Background of the Invention
Transforming growth factor-beta (TGF-~) is a prototype for a large family of
growth and differentiation factors that regulate development. TGF-~ family members
activate transmembrane serine/threonine receptor kinases, thereby initiating a signaling
cascade via Smads, a novel class of intracellular signaling effectors that regulate gene
expression. TGF-~ is a potent inducer of growth arrest in many cell types, including
epithelial cells. This activity is the basis of the tumor suppressor role of the TGF-~
signaling system in carcinomas. Other activities, including TGF-~-induced epithelial-tomesenchymal
differentiation, contribute to cancer progression.
TGF-~ family signaling is of special relevance in mesenchymal differentiation,
including bone development. Deregulated expression or activation of components of this
signaling system can contribute to skeletal diseases, e.g. osteoarthritis. See Wakefield, et
al. (2002) Current Opinion in Genetics & Development 12:22-29; Siegel, et al. (2003)
Nature Reviews (Cancer) 3:807-820; Dumont, et al. (2003) Cancer Cell3:531-536.
PCT patent application WO 02/0948332 describes a genus of
dihydropyrrolopyrazole compounds useful for treating disorders associated with enhanced
TGF-~ signaling activity or overproduction. PCT patent application WO 04/04382
describes an anhydrous form of 2-(6-methyl-pyridin-2-yl)-3-[6-amido-quinolin-4-yl)-5,6-
dihydro-4H-pyrrolo [ 1 ,2-b ]pyrazole.
It has been surprisingly discovered that 2-(6-methyl-pyridin-2-yl)-3-[6-amidoquinolin-
4-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazole can be prepared in a crystalline
monohydrate form having the advantageous properties relative to the anhydrous 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.
A manufacturing process for the new form has also been discovered.
Summary of the Invention
The present invention provides 2-(6-methyl-pyridin-2-yl)-3-[6-amido-quinolin-4-
yl)-5,~-dihydro-4H-pyrrolo[1,2-b]pyrazole monohydrate, i.e., Formula I.
Formula I.
The present invention provides 2-(6-methyl-pyridin-2-yl)-3-[6-amido-quinolin-4-
yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazole monohydrate in a crystalline form.
The present invention provides a pharmaceutical composition comprising 2-(6-
methyl-pyridin-2-yl)-3-[6-amido-quinolin-4-yl)-5,6-dihydro-4H-pyrrolo[l,2-b]pyrazole
monohydrate and a pharmaceutically acceptable excipient, diluent or carrier.
The present invention provides a method of inhibiting TGF-f3 signaling in a
mammal comprising administering to a mammal in need of such treatment an effective
amount of 2-(6-methyl-pyridin-2-yl)-3-[6-amido-quinolin-4-yl)-5,6-dihydro-4Hpyrrolo[
1 ,2-b ]pyrazole monohydrate.
The present invention further provides a method of treating conditions resulting
from excessive TGF-f3 production in a mammal comprising administering to a mammal in
need of such treatment a TGF-f3 signal-suppressing amount of 2-(6-methyl-pyridin-2-yl)-
3-[6-amido-quinolin-4-yl)-5,6-dihydro-4H-pyrrolo[l,2-b]pyrazole monohydrate.
Because the compounds of the present invention are inhibitors of TGF-f3
signaling, the compounds of the present invention are useful for the treatment of a variety
of disorders including the treatment of susceptible neoplasms.
In one of its method aspects, this invention is directed to a method for treating
susceptible neoplasms comprising administering to a patient in need thereof an effective
amount of 2-(6-methyl-pyridin-2-yl)-3-[6-amido-quinolin-4-yl)-5,6-dihydro-4Hpyrrolo[
1 ,2-b ]pyrazole monohydrate.
The present invention provides 2-(6-methyl-pyridin-2-yl)-3-[6-amido-quinolin-4-
yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazole monohydrate for use in therapy. The present
invention provides for the use of 2-(6-methyl-pyridin-2-yl)-3-[6-amido-quinolin-4-yl)-
5,6-dihydro-4H-pyrrolo[1,2-b]pyrazole monohydrate for the manufacture of a
medicament for the treatment of disorders associated with enhanced TGF-P signaling
activity or overproduction.
In another embodiment this invention provides a process for making 2-(6-methylpyridin-
2-yl)-3-[6-amido-quinolin-4-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazole
monohydrate and novel intermediates useful for making crystalline 2-(6-methyl-pyridin-
2-yl)-3-[6-amido-quinolin-4-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazole monohydrate: 6-
cyano-4-methyl-quinoline hydrochloride, 2-( 6-cyano-quinolin-4-yl)-1-( 6-methyl-pyridin-
2-yl)-ethanone; 1-amino )-2-pyrrolidinone, p-toluene sulfonate; 1-[ (6-methyl-pyridin-2-
yl)-2-(6-cyano-quinolin-4-yl)-ethylideneamino]-pyrrolidin-2-one; and 3-(6-cyanoquinolin-
4-yl)-2-(6-methyl-pyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazole.
Detailed Description of the Invention
As used herein, the term "6-cyano-4-methyl-quinoline hydrochloride" refers to
NC~
U"N) HCl
NC
to
The term "2-(6-cyano-quinolin-4-yl)-1-(6-methyl-pyridin-2-yl)-ethanone" refers
0
The term "1-amino)-2-pyrrolidinone, p-toluene sulfonate" refers to Q,NH,
O ~S02H H
The term "1-[(6-methyl-pyridin-2-yl)-2-(6-cyano-quinolin-4-yl)ethylideneamino]-
pyrrolidin-2-one" refers to
R ko I
The term "3-(6-cyano-quinolin-4-yl)-2-(6-methyl-pyridin-2-yl)-5,6-dihydro-4Hpyrrolo[
1,2-b]pyrazole" refers to
NC
The term "effective amount" of a compound of the present invention refers to an
amount, that is, the dosage which is effective in treating the disorders described herein.
An effective amount can be readily determined by the attending diagnostician, as
one skilled in the art, by the use of conventional techniques and by observing results
obtained under analogous circumstances. In determining an effective amount, the dose of
a compound of the present invention, a number of factors are considered by the attending
diagnostician, including, but not limited to: the compound of the present invention to be
administered; the co-administration of other therapies, if used; the species of mammal; its
size, age, and general health; the specific disorder involved; the degree of involvement or
the severity of the disorder; the response of the individual patient; the mode of
administration; the bioavailability characteristics of the preparation administered; the
dose regimen selected; the use of other concomitant medication; and other relevant
circumstances.
An effective amount of a compound of the present invention is expected to be
delivered QD or BID at 10 mg to 1 g total daily dose, preferably from 100-200 mg
preferred flat dose. An effective amount could also be once a day or once a week dosing
with sustained release formulations. More preferred amounts can be determined by one
skilled in the art.
It is understood that TGF-P includes both TGF-P 1 and TGF-P2.
Conditions "characterized by enhanced TGF-P activity" include those wherein
TGF-P synthesis is stimulated so that TGF-P is present at increased levels or wherein
TGF-~ latent protein is undesirably activated or converted to active TGF-~ protein or
wherein TGF-~ receptors are upregulated or wherein the TGF-~ protein shows enhanced
binding to cells or extracellular matrix in the location of the disease. Thus, in either case
"enhanced activity" refers to any condition wherein the biological activity of TGF-~ is
undesirably high, regardless of the cause.
There are several types of cancer, i.e., susceptible neoplasms, where TGF-~ 1
produced by the tumor may be deleterious. These include prostrate cancer (e.g., Steiner
and Barrack (1992) Mol. Endocrinol 6: 15-25), colorectal cancer (e.g., Neurath et al.
(2004) Immunity. 21:491-501), breast cancer (e.g.,Arteaga, et al. (1993) Cell Growth and
Differ. 4:193-201), non-small cell lung cancer (e.g., Ready et al., (Apr 2005) Semin
Oncol. 32(2 Supp13):S35-41, ovarian cancer (e.g., Dr. Gustavo C. Rodriguez, (March 2-
7, 2001) 32nd Annual Meeting of the Society of Gynecologic Oncologists. Nashville,
TN), endometrial cancer (e.g., Dowdy et al, (Feb 2005) Gynecol Oncol. 96(2):368-73),
testicular cancer (e.g., Morera et al., (1992) Endocrinology. 130:831-836), osteosarcoma
(e.g., Kloen et al., (Aug 1, 1994) Int J Cancer. 58(3):440-5), and multiple myeloma (e.g.,
Cook et al., (1999) Journal of Leukocyte Biology, Vol 66, Issue 6:981-988). See also
PCT patent application WO 02/0948332.
Another embodiment of the present invention is the crystalline monohydrate form
of 2-(6-methyl-pyridin-2-yl)-3-[6-amido-quinolin-4-yl)-5,6-dihydro-4H-pyrrolo[1,2-
b]pyrazole. This compound has been characterized as described below.
X-Ray Powder Diffraction
The X-ray powder diffraction (XRD) pattern of the crystalline monohydrate was
obtained on a Siemens D5000 X-ray powder diffractometer, equipped with a CuKa.
source (A= 1.54056 A) and a Kevex solid state Si(Li) detector, operating at 50 kV and 40
rnA. Each sample was scanned between 3 and 40 in 28, with a step size of 0.02° in 26
and a scan rate of 9.0 seconds/step, and with 1 mm divergence and receiving slits and a
0.1 mm detector slit. The dry powder was packed into recessed top-loading sample
holder and a smooth surface was obtained using a glass slide. The monohydrate crystal
form diffraction pattern, collected at ambient temperature and relative humidity, was
adjusted based on the NIST 675 standard peak at 8.85 degrees 2-theta.
