CRYSTALLINE (R)-(E)-2-(4-(2-(5-(l-(3,5-DICHLOROPYRIDTN-4-YL)ETHOXY)-
1H-TNDAZOL-3 -YL)VINYL)- 1H-PYRAZOL- 1-YL)ETHANOL
Fibroblast growth factor (FGF) has been recognized as an important mediator
of many physiological processes, such as morphogenesis during development and
angiogenesis. The fibroblast growth factor receptor (FGFR) family consists of four
members (FGFR1-FGFR4), which are glycoproteins composed of extracellular
immunoglobulin (Ig)-like domains, a hydrophobic transmembrane region and a
cytoplasmic part containing a tyrosine kinase domain. FGF binding leads to FGFR
dimerization, followed by receptor autophosphorylation and activation of downstream
signaling pathways. Receptor activation is sufficient for the recruitment and
activation of specific downstream signaling partners that participate in the regulation
of diverse processes such as cell growth, cell metabolism and cell survival. Thus, the
FGF/FGFR signaling pathway has pleiotropic effects on many biological processes
critical to tumor cell proliferation, migration, invasion, and angiogenesis.
Vinyl indazoles are known in the art for the treatment of cancer. See for
example, WO200210137 and WO2003 101968. FGFR inhibitors are also known in
the art. See for example, WO2002022598.
PCT/US2010/033487 discloses an amorphous form of (R)-(E)-2-(4-(2-(5-(l-
(3,5-dichloropyridin-4-yl)ethoxy)- 1H-indazol-3 -yl)vinyl)- 1H-pyrazol- 1-yl)ethanol
that is poorly crystalline and is useful as an inhibitor of FGFR.
The present invention provides a crystalline ((R)-(E)-2-(4-(2-(5-(l-(3,5-
dichloropyridin-4-yl)ethoxy)-l H-indazol-3 -yl)vinyl)-l H-pyrazol- l-yl)ethanol that is a
potent inhibitor of FGFR and may offer the advantageous properties relative to the
prior form of superior solid handling properties on a large scale, ease of purification
by crystallization, and thermodynamic stability under conditions of pharmaceutical
processing and storage. In one embodiment, the crystalline ((R)-(E)-2-(4-(2-(5-(l-
(3,5-dichloropyridin-4-yl)ethoxy)- 1H-indazol-3 -yl)vinyl)- 1H-pyrazol- 1-yl)ethanol is
the monohydrate form.
The present invention also provides crystalline (R)-(E)-2-(4-(2-(5-(l-(3,5-
dichloropyridin-4-yl)ethoxy)- 1H-indazol-3 -yl)vinyl)- 1H-pyrazol- 1-yl)ethanol
characterized by the X-ray powder diffraction pattern (Cu radiation, l = 1.54059 A)
comprising apeak at 14.65, and one or more peaks at 3.54, 12.51, or 19.16 (2Q+/-
0.1°).
The present invention provides a method of treating cancer wherein the cancer
is selected from the group consisting of breast cancer, non-small cell lung ( SCL)
cancer, bladder cancer, gastric cancer, pancreatic cancer, prostate cancer, colon
cancer, multiple myeloma, liver cancer, melanoma, head and neck cancer, thyroid
cancer, renal cell cancer, glioblastoma, and testicular cancer in a mammal comprising
administering to a mammal in need of such treatment an effective amount of a
compound or salt of the present invention.
This invention also provides pharmaceutical compositions comprising a
compound or salt of the present invention in combination with one or more
pharmaceutically acceptable carriers, diluents, or excipients. In a particular
embodiment the composition further comprises one or more other therapeutic agents.
This invention also provides a compound or salt of the present invention for
use in therapy. Additionally, this invention provides use of a compound or salt of the
present invention in the manufacture of a medicament for treating cancer.
Additionally, this invention provides for use of a compound or salt of the present
invention for use in the treatment of cancer. In particular these cancers are selected
from the group consisting of breast cancer, lung cancer, bladder cancer, gastric
cancer, pancreatic cancer, prostate cancer, colon cancer, multiple myeloma AML,
liver cancer, melanoma, head and neck cancer, thyroid cancer, renal cell cancer,
glioblastoma, and testicular cancer. More particularly, the cancers are selected from
the group consisting of breast cancer, non-small cell lung cancer, bladder cancer,
gastric cancer, pancreatic cancer, prostate cancer, colon cancer, multiple myeloma,
liver cancer, melanoma, head and neck cancer, thyroid cancer, renal cell cancer,
glioblastoma, and testicular cancer. Most particularly the cancer is non-small cell
lung cancer. Most particularly the cancer is gastric cancer. Most particularly the
cancer is multiple myeloma. Furthermore, this invention provides a pharmaceutical
composition for treating cancer selected from the group consisting of breast cancer,
non-small cell lung cancer, bladder cancer, gastric cancer, pancreatic cancer, prostate
cancer, colon cancer, multiple myeloma, liver cancer, melanoma, head and neck
cancer, thyroid cancer, renal cell cancer, glioblastoma, and testicular cancer
comprising a compound or salt of the present invention as an active ingredient.
