U.S. patent application number 10/571100 was filed with the patent office on 2007-01-25 for diaryl ureas with kinase inhibiting activity.
Invention is credited to Jacques Dumas, Gaetan Ladouceur, Mark Lynch, William J. Scott, Scott Wilhelm.
Application Number | 20070020704 10/571100 |
Document ID | / |
Family ID | 33545309 |
Filed Date | 2007-01-25 |
United States Patent
Application |
20070020704 |
Kind Code |
A1 |
Wilhelm; Scott ; et
al. |
January 25, 2007 |
Diaryl ureas with kinase inhibiting activity
Abstract
The present invention provides methods of using aryl ureas to
treat diseases and conditions associated with signal transduction
pathways comprising at least one of raf, VEGFR, PDGFR, p38 and/or
FLT-3. The present invention also provides compositions and methods
for identifying conditions and diseases which can be modulated with
compounds of the present invention. These methods facilitate the
selection of subjects who can be efficiently treated with compounds
of the present invention. Additionally, the invention provides
methods for monitoring subjects who have been administered a
compound of the present invention.
Inventors: |
Wilhelm; Scott; (Orange,
CT) ; Dumas; Jacques; (Bethany, CT) ;
Ladouceur; Gaetan; (Guilford, CT) ; Lynch; Mark;
(Madison, CT) ; Scott; William J.; (Guilford,
CT) |
Correspondence
Address: |
MILLEN, WHITE, ZELANO & BRANIGAN, P.C.
2200 CLARENDON BLVD.
SUITE 1400
ARLINGTON
VA
22201
US
|
Family ID: |
33545309 |
Appl. No.: |
10/571100 |
Filed: |
May 19, 2004 |
PCT Filed: |
May 19, 2004 |
PCT NO: |
PCT/US04/15655 |
371 Date: |
July 28, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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60471735 |
May 20, 2003 |
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60520399 |
Nov 17, 2003 |
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60556062 |
Mar 25, 2004 |
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Current U.S.
Class: |
435/7.21 ;
435/23; 514/114; 514/616 |
Current CPC
Class: |
A61P 31/22 20180101;
A61P 35/02 20180101; A61P 11/06 20180101; A61P 9/00 20180101; A61K
31/4412 20130101; A61P 31/18 20180101; A61K 31/443 20130101; A61P
31/06 20180101; A61K 31/4418 20130101; A61P 3/10 20180101; A61P
29/00 20180101; A61P 19/02 20180101; A61P 25/00 20180101; G01N
33/57438 20130101; A61P 13/08 20180101; G01N 33/5743 20130101; A61P
11/00 20180101; A61K 31/00 20130101; A61P 33/06 20180101; A61K
45/06 20130101; A61P 35/04 20180101; A61P 37/00 20180101; A61P
37/06 20180101; A61P 43/00 20180101; A61P 39/02 20180101; A61P 1/02
20180101; A61K 31/4412 20130101; A61P 25/28 20180101; A61P 35/00
20180101; A61K 31/66 20130101; A61K 31/4418 20130101; A61P 9/12
20180101; A61P 31/04 20180101; A61P 13/12 20180101; A61P 5/14
20180101; A61L 2300/416 20130101; A61K 31/443 20130101; A61P 13/10
20180101; A61K 31/165 20130101; A61P 17/00 20180101; A61P 1/16
20180101; A61P 17/06 20180101; A61L 2300/204 20130101; A61P 33/02
20180101; A61P 37/02 20180101; A61P 37/04 20180101; A61L 31/16
20130101; A61P 9/10 20180101; A61P 9/08 20180101; A61P 19/00
20180101; A61P 15/00 20180101; A61P 27/02 20180101; A61P 37/08
20180101; G01N 33/5041 20130101; A61P 7/00 20180101; A61P 9/04
20180101; A61P 19/10 20180101; A61P 7/02 20180101; A61P 1/04
20180101; A61P 27/06 20180101; A61K 31/16 20130101; A61P 17/02
20180101; A61P 7/06 20180101; A61K 2300/00 20130101; A61K 2300/00
20130101; A61P 31/16 20180101; A61K 2300/00 20130101 |
Class at
Publication: |
435/007.21 ;
435/023; 514/114; 514/616 |
International
Class: |
G01N 33/567 20060101
G01N033/567; C12Q 1/37 20060101 C12Q001/37; A61K 31/165 20070101
A61K031/165; A61K 31/16 20060101 A61K031/16; A61K 31/66 20060101
A61K031/66 |
Claims
1. A method of assessing the efficacy of a compound of formula I in
treating a treating a disease in a mammalian subject, or a cell
derived therefrom, comprising: measuring the expression or activity
of Raf, VEGFR-2, VEGFR-3, p38, PDGFR-beta, and/or Flt-3 in a sample
obtained from said subject who has been treated with a compound of
formula I, and determining the effects of said compound on said
expression or activity, wherein said compound of formula I is:
B--NH--C(O)--NH-L-M-L.sup.1-(Q).sub.1-3 (I) wherein B is (i)
phenyl, optionally substituted with 1-3 substituents independently
selected from the group consisting of R.sup.1, OR.sup.1,
NR.sup.1R.sup.2, S(O).sub.qR.sup.1, SO.sub.2NR.sup.1R.sup.2,
NR.sup.1SO.sub.2R.sup.2, C(O)R.sup.1, C(O)OR.sup.1,
C(O)NR.sup.1R.sup.2, NR.sup.1C(O)R.sup.2, NR.sup.1C(O)OR.sup.2,
halogen, cyano, and nitro; (ii) naphthyl, optionally substituted
with 1-3 substituents independently selected from the group
consisting of R.sup.1, OR.sup.1, NR.sup.1R.sup.2,
S(O).sub.qR.sup.1, SO.sub.2NR.sup.1R.sup.2,
NR.sup.1SO.sub.2R.sup.2, C(O)R.sup.1, C(O)OR.sup.1,
C(O)NR.sup.1R.sup.2, NR.sup.1C(O)R.sup.2, NR.sup.1C(O)OR.sup.2,
halogen, cyano, and nitro; (iii) a 5 or 6 membered monocyclic
heteroaryl group, having 1-3 heteroatoms independently selected
from the group consisting of O, N and S, optionally substituted
with 1-3 substituents independently selected from the group
consisting of R.sup.1, OR.sup.1, NR.sup.1R.sup.2,
S(O).sub.qR.sup.1, SO.sub.2NR.sup.1R.sup.2,
NR.sup.1SO.sub.2R.sup.2, C(O)R.sup.1, C(O)OR.sup.1,
C(O)NR.sup.1R.sup.2, NR.sup.1C(O)R.sup.2, NR.sup.1C(O)OR.sup.2,
halogen, cyano, oxo, and nitro; or (iv) an 8 to 10 membered
bicyclic heteroaryl group in which the first ring is bonded to the
NH of FIG. 1 and contains 1-3 heteroatoms independently selected
from the group consisting of O, N, and S, and the second ring is
fused to the first ring using 3 to 4 carbon atoms. The bicyclic
heteroaryl group is optionally substituted with 1-3 substituents
independently selected from the group consisting of R.sup.1,
OR.sup.1, NR.sup.1R.sup.2, S(O).sub.qR.sup.1,
SO.sub.2NR.sup.1R.sup.2, NR.sup.1SO.sub.2R.sup.2, C(O)R.sup.1,
C(O)OR.sup.1, C(O)NR.sup.1R.sup.2, NR.sup.1C(O)R.sup.2,
NR.sup.1C(O)OR.sup.2, halogen, cyano, oxo, and nitro. L is (i)
phenyl, optionally substituted with 1-3 substituents independently
selected from the group consisting of C.sub.1-C.sub.5 linear or
branched alkyl, C.sub.1-C.sub.5 linear or branched haloalkyl,
C.sub.1-C.sub.3 alkoxy, hydroxy, amino, C.sub.1-C.sub.3 alkylamino,
C.sub.1-C.sub.6 dialkylamino, halogen, cyano, and nitro; (ii)
naphthyl, optionally substituted with 1-3 substituents
independently selected from the group consisting of C.sub.1-C.sub.5
linear or branched alkyl, C.sub.1-C.sub.5 linear or branched
haloalkyl, C.sub.1-C.sub.3 alkoxy, hydroxy, amino, C.sub.1-C.sub.3
alkylamino, C.sub.1-C.sub.6 dialkylamino, halogen, cyano, and
nitro; (iii) a 5 or 6 membered monocyclic heteroaryl group, having
1-3 heteroatoms independently selected from the group consisting of
O, N and S, optionally substituted with 1-3 substituents
independently selected from the group consisting of C.sub.1-C.sub.5
linear or branched alkyl, C.sub.1-C.sub.5 linear or branched
haloalkyl, C.sub.1-C.sub.3 alkoxy, hydroxy, amino, C.sub.1-C.sub.3
alkylamino, C.sub.1-C.sub.6 dialkylamino, halogen, cyano, and
nitro; or (iv) an 8 to 10 membered bicyclic heteroaryl group having
1-6 heteroatoms independently selected from the group consisting of
O, N and S, optionally substituted with 1-3 substituents
independently selected from the group consisting of C.sub.1-C.sub.5
linear or branched alkyl, C.sub.1-C.sub.5 linear or branched
haloalkyl, C.sub.1-C.sub.3 alkoxy, hydroxy, amino, C.sub.1-C.sub.3
alkylamino, C.sub.1-C.sub.6 dialkylamino, halogen, cyano, and
nitro. M is (a) --(CH.sub.2).sub.m--O--(CH.sub.2).sub.l--, (b)
--(CH.sub.2).sub.m--(CH.sub.2).sub.l--, (c)
--(CH.sub.2).sub.m--C(O)--(CH.sub.2).sub.l--, (d)
--(CH.sub.2).sub.m--NR.sup.3--(CH.sub.2).sub.l--, (e)
--(CH.sub.2).sub.m--NR.sup.3C(O)--(CH.sub.2).sub.l--, (f)
--(CH.sub.2).sub.m--S--(CH.sub.2).sub.l--, (g)
--(CH.sub.2).sub.m--C(O)NR.sup.3--(CH.sub.2).sub.l--, (h)
--(CH.sub.2).sub.m--CF.sub.2--(CH.sub.2).sub.l--, (i)
--(CH.sub.2).sub.m--CCl.sub.2--(CH.sub.2).sub.l--, (j)
--(CH.sub.2).sub.m--CHF--(CH.sub.2).sub.l--, (k)
--(CH.sub.2).sub.m--CH(OH)--(CH.sub.2).sub.l--; (l)
--(CH.sub.2).sub.m--C.ident.C--(CH.sub.2).sub.l--; (m)
--(CH.sub.2).sub.m--C.dbd.C--(CH.sub.2).sub.l--; (n)
--(CH.sub.2).sub.m--CR.sup.4R.sup.5--(CH.sub.2).sub.l--; or (o) a
single bond, where m and l are 0; wherein the variables m and l are
integers independently selected from 0-4, L' is (i) phenyl,
optionally substituted with 1-2 additional substituents other than
Q, independently selected from the group consisting of R.sup.1,
OR.sup.1, NR.sup.1R.sup.2, S(O).sub.qR.sup.1,
SO.sub.2NR.sup.1R.sup.2, NR.sup.1SO.sub.2R.sup.2,
NR.sup.1C(O)R.sup.2, NR.sup.1C(O)OR.sup.2, halogen, cyano and
nitro; (ii) naphthyl, optionally substituted with 1-2 additional
substituents other than Q, independently selected from the group
consisting of R.sup.1, OR.sup.1, NR.sup.1R.sup.2,
S(O).sub.qR.sup.1, SO.sub.2NR.sup.1R.sup.2,
NR.sup.1SO.sub.2R.sup.2, NR.sup.1C(O)R.sup.2, NR.sup.1C(O)OR.sup.2,
halogen, cyano and nitro; (iii) a 5 and 6 membered monocyclic
heteroaryl group, having 1-3 heteroatoms independently selected
from the group consisting of O, N and S, optionally substituted
with 1-2 additional substituents other than Q, independently
selected from the group consisting of R.sup.1, OR.sup.1,
NR.sup.1R.sup.2, S(O).sub.qR.sup.1, SO.sub.2NR.sup.1R.sup.2,
NR.sup.1SO.sub.2R.sup.2, NR.sup.1C(O)R.sup.2, NR.sup.1C(O)OR.sup.2,
halogen, cyano and nitro and also oxides (e.g. .dbd.O, --O.sup.- or
--OH); (iv) an 8 to 10 membered bicyclic heteroaryl group, having
1-6 heteroatoms independently selected from the group consisting of
O, N and S, optionally substituted with 1-2 additional substituents
other than Q, independently selected from the group consisting of
R.sup.1, OR.sup.1, NR.sup.1R.sup.2, S(O).sub.qR.sup.1,
SO.sub.2NR.sup.1R.sup.2, NR.sup.1SO.sub.2R.sup.2,
NR.sup.1C(O)R.sup.2, NR.sup.1C(O)OR.sup.2, halogen, cyano and nitro
and also oxides (e.g. .dbd.O, --O.sup.- or --OH). (v) a saturated
and partially saturated C.sub.3-C.sub.6 monocyclic carbocyclic
moiety optionally substituted with 1-2 additional substituents
other than Q, independently selected from the group consisting of
R.sup.1, OR.sup.1, NR.sup.1R.sup.2, S(O).sub.qR.sup.1,
SO.sub.2NR.sup.1R.sup.2, NR.sup.1SO.sub.2R.sup.2,
NR.sup.1C(O)R.sup.2, NR.sup.1C(O)OR.sup.2, halogen, cyano and,
nitro; (vi) a saturated and partially saturated C.sub.8-C.sub.10
bicyclic carbocyclic moiety, optionally substituted with 1-2
additional substituents other than Q, independently selected from
the group consisting of R.sup.1, OR.sup.1, NR.sup.1R.sup.2,
S(O).sub.qR.sup.1, SO.sub.2NR.sup.1R.sup.2,
NR.sup.1SO.sub.2R.sup.2, NR.sup.1C(O)R.sup.2, NR.sup.1C(O)OR.sup.2,
halogen, cyano and nitro; (vii) a saturated and partially saturated
5 and 6 membered monocyclic heterocyclic moiety, having 1-3
heteroatoms independently selected from the group consisting of O,
N and S, optionally substituted with 1-2 additional substituents
other than Q, independently selected from the group consisting of
R.sup.1, OR.sup.1, NR.sup.1R.sup.2, S(O).sub.qR.sup.1,
SO.sub.2NR.sup.1R.sup.2, NR.sup.1SO.sub.2R.sup.2N,
R.sup.1C(O)R.sup.2, NR.sup.1C(O)OR.sup.2, halogen, cyano and nitro,
and also oxides (e.g. .dbd.O, --O.sup.- or --OH); or (viii) a
saturated and partially saturated 8 to 10 membered bicyclic
heterocyclic moiety, having 1-6 heteroatoms independently selected
from the group consisting of O, N and S, optionally substituted
with 1-2 additional substituents other than Q, independently
selected from the group consisting of R.sup.1, OR.sup.1,
NR.sup.1R.sup.2, S(O).sub.qR.sup.1, SO.sub.2NR.sup.1R.sup.2,
NR.sup.1SO.sub.2R.sup.2, NR.sup.1C(O)R.sup.2, NR.sup.1C(O)OR.sup.2,
halogen, cyano and nitro, and also oxides (e.g. .dbd.O, --O.sup.-
or --OH); and each Q is independently C(O)R.sup.4, C(O)OR.sup.4 and
C(O)NR.sup.4R.sup.5; wherein each R.sup.1-R.sup.5 is independently
selected from: (a) hydrogen, (b) C.sub.1-C.sub.5 linear, branched,
or cyclic alkyl, (c) phenyl, (d) C.sub.1-C.sub.3 alkyl-phenyl,
wherein the alkyl moiety is optionally substituted with halogen up
to per-halo; (e) up to per-halo substituted C.sub.1-C.sub.5 linear
or branched alkyl. Or (f) --(CH.sub.2).sub.q--X, where X is a 5 or
6 membered monocyclic heterocyclic ring, containing 1-4 atoms
selected from oxygen, nitrogen and sulfur, which is saturated,
partially saturated, or aromatic, or a 8-10 membered bicyclic
heteroaryl having 1-4 heteroatoms selected from the group
consisting of O, N and S; and wherein said alkyl moiety is
optionally substituted with halogen up to per-halo, wherein each
R.sup.1-R.sup.5, other than per-halo substituted C.sub.1-C.sub.5
linear or branched alkyl, is optionally substituted with 1-3
substituents independently selected from the group consisting of
C.sub.1-C.sub.5 linear or branched alkyl, up to perhalo substituted
C.sub.1-C.sub.5 linear or branched alkyl, C.sub.1-C.sub.3 alkoxy,
hydroxy, carboxy, amino, C.sub.1-C.sub.3 alkylamino,
C.sub.1-C.sub.6 dialkylamino, halogen, cyano, and nitro; wherein
the variable p is an integer selected from 0, 1, or 2 and the
variable q is an integer selected from 0, 1, 2, 3, or 4.
2. A method of claim 1, wherein said measuring the expression is
determining the amounts of mRNA corresponding to Raf, VEGFR-2,
VEGFR-3, p38, PDGFR-beta, and/or Flt-3.
3. A method of claim 1, wherein said measuring the expression is
determining the amounts of polypeptide corresponding to Raf,
VEGFR-2, VEGFR-3, p38, PDGFR-beta, and/or Flt-3.
4. A method of claim 3, wherein said measuring the activity is
determining the amounts of phospho-ERK.
5. A method of claim 4, wherein said subject has cancer, and
measuring the activity is determining the amounts of phospho-ERK in
peripheral blood lymphocytes or a tissue biopsy of a cancer.
6. A method of claim 1, further comprising comparing the expression
or activity in said sample to a normal control.
7. A method of claim 1, further comprising comparing the expression
or activity in at least one sample before treating with said
compound and in at least one sample after treating with said
compound.
8. A method of claim 1, further comprising measuring expression in
at least two different samples collected at different timepoints in
the treatment regimen with said compound.
9. A method of claim 1, wherein a reduction in expression or
activity indicates that said compound is effective in treating said
disease.
10. A method of claim 1, wherein said disease is renal cell
carcinoma or melanoma.
11. A method of claim 1, wherein said sample comprises tumor
cells.
12. A method of claim 1, wherein said sample comprises peripheral
blood cells.
13. A method of claim 1, further comprising administering a
compound of formula I at a plurality of timepoints.
14. A method of selecting subjects having a disease for treatment
with a compound of formula I, comprising: measuring the expression
or activity of Raf, VEGFR-2, VEGFR-3, p38, PDGFR-beta, and/or
Flt-3, in a sample obtained from a subject having a disease, and
administering said compound of formula I to subjects who are
identified as having high levels of expression or activity, where
said compound is a compound of formula I of claim 1.
15. A method of selecting subjects having a disease for treatment
with a compound of formula I, comprising: determining the presence
of a Raf, VEGFR-2, VEGFR-3, p38, PDGFR-beta, and/or Flt-3 gene
mutation in a sample obtained from a subject, wherein said mutation
is associated with a disease, and administering said compound of
formula I of claim 1 to subjects who are identified as having said
mutation.
16. A method of claim 15, wherein said mutation is in the BRAF
gene.
17. A method of claim 16, wherein said BRAF mutation is at amino
acid position 599 of the coding sequence of said gene.
18. A method of claim 17, wherein said BRAF mutation is V599E.
19. A method of claim 15, wherein said disease is melanoma.
20. (canceled)
21. (canceled)
22. (canceled)
23. (canceled)
24. A method of inhibiting lymphangiogenesis, comprising:
administering administering a compound of formula I of claim 1 to a
subject in need thereof.
25. A method of inhibiting angiogenesis, comprising: administering
administering a compound of formula I of claim 1 to a subject in
need thereof.
26. (canceled)
27. A method treating a tumor in a subject in need thereof,
comprising: administering an effective amount of a compound of
formula I of claim 1 to a subject in need thereof, wherein said
amount is effective to inhibit tumor cell proliferation and
neovascularization.
28. (canceled)
29. (canceled)
30. (canceled)
31. A method of assessing the efficacy of a compound in treating a
disease in a mammalian subject, or a cell derived therefrom,
comprising: measuring the expression or activity of Raf, VEGFR-2,
VEGFR-3, p38, PDGFR-beta, and/or Flt-3 in a sample obtained from
said subject who has been treated with said compound, and
determining the effects of said compound on said expression or
activity, wherein said compound is:
N-(4-chloro-3-(trifluoromethyl)phenyl)-N'-(4-(2-(N-methylcarbamoyl)-4-pyr-
idyloxy)phenyl)urea,
N-(4-bromo-3-(trifluoromethyl)phenyl)-N'-(4-(2-(N-methylcarbamoyl)-4-pyri-
dyloxy)phenyl)urea,
N-(4-bromo-3-(trifluoromethyl)phenyl)-N'-(4-(2-(N-methylcarbamoyl)-4-pyri-
dyloxy)-2-chlorophenyl)urea,
N-(4-chloro-3-(trifluoromethyl)phenyl)-N'-(4-(2-carbamoyl-4-pyridyloxy)ph-
enyl)urea,
N-(4-chloro-3-(trifluoromethyl)phenyl)N'-(4-(1-hydroxy-2-carbamoyl-4-pyri-
dyloxy)phenyl)urea,
N-(4-chloro-3-(trifluoromethyl)phenyl)N'-(4-(1-hydroxy-2-(N-methylcarbamo-
yl)-4-pyridyl oxy)phenyl)urea,
N-(4-chloro-3-(trifluoromethyl)phenyl)N'-(4-(2-(N-methylcarbamoyl)-4-pyri-
dyl oxy)-2-fluorophenyl)urea,
N-(4-bromo-3-(trifluoromethyl)phenyl)-N'-(4-(2-(N-methylcarbamoyl)-4-pyri-
dyl oxy)-2-fluorophenyl)urea,
N-(4-fluoro-3-(trifluoromethyl)phenyl)-N'-(4-(2-(N-methylcarbamoyl)-4-pyr-
idyl oxy)-2-fluorophenyl)urea,
N-(4-chloro-3-(trifluoromethyl)phenyl)-N'-(4-(2-(N-methylcarbamoyl)-4-pyr-
idyl oxy)-2-chlorophenyl)urea,
N-(6-(2,2,4,4-tetrafluoro-4H-benzo[1,3]dioxinyl))N'-(4-(2-cyano-4-pyridyl-
oxy)phenyl)urea, or
N-(6-(2,2,4,4-tetrafluoro-4H-benzo[1,3]dioxinyl))N'-(4-(2-cyano-4-pyridyl-
oxy)-2-fluorophenyl)urea.
32. (canceled)
33. (canceled)
34. (canceled)
35. (canceled)
36. (canceled)
37. (canceled)
38. (canceled)
39. (canceled)
40. (canceled)
41. (canceled)
42. (canceled)
43. (canceled)
44. (canceled)
45. (canceled)
46. (canceled)
47. (canceled)
48. A method of treating acute myeloid leukemia, comprising
administering an effective amount of a compound of formula I of
claim 1.
49. A method of treating a hematopoietic cell cancer, comprising
administering an effective amount of a compound of formula I of
claim 1.
50. A method of claim 49, wherein said cancer is associated with a
mutation in Flt-3.
51. A method of claim 50, wherein said mutation is FLT3 ITD.
52. A method of claim 15, wherein said disease is a cancer and the
presence of a Flt-3 mutation is determined.
Description
[0001] This application claims the benefit of U.S. Provisional
Application Nos. 60/556,062, filed Mar. 25, 2004, 60/520,399, filed
Nov. 17, 2003, and 60/471,735, filed May 20, 2003, each of which
are hereby incorporated by reference in their entirety.
BACKGROUND OF THE INVENTION
[0002] Activation of the ras signal transduction pathway indicates
a cascade of events that have a profound impact on cellular
proliferation, differentiation, and transformation. Raf kinase, a
downstream effector of ras, is a key mediator of these signals from
cell surface receptors to the cell nucleus (Lowy, D. R. and
Willumsen, B. M. Ann. Rev. Biochem. 1993, 62, 851; Bos, J. L.
Cancer Res. 1989, 49, 4682). It has been shown that inhibiting the
effect of active ras by inhibiting the raf kinase signaling pathway
by administration of deactivating antibodies to raf kinase or by
co-expression of dominant negative raf kinase or dominant negative
MEK, the substrate of raf kinase, leads to the reversion of
transformed cells to the normal growth phenotype (e.g., Daum et al.
Trends Biochem. Sci. 1994, 19, 474-80; Fridman et al. J. Biol.
Chem. 1994, 269, 30105-8). Kolch et al. (Nature 1991, 349, 426-28)
have further shown that inhibition of raf expression by antisense
RNA blocks cell proliferation in membrane-associated oncogenes.
Similarly, inhibition of raf kinase (by antisense
oligodeoxynucleotides) has been correlated in vitro and in vivo
with inhibition of the growth of a variety of human tumor types
(Monia et al., Nat. Med. 1996, 2, 668-75). Thus, small molecule
inhibitors of Raf kinase activity are important agents for the
treatment of cancer (Naumann, U.; Eisemnann-Tappe, I.; Rapp, U. R.
Recent Results Cancer Res. 1997, 143, 237; Monia, B. P.; Johnston,
J. F.; Geiger, T.; Muller, M.; Fabbro, D. Nature Medicine 1996, 2,
668).
[0003] To support progressive tumor growth beyond the size of 1-2
mm.sup.3, tumor cells require a functional stroma, a support
structure consisting of fibroblast, smooth muscle cells,
endothelial cells, extracellular matrix proteins, and soluble
factors (Folkman, J., Semin Oncol, 2002. 29(6 Suppl 16), 15-8).
Tumors induce the formation of stromal tissues through the
secretion of soluble growth factor such as PDGF and transforming
growth factor-beta (TGF-beta), which in turn stimulate the
secretion of complimentary factors by host cells such as fibroblast
growth factor (FGF), epidermal growth factor (EGF), and vascular
endothelial growth factor (VEGF). These stimulatory factors induce
the formation of new blood vessels, or angiogenesis, which brings
oxygen and nutrients to the tumor and allows it to grow and
provides a route for metastasis.
[0004] There is a need of developing a novel agent with pluripotent
activity against a number of key signaling pathways utilized by
tumors to induce angiogenesis in the host stroma. These include
PDGF, a potent stimulator of stroma formation (Ostman, A. and C. H.
Heldin, Adv Cancer Res, 2001, 80, 1-38), FGF, a chemo-attractant
and mitogen for fibroblasts and endothelial cells, and VEGF, a
potent regulator of vascularization.
[0005] One of the key regulators of stromal formation is PDGF,
which is secreted by many tumors in a paracrine fashion and
promotes the growth of fibroblasts, smooth muscle and endothelial
cells, promoting stroma formation and angiogenesis. PDGF was
originally identified as the v-sis oncogene product of the simian
sarcoma virus (Heldin, C. H., et al., J Cell Sci Suppl, 1985, 3,
65-76). The growth factor is made up of two peptide chains,
referred to as A or B chains which share 60% homology in their
primary amino acid sequence. The chains are disulfide cross linked
to form the 30 kDa mature protein composed of either AA, BB or AB
homo- or heterodimmers. PDGF is found at high levels in platelets,
and is expressed by endothelial cells and vascular smooth muscle
cells. In addition, the production of PDGF is up regulated under
low oxygen conditions such as those found in poorly vascularized
tumor tissue (Kourembanas, S., et al., Kidney Int, 1997, 51(2),
438-43). PDGF binds with high affinity to the PDGF receptor, a 1106
amino acid 124 kDa transmembrane tyrosine kinase receptor (Heldin,
C. H.; A. Ostman, and L. Ronnstrand, Biochim Biophys Acta, 1998.
1378(1), 79-113). PDGFR is found as homo- or heterodimer chains
which have 30% homology overall in their amino acid sequence and
64% homology between their kinase domains (Heldin, C. H., et al.
Embo J, 1988, 7(5), 1387-93). PDGFR is a member of a family of
tyrosine kinase receptor with split kinase-domains that includes
VEGFR2 (KDR), c-Kit, and FLT3 which have all been found to have a
role in promoting tumor angiogenesis, growth and survival. The PDGF
receptor is expressed primarily on fibroblast, smooth muscle cells,
and pericytes and to a lesser extent on neurons, kidney mesangial,
Leydig, and Schwann cells of the central nervous system. Upon
binding to the receptor, PDGF induces receptor dimerization and
undergoes auto- and trans-phosphorylation of tyrosine residues
which increase the receptors' kinase activity and promotes the
recruitment of downstream effectors through the activation of SH2
protein binding domains. A number of signaling molecules form
complexes with activated PDGFR including PI-3-kinase, phospholipase
C-gamma, src and GAP (GTPase activating protein for p21-ras)
(Soskic, V., et al. Biochemistry, 1999, 38(6), 1757-64). Through
the activation of PI-3-kinase, PDGF activates the Rho signaling
pathway inducing cell motility and migration, and through the
activation of GAP, induces mitogenesis through the activation of
p21-ras and the MAPK signaling pathway.
[0006] In adults, the major function of PDGF is to facilitate and
increase the rate of wound healing and to maintain blood vessel
homeostasis (Baker, E. A. and D. J. Leaper, Wound Repair Regen,
2000. 8(5), 392-8; Yu, J., A. Moon, and H. R. Kim, Biochem Biophys
Res Commun, 2001. 282(3), 697-700). PDGF is found at high
concentrations in platelets and is a potent chemoattractant for
fibroblast, smooth muscle cells, neutrophils and macrophages. In
addition to its role in wound healing PDGF helps maintain vascular
homeostasis. During the development of new blood vessels, PDGF
recruits pericytes and smooth muscle cells that are needed for the
structural integrity of the vessels. PDGF is thought to play a
similar role during tumor neovascularization. As part of its role
in angiogenesis PDGF controls interstitial fluid pressure,
regulating the permeability of vessels through its regulation of
the interaction between connective tissue cells and the
extracellular matrix. Inhibiting PDGFR activity can lower
interstitial pressure and facilitate the influx of cytotoxics into
tumors improving the anti-tumor efficacy of these agents (Pietras,
K., et al. Cancer Res, 2002. 62(19), 5476-84; Pietras; K., et al.
Cancer Res, 2001. 61(7), 2929-34).
[0007] PDGF can promote tumor growth through either the paracrine
or autocrine stimulation of PDGFR receptors on stromal cells or
tumor cells directly, or through the amplification of the receptor
or activation of the receptor by recombination. Over expressed PDGF
can transform human melanoma cells and keratinocytes (Forsberg, K.,
et al. Proc Natl Acad Sci USA., 1993. 90(2), 393-7; Skobe, M. and
N. E. Fusenig, Proc Natl Acad Sci USA, 1998. 95(3), 1050-5), two
cell types that do not express PDGF receptors, presumably by the
direct effect of PDGF on stroma formation and induction of
angiogenesