-TGFPR1(200-503)(T204D) containing fractions. Determine the
protein concentration by UV280. Aliquot the protein and store at -80 °C.
TR-FRET Assay Conditions
Pre-incubate compounds with recombinant His-TGFpRl(200-503)(T204D) , and
Eu-anti-HIS detection antibodies (InVitrogen, Cat# PV5597) in half-area black plates.
Prepare compound serial dilutions from ImM stock test compounds in DMSO. Serially
dilute the stock solution 3-fold in DMSO to obtain a ten-point dilution curve with final
compound concentrations ranging from 2 to 0.1 nM. The final DMSO concentration
in the assay is 4%. Initiate the reaction with the addition of kinase tracer (Kinase Tracer
178, Life Technologies PR9080A, InVitrogen). After 45-60 minutes, read the
fluorescence on a plate reader.
Calculate percent inhibition of compound treated groups relative to the minimum
inhibition group (DMSO alone, untreated). Calculate absolute IC50 using a 4-parameter
nonlinear logistic equation where absolute IC50 = concentration causing 50% inhibition
using ActivityBase data analysis software. The results of these assays demonstrate that
the exemplified compounds are effective inhibitors of TGFpRl. For example, all
exemplified compounds demonstrate IC50 values less than 1 . Specifically, the IC50
for Example 1 is 0.027 .
Cell-Based Luciferase Reporter Assay for TGFBRl Activity
The purpose of this assay is to identify compounds that selectively interfere with
SMAD 2,3-dependent gene expression in cell-based assays demonstrating that they
inhibit TGFpRl at the cellular level.
Engineer HEK293 cells (ATCC, CRL-1573) to express firefly luciferase from a
SMAD 2,3- responsive promoter in response to TGF stimulation. Such a cell line may
be generated via infection with lentiviral particles (SA Biosciences) and selection for
puromycin resistance. Plate the HEK293 SMAD 2/3 cells from assay-ready frozen
stocks at 15,000 cells per well in 96-well plates in OPTI-MEM® medium containing 10%
fetal bovine serum. After 72 hours, change the medium to OPTI-MEM® containing
0.1% bovine serum albumin. Prepare test compounds in DMSO to make 10 mM stock
solutions. Serially dilute the stock solutions 3-fold in DMSO to obtain a ten-point
dilution curve with final compound concentrations ranging from 20 to 1 nM with the
final DMSO concentration in the assay is 0.5%. Add the test compounds and after a one
hour equilibration, add TGF (final concentration = 2 nM, R&D Systems).
After 24 hours, add lysis buffer [Glo Lysis Buffer (Cat #E2661)] and luciferase
reagent [Promega Bright Glo Luciferase Reagent (Cat #E2620)] to each well to double
the well volume. Transfer aliquots (80 ) to white solid bottom plates for reading
luminescence on a plate reader (Emission filter: Luminescence 700, 1 second read).
Calculate percent inhibition of compound treated groups relative to the minimum
inhibition group (DMSO alone, untreated). Calculate the relative IC50 for each compound
from a dose response study and is the concentration necessary to achieve 50% inhibition.
Fit the data generated from the dose-response studies to a four-parameter logistic equation
using ActivityBase data analysis software. The results of these assays demonstrate that
the exemplified compounds are effective inhibitors of luciferase reporter activity from
TGFp-stimulated HEK293 SMAD2/3 cells. For example, all exemplified compounds
demonstrate IC50 values less than 1 . Specifically, the IC50 for Example 1 is 0.0824
(±0.005, n=2).
IVTI Assay
The purpose of this assay is to measure the ability of a test compound to inhibit
the pSMAD2 expression in tumors in an EMT6-LM2 syngeneic animal model, in other
words, the assay measures the ability of a test compound to inhibit TGF Rl signaling in a
solid tumor animal model.
EMT6-LM2 Cell Generation
Implant EMT-6 cells (ATCC, CRL-2755) subcutaneously (5 x 10 /animal) to the
flank of immune competent BALB/cAnNHsd mice (Harlan Laboritories). When tumors
reach approximately 3000 mm3, sacrifice the animals by C0 2 asphyxiation. Isolate the
lungs from tumor bearing animals and place in culture. Gently homogenize the lungs to
create a single cell suspension. Grow cells in culture media (IMDM, 10% FBS) and
isolate the tumor cells to give EMT6-LM1 cells. Repeat the above process by using
EMT6-LM1 cells for implantation to generate EMT-LM2 cells.