It will be understood by the skilled reader that all of the compounds of the
present invention are capable of forming salts. The 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, et al, "Pharmaceutical Salts", Journal of
Pharmaceutical Sciences, Vol 66, No. 1, January 1977.
As used herein, the term "isolated" means crystalline (R)-(E)-2-(4-(2-(5-(l-
(3,5-dichloropyridin-4-yl)ethoxy)- 1H-indazol-3 -yl)vinyl)- 1H-pyrazol- 1-yl)ethanol
that is 99% pure.
The compounds of the present invention can be prepared essentially as
illustrated in the preparations and examples below. The naming of the following
preparations and examples is done using the Struct=Name naming feature in
ChemDraw® Ultra 10.0.
Preparation 1
1-(3 ,5-Dichloropyridin-4-yl)ethanol
To a 3-neck 12 L round bottom flask add tetrahydrofuran (THF, 3 L) and
diisopropylamine (DIPA, 315 mL, 2.24 mol) and cool to -78 °C. Add slowly nbutyllithium
(1.6 M in hexanes, 1400 mL, 2.24 mol). After the addition is complete
and the temperature has settled at -78 °C slowly add a solution of 3,5-dichloropyridine
(296.7 g, 2.00 mol) which immediately forms a yellow solution that changes to a rust
colored suspension. After the addition is complete and the temperature has settled at
-78 °C slowly add acetaldehyde (230 mL, 4.05 mol) in THF (600 mL). Continue
stirring at -78 °C. After 3 hours, remove the dry ice bath and begin quenching the
reaction by the dropwise addition of saturated aqueous ammonium chloride ( 1
L). Allow the reaction to warm to room temperature ( T) overnight with stirring.
Dilute the mixture with methyl-tert-butylether (MTBE, 2 L), saturated aqueous
ammonium chloride ( 1 L) and water (2 L). Partition and wash organics with saturated
aqueous sodium chloride (brine). Extract the aqueous phase with MTBE
(1.5 L). Combine the organic layers, dry over sodium sulfate, filter and concentrate in
vacuo. Purify the residue by silica gel chromatography [25% ethylacetate (EA) in
hexanes] to give the title compound as a red oil. Yield: 352 g (90%). MS (ES) m/z
192 [M+l] +.
Preparation 2
(S)-l-(3,5-Dichloropyridin-4-yl)ethanol
Separate the mixture of stereoisomers obtained in Preparation 1 on a
CHIRALPAK® AD-H column eluting with 90% heptanes/ 10% ethanol. Peak 2 is the
desired enantiomer. To establish the absolute configuration dissolve a sample of the
product in CDCI3 (final concentration 100 mg/mL). Obtain the vibrational circular
dichroism (VCD) and infra red (IR) spectra with a resolution of 4 cm-1 using a
ChirallR FT VCD spectrometer (BioTools Inc ®) with an IR cell equipped with BaF2
windows and a path length of 100 mm. Collect the VCD and IR for 6 hours with
150 m of the sample. Present the data without smoothing or further data processing.
Obtain vibrational frequencies and absorption and VCD intensities by optimizing the
lowest energy conformer by Gaussian at the B3PW9 1/6-3 1G** level on a Linux
cluster, and simulate the corresponding spectra using a Lorentzian bandwidth of 6 cm-
1 vibrational circular dichroism. The above analysis shows the product to be the Sisomer.
Yield: 84.37 g (27%). MS (ES) m/z 192 [M+l] +.
Preparation 3
(S)-l-(3,5-Dichloropyridin-4-yl)ethyl methanesulfonate
Dissolve (S)-l-(3,5-dichloropyridin-4-yl)ethanol (5.02 g, 26.14 mmol) in
dichloromethane (DCM, 100 mL) and cool the flask in an ice bath. Add triethylamine
(TEA, 3.5 mL, 25.1 1 mmol) followed by the dropwise addition of methanesulfonyl
chloride (2.2 mL, 28.42 mmol). Remove the ice bath and allow the reaction to warm
to RT. After 4 hours, quench the reaction with water (100 mL) and separate
layers. Extract the aqueous layer with DCM (50 mL) followed by 20% isopropyl
alcohol (IPA)/chloroform (50 mL). Combine the organic extracts, dry over anhydrous
sodium sulfate, filter and concentrate in vacuo. Yield: 7.15 g, (100%). MS (ES) m/z
270 [M+l] +.
Preparation 4
4-Iodo-l-(2-(tetrahydro-2H-pyran-2-yloxy)ethyl)-lH-pyrazole
In a 1 L 3-neck flask equipped with magnetic stir bar, nitrogen blanket and
internal temperature probe dissolve 2-(2-bromoethoxy)tetrahydro-2H-pyran (34 g,
156 mmol) in acetonitrile (ACN, 400 mL). Add 4-iodopyrazole (29.34 g, 149.74
mmol) followed by cesium carbonate (73.4 g, 223.02 mmol). Stir the mixture at RT
for 18 hours. Filter the reaction mixture through CELITE®, wash the filter cake with
ACN and concentrate the filtrate to a golden oil. Use without further purification.
Yield: 47.819 g (99%). MS (ES) m/z 323 [M+l] +.
Preparation 5
5-(tert-Butyldimethylsilyloxy)-lH-indazole
Charge a 10 L reaction vessel with N ,N-dimethylformamide (DMF, 2.50 L),
5-hydroxyindazole (150.20 g, 1.12 mol) and lH-imidazole ( 114.35 g, 1.68 mol).
Cool the mixture to 0 °C and add tert-butyldimethylchlorosilane (253.16 g, 1.68 mol)
over 0.5 hours. Stir the mixture at 18 °C for 3 hours. Add water (2.5 L) to the
reaction slowly with an ice bath at 5 °C to maintain an internal temperature at around
20 °C. Transfer the mixture to a separating funnel and extract with EA (2 x 2.5 L).
Combine the extracts and wash with water (3 x 2.5 L) and brine. Dry the organic
solutions over anhydrous sodium sulfate, filter, and evaporate to a red oil. Pass the oil
through a silica gel pad and elute with eluent (0% to 30% EA in hexane) to afford the
title compound as an orange oil which crystallizes. Yield: 300 g (100%). MS (ES)
m/z 249 [M+l] +.
Preparation 6
5-(tert-Butyldimethylsilyloxy)-3-iodo-lH-indazole
Cool a solution of 5-(tert-butyldimethylsilyloxy)-lH-indazole (300.00 g,
1.21 mol) in DCM (4.00 L) to 10 °C in a 10 L jacketed reactor vessel. To the
resulting solution add N-iodosuccinimide (298.89 g, 1.33 mol) in portions over 0.5
hours. Stir the mixture at RT for 3 hours to give complete conversion as indicated by
liquid chromatography mass spectrometry (LC-MS) and thin layer chromatography
(TLC). Cool the mixture to 10 °C and quench with water (2.5 L). Transfer the
mixture to a separatory funnel and extract the aqueous layer into DCM (2.5 L). Wash
the combined organic extracts with a 10% aqueous sodium thiosulfate solution (5 L)
and brine. Dry the organic solution over magnesium sulfate, filter and concentrate in
vacuo to afford the title compound as an orange solid. Yield: 388 g (90%). MS (ES)
m/z 375 [M+l] +.
Preparation 7
5-(tert-Butyldimethylsilyloxy)-3-iodo-l-(tetrahydro-2H-pyran-2-yl)-lH-indazole
Cool a solution of 5-(tert-butyldimethylsilyloxy)-3-iodo-lH-indazole
(387.00 g, 1.08 mol) in DCM (2.50 L) and THF (1.00 L) to 10 °C in a 10 L jacketed
reactor vessel. To the resulting mixture add methanesulfonic acid (14.0 mL,
216.02 mmol), followed by 3,4-dihydro-2H-pyran (296 mL, 3.24 mol) over 0.5 hours,
observing a slight exotherm. Stir the mixture at RT for 3 hours. Cool the reaction to
10 °C and quench with saturated aqueous sodium bicarbonate (2 L). Dilute the
mixture with water (2 L) and extract the aqueous layer with DCM (2 L). Wash the
combined organic extracts with water (2 L) and brine. Dry the organic mixture over
anhydrous sodium sulfate, filter and concentrate in vacuo. Elute the residue through a
silica gel pad with eluent (0 to 10% EA/hexanes) to give the title compound. Yield:
150 g (3 1%). MS (ES) m/z 459 [M+l .
Preparation 8
(E)-l-(Tetrahydro-2H-pyran-2-yl)-3-(2-(l-(2-(tetrahydro-2H-pyran-2-yloxy)ethyl)-
lH-pyrazol-4-yl)vinyl)-lH-indazol-5-ol
Sparge with nitrogen a mixture of 5-(tert-butyldimethylsilyloxy)-3-iodo-l-
(tetrahydro-2H-pyran-2-yl)-lH-indazole (14 g, 30.54 mmol) in DMF (150 mL) in a
500 mL 3-neck round bottom flask equipped with magnetic stirring, temperature
probe, and condenser with septa for 10 minutes. To the resulting solution add
tributylamine (TBA, 6.7 g, 36.1 mmol) and 4,4,5, 5-tetramethyl-2-vinyl-l, 3,2-
dioxaborolane (7.0 g, 43.18 mmol) and continue sparging for 10 minutes. To the
resulting mixture add bis(triphenylphosphine) palladium (II) chloride (0.45 g, 0.63
mmol) and continue to sparge for an additional 0.5 hours. Heat the mixture at
95-100 °C for 18 hours. Cool the reaction mixture to below 40 °C and charge with
4-iodo-l-(2-(tetrahydro-2H-pyran-2-yloxy)ethyl)-lH-pyrazole (9.8 g, 30.42 mmol).
To the resulting mixture add barium hydroxide octahydrate (19.3 g, 60.3 mmol) and
water (13 mL) and continue sparging for 10 minutes. Add 1,1'-
bis(diphenylphosphino)ferrocene palladium (II) chloride DCM complex (1.3 g, 1.56
mmol) to the reaction and continue sparging 0.5 hours. Heat the mixture at 95 °C
under nitrogen for 3 hours. Dilute the mixture with EA and filter through a Celite®
pad. Wash the pad with brine (400 mL) and separate the filtrate layers. Wash the
organic layer with brine and extract the combined aqueous layers with EA. Combine
the organic solutions and concentrate to a brown oil. Dissolve the oil in DCM
(100 mL) and add to a silica gel pad. Elute the pad with eluent (50% EA in hexanes
followed by 70% EA in hexanes) to afford a light brown oil. Triturate with MTBE
(100 mL) to afford the title compound as a solid. Yield: 5 g (37%). MS (ES) m z
439 [M+l] +.
Preparation 9
5-((R)- 1-(3 ,5-Dichloropyridin-4-yl)ethoxy)- 1-(tetrahydro-2H-pyran-2-yl)-3 -((E)-2-( 1-
(2-(tetrahydro-2H-pyran-2-yloxy)ethyl)-lH-pyrazol-4-yl)vinyl)-lH-indazole
In a 3-neck 250 mL round bottom flask equipped with an internal temperature
probe, reflux condenser, nitrogen blanket and magnetic stir bar, slurry (E)-l(
tetrahydro-2H-pyran-2-yl)-3-(2-(l-(2-(tetrahydro-2H-pyran-2-yloxy)ethyl)-lHpyrazol-
4-yl)vinyl)-lH-indazol-5-ol (10.0 g, 22.83 mmol) and cesium carbonate
(7.88 g, 23.94 mmol) in ACN (92 mL) and warm to 60 °C. To the suspension, add
(S)-l-(3,5-dichloropyridin-4-yl)ethyl methanesulfonate (7.03 g, 26.02 mmol) and stir
overnight. Cool the reaction mixture to RT, filter and wash solids with
ACN. Concentrate the filtrate and purify the residue by silica gel chromatography
(2-4% (2 M ammonia in methanol) /DCM). Combine product fractions and
concentrate in vacuo to a white foam. Yield: 12.5 g (86%). MS (ES) m/z 612
[M+l] +.
Preparation 10 (the amorphous form)
(R)-(E)-2-(4-(2-(5-( 1-(3 ,5-Dichloropyridin-4-yl)ethoxy)- 1H-indazol-3 -yl)vinyl)- 1Hpyrazol-
1-yl)ethanol
Charge a 3-neck, 250 mL round bottom flask equipped with an addition
funnel, nitrogen inlet, internal temperature probe and magnetic stirrer with methanol
(57 mL) and cool in an ice bath. To the resulting solution, add acetyl chloride (20
mL, 281.03 mmol) slowly through an addition funnel. To the solution, add 5-((R)-l-
(3,5-dichloropyridin-4-yl)ethoxy)-l-(tetrahydro-2H-pyran-2-yl)-3-((E)-2-(l-(2-
(tetrahydro-2H-pyran-2-yloxy)ethyl)-lH-pyrazol-4-yl)vinyl)-lH-indazole (7.1 g,
11.59 mmol) dissolved in methanol (40 mL) via addition funnel. After addition is
complete, remove the ice bath, warm to RT and stir the mixture for 4 hours.
Concentrate the reaction mixture in vacuo to a yellow foam. Dissolve the yellow
foam in methanol (10 mL) and add slowly to a saturated aqueous sodium bicarbonate
solution (120 mL). Stir the mixture at RT for 30 minutes. Filter the mixture, wash
the solid with water (100 mL), and dry under vacuum. Recrystallize the solid from
hot EA/methanol/hexanes to give the title compound as a white solid. Yield: 2.1 g
(41%). MS (ES) m/z 444 [M+l] +.
Example 1
(R)-(E)-2-(4-(2-(5-( 1-(3 ,5-Dichloropyridin-4-yl)ethoxy)- 1H-indazol-3 -yl)vinyl)- 1Hpyrazol-
l-yl)ethanol monohydrate Form 1:
A reaction vessel is purged with nitrogen and charged with (R)-(E)-2-(4-(2-(5-
(1-(3 ,5-dichloropyridin-4-yl)ethoxy)- 1H-indazol-3 -yl)vinyl)- 1H-pyrazol- 1-yl)ethanol
and a solvent mixture consisting of 11% water/acetonitrile. The resulting suspension
is heated to an internal temperature of 66- 68 °C producing a solution. The solution is
cooled slowly to 56-58 °C, then seeded with a suspension of seed crystals in 11%
water/acetonitrile mixture and stirred slowly. The reaction mixture is initially cooled
down to 48-50 °C and then to 19-20 °C. The product is isolated by filtration in the
presence of nitrogen stream with a relative humidity of at least 80 % passing through
the solid cake. The humidity level in the nitrogen stream is then subsequently changed
to 40 % and the drying continued, resulting in the production of the title compound.
Note that the seed crystal is similarly obtained as follows: A reaction vessel is purged
with nitrogen and charged with (R)-(E)-2-(4-(2-(5-(l-(3,5-dichloropyridin-4-
yl)ethoxy)-l H-indazol-3 -yl)vinyl)-l H-pyrazol- l-yl)ethanol and a solvent mixture
consisting of 11% water/acetonitrile. The resulting suspension is heated to an internal
temperature of 70 °C producing a solution. The solution is then cooled slowly,
allowed to crystallize, filtered and dried. This compound is a potent inhibitor of
FGFR and may have the advantageous properties relative to the prior form of superior
solid handling properties on a large scale, ease of purification by crystallization, and
thermodynamic stability under conditions of pharmaceutical processing and storage.
The Compound of Example 1 XRPD
The XRPD pattern is collected using a PANalytical X'Pert™ Pro MPD
PW3040 Pro diffractometer, equipped with a CuKa radiation (l = 1.54059 A
(voltage: 45kV and amperage: 40 mW)) produced using an Optix long fine-focus
source. The specimen is sandwiched between 3-micron thick films, analyzed in
transmission geometry, and rotated at 1 revolution per second to optimize orientation
statistics. Prior to the analysis a silicon specimen (NIST standard reference material
640c) is analyzed to verify the position of the silicon 1 1 1 peak. One Panalytical
pattern is analyzed for this material, and preferred orientation and particle statistic
effects are assessed through comparison of the simulated XRPD pattern from single
crystal analysis. An elliptically graded multilayer mirror is used to focus the Cu K
X-rays of the source through the specimen and onto the detector. Diffraction patterns
are collected using a scanning position-sensitive detector (X'Celerator) located 240
mm from the specimen. Data are collected from 1.01 to 39.99 degree 2Qwith a step
size of 0.017 degree 2Qand a scan speed of 1.2 degree/min and with a 0.5 degree
divergence slit and a 0.25 degree scattering slit. A beam-stop is used to minimize the
background generated by air scattering. Soller slits are used for the incident and
diffracted beams to minimize axial divergence. Observed peaks are shown in the
Table 1. An intensity threshold of 5% is used.
Table 1. Observed peaks for the Compound of Example 1, XRPD .
°2Q d space (A) Intensity (%)
3.54 ± 0.10 24.975 ± 0.726 45
7.08 ± 0.10 12.485 ± 0.179 10
10.62 ± 0.10 8.328 ± 0.079 9
12.51 ± 0.10 7.075 ± 0.057 78
13.00 ± 0.10 6.812 ± 0.053 37
13.60 ± 0.10 6.512 ± 0.048 16
14.18 ± 0.10 6.245 ± 0.044 13
14.65 ± 0.10 6.046 ± 0.041 100
14.97 ± 0.10 5.919 ± 0.040 29
15.49 ± 0.10 5.722 ± 0.037 16
16.24 ± 0.10 5.459 ± 0.034 35
16.59 ± 0.10 5.344 ± 0.032 24
17.06 ± 0.10 5.198 ± 0.030 26
17.76 ± 0.10 4.995 ± 0.028 20
18.49 ± 0.10 4.798 ± 0.026 9
19.16 ± 0.10 4.632 ± 0.024 68
20.37 ± 0.10 4.361 ± 0.021 44
21.67 ± 0.10 4.101 ± 0.019 8
21.89 ± 0.10 4.061 ± 0.018 8
22.17 ± 0.10 4.010 ± 0.018 22
23.02 ± 0.10 3.863 ± 0.017 54
24.33 ± 0.10 3.659 ± 0.015 15
25.25 ± 0.10 3.528 ± 0.014 27
25.93 ± 0.10 3.436 ± 0.013 49
26.16 ± 0.10 3.406 ± 0.013 16
26.77 ± 0.10 3.331 ± 0.012 10
27.23 ± 0.10 3.275 ± 0.012 15
28.25 ± 0.10 3.159 ± 0.01 1 10
28.59 ± 0.10 3.123 ± 0.01 1 1 1
29.56 ± 0.10 3.022 ± 0.010 13
Thus, a properly prepared sample of Example 1 may be characterized by Xray
diffraction pattern using CuK radiation as having diffraction peaks (2-theta
values) as described in Table 1, and in particular having peaks at 14.65 in
combination with one or more of the peaks at 3.54, 12.51, and 19.16; and more
particularly having a peak at 14.65; with a tolerance for the diffraction angles of 0.1
degrees, more preferably 0.01 degrees.
