FIELD OF INVENTION
The invention relates to a quinolinyl-pyrrolopyrazole compound and
pharmaceutical composition thereof, in particular their use as TGF-beta signal
transduction inhibitors.
BACKGROUND OF THE INVENTION
The transforming growth factor-beta (TGF-beta) ("TGF-P") polypeptides
influence growth, differentiation, and gene expression in many cell types. The first
polypeptide of this family that was characterized, TGF-P 1, has two identical 112 amino
acid subunits that are covalently hnked. TGF-P 1 is a highly conserved protein with only
a single amino acid difference distinguishing humans from mice. There are two other
members of the TGF-P gene family that are expressed in mammals. TGF-P2 is 71%
homologous to TGF-P 1 (de Martin, et al. (1987) EMBO J. 6:3673-3677), whereas
TGF-P3 is 80% homologous to TGF-pi (Deiynck, et al. (1988) EMBO J 7:3737-3743).
The structural characteristics of TGF-P 1 as determined by nuclear magnetic resonance
(Archer, et al. (1993) Biodiemistry 32:1164-1171) agree with the crystal structure of
TGF-P2 (Daopin, ct al. (1992) Science 257:369-374; Schlunegger and Grutter (1992)
Nature 358:430-434).
There are at least three different extracellular TGF-P receptors, Type 1,11 and III
tliat are involved in the biological functions of TGF-P 1, -P2 and -P3 (For reviews, see
Derynck (1994) TIBS 19:548-553 and Massague (1990) Ann. Rev. Cell Biol. 6:597-641).
The Type I and Type II receptors are transmembrane serine/threonine kinases, which in
the presence of TGF-P form a heteromeric signaling complex (Wrana, et al (1992)
Cell 71: 1003-1014).
The mechanism of activation of the heteromeric signaling complex at the cell
surface has been elucidated (Wrana, et al. (1994) Nature 370: 341-347). TGF-P first
binds the type II receptor that is a constitutively active transmembrane serine/threonine
kinase. The type I receptor is subsequently recruited into the complex, phoshorylated at
the GS domain and activated to phosphoryiate downstream signaling components (e.g.
Smad proteins) to initiate the intracellular signaling cascade. A constitutively active type I
receptor (T2G4D mutant) has been shown to effectively transduce TGF-P responses, thus
bypassing the requirement for TGF-p and the type 11 receptor (Wieser, et al. (1995)
EMBO J 14: 2199-2208). Although no signaling function has been discovered for the
type III receptor, it does increase TGF-P2's affinity for the type II receptor making it
essentially equipotent with TGF-pl and TGF-p3 (Lopez-Casillas, et al. (1993) Cell
73:1435-1444).
Vascular endothelial cells lack the Type III receptor. Instead endothelial cells
express a structurally related protein called endoglin (Cheifetz, et al. (1992) J. Biol.
Chem. 267:19027-19030), which only binds TGF-pl and TGF-p3 with high affinity.
Thus, the relative potency of the TGF-P's reflects the type of receptors expressed in a cell
and organ system. In addition to the regulation of the components in the multi-factorial
signaling pathway, the distribution of the synthesis of TGF-P polypeptides also affects
physiological function. The distribution of TGF-P2 and TGF-P3 is more lunited
(Derynck, et al. (1988) EMBO J 7:3737-3743) than TGF-pl, e.g., TGF-p3 is limited to
tissues of mesenchymal origin, whereas TGF-pl is present in both tissues of
mesenchymal and epithelial origin.
TGF-pl is a multifunctional cj'tokine critical for tissue repair. High
concentrations of TGF-P 1 are delivered to the site of injury by platelet granules (Assoian
and Spom (1986) J. Cell Biol. 102:1217-1223). TGF-pl initiates a series of events that
promote healing including chemo taxis of cells such as leukocytes, monocytes and
fibroblasts, and regulation of growth factore and cytokines involved in angiogenesis, cell
division associated with tissue repair and inflammatory responses. TGF-pl also
stimulates the synthesis of extracellular matrix components (Roberts, et al. (1986) Proc.
Natl. Acad. Sci. USA 83:4167-4171; Spom, et al. (1983) Science 219:1329-1330;
Massague (1987) Cell 49:437-438) and most importantly for understanding the
pathophysiology of TGF-Pl, TGF-pi autoregulates its own synthesis (Kim, et al. (1989)
J. Biol. Chem. 264:7041-7045).
The compounds disclosed herein may also exhibit other kinase activity, such as
p38 kinase inhibition and/or KDR (VEGFR2) kmase inhibition. Assays to determine such
kinase activity are known in the art and one skilled in the art would be able to test the
disclosed compounds for such activity.