Purified phospho HIS-SMAD2 >SMAD2
Insert the nucleotide sequence encoding full-length human SMAD2
(NM 005901.5) into PFASTBACHTA™ (Invitrogen, Cat # 10584-027) -vector, resulting
in the baculovirus construct for expressing HIS-SMAD2 protein. Insert the nucleotide
sequence encoding amino acids 148-503 of human TGFpRl (NM 004612.2) with amino
acid Thr at position 204 changed to Asp into PFASTBACHTA™ (Invitrogen, Cat #
10584-027) vector, resulting in the baculovirus construct for expressing HISTGFPR1(
148-503)(T204D) protein. Generate baculovirus according to the protocol of
the BAC-TO-BAC® Baculovirus Expression System (Invitrogen). Infect Sf9 cells at 1.5
x 106 cells/mL using 10 mL PI virus of HIS-SMAD2 and PI virus of HIS-TGFpRl(148-
503)(T204D) per liter of culture and incubate at 28°C for 45 hours. Add okadaic acid to a
final concentration of 0.1 . After an additional three hours of incubation, harvest the
cells and store at -80°C for subsequent protein purification. Conduct protein purification
at 4°C. Lys frozen cell pellets from 6 L culture by incubation with stirring in 300 mL of
cold buffer A (50 mM sodium phosphate, pH7.5, 300 mM NaCl, 2mM -
mercaptoethanol, 5 mM imidazol, 10% glycerol, 0.1 okadaic acid) containing 0.1%
TRITON® X-100 and Roche complete EDTA-free protease inhibitor cocktail and
homogenization. Clarify cell lysates by centrifugation in a Bechman JA-18 rotor for 45
minutes at 16,500 rpm. Incubate the supernatant with 10 mL of TALON metal affinity
resin (Clontech, Cat# 635504) for two hours. Wash the batch with 100 mL of buffer A
containing 0.1% TRITON® X-100. Pack the resin onto a column and wash with buffer
A. Elute the HIS-SMAD2 protein with a 0-100 mM imidazole gradient in buffer A. Pool
the fractions containing phospho HIS-SMAD2 and supplement with 0.1 okadiac acid
and 5 mM EDTA. Determine the protein concentration by the BioRad protein assay
(BioRad DC Protein Assay kit #500-01 16) using BSA as standard. Aliquot the protein
and store at -80°C.
Live Phase
Culture EMT6-LM2 cells in Iscoves Modified Dulbecco's Media (MDM)
supplemented with 10% FBS, 2 mM Glutamax and 0.1 mM non-essential amino acids
and incubate at 37°C in 5% C0 2. Trypsinize and isolate the cells from culture.
Resuspend the cells in Hank's balanced salt solution (HBSS), then mix with
MATRIGEL® (1:1). Implant the cells (5 x 10 /animal) subcutaneously into the rear flank
of the mice (female BALB/c mice, Harlan). Measure the tumor volume with a caliper
and the body weight twice a week. After tumor volume reaches approximately 200-250
mm3, randomize animals and group into vehicle control and compound treatment groups.
Administer the compound (formulated in 1% hydroxyethylcellulose (HEC) and 0.25%
TWEEN® 80 and 0.05% Antifoam) and vehicle control (1% HEC and 0.25% TWEEN®
80 and 0.05% Antifoam) by oral gavage. Generate dose response by testing compounds
at a single time point (2 hours) following a single dose of: 2.7, 8.3, 25, 75, or 150 mg/kg.
Perform a time course at the calculated (method detailed below) TED50 or TED 0 dose
from a dose response study by sacrificing the mice at multiple time points between 1 hour
and 16 hours after a single dose.
Tissue Processing
Harvest tumor tissues and homogenize as described below. Freeze tumor tissues
(-100 mg each) in liquid nitrogen and pulverize with a pestle. Place pulverized tissue
into a tube (Lysing Matrix A tube, MPBio # 6910-100) on dry ice and homogenize in a
lysis buffer (0.6 mL each) (150 mM NaCl; 20 mM Tris, pH 7.5; 1mM
ethylenediaminetetraacetic acid (EDTA); 1mM ethylene glycol tetraacetic acid (EGTA);
1% TRITON® X-100; Protease Inhibitor cocktail (Sigma P8340); Phosphatase Inhibitor
Cocktail II (Sigma P5726); Phosphatase Inhibitor Cocktail III (Sigma P0044)) for 25
seconds using a BiolOl FASTPREP® FP120 homogenizer (setting 4.5). Pellet cellular
debris and beads by centrifugation at 14,000 x g for 10 minutes at 4°C. Transfer the
lysate to a new microfuge tube and centrifuge again, at 14,000 x g for 10 minutes at 4°C.
Transfer centrifuged lysate to a deep-well 96-well plate and keep on ice. Determine the
protein concentration for each lysate using a BioRad protein assay (BioRad DC Protein
Assay kit #500-01 16) as follows. Prepare the working reagent by adding kit reagent S
(20 ) to every lmL of kit reagent A needed for the assay. Prepare 3-5 dilutions of a
protein standard from 0.2 mg/mL to 1.5 mg/mL protein and generate a standard curve.
Pipet 5 ΐ of standards and samples into a clean, dry microtiter plate. Add 25 ΐ of
working reagent to each well. Add 200 ΐ of reagent B into each well and agitate for 5
seconds. After 15 minutes, read the absorbance of each well at 750 nM. Protein levels
for each well are determined by comparing the absorbance of the sample wells to the
standard curve derived from the standard wells. Normalize the tumor lysates to 10
mg/mL with lysis buffer in preparation for analysis of pSMAD2 and total SMAD2/3 by
ELISA as method described below.
SMAD ELISA
Tumor lysates are assayed using independent ELISA plates, where one plate is
used to determine the total SMAD 2/3 levels and the other plate is used to determine the
phospho SMAD 2 levels. While the coating antibody is the same for both plates, the
secondary antibody is specific for total SMAD 2/3 or phospho SMAD 2. These plates are
referred to collectively as "ELISA plates" and separately as "Total ELISA plate" or
"phospho ELISA plate", respectively. Prepare the coating antibody at 2.5 g/mL in BupH
Carbonate-Bicarbonate buffer (anti-SMAD 2/3 monoclonal antibody, BD Biosciences
#610843; BupH Carbonate-Bicarbonate from Pierce #28382) and add at 100 ΐ per well
to 96-well immunoplates (Thermo Scientific #439454) and incubate overnight at 4°C on a
platform shaker to generate the ELISA plates. Next, wash the ELISA plates four times
with wash buffer (0.5% TWEEN® 20 in tris buffered saline (TBS), pH 8.0 from Sigma
#T-9039) and subsequently block with 200 ΐ per well of blocking buffer (1% bovine
serum albumin (BSA) in l x TBS) at room temperature on a platform shaker for two
hours. Wash four times with wash buffer. To the phospho SMAD ELISA plate, add 100
ΐ per well of tumor lysate or vehicle lysate at 10 mg/ml to the appropriate wells. To the
Total ELISA plate, add 98 ΐ per well of lysis buffer and 2 ul per well of 10 mg/ml
tumor lysate or vehicle lysate to the appropriate wells (0.02 mg protein lysate final). A
standard curve is also added to each ELISA plate (phospho and total both) using purified
pSMAD2. Incubate overnight. Wash the ELISA plates again four times with wash
buffer. Prepare secondary antibodies (Millipore anti-phospho SMAD2 rabbit monoclonal
antibody #04-953; Millipore anti-SMAD2/3 rabbit polyclonal antibody #07-408) at 1:500
dilution in lysis buffer supplemented with 1% BSA and add 100 per well to the
appropriate plate. Incubate the plates at room temperature for two to three hours. Wash
four times with wash buffer and add 100 per well of reporter antibody (anti-rabbit
HRP, GE Healthcare #NAV934V, diluted 1:10,000 in blocking buffer) to the plates.
Incubate for one hour at room temperature and wash the plates a final four times with
wash buffer and add 100 per well of room temperature 3, 3', 5, 5'-
tetramethylbenzidine (TMB; Surmodics/BioFX #TMBW-0100-01). Incubate the plates at
37°C for up to thirty minutes. Stop the reaction with the addition of 100 of Stop
solution (IN H2S0 4). Read the absorbance (OD) at 450 nm on a plate reader.
Use the ratio of total SMAD (tSMAD) to phospho SMAD (pSMAD) for the
vehicle group to determine the minimum inhibition (0%) of pSMAD signal. Calculate the
percent inhibition for compound treated groups relative to the minimum pSMAD
inhibition of the vehicle group. Calculate TED50 and TED 0 from a dose response study
(dose necessary to achieve 50% and 80% inhibition at this time point, respectively) by
using NLIN procedure in SAS (Version 9.3, Cary, NC). This assay demonstrates that
Example 1 has a TED50 value of 10.8 mg/kg 2 hours after 1 dose and a TED 0 of 24.1
mg/kg. In the time course study at the TED50 dose ( 11 pmk), Example 1 demonstrates
48% inhibition at one hour and 39% inhibition at two hours after dosing. In the time
course study at (25 mpk), Example 1 demonstrates 1% inhibition at one hour and 70%
inhibition at two hours after dosing.
The compounds of the present invention are generally effective over a wide
dosage range. For example, dosages per day normally fall within the daily range of about
1-2000 mg. Preferably such doses fall within the daily range of 10-1000 mg. More
preferably such doses fall within the daily range of 10-100 mg. Even more preferably
such doses fall within the daily range of 10-80 mg. Most preferably such doses fall
within the daily range of 10-50 mg. In some instances dosage levels below the lower
limit of the aforesaid ranges may be more than adequate, while in other cases still larger
doses may be employed, and therefore the above dosage ranges are not intended to limit
the scope of the invention in any way. It will be understood that the amount of the
compound actually administered will be determined by a physician, in the light of the
relevant circumstances, including the condition to be treated, the chosen route of
administration, the actual compound or compounds administered, the age, weight, and
response of the individual patient, and the severity of the patient's symptoms.
We Claim:
1. A compound of the formula:
wherein:
R1 is hydrogen, isopropyl, difluoromethyl, difluoroethyl, or cyclopropyl;
R2 is ethyl, tert-butyl, pyridin-2-yl, tetrahydropyran-4-yl, tetrahydrofuran-3-yl,
cyclopropyl, or cyclobutyl; and
R3 is carbamoylphenyl, pyridine-2-yl, ( 1-hydroxy- l-methylethyl)pyridinyl, 1-
methyl-2-oxo-lH-pyridin-4-yl, 1-methylpyrazolyl, pyrazin-2-yl, 2-methoxypyrimidin-4-
yl, l-methyl-2-oxo-lH-pyrimidin-4-yl, pyridazin-3-yl, 6-chloropyridazin-3-yl, 6-
methylpyridazin-3-yl, or 6-methoxypyridazin-3-yl;
or a pharmaceutically acceptable salt thereof.
2. The compound according to Claim 1 which is 2-{4-[(4-{[l-cyclopropyl-3-
(tetrahydro-2H-pyran-4-yl)-lH-pyrazol-4-yl]oxy}pyridin-2-yl)amino]pyridin-2-
yl}propan-2-ol or a pharmaceutically acceptable salt thereof.
3. The compound or salt according to Claim 1 or 2 which is 2-{4-[(4-{[lcyclopropyl-
3-(tetrahydro-2H-pyran-4-yl)-lH-pyrazol-4-yl]oxy}pyridin-2-
yl)amino]pyridin-2-yl}propan-2-ol 4-methylbenzenesulfonate.
4. The compound or salt according to any one of Claims 1-3 which is
crystalline 2-{4-[(4-{[l-cyclopropyl-3-(tetrahydro-2H-pyran-4-yl)-lH-pyrazol-4-
yl]oxy}pyridin-2-yl)amino]pyridin-2-yl}propan-2-ol 4-methylbenzenesulfonate.
5. The compound or salt according to any one of Claims 1-4 which is
crystalline 2-{4-[(4-{[l-cyclopropyl-3-(tetrahydro-2H-pyran-4-yl)-lH-pyrazol-4-
yl]oxy}pyridin-2-yl)amino]pyridin-2-yl}propan-2-ol 4-methylbenzenesulfonate
comprising at least one peak at 17.8° in combination with one or more of the peaks
selected from the group consisting of 19.7°, 18.4°, and 22.0° (2± 0.2°).
6. A pharmaceutical composition comprising a compound or salt according
to any one of Claims 1-5 and one or more pharmaceutically acceptable excipients,
carriers, or diluents.
7. A method of treating cancer in a patient in need of such treatment
comprising administering the patient an effective amount 2-{4-[(4-{[l-cyclopropyl-3-
(te1rahydro-2H-pyran-4-yl)-lH-pyrazol-4-yl]oxy}pyridin-2-yl)amino]pyridin-2-
yl}propan-2-ol or a pharmaceutically acceptable salt thereof.
8. The method according to Claim 7 wherein the salt is a 4-
methylbenzenesulfonate.
9. The method according to Claim 7 or 8 wherein the cancer is selected from
the group consisting of colon cancer, melanoma, hepatocellular carcinoma, renal cancer,
glioblastoma, pancreatic cancer, myelodysplasia syndrome, lung cancer, and gastric
cancer.
10. A method of treating fibrosis in a patient in need of such treatment
comprising administering the patient an effective amount of 2- {4- [(4- {[1-cyclopropyl-3 -
(te1rahydro-2H-pyran-4-yl)-lH-pyrazol-4-yl]oxy}pyridin-2-yl)amino]pyridin-2-
yl}propan-2-ol or a pharmaceutically acceptable salt thereof.
11. The method according to Claim 10 wherein the salt is a 4-
methylbenzenesulfonate.
12. The method according to Claim 10 or 11 wherein the fibrosis is selected
from the group consisting of liver fibrosis and chronic kidney disease.
13. A compound or salt according to any one of Claims 1-5 for use in therapy.
14. A compound or salt according to any one of Claims 1-5 for use in the
treatment of cancer.
15. A compound or salt for use according to Claim 14 wherein the cancer is
selected from the group consisting of colon cancer, melanoma, hepatocellular carcinoma,
renal cancer, glioblastoma, pancreatic cancer, myelodysplastic syndrome, lung cancer,
and gastric cancer.
16. A compound or salt according to any one of Claims 1-5 for use in the
treatment of fibrosis.
17. A compound or salt for use according to Claim 16 wherein the fibrosis is
selected from the group consisting of liver fibrosis and chronic kidney disease.