Aberrant regulation of the FGF/FGFR pathway has been implicated in many
forms of human malignancies. FGFRs and FGFs are often over-expressed in
numerous cancers, and their expression often correlates with poor prognosis. The
activating mutations in the FGFR kinase domain have been found in several types of
tumors, including breast, NSCLC, bladder, gastric, prostate, colon, and multiple
myeloma. Genomic amplification of FGFR locus was also detected in many breast,
gastric, and lung cancer patients. Over-expression of FGFRs or FGFs has also been
found in many different types of tumors such as bladder, multiple myeloma, prostate,
and lung cancers. Other cancers that might benefit from FGFR family pathway
inhibitor therapy include AML, liver cancer, melanoma, head and neck cancer,
thyroid cancer, pancreatic cancer, renal cell cancer, glioblastoma, and testicular
cancer. In addition to their roles in tumor formation and progression, FGFs and
FGFRs are also key regulators of angiogenesis, especially during tumor growth. The
FGF/FGFR axis also plays an important role in augmenting other tumor stromal cells
such as cancer associated fibroblasts. Up-regulation of FGFs also leads to resistance
to anti-angiogenic and other chemo-therapies. Finally, small molecule inhibitors of
FGFRs have demonstrated anti-tumor activities in several preclinical tumor models
and are being explored in the clinic. Taken together, the FGF/FGFR pathway is
essential to several important cellular processes in cancer cells. For these reasons,
therapies based on targeting FGFRs and/or FGF signaling may affect both the tumor
cells directly and tumor angiogenesis.
Preparation 10 is tested essentially as described below in the following assays:
FGFR1 Enzyme Assay (Filter Binding), the FGFR3 Enzyme Assay (Filter Binding),
FGF9 induced p-ERK in RT-1 12 cell based assay (in the presence of BSA), and the
AlphaScreen SureFire Detection of ERK phosphorylation (Thr202/Tyr204) in Human
Umbilical Vein Endothelial Cells (HUVEC) cell based assays. These assays
demonstrate that Preparation 10 is an FGFR family pathway inhibitor and has anticancer
activity. Thus, results with the amorphous form of (R)-(E)-2-(4-(2-(5-(l-(3,5-
Dichloropyridin-4-yl)ethoxy)- 1H-indazol-3 -yl)vinyl)- 1H-pyrazol- 1-yl)ethanol are
indicative of results with the compound of the present invention. Crystal forms of the
compound are still advantageous because they may offer the properties relative to the
prior form of superior solid handling properties on a large scale, ease of purification
by crystallization, and thermodynamic stability under conditions of pharmaceutical
processing and storage.
FGFR1 and FGFR3 Enzyme Assay (Filter Binding)
FGFR1 or FGFR3 kinase (0.15 ng/m human FGFR1 or 0.32 ng/m human
FGFR3) is incubated in 50 m of a buffer containing 10 mM 4-(2-hydroxyethyl)-lpiperazineethane-
sulfonic acid (HEPES ) pH 7.5, 8 mM
tris(hydroxymethyl)aminomethane (Tris-HCl), pH 7.5, 5.0 mM dithiothreitol (DTT),
10.0 mM adenosine triphosphate (ATP), 10 mM MnCl2, 150 mMNaCl, 0.01%
TRITON® X-100, 0.5 m P-ATP, and 0.05 mg/mL Poly(Glu-Tyr). The reaction is
carried out in a volume of 50 mΐ at RT for 30 minutes and then quenched by adding
130 mΐ of 10% H3PO4. The reaction (120 m ) is transferred to a 96 well 1.0 mih glass
fiber filter plate, incubated at RT for 20-30 minutes and then washed 3x on a
TITERTEK® Zoom with 0.5% H3PO4. Wells are air dried before addition of 40 mΐ
TM of MicroScint 20 (Packard) and then counted on a Wallac Micobeta counter. For
compound inhibition, the compound is provided as 10 mM stocks in dimethyl
sulfoxide (DMSO). The compound is serially diluted 1:3 in 20% DMSO to create a
10 point concentration-response curve and diluted 1:5 (20 mM to 0.001 mM final in
4% final DMSO concentration) into the reaction plate prior to addition of the reaction
mixture in the filter plate to determine compound activity. Control wells contain 4%
DMSO only while the baseline is established by control wells containing 0.1M
ethylenediaminetetraacetic acid (EDTA). The percent inhibition values for each of
the 10 concentrations are calculated from control wells on each plate and the 10-point
concentration response data are subsequently analyzed using ActivityBase software
(IDBS) using a 4-parameter logistic equation and absolute IC 0 values estimated from
the resulting curve fit. The FGFR1 and FGFR3 enzyme assays have Minimum
Significant Ratios (MSR) for the estimated IC50 of 1.38 and 1.47, respectively. The
IC 0 results for Preparation 10 for FGFR1 and FGFR3 in these assays are estimated to
be 0.0077 and 0.0064 mM, respectively. This data demonstrates that Preparation 10 is
a potent FGFR1 and FGFR3 enzyme inhibitor.
FGF9 induced p-ERK with BSA
Human RT1 12 bladder carcinoma cells are seeded at a density of 5,000 cells
per well in 100 mΐ RPMI 1640 (Gibco 11875-085) supplemented with 10% fetal
bovine serum (FBS, Gibco 10082-147) and 1% of a penicillin/streptomycin solution
(Gibco 15 140-122) in CELLBIND® 96 well plates (Corning 3340) and incubated
overnight at 37 °C. The next morning the growth medium is removed and replaced
with 100 m RPMI 1640 supplemented with 20 mg/mL bovine serum albumin (BSA).
After 3 hours of incubation at 37 °C, 20 mΐ of 3x serially diluted compounds in RPMI
1640 with 20 mg/mL BSA in 6% DMSO are added to each well. This yielded a 10
point dose-response curve ranging from 10 - 0.005 mM in 1% DMSO. The
incubation is continued for 1 hour at 37 °C. The cells are stimulated with 50 m of a
50 mg/mL FGF9 (R&D Systems 273 -F9) solution in serum free RPMI to give a final
concentration of 500 ng/niL FGF9. Cells are fixed by the addition of 30 m of a 25%
formaldehyde solution in phosphate buffered saline (PBS) (3.7% formaldehyde final
concentration), and incubated 30 minutes at RT. Cells are washed 3x with PBS,
followed by the addition of 100 mΐ of cold methanol and incubated for 30 minutes at
-20 °C. The methanol is removed and the cells are treated with PBS containing 0.1%
TRITON® X-100 (PBST), washed 3x with PBS, and incubated 15 minutes at RT.
Cells are then incubated overnight at 4 °C with gentle shaking in 50 m of a 1:400
dilution of the p-p44/42 MAPK primary antibody (Cell Signaling 910 1S) in PBS
supplemented with 2% BSA, 0.01% Phosphatase Inhibitor Cocktail 1 (Sigma P2850),
0.01% Phosphatase Inhibitor Cocktail 2 (Sigma P5726), and 0.01% Protease Inhibitor
Cocktail (Sigma P8340). The next morning, plates are washed 2x with PBST and 2x
with PBS, followed by a 1 hour RT incubation in the dark in 80 uL of a 1:1000
dilution of the Alexa Fluor 488 goat anti-rabbit IgG H+L secondary antibody
(Invitrogen Al 1034) in PBS with 1% BSA and 0.1% of Phosphatase Inhibitor
Cocktail 1, 0.01% Phosphatase Inhibitor Cocktail 2, and 0.01% Protease Inhibitor
Cocktail. Cells are washed 3x with PBS, followed by the addition of 100 m of a
1:200 dilution of propidium iodide (PI) (Molecular Probe P-3566) in PBS and then
incubated in the dark for 1 hour. The p-ERK positive cells and total cells per well are
identified with the ACUMEN EXPLORER™ (TTP LabTech Ltd) using optical filter
500-530 nM and 575-640 nM for Alexa 488 and PI, respectively. The total mean
intensity for pERK/well using the Alexa 488 values is subsequently converted to
percent inhibition using values obtained from MGN (10 mM positive control
compound in DMSO) and MAX (DMSO alone) controls run on the same plate. The
percentage inhibition values and the 10-point concentration response data are
subsequently analyzed using a 4-parameter sigmoidal dose response equation and
relative IC50 values are estimated from the resulting curve. The FGF9 induced p-ERK
with BSA assay has a Minimum Significant Ratio (MSR) for the estimated IC50 of
2.7. The IC50 for Preparation 10 in this assay is estimated to be 0.0004 mM. This data
demonstrates that Preparation 10 is a potent inhibitor of FGF9 induced ERK
phosphorylation in human cancer cells.
AlphaScreen SureFire Detection of ERK phosphorylation (Thr202/Tyr204) in Human
Umbilical Vein Endothelial Cells (HUVEC)
The effect of compound on the inhibition of FGF receptor 1 is measured by
monitoring the phosphorylation of ERK (pERK) in response to basic-Fibroblast
growth factor (b-FGF) stimulation in Human Umbilical Endothelial cells (HUVEC).
The levels of pERK formed are measured using the ALPHASCREEN® SUREFIRE
® system (TGR Biosciences, TGRES50K). This is a homogeneous assay format
utilizing the immuno-sandwich capture of the phosphorylated analyte followed by
detection using antibody-coated ALPHASCREEN® beads (Perkin Elmer) to generate
an amplified signal.
HUVEC cells are recovered and maintained in growth medium consisting of
endothelial cell basal medium (Clonetics, CC-3132) supplemented with 10% FBS
0.4% bovine brain extract 0.1% hydrocortisone, 0.1% gentamicin sulfate
amphotericin-B, and 0.1% epidermal growth facter, human recombination until
passage 7. For the assay, cells are harvested by standard procedures and then
counted. Cells (20,000/well) are plated in 100 of growth medium into 96 well
Poly-D-Lysine coated plates (BD, 354640). Plates are incubated overnight at 37 °C,
5% C0 2.
On the day of the assay, cells are serum starved in 100 m EBM (endothelial
cell basal) medium containing 1.5% FBS and 20 mg/mL BSA for 3 hours at 37 °C,
5% CO2, then treated with 20 mM of 3x serially diluted compound in starvation
medium for 1 hour at 37 °C. This yielded a 10 point concentration-response curve
ranging from 10-0.005 mM in 1% DMSO. After 1 hour compound treatment, cells are
stimulated with 50 m b-FGF (Sigma, F0291, final b-FGF concentration 50 ng/niL) at
37 °C for 15 minutes. In the wells containing cells and 50 m stimulator b-FGF yields
MAX signal, and cells with 10 mM positive control compound and 50 ul stimulator b-
FGF as MGN. The medium then is removed and 50 m of lx SUREFIRE® Lysis
Buffer (TGR Biosciences SUREFIRE ® Kit component) is added/well and incubation
continued at RT for 10 minutes with gentle shaking. For pERK detection, 6 m lysate
and 10 m reaction mixture (60 parts reaction buffer/10 parts activation buffer/0.6 part
each of donor and acceptor beads, Perkin Elmer, 67606 17R) are transferred to a 384
well proxiplate (Perkin Elmer, 6006280). The plate is sealed and incubated at RT for
2 hours with gentle shaking and then read on Perkin Elmer EnVision plate reader
equipped with a TurboModule using standard ALPHASCREEN® settings (Ex 80nm
and Em 2o-620nm). The emission data is converted to percent inhibition determined
from MAX (DMSO alone) and MGN (10 mM positive control compound in DMSO)
controls on each plate and ten-point compound concentration data are then fit to a
four-parameter logistic equation using ACTIVITYBASE® 4.0 and the IC 0 is
estimated. The ALPHASCREEN® SUREFIRE® Detection of ERK phosphorylation
((Thr202/Tyr204) assay has a Minimum Significant Ratio (MSR) for the IC 0 of 2 . 1.
The IC5 0 of Preparation 10 in this assay is estimated to b e 0.0006 mM. This data
demonstrates that Preparation 10 is a potent inhibitor of bFGF induced ERK
phosphorylation in Human Umbilical Endothelial cells.
The compound of the present invention is preferably formulated as a
pharmaceutical composition administered by a variety of routes. Most preferably,
such compositions are for oral or intravenous administration. Such
pharmaceutical compositions and processes for preparing same are well known in
the art. See, e.g., REMINGTON: THE SCIENCE AND PRACTICE OF
PHARMACY (D. Troy, et al, eds., 2 1st ed., Lippincott Williams & Wilkins,
2005).
The compound of the present invention is generally effective over a wide
dosage range. For example, dosages per day normally fall within the range of about
0.5 to about 100 mg/kg of body weight. In some instances dosage levels below the
lower limit of the aforesaid range may be more than adequate, while in other cases
still larger doses may b e employed without causing any harmful side effect, and
therefore the above dosage range is not intended to limit the scope of the invention in
any way. It will b e understood that the amount of the compound actually
administered will b e determined by a physician, in the light of the relevant
circumstances, including the condition to b e 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 which is crystalline ((R)-(E)-2-(4-(2-(5-(l-(3,5-
dichloropyridin-4-yl)ethoxy)- 1H-indazol-3 -yl)vinyl)- 1H-pyrazol- 1-yl)ethanol.
2. The compound according to claim 1 which is crystalline ((R)-(E)-2-(4-(2-
(5-(1-(3 ,5-dichloropyridin-4-yl)ethoxy)- 1H-indazol-3 -yl)vinyl)- 1H-pyrazol- 1-
yl)ethanol monohydrate.
3. The compound according to claim 1 or claim 2, which is isolated.
4. The compound according to any one of claims 1 to 3, wherein the
compound is characterized by the X-ray powder diffraction pattern (Cu radiation, l =
1.54059 A) comprising a peak at 14.65.
5. The compound according to claim 4, further comprising a peak at 3.54
(2Q+/- 0.1 0) .
6. The compound according to claim 4 or claim 5, further comprising a peak
at 12.51 (2Q+/- 0.1 0) .
7. The compound according to any one of claims 4 to 6, further comprising a
peak at 19.16 (2Q+/- 0.1°).
8. A pharmaceutical composition comprising the compound as in any one of
the preceding claims in combination with a pharmaceutically acceptable carrier,
diluent or excipient.