Angle(+/- 0.01 degrees) d-spacing
9.05 9.76
10.25 8.63
11.02 8.02
11.95 7.40
12.37 7.15
13.49 6.56
14.84 5.96
17.48 5.07
20.11 4.41
20.77 4.27
24.13 3.69
25.38 3.51
26.00 3.42
26.73 3.33
28.79 3.10
29.91 2.98
31.84 2.81
Thus, a properly prepared sample of 2-(6-methyl-pyridin-2-yl)-3-[6-amidoquinolin-
4-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazole monohydrate may be characterized
by X-ray diffraction pattern using CuKa radiation as having diffraction peaks (2-theta
values) as described in Table 1, and in particular having peaks at 9.05 in combination
with one or more of the peaks at 11.02, 11.95, and 14.84; and more particularly having a
peak at 9.05; with a tolerance for the diffraction angles of 0.1 degrees, more preferably
.01 degrees.
Solid-State 13C NMR Spectroscopy
13C Cross polarization I magic angle spinning (CP/MAS) NMR (solid-state NMR
or SSNMR) spectra were obtained for the crystalline monohydrate using a Varian Unity
In ova 400 MHz NMR spectrometer operating at a carbon frequency of 100.573 MHz and
equipped with a complete solids accessory and a Chemagnetics 4.0 mm T3 probe.
Ramped-amplitude cross-polarization (RAMP-CP) at 62 kHz and two-pulse phase
modulation (TPPM) decoupling at 70 kHz were used. Acquisition parameters were as
follows: 90° proton radio frequency pulse width 4.0 jlS, contact time 2.0 ms, pulse
repetition time 60 s, MAS frequency 10 kHz, spectral width 50 kHz, and acquisition time
50 ms. Chemical shifts were referenced to the methyl group of hexamethylbenzene (o ==
17.3 ppm) by sample replacement.
13C chemical shifts of monohydrate crystal form: 20.5, 22.5, 26.3, 48.7, 108.8, 115.6,
122.6, 127.9, 128.8, 130.5, 136.4, 146.8, 149.0, 151.3, 152.0, 153.2, 157.9, and 171.0 (+/-
0.2) ppm.
Thus, crystalline 2-(6-methyl-pyridin-2-yl)-3-[6-amido-quinolin-4-yl-5,6-dihydro-
4H-pyrrolo[1,2-b]pyrazole monohydrate may be characterized by solid state 13C nuclear
magnetic resonance having chemical shift (ppm) of 108.8, 115.6, 122.6, and 171.0 (+/-
0.2) ppm.
In another embodiment this invention provides a process for preparing 2-(6-
methyl-pyridin-2-yl)-3-[6-amido-quinolin-4-yl-5,6-dihydro-4H-pyrrolo[l,2-b]pyrazole
monohydrate comprising crystallizing 2-(6-methyl-pyridin-2-yl)-3-[6-amido-quinolin-4-
yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazole from an appropriate solvent containing an
organic solvent to water ratio of 0-90% under conditions which yield 2-(6-methylpyridin-
2-yl)-3-[6-amido-quinolin-4-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazole
monohydrate.
2-(6-methyl-pyridin-2-yl)-3-[6-amido-quinolin-4-yl-5,6-dihydro-4H-pyrrolo[1,2-
b]pyrazole monohydrate may be prepared by crystallization under controlled conditions.
In particular, the monohydrate of the present invention can be prepared by crystallization
from an aqueous solvent. A suitable solvent is one that has an organic solvent to water
ratio of 0-90% organic solvent. Preferred is an organic solvent to water ratio between
60:40 to 85:15. More preferred is an organic solvent to water ratio of 75:25. Preferred
organic solvents are acetonitrile, acetone, tetrahydrafuran (THF), methyl ethyl ketone,
dimethyl sulfoxide (DMSO), dimethyl formamide (DMF), N,N-dimethyl acetamide, and
N-methyl pyrrolidinone. In practice, it has been found that acetone is most preferred.
After suspending anhydrous, monohydrated, or partially hydrated 2-(6-methyl-pyridin-2-
yl)-3-[6-amido-quinolin-4-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazole in a suitable solvent,
the mixture is stirred at 20 to 100 ac, preferably 60 °C. For lower boiling point solvents
such as acetonitrile, acetone, and methyl ethyl ketone, the volume of the resulting solution
is reduced, preferably by distillation, to between 35-70% of the initial volume, preferably
50%. For higher boiling point solvents, an appropriate amount of additional water may
need to be added to precipitate the product. During the reduction, seed crystals of
monohydrate may be added. Slowly cool the solution to 0-5 °C, preferably in two stages
wherein the solution is first cooled to 20-25 oc over 90 minutes followed by cooling to 0-
5 oc over 30-40 minutes. Hold the slurry at 0-5 oc for an additional 30 minutes to 25
hours, preferably 2-3 hours. Filter the slurry and rinse the product, preferably with water
or aqueous organic solvent. Dry the product, preferably at 45 °C.
In another embodiment 2-(6-methyl-pyridin-2-yl)-3-[6-amido-quinolin-4-yl)-5,6-
dihydro-4H-pyrrolo[l,2-b]pyrazole monohydrate can be prepared by reslurrying 2-(6-
methyl-pyridin-2-yl)-3-[6-amido-quinolin-4-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazole
in water or aqueous DMSO.
2-(6-Methyl-pyridin-2-yl)-3-[6-amido-quinolin-4-yl-5,6-dihydro-4H-pyrrolo[1,2-
b]pyrazole monohydrate may be prepared by stirring in 10 volumes of 0-90% organic
solvent to water at room temperature for 1-2 hours, filtering, and drying at 35-45 °C,
preferably 45 oc under vacuum. In cases where organic solvent is used, the reaction
mixture may be diluted with water (2-5 times the volume of organic solvent used) to
improve recovery.
Crystalline 2-(6-methyl-pyridin-2-yl)-3-[6-amido-quinolin-4-yl)-5,6-dihydro-4Hpyrrolo[
1,2-b]pyrazole monohydrate can be prepared by a variety of procedures, some of
which are illustrated in the examples below.- It will be recognized by one of skill in the
art that the order of the individual steps in the following may be varied to provide
crystalline 2-(6-methyl-pyridin-2-yl)-3-[6-amido-quinolin-4-yl)-5,6-dihydro-4Hpyrrolo[
1 ,2-b ]pyrazole monohydrate:
Preparation 1:
Preparation of 2-(6-methyl-pyridin-2-yl)-3-[6-amido-quinolin-4-yl)-5,6-dihydro-4Hpyrrolo[
1,2-b]pyrazole monohydrate seed crystals
To a flask equipped with mechanical stirrer add 3-(6-cyano-quinolin-4-yl)-2-(6-
methyl-pyridin-2-yl)-5,6-dihydro-4H-pyrrolo[l,2-b]pyrazole (500.2 g), potassium
carbonate (39.4 g), and DMSO (3,000 mL) to form a slurry. In a separate flask, combine
35% hydrogen peroxide (154 mL) and deionized water (250 mL). Add the dilute
hydrogen peroxide solution to the above slurry over 30 to 45 minutes in such a way that
the reaction temperature is in the range of 22 to 34 °C. After completion of addition, stir
the reaction mixture until the reaction is complete as judged by HPLC.
Prepare a solution of sodium sulfite (107.6 g) in deionized water (4,500 mL). Add
the reaction mixture slowly to the sodium sulfite solution to quench the reaction solution
while maintaining the temperature from 22 to 40 °C. Stir the reaction mixture for 40-60
minutes and add concentrated (37.5%) HCl acid (450 mL). To the resulting solution add
activated charcoal (56.4 g) and stir for 10-15 minutes. Filter the solution through
dia.tomaceous earth to remove the activated charcoal. Add methanol (525 mL) to the
filtrate and then add sodium hydroxide (1,700 mL) over 35 minutes. Stir the resulting
slurry overnight and filter.
Suspend the wet cake in a 66.75% acetonitrile in water solution (10,000 mL).
Heat the resulting mixture to reflux ( ~ 77 to 81 °C) and stir for 20 minutes. Cool the
mixture to 40 oc and add deionized water (2,500 mL) and then cool to 0-5 °C and stir for
2 to 3 hours. Filter the slurry wash the cake with deionized water (500 mL). Dry the
cake overnight at 40 oc in a vacuum oven to furnish 404.2 g of product.
Preparation 2:
Preparation of 2-(6-cyano-quinolin-4-yl)-1-(6-methyl-pyridin-2-yl)-ethanone seed
crystals
To a flask equipped with an Nz purge and overhead stirring, charge 6-cyano-6-
methyl quinoline hydrochloride (10 g, 1 equiv) and THF (100 mL, 10 vol), then cool the
mixture to 20-25 °C. Add NaOt-bu (15.497 g, 3.3 equiv) in portions over 30 minutes to
control the exotherm, keeping the temperature ::S25 °C. Then add a solution of methyl-6-
methyl pyridine-2-carboxylate (11.08 g, 1.5 equiv) in THF (20 mL, 2 vol) dropwise to
keep the temperature between 20 °C and 25 °C. Monitor reaction completion by HPLC
analysis (~2 h). Once complete, cool the mixture to below 15 oc and add 1 N HCI (70
mL). Adjust pH to 8.0-9.0 with 5N NaOH (final pH at 8.8). Add EtOAc (70 mL) and
separate the aqueous layer and wash the organic layer with saturated aqueous sodium
chloride (35 mL) and saturated bicarbonate (35 mL). Concentrate the organic layer in
vacuo to ~5 vol and slowly add MeOH (10 vol), then distill off 10 vol and add back
MeOH (10 vol). Cool the mixture to 5 oc, filter and rinse the cake with MeOH (5 vol),
then dry the cake in vacuo at 40 °C to furnish 11.5 g of product.
Example 1:
Preparation of 2-(6-methyl-pyridin-2-yl)-3-[6-amido-quinolin-4-yl-5,6-dihydro-4Hpyrrolo[
1,2-b ]pyrazole monohydrate
Step!
NC~
VNH
2
0
Vl.._
S=e 0)=("0
Step2
CJ CJ NC~
-----· U,N) HCJ
EtOH, HCI, heat
THF
Meo\y t ~ NC ...., ....,
--- I~,..,
NaOBut, THF N
Q Ph Step3a
Step4
Acetone/water
Recryst
N·N=<
Ph
0
i'N-NH2 ~ TsOH
Step3c
~co3
Step 1: Preparation of 6-cyano-4-methyl-quinoline hydrochloride.
Step3b
2,6-Lutidine/ Toluene/DMF
Reflux
Add 95% ethanol (EtOH) (270 L, 9 vol.), 4-aminobenzonitrile (30.0 kg, 1 equiv)
and 2,3,5,6-tetrachloro-2,5-cyclohexadiene-1,4-dione, (66.81 kg 1.07 equiv) to a 200
gallon reaction vessel equipped with nitrogen purge, condenser, thermocouple, and
overhead agitation. Stir for 2-5 min, then add concentrated hydrochloric acid (HCl)
(62.56 L, 3.0 equiv), then heat to 75 °C. Dilute methyl vinylketone (33.06 L, 1.5 equiv)
in 95% EtOH (30 L, 1 vol.) then add slowly to reaction mixture over 30 min. Monitor
for reaction completion by high performance liquid chromatography (HPLC). Add
tetrahydrofuran (THF) (11 vol., 330 L), at 75 °C, then stir for 1 hour at 60 °C. Cool to
room temperature and stir for 1 additional hour. Filter on agitated filter/dryer, then rinse
with THF (240 L, 8 volumes). Dry overnight under vacuum at 70 oc to give the title
compound (42.9 kg, 82.55%).
1H NMR (DMSO d6): o = 9.047 ppm (d, 4.4 Hz, 1H); 8.865 ppm (d, 1.6 Hz, lH); 8.262
ppm (d, 8.8 Hz, lH); 8.170 ppm (dd, 2.2 Hz, 8.8 Hz, 1H); 7.716 ppm (d, 4.4 Hz, 1H);
2.824 ppm (s, 3H). MS ES+: 169.1; Exact: 168.07.
Step 2: Preparation of 2-(6-cyano-quinolin-4-yl)-1-(6-methyl-pyridin-2-yl)-ethanone.
Combine the 6-cyano-4-methyl-quinoline (28 kg) and THF (9.5 vol.) and cool to
5 °C. Add sodium t-butoxide solid (3.3 equiv.) in portions to the cooled slurry to keep the
batch temperature ::;25 °C. Stir the resulting mixture at 20 oc for 30 min. To a separate
vessel, charge with liquid 6-methyl-2-pyridinecarboxylic acid, methyl ester (1.5 equiv.)
and dilute with THF (2.0 vols.). The 6-methyl-2-pyridinecarboxylic acid, methyl ester
solution is slowly added (20-40 min) while maintaining a temperature of::; 25 °C. Stir the
reaction mixture for 2 hours at 20 oc and monitor by HPLC/TLC (thin layer
chromatography on silica gel) to confirm reaction completion. In a separate vessel, dilute
1.03 kg cone. HCl per kg of 2-(6-cyano-quinolin-4-yl)-1-(6-methyl-pyridin-2-yl)ethanone
with 7.7 vol water. Cool both the reaction mixture and the HCI solution to 5 °C.
Perform a pH adjustment on the reaction mixture by the slow addition of the acid
solution, keeping the temperature <15 °C. Acid solution is added until the pH of the
mixture is 8.0~9.0. After the pH endpoint is obtained, extract the mixture with ethyl
acetate (7 vol.). Wash the organic layer with an aqueous sodium chloride/sodium
bicarbonate solution [0.78 kg sodium chloride per kg of 2-(6-cyano-quinolin-4-yl)-1-(6-
methyl-pyridin-2-yl)-ethanone, and 0.20 kg of sodium bicarbonate (NaHC03) per kg of
2-(6-cyano-quinolin-4-yl)-1-(6-methyl-pyridin-2-yl)-ethanone in 6.6 vol.]. Distill the
organic layer at one atmosphere to remove THF and ethylacetate (EA) until 5 vol. of
concentrated solution remains. Using methanol (10 vol.) perform a solvent exchange to
methanol using a constant add/distill operation while maintaining 5 vol.. Add warm
methanol (MeOH) (1 0 vol. @ 60 °C). Cool the solution to 50 oc, then add seed crystals
obtained by Preparation 2. Cool the mixture gradually to 5 °C, stir for 1 hour, and filter.
Wash the product cake with chilled methanol (5 vols. @ 5 °C) and dry under vacuum at
40 oc until a loss on drying (LOD) specification of <1% is satisfied. Gives the title
compound (31.6 kg, 81% ).
1H NMR (CDCh): o = 8.978 ppm (d, 4.4 Hz, 1H); 8.627 ppm (d, 1.6 Hz, 1H); 8.199 ppm
(d, 8.8 Hz, 1H); 7.874 ppm (d, 7.7 Hz, 1H); 7.837 ppm (dd, 2.2 Hz, 8.8 Hz, 1H); 7.759
ppm (t, 7.7 Hz, 1H); 7.546 ppm (d, 4.4 Hz, lH); 7.416 ppm (d, 7.7 Hz, lH); 5.036 ppm (s,
2H); 2.720 ppm (s, 3H). MS ES+: 288.1; Exact: 287.11.
Step 3a: Preparation of 1-(amino)-2-pyrrolidinone, p-toluene sulfonate.
Combine 1-[(Diphenylmethylene)amino]-2-pyrrolidinone (35.36 g,134 mmoles)
with 15 volumes of toluene (530 mL) in a 1 L reaction flask, add 1 equiv of water (2.43 g,
134.9 mmoles) and heat to 40 °C. Add 1 equiv of p-toluensulfonic acid monohydrate
(25.978 g, 133.8 mmoles). Monitor reaction by TLC, then cool to 20-25 °C. Filter the
slurry and rinse the filter cake with 3 volumes of toluene (105 mL). Dry to a constant
weight in a vacuum dryer at 50 °C to give the title compound (36.14 g, 99.2%).
1H NMR (DMSO): & = 7.472 ppm (dt, 8.2 Hz, 1.9Hz, 2H); 7.112 ppm (m, 2H); 3.472
ppm (t, 7.0 Hz, 2H); 2.303 ppm (m, 5H); 2.012 ppm (m, 2H). MS: ES+ = 179; 157. ES=
171. Exact: 272.08.
Step 3b and 3c: Preparation of intermediates 1-[(6-methyl-pyridin-2-yl)-2-(6-cyanoquinolin-
4-yl)-ethylideneamino]-pyrrolidin-2-one and 3-(6-cyano-quinolin-4-yl)-2-(6-
methyl-pyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazole.
Into a 3-neck, 1 L flask equipped with mechanical stirring, a Dean-Stark
condenser, thermocouple and N2 purge charge 2-(6-cyano-quinolin-4-yl)-1-(6-methylpyridin-
2-yl)-ethanone (25 g, 1 equiv), 1-(amino)-2-pyrrolidinone, p-toluene sulfonate
(27.3 g, 1 equiv), dimethylformamide (DMF) (150 mL, 6 vol), toluene (250 mL, 10 vol)
and 2,6-lutidine (26 mL, 1 vol). Heat the mixture to reflux and periodically remove
water from the trap. Monitor the reaction by HPLC or TLC analysis (5%
MeOH/methylene chloride, silica). After 4 hours, most of the ketone is converted into 1-
[(6-methyl-pyridin-2-yl)-2-(6-cyano-quinolin-4-yl)-ethylideneamino]-pyrrolidin-2-one as
indicated by TLC.
Cool the reaction mixture to 50 to 55 oc and charge potassium carbonate (K2C03)
(20.42 g, 1.66 equiv) into the reaction mixture over a couple of minutes and heat the
reaction mixture back up to reflux. Continue to remove the water collected in the trap
and monitor the reaction by HPLC for the disappearance of hydrazone. After completion
of reaction distill off most of the toluene (total distillate is 350 mL) until the reaction
mixture reaches a temperature of 145 °C. Cool the reaction mixture to ~30 °C and dilute
with water (450 mL) and stir for 1.5 hours at room temperature (RT). Filter the formed
product by filtration and rinse the cake with water 200 mL. After 1 hour under vacuum,
and then dried in a vacuum oven at 70 °C to a consistent weight. The dried solid weighed
28.5 g, 93.2% yield and the purity by HPLC is 97%. The product is used as is in the next
step.
1H NMR (CDCh): o = 9.018 ppm (d, 4.5 Hz, 1H); 8.233 ppm (d, 8.7 Hz, 1H); 8.198 ppm
(dd, 0.5 Hz, 1.8 Hz, 1H); 7.808 ppm (dd, 1.8 Hz, 8.8 Hz, 1H); 7.483-7.444 ppm (m, 2H);
7.380 ppm (d, 7.9 Hz, 1H); 6.936 ppm (d, 7.6 Hz, 1H); 4.422 ppm (t, 7.2 Hz, 2H); 2.970-
2897 ppm (m, 2H); 2.776 ppm (p, 7.2 Hz, 2H); 2.065 ppm (s, 3H). MS ES+: 352.4
Exact: 351.15.
Step 4: Preparation of 2-(6-methyl-pyridin-2-yl)-3-(6-amido-quinolin-4-yl)-5,6-dihydro-
4H-pyrrolo[1,2-b]pyrazole, monohydrate.
Slurry 3-(6-cyano-quinolin-4-yl)-2-(6-methyl-pyridin-2-yl)-5,6-dihydro-4Hpyrrolo[
1,2-b]pyrazole (25.515 kg) and potassium carbonate (0.2 eq.) in 6 volumes of
dimethyl sulfoxide (DMSO). Add dilute hydrogen peroxide solution [35% hydrogen
peroxide (1.25 eq.) to 0.5 volumes of purified water] to the slurry over 2-3.3 hours while
maintaining the temperature between 20-38 °C. Monitor the reaction by HPLC (1 hour).
Add sodium sulfite (0.6 eq.) to 9.1 volumes of purified water. Add the product slurry to
dilute sodium sulfite solution [sodium sulfite (0.6 eq.) in 9.1 volumes of purified water]
while maintaining a temperature of 20-39 °C, stir this slurry for 1-2 hours to ensure all
remaining hydrogen peroxide is completely neutralized. Check for peroxide. Add 1.08
vol. of 32.1% HCl Food Grade to this slurry and stir for 20-30 min. Add activated
charcoal (10% by wt.) to the solution and stir for 20-40 minutes. Filter the crude product
(mostly monohydrate), rinsing the cake with purified water. Add 1.05 vol. of methanol to
the filtrate. Add 5.5 vol. of 2N sodium hydroxide to the filtrate while maintaining a
temperature of 20-30 °C. Stir the slurry for 20-30 min. Ensure pH is> 8.
Filter the slurry, and rinse the cake with purified water. Suspend the wet cake in
28 vol. of a 75%/25% acetone/purified water solution. Heat this slurry to reflux (60 °C)
and stir for 30-45 minutes after the product dissolves. Filter the product solution. Start
the distillation, and add milled seed when the pot temperature reaches 63 °C. Continue
distilling until the distillate volume is 50% of the initial volume. Cool the slurry to 20-
250C over 90 minutes. Then cool the slurry to 0-5 oc over 30-40 minutes. Stir for 2-3
hours at 0-5 °C. Filter the slurry and rinse the product cake on the filter with purified
water. Dry the product under vacuum at 45 octo furnish the title compound (25.4 kg,
90%). Water content by Karl Fischer of 4.6% in monohydrate. Theory: 4.65%.
1H NMR (CDCb): 8 = 9.0 ppm (d, 4.4 Hz, 1H); 8.23-8.19 ppm (m, 2H); 8.315 ppm (dd,
1.9 Hz, 8.9 Hz, 1H); 7.455 ppm (d, 4.4 Hz, 1H); 7.364 ppm (t, 7.7 Hz, 1H); 7.086 ppm (d,
8.0 Hz, 1H); 6.969 ppm (d, 7.7 Hz, 1H); 6.022 ppm (m, 1H); 5.497 ppm (m, 1H); 4.419
ppm (t, 7.3 Hz, 2H); 2.999 ppm (m, 2H); 2.770 ppm (p, 7.2 Hz, 7.4 Hz, 2H); 2.306 ppm
(s, 3H); 1.817 ppm (m, 2H). MS ES+: 370.2; Exact: 369.16.
Alternatively, the monohydrate of the present invention can be prepared by
recrystallization of 2-(6-Methyl-pyridin-2-yl)-3-[6-amido-quinolin-4-yl)-5,6-dihydro-4Hpyrrolo
[ 1 ,2-b ]pyrazole.
Example 2:
· 2-(6-Methyl-pyridin-2-yl)-3-[6-amido-quinolin-4-yl)-5,6-dihydro-4H-pyrrolo[1,2-
b]pyrazole monohydrate
Suspend 2-(6-methyl-pyridin-2-yl)-3-[6-amido-quinolin-4-yl)-5,6-dihydro-4Hpyrrolo[
1,2-b]pyrazole in 28 vol. of a 75%/25% acetone/purified water solution. Heat
this slurry to reflux (60° C) and stir for 30-45 minutes after the product dissolves. Filter
the product solution. Start the distillation, and add milled seed when the pot temperature
reaches 63 °C. Continue distilling until the distillate volume is 50% of the initial volume.
Cool the slurry to 20-25 oc over 90 minutes. Then cool the slurry to 0-5 °C over 30-40
minutes. Stir for 2-3 hours at 0-5°C. Filter the slurry and rinse the product cake on the
filter with purified water. Dry the product under vacuum at 45 °C to furnish the title
compound. The reaction yield is >80%. Product purity is >98% with low total related
substances.
Alternatively, the monohydrate of the present invention can be prepared by
reslurrying of 2-(6-Methyl-pyridin-2-yl)-3-[6-amido-quinolin-4-yl)-5,6-dihydro-4Hpyrrolo[
1,2-b]pyrazole.
Example 3:
2-(6-Methyl-pyridin-2-yl)-3-[6-amido-quinolin-4-yl)-5,6-dihydro-4H-pyrrolo[1,2-
b]pyrazole monohydrate
Prepare 2-(6-Methyl-pyridin-2-yl)-3-[6-amido-quinolin-4-yl-5,6-dihydro-4Hpyrrolo[
I,2-b]pyrazole monohydrate by stirring the compound or active pharmaceutical
ingredient (API) in IO volumes of water at room temperature for I-2 hours, filtering, and
drying at 45 °C under vacuum.
TGF-Bl RECEPTOR I PURIFICATION AND IN VITRO KINASE
REACTIONS
For TGF-~ I Type I (RIT204D) Receptors:
The 6X-HIS tagged cytoplasmic kinase domain of each receptor was expressed
and purified from Sf9 insect celllysates as briefly described below:
Celllysates were clarified by centrifugation and 0.45 uM filtered prior to
purification by Ni/NTA affinity chromatography (Qiagen).
Chromatography Protocol:
Equilibrate with IO CV ofLB, load sample, wash with 10 CV RIPA buffer (50
mM Tris pH 7.5, I50 mM NaCl, I% NP40, ImM EDTA, 0.25% sodium deoxycholate,
added fresh 20 mM ~-mercaptoethanol, I mM PMSF), wash with IO CV LB, wash with
10 CV IX KB (50 mM Tris pH 7.5, I50 mM NaCl, 4 mM MgClz, I mM NaF, 2 mM
~-mercaptoethanol), elute with a linear gradient of IX KB containing 200 mM Imidazole.
Both enzymes were approximately 90% pure and had autophosphorylation
activity.
Reactions: I70-200 nM enzyme in IX KB, compound dilution series in IX
KB/16% DMSO (20 ~tM to I nM final concentration with 4% DMSO final
concentration), reactions are started by adding ATP mix (4 ~tM ATP/1 ~tCi 33P-a-ATP
final concentrations) in IX KB.
Reactions are incubated at 30 °C for I hour. Reactions are stopped and
quantitated using standard TCA/BSA precipitation onto Millipore FB glass fiber filter
plates and by liquid scintillation counting on a MicroBeta JET.
The compound disclosed herein inhibits the TGF-~1 Type I (RIT204D) receptor
kinase domain with an ICso value of 56 nM.
The compounds of the present invention are preferably formulated as
pharmaceutical compositions administered by a variety of routes. Most preferably, such
compositions are for oral 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 (A. Gennaro, et al., ed., 19th ed., Mack
Publishing Co., 1995).
The compounds of the present invention are generally effective over a wide
dosage range. For example, dosages per day normally fall within the range of about
0.0001 to about 30 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 be 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 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.
18

We Claim:
1. A crystalline 2-(6-Methyl-pyridin-2-yl)-3-[6-amido-quinolin-4-yl)-5,6-dihydro-
4H- pyrrolo [ 1 ,2-b] pyrazole monohydrate of formula 1
2. The crystalline 2-(6-methyl-pyridin-2-yl)-3-[6-amido-quinolin-4-yl)-5,6- dihydro-
4H-pyrrolo [l,2-b] pyrazole monohydrate as claimed in claim 1, wherein the Xray
powder diffraction pattern (Cu radiation, λ = 1.54056 A) comprising a peak at
9.05, and one or more peaks selected from the group comprising 11.02, 11.95, and
14.84 (20 +/- 0.10).
3. The crystalline 2-(6-methyl-pyridin-2-yl)-3-[6-amido-quinolin-4-yl)-5,6- dihydro-
4H-pyrrolo[l,2-b] pyrazole monohydrate as claimed in claim 1, wherein the X-ray
powder diffraction pattern (Cu radiation, λ = 1.54056 Å) comprising a peak at
9.05 (20 +/- 0.1°).
4. The crystalline 2-(6-methyl-pyridin-2-yl)-3-[6-amido-quinolin-4-yl)-5,6- dihydro-
4H-pyrrolo[l,2-b]pyrazole monohydrate as claimed in claim 1, characterized by
the solid state 13 C nuclear magnetic resonance having a chemical shift (ppm) of
108.8, 115.6, 122.6, and 171.0 (+/- 0.2) ppm.
5. A pharmaceutical composition comprising any of the compounds as claimed in
claims 1-4 with a pharmaceutically acceptable excipient, diluent, or carrier.
19
6. The compound as claimed in any of claims 1-5, for the manufacture of a
medicament for the treatment of conditions resulting from excessive TGF-β
production in a mammal.
7. The compound as claimed in any one of claims 1-6 for use in the treatment of
prostrate cancer, colorectal cancer, breast cancer, non-small cell lung cancer,
endometrial cancer, testicular cancer, osteosarcoma, or multiple myeloma.