SUMMARY OF THE INVENTION
The disclosed invention also relates to the select compound of Formula 11:
Formula II
2-(6-raethyl-pyridin-2-yI)-3-{6-aniido-quinolin-4-yl)-5,6-dihydro-4H-pyrrolo[l,2-
b]pyrazoIe
and the pharraaceutically acceptable salts thereof.
The compound above is generically disclosed and claimed in PCT patent application
PCT/US02/11884, filed^l3 May 2002, which claims priority from U.S. patent application
U.S.S.N. 60/293,464, filed 24 May^OOl, and incorporated herein by reference. The
above compound has been selected for having a surprisingly superior toxicology profile
over the compounds specifically disclosed in application cited above.
DETAILED DESCRIPTION OF THE INVENTION
The term "effective amount" as used in "an effective amount of a compound of
Formula I," for example, refers to an amount of a compound of the present invention that
is capable of inhibiting TGF-beta.
The term pM refers to micromolar.
The general chemical terms used herein have their usual meanings.
The following abbreviations are used throughout the synthesis Schemes and
Examples:
DMF refers to dimethyl formamide
THF refers to tetrahydrofiiran
Ms refers to mesyl which is methylsulfonyl
THP refers to tetrahydropyran
The compounds disclosed herein can be made according to the following schemes
and examples. The examples should in no way be understood to be limiting in any way
as to how the compounds may be made.
The following scheme illustrates the preparation of the compound of Formula I.
The following scheme illustrates the preparation of the compound of Formula II.
The following examples further illustrate the preparation of the compounds of this
invention as shown schematically in Schemes I and II.
Example 1
Preparation of 7-(2-morpholin-4-y]-ethoxy)-4-(2-pyridin-2-yI-5,6-dihydro-4H-
pyrrolofl,2-bjpyrazol-3-yl)-quinoline
A. Preparation of 4-{2-pyridin-2-yl-5,6-dihydro-4H-pyrrolo[l,2-bIpyrazol-3-yO-
7-[2-(tetrahydropyran-2-yloxy)ethoxy]quinoline
Heat 4-(2-pyridin-2-yl-5,6-dihydro-4H-pyrrolo[l ,2-b]pyi-azol-3-yl)-quinolin-7-ol (376
mg, 1.146 rnmol), cesium carbonate (826 mg, 2.54 mmol), and 2-(2-
bromoethoxy)tetrahydro-2H-pyran (380 [lL, 2.52 mmol) in DMF (5 mL) at 120 °C for 4
hours. Quench the reaction with saturated sodium chloride and then extract with
chloroform. Dry the organic layer over sodium sulfate and concentrate in vacuo. Purify
the reaction mixture on a silica gel column eluting with dichloromethane to 10%
methanol in dichloromethane to give the desired subtitled intermediate as a yellow oil
(424 mg, 81%). MS ES^m/e 457.0 (M+1).
B. Preparation of 2-[4-(2-pyridin-2-yI-5,6-dihydro-4H-pyrrolo[l,2-b]pyrazol-3-
yl)-quinoKn-7-yloxy]-ethanoI
Heat a solution of 4-(2-pyridin-2-yl-5,6-dihydro-4H-pyrrolo[l,2-b]pyrazol-3-yl)-
7-[2-(tetrahydropyran-2-yloxy)ethoxy]quinoline (421 mg, 0.92 mmol) in acetic acid:
tetrahydrofuran: water (4:2:1) (20 mL). Remove the solvent in vacuo and recover the
residue with chloroform:isopropyl (3:1). Wash the organic layer with saturated sodium
bicarbonate and dry over sodium sulfate. Concentrate in vacuo. The residue will be pure
enough for the next step in the scheme (425 mg, 100%). MS ES'^m/e 373.1 (M+1).
C. Preparation of methanesulfonic acid 2-[4-(2-pyridin-2-yI-5,6-dihydro-4H-
pyrrolo [1,2-b]pyrazol-3-yI)-quinolin-7-yloxyl-ethyl ester
Stir a solution of 2-[4-(2-pyridin-2-yl-5,6-dihydro-4H-pyrrolo[l,2-b]pyrazol-3-
yl)-quinolin-7-yloxy]-ethanol (293 mg, 0.78 nunol) and methane sulfonyl chloride (68
|iL, 0.81 ml) in dried pyridine (5 mL) for 2 hours. Remove the pyridine in vacuo and
recover the residue with chloroform. Wash the organic layer with saturated sodium
bicarbonate and dry over sodium sulfate to give the desired subtitled intermediate as a
white foam (425 mg, 100%). MS ES*m/e 451.1 (M+1).
D. Preparation of 7-(2-morphoIin-4-yl-etlioxy)-4-(2-pyridin-2-yI-5,6-dihydro-
4H-pyrroIo[l,2-b]pyrazol-3-yI)-quinoIine
Heat raethanesulfonic acid 2-[4-(2-pyridin-2-yl-5,6-dihydro-4H-pyrrolo[l,2-
b]pyrazol-3-yl)-quinolin-7-yloxy]-ethyl ester (87 mg, 0.19 mmol) with morpholine (1
mL) at 50 "C for 4 hours. Remove the morpholine in vacuo and then extract the product
with isopropyl alcohol:chloroform (1:3). Wash the organic layer with sodium chloride
and dry over sodium sulfate. Concentrate in vacuo to give the desired title product as a
slight yellow solid (83 mg, 100%). MS ES^m/e 442.0 (M+1).
EXAMPLE 2
Preparation of
2-(6-niethyl-pyridln-2-yI)-3-[6-amido-quinolin-4-yl)-5,6-dihydro-4H-pyrroIo[l,2-
bjpyrazole
A. Preparation of 6-bromo-4-methyI-quinoline
Stir a solution of 4-bromo-phenylamine (1 eq), in 1,4-dioxane and cool to
approximately 12 °C. Slowly add sulfuric acid (2 eq) and heat at reflux. Add
methylvinyl ketone (1.5 eq) dropwise into the refluxing solution. Heat the solution for 1
hour after addition is complete. Evaporate the reaction solution to dryness and dissolve in
methylene chloride. Adjust the solution to pH 8 with 1 M sodium carbonate and extract
three times with water. Chromatograph the residue on SiOa (70/30 hexane/ethyl acetate)
to obtain the desired subtitled intermediate.
MS ES+ ra/e = 158.2 (M+1).
B. Preparation of 6-methyI-pyridinc-2-carboxyIic acid methyl ester
Suspend 6-methyI-pyridine-2-carboxylic acid (10 g, 72.9 rainol) in methylene
cWoride (200 mL). Cool to 0 °C. Add methanol (10 mL), 4-dimethylaminopyridine (11.6
g, 94.8 mmol), and l-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC)
(18.2 g, 94.8 mmol). Stir the mixture at room temperature for 6 hours, wash with water
and brine, and dry over sodium sulfate. Filter the mixture and concentrate in. vacuo.
Chromatograph tlie residue on SiOa (50% ethyl acetate/hexanes) to obtain the desired
subtitled intermediate, 9.66 g (92%), as a colorless liquid.
'H NMR (CDCI3) 6 7.93-7.88 (m, IH), 7.75-7.7 (m, IH), 7.35-7.3 (m, IH), 4.00 (s, 3H),
2.60 (s, 3H).
C. Preparation of 2-(6-bromo-quinolin-4-y])-l-(6-nietby]-pyridiii-2-yl)-ethanone
Dissolve 6-bromo-4-methyl-quinoline (38.5 g, 153 mmol) in 600 mL dry THF.
Cool to -70° C and treat with the dropwise addition of 0.5 M potassium
hexamethyldisilazane (KN(SiMe3)2 (400 mL, 200 mmol) over 2 hours while keeping the
temperature below -65 °C. Stir the resultant solution at -70°C for 1 hour and add a
solution of 6-methylpyridine-2-carboxylic acid methyl ester (27.2,180 mmol) in 100 mL
dry THF dropwise over 15 minutes. During the addition, the mixture will turn from dark
red to pea-green and form a precipitate. Stir the mixture at -70°C over 2 hours then allow
it to warm to ambient temperature with stirring for 5 hours. Cool the mixture then quench
with 12 N HCl to pH=], Raise the pH to 9 with solid potassium carbonate. Decant the
solution from the solids and extract twice with 200 mL ethyl acetate. Combine the
organic extracts, wash with water and dry over potassium carbonate. Stir the solids in
200 mL water and 200 mL ethyl acetate and treat with additional potassium carbonate.
Separate the organic portion and dry with the previous etliyl acetate extracts. Concentrate
the solution in vacuo to a dark oil. Pass the oil through a 300 mL silica plug with
methylene chloride then ethyl acetate. Combine the appropriate fractions and concentrate
in vacuo to yield an amber oil. Rinse the oil down the sides of the flask with methylene
chloride then dilute with hexane while swirling the flask to yield 38.5 g (73.8 %) of the
desired subtitled intermediate as a yellow solid.
MSES+ = 341(M+1).

D. Preparation of l-[2-(6-bromo-quinoIin-4-yI)-l-(6-methyl-pyridin-2-yl)-
ethyHdeneaniino]-pyrrolidin-2-one
Stir a mixture of 2-(6-bromo-quinolin-4-yl)-l-(6-methyl-pyridin-2-yl)-ethanone
(38.5 g, 113 mmol) and I-ammopyrrolidinone hydrochloride (20 g, 147 nrniol) in 115
mL pyridine at ambient temperature for 10 hours. Add about 50 g 4 A unactivated
sieves. Continue stirring an additional 13 h and add 10-15 g silica and filter the
mixture through a 50 g silica plug. Elute the silica plug with 3 L ethyl acetate.
Combine the filtrates and concentrate in vacuo. Collect the hydrazone precipitate by
filtration and suction dry to yield 33.3 g (69.7%) of the desired subtitled intermediate
as an off-white solid.
MSES'' = 423(M+1).
E. Preparation of 6-bromo-4-[2-(6-methyl-pyridin-2-yl)-5,6-dihydro-4H-
pyrrolo{l,2-b|p>Tazol-3-yl|-quinonne
To a mixture of (1.2 eq.) cesium carbonate and l-[2-(6-bromo-quinolin-4-yl)-l-
(6-methyl-pyridin-2-yl)-ethylideneamino]-pyn-olidin-2-one (33.3 g, 78.7 mmol) add
300 mL dry N,N-dimethylfonnamide. Stir the mixture 20 hours at 100°C. The
mixture may turn dark during the reaction. Remove the N,N-dimethylfonnamide in
vacuo. Partition the residue between water and methylene chloride. Extract the
aqueous portion with additional methylene chloride. Filter the organic solutions
through a 300 mL silica plug, eluting with 1.5 L methylene chloride, 1.5 L ethyl
acetate and 1.5 L acetone. Combine the appropriate firactions and concentrate in
vacuo. Collect the resulting precipitate by filtration to yield 22.7 g (71.2%) of the
desired subtitled intermediate as an off-white solid.
MSES* = 405(M+1).
F. Preparation of 4-p-(6-methyl-pyridin-2-yl)-S,6-dihydro-4H-pyrrolo[l,2-
b]pyrazol-3-y]I-quinoIine-6-carboxyUc acid methyl ester
Add6-bromo-4-[2-(6-methyl-pyridin-2-yI)-5,6-dihydro-4H-pyrrolo[l,2-
b]pyrazol-3-yl]-quinoline (22.7 g, 45 mmol) to a mixture of sodium acetate (19 g, 230
mmol) and the palladium catalyst [1,1'-
bis(diphenylphosphino)ferroceneJdichloropalladium(II), complex with

dicUoromethajne (1:1) (850 mg, 1.04 mmol) in 130 mL methanol. Place the mixture
under 50 psi carbon monoxide atmosphere and stir wliile wanning to 90° C over 1 hour
and with constant charging with additional carbon monoxide. Allow the mixture to
cool over 8 hours, recharge again with carbon monoxide and heat to 90 °C. The
pressure may rise to about 75 PSI. The reaction is complete in about an hour when the
pressure is stable and tic (1:1 toluene/acetone) shows no remaining bromide. Partition
the mixture between methylene chloride (600 roL) and water (1 L). Extract the
aqueous portion with an additional portion of methylene chloride (400 mL.) Filter the
organic solution through a 300 mL silica plug and wash with 500 mL methylene
chloride, 1200 mL ethyl acetate and 1500 mL acetone. Discard the acetone portion.
Combine appropriate fractions and concentrate to yield 18.8 g (87.4%) of the desired
subtitled intermediate as a pink powder.
MSES"' = 385(M+1).
G. Preparation of 2-(6-methyl-pyridin-2-yI)-3-{6-amido-quinolin-4-y0-5,6-
dihydro-4H-pyrrolo[l,2-b]pyrazole
Warm a mixture of 4-[2-(6-methyl-pyridin-2-yl)-5,6-dihydro-4H-py^rolo[l,2-
b]pyrazol-3-yl]-quinoline-6-carboxylic acid methyl ester in 60 mL 7 N ammonia in
methanol to 90 "C in a stainless steel pressure vessel for 66 hours. The pressure will rise
to about 80 PSL Maintain the pressure for the duration of the reaction. Cool the vessel
and concentrate the brown mixture in vacuo. Purify the residual solid on two 12 g Redi-
Pak cartridges coupled in series eluting with acetone. Combine appropriate jfractions and
concentrate in vacuo. Suspend the resulting nearly white solid in metliylene chloride,
dilute with hexane, and filter. Tlie collected off-white solid yields 1.104 g (63.8%) of the
desired title product.
MSES* = 370(M+1).

The compounds disclosed herein were tested by the following protocols for TGF-
P inhibition, as described below in the protocol description.
TGF-6 RECEPTOR I PURIFICATION AND IN VITRO KINASE
REACTIONS
For TGF-p Type I (RIT204D) Receptors:
The 6X-HIS tagged cytoplasmic kinase domain of each receptor was expressed
and purified from Sf9 insect cell lysates as briefly described below:
Cell pellets after 48-72 hours of infection were lysed in lysis buffer (LB: 50 mM
Tris pH 7.5, 150 mM NaCI, 50 mM NaF, 0.5% NP40 with freshly added 20 mM
p-mercaptoethanol, 10 mM imidazole, 1 mM PMSF, IX EDTA-free Complete Protease
Inhibitor(Boehringer Mannheim).
Cell lysates were clarified by centrifugation and 0.45 uM filtered prior to
purification by Ni/NTA affinity chromatography (Qiagen).
Chromatography Protocol:
Equilibrate with 10 CV of LB, load sample, wash with 10 CV RIPA buffer (50
mM Tris pH 7.5, 150 mM NaCI, 1% NP40, ImM EDTA, 0.25% sodium deoxycholate,
added fresh 20 mM P-mercaptoethanol, 1 mM PMSF), wash with 10 CV LB, wash with
10 CV IX KB (50 mM Tris pH 7.5. 150 mM NaCI, 4 mM UgCh, 1 mM NaF. 2 mM
P-mercaptoethanoI), elute with a linear gradient of IX KB containing 200 mM Imidazole.
Both enzymes were approximately 90% pure and had autophosphorylation
activity.
Reactions: 170-200 nM enzyme in IX KB, compound dilution series in IX
KB/16% DMSO (20 nM to 1 nM final concentration with 4% DMSO final
concentration), reactions started by adding ATP mix (4 iiM ATP/1 nCi ^¥-Y-ATP final
concentrations) in IX KB.
Reactions were incubated at 30 "C for 1 hour. Reactions were 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 compounds disclosed herein inhibit the TGF-p Type I (RJT204D) receptor
kinase domain with IC50 values <20 fxM, while exhibiting less toxicity in vivo than
structurally related compounds as disclosed in PCT patent application PCT/US02/11S84
identified above.
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-P latent protein is undesirably activated or converted to active TGF-P protein or
wherein TGF-p receptors are upregulated or wherein the TGF-p 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 tlie biological activity of TGF-P is
undesirably high, regardless of the cause.
A number of diseases have been associated with TGF-p 1 over production.
Inhibitors of TGF-B intracellular signaling patliway are useful treatments for
fibroproliferative diseases. Specifically, fibroproliferative diseases include kidney
disorders associated with unregulated TGF- B activity and excessive fibrosis including
glomerulonephritis (GN), such as mesangial proliferative GN, immune GN, and
crescentic GN. Other renal conditions include diabetic nephropathy, renal interstitial
fibrosis, renal fibrosis in transplant patients receiving cyclosporin, and HIV-associated
nephropathy. Collagen vascular disorders include progressive systemic sclerosis,
polymyositis, scleroderma, demiatomyositis, eosinophilic fascitis, morphea, or those
associated with the occurrence of Raynaud's syndrome. Lung fibroses resulting from
excessive TGF- B activity include adult respiratory distress syndrome, idiopathic
pulmonary fibrosis, and interstitial pulmonary fibrosis often associated with autoimmune
disorders, such as systemic lupus erythematosus and scleroderma, chemical contact, or
allergies. Another autoimmune disorder associated with fibroproliferative characteristics
is rheumatoid arthritis.
Eye diseases associated with a fibroproliferative condition include retinal
reattachment surgery accompanying proliferative vitreoretinopathy, cataract extraction
with intraocular lens implantation, and post glaucoma drainage surgery are associated
with TGF-P 1 overproduction.
Fibrotic diseases associated with TGF-pl overproduction can be divided into
chronic conditions such as fibrosis of the kidney, lung and liver and more acute
conditions such as dennal scarring and restenosis (Chamberlain, J. Cardiovascular Drug
Reviews, 19(4):329-344). Synthesis and secretion of TGF-pl by tumor cells can also
lead to immune suppression such as seen in patients with aggressive brain or breast
tumors (Arteaga, et al. (1993) J. Clin. Invest. 92:2569-2576). The course of Leishmanial
infection in mice is drastically altered by TGF-Pl (Barral-Netto, ct al. (1992) Science
257:545-547). TGF-PI exacerbated .the disease, whereas TGF-pi antibodies halted the
progression of the disease in genetically susceptible mice. Genetically resistant mice
became susceptible to Leishmanial infection upon administration of TGF-(5l.
The profound effects of TGF-^l on extracellular matrix deposition have been
reviewed (Rocco and Ziyadeh (1991) in Contemporary Issues in Nephrology v.23.
Hormones, autocoids and the kidney, ed. Jay Stein, Churchill Livingston, New York
pp.391-410; Roberts, et al. (1988) Rec. Prog. Hormone Res. 44:157-197) and include the
stimulation of the synthesis and the inhibition of degradation of extracellular matrix
components. Since the structure and filtration properties of the glomerulus are largely
determined by the extracellular matrix composition of the mesangium and glomerular
membrane, it is not surprising that TGF-P1 has profound effects on the kidney. The
accumulation of mesangial matrix in proliferative glomerulonephritis (Border, et al.
(1990) Kidney Int. 37:689-695) and diabetic nephropathy (Mauer, et al. (1984) J. Clin.
Invest. 74:1143-1155) are clear and dominant pathological features of the diseases. TGF-
Pl levels are elevated in human diabetic glomerulosclerosis (advanced neuropathy)
(Yamaraoto, et al. (1993) Proc. Natl. Acad. Sci. 90:1814-1818). TGF-jil is an important
mediator in the genesis of renal fibrosis in a number of animal models (Phan, et al. (1990)
Kidney Int. 37:426; Okuda, et al. (1990) J. Clin. Invest. 86:453). Suppression of
experimentally induced glomerulonephritis in rats has been demonstrated by antiserum
against TGF-pi (Border, et al. (1990) Nature 346:371) and by an extracellular matrix
protein, decorin, which can bind TGF-Pl (Border, et al. (1992) Nature 360:361-363).
Too much TGF-Pl leads to dennal scar-tissue formation. Neutralizing TGF-(31
antibodies injected into the margins of healing wounds in rats have been shown to inhibit
scarring without interfering with the rate of wound healing or the tensile strength of the
wound (Shah, et al. (1992) Lancet 339:213-214). At the same time there was reduced
angiogenesis, reduced number of macrophages and monocytes in the wound, and a
reduced amount of disorganized collagen fiber deposition in the scar tissue.
TGF-pI maybe a factor in the progressive thickening of the arterial wall which
results from the proliferation of smooth muscle cells and deposition of extracellular
matrix in the artery after balloon angioplasty. The diameter of the restenosed artery may
be reduced 90% by this tliickening, and since most of the reduction in diameter is due to
extracellular matrix rather than smooth muscle cell bodies, it may be possible to open
these vessels to 50% simply by reducing extensive extracellular matrix deposition. In
uninjured pig arteries transfected in vivo with a TGF-pi gene, TGF-Pl gene expression
was associated with both extracellular matrix synthesis and hyperplasia (Nabel, et al.
(1993) Proc. Natl. Acad. Sci. USA 90:10759-10763). Tlie TGF-pI induced hyperplasia
was not as extensive as that induced with PDGF-BB, but the extracellular matrix was
more extensive with TGF-Pl transfectants. No extracellular matrix deposition was
associated with FGF-1 (a secreted form of FGF) induced hyperplasia in this gene transfer
pig model (Nabel (1993) Nature 362:844-846).
There are several types of cancer where TGF-jil produced by the tumor may be
deleterious. 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 TGF-pl. TGF-pl 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-3
(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-pl 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

TGF-Pl 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 TGF-Pl also showed decreased NK activity and increased tumor growth in
nude mice (Wallick, et al. (1990) J. Exp. Med. 172:1777-1784). Thus, TGF-P secreted by
breast tumors may cause an endocrine immune suppression. High plasma concentrations
of TGF-pi 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 TGF-P 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 TGF-pl
can be used to identify at risk patients and 2) that reduction of TGF-P 1 could decrease the
morbidity and mortality of these common treatments for breast cancer patients.
Many malignant cells secrete transforming growth factor-P (TGF-P), a j>otent
immunosuppressant, suggesting that TGF-P production may represent a significant tumor
escape mechanism from host immunosurveilJance. Establishment of a leukocyte sub-
population with disrupted TGF-P signaling in the tumor-bearing host offers a potential
means for immunotherapy of cancer. A transgenic animal model with disrupted TGF-P
signaling in T cells is capable of eradicating a normally lethal TGF-P over expressing
lymphoma tumor, EL4 (Gorelik and Flavell, (2001) Nahire Medicine 7(10): 1118-1122).
Down regulation of TGF-P secretion in tumor cells results in restoration of
immunogenicity in the host, while T-cell insensitivity to TGF-P results in accelerated
differentiation and autoimmunity, elements of which may be required in order to combat
self-antigen-expressing tumors in a tolerized host. The immunosuppressive effects of
TGF-P 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 TGF-P neutralizing antibody was capable of
reversing the effect in culture, indicating that TGF-p signaling inhibitors may have utility
in reversing the immune suppression present in this subset of HIV patients.

During the earliest stages of carcinogenesis, TGF-(31 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, tiunor cells
appear to escape from TGF-p-dependent growth inhibition in parallel with the appearance
of bioactive TGF-p in the microenvironment. The dual tumor suppression/tumor
promotion roles of TGF-P have been most clearly elucidated in a transgenic system over
expressing TGF-P in keratinocytes, Wliile the transgenics were more resistant to
formation of benign skin lesions, tlie rate of metastatic conversion in the transgenics was
dramatically increased (Cui, et al (1996) Cell 86(4):53I-42). The production of TGF-pi
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 TGF-P by human cancers occurs as a relatively late event during tumor
progression. Further, this tumor-associated TGF-p provides the tumor cells with a
selective advantage and promotes tumor progression. The effects of TGF-P on cell/ceD
and cell/stroma interactions result in a greater propensity for invasion and metastasis.
Tumor-associated TGF-P may allow tumor cells to escape from immune surveillance
since it is a potent inhibitor of the clonal expansion of activated lymphocytes. TGF-P has
also been shown to inliibit the production of angiostatin. Cancer therapeutic modalities
such as radiation therapy and chemotherapy induce the production of activated TGF-P in
the tumor, thereby selecting outgrowth of malignant cells that are resistant to TGF-P
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 TGF-p-mediated signal transduction might be a very effective
therapeutic strategy. The resistance of tumor cells to TGF-p has beeii shown to negate
much of the cjtotoxic effects of radiation therapy and chemotherapy and the treatment-
dependent activation of TGF-P in the stroma may even be detrimental as it can make the
microenviroiunent more conducive to tumor progression and contributes to tissue damage
leading to fibrosis. The development of a TGF-p signal transduction inhibitors is likely
to benefit the treatment of progressed cancer alone and in combination with other
therapies.
The compounds are usefiil for the treatment of cancer and other disease states
influenced by TGF-p by inhibiting TGF-P in a patient in need thereof by administering
said compound(s) to said patient. TGF-P would also be useful against atherosclerosis
(T.A. McCaffrey: TGF-ps and TGF-p Receptors in Atherosclerosis: Cytokine and Growth
Factor Reviews 2000,11, 103-114) and Alzheimer's (Masliah, E.; Ho, G.; Wyss-Coray,
T.: Functional Role of TGF-p in Alzheimer's Disease Microvascular Injury: Lessons from
Transgenic Mice: Neurochemistiy International 2001, 39, 393-400) diseases.
PHARMACEUTICAL COMPOSITIONS
The compositions of the present invention are therapeutically effective amounts of
the TGF-P antagonists, noted above. The composition may be formulated with common
excipients, diluents or carriers, and compressed into tablets, or formulated elixirs or
solutions for convenient oral adnunistration or administered by intramuscular intravenous
routes. The compounds can be administered transdermally and maybe formulated as
sustained release dosage forms and the like.
The method of treating a human patient according to the present invention
includes administration of the TGF-P antagonists. The TGF-P antagonists are formulated
into formulations which may be administered by the oral and rectal routes, topically,
parenterally, e.g., by injection and by continuous or discontinuous intra-arterial infusion,
in the form of, for example, tablets, lozenges, sublingual tablets, sachets, cachets, elixirs,
gels, suspensions, aerosols, ointments, for example, containing from 1 to 10% by weight
of the active compound in a suitable base, soft and hard gelatin capsules, suppositories,
injectable solutions and suspensions in physiologically acceptable media, and sterile
packaged powders adsorbed onto a support material for making injectable solutions.
Advantageously for tliis purpose, compositions may be provided in dosage urut form,
preferably each dosage imit containing from about 5 to about 500 mg (from about 5 to 50
mg in the case of parenteral or inhalation administration, and from about 25 to 500 mg in
the case of oral or rectal administration) the compounds. Dosages from about 0.5 to about
300 mg/kg per day, preferably 0.5 to 20 mg/kg, of active ingredient may be administered
although it will, of course, readily be imderstood that the amount of the compound
actually to be administered will be determined by a physician, in the light of all the
relevant circumstances including the condition to be treated, the choice of compound to
be administered and the choice of route of administration and therefore the above
preferred dosage range is not intended to limit the scope of the present invention in any
way.
The formulations useful for separate administration of the TGF-P antagonists will
normally consist of at least one compound selected from the compounds specified herein
mixed with a carrier, or diluted by a carrier, or enclosed or encapsulated by an ingestible
carrier in the form of a capsule, sachet, cachet, paper or other container or by a disposable
container such as an ampoule. A carrier or diluent may be a soh'd, semi-solid or liquid
material which serves as a vehicle, excipient or medium for the active therapeutic
substance. Some examples of the diluents or carrier which may be employed in the
pharmaceutical compositions of the present invention are lactose, dextrose, sucrose,
sorbitol, maimitol, propylene glycol, liquid paraffin, white soft paraffin, kaolin, fumed
silicon dioxide, microcrystalline cellulose, calcium silicate, silica, polyvinylpyrrolidone,
cetostearyl alcohol, starch, modified starches, gum acacia, calcium phosphate, cocoa
butter, ethoxylated esters, oil of theobroma, arachis oil, alginates, tragacanth, gelatin,
syrup, methyl cellulose, polyoxyethylene sorbitan monolaurate, ethyl lactate, methyl and
propyl hydroxybenzoate, sorbitan trioleate, sorbitan sesquioleate and oleyl alcohol and
propellants such as trichloromonofluororaethane, dichlorodifluoromethane and
dichlorotetrafluoroethane. In the case of tablets, a lubricant may be incorporated to
prevent sticking and binding of the powdered ingredients in the dies and on the punch of
the tableting machine. For such purpose there may be employed for instance aluminum,
magnesium or calcium stearates, talc or mineral oil.
Preferred pharmaceutical forms of the present invention are capsules, tablets,
suppositories, injectable solutions, creams and ointments. Especially preferred are
formulations for inhalation application, such as an aerosol, for injection, and for oral
ingestion.
We Claim:
1. A Quinolinyl-Pyrrolopyrazole compound according to formula II
Formula II
and pharmaceutically acceptable salts thereof.
2. The Quinolinyl-Pyrrolopyrazole compound as claimed in claim 1, wherein the
compound is 2- (6-methyl-pyridin-2-yl)-3- [6-amido-quinolin-4-yl) -5,6-dihydro-
4H-pyrrolo [1, 2-b] pyrazole and pharmaceutically acceptable salts thereof.
3. A pharmaceutical composition comprising a compound as claimed in Claim 1
or a pharmaceutically acceptable salt or ester thereof as an active ingredient
together with a pharmaceutically acceptable diluent or carrier wherein the
amount of active ingredient is present in an amount ranging from 5 to 500 mg.

A compound according to formula II and the pharmaceutically acceptable salts
thereof and the method of treating cancer in a patient in need thereof by
administration of said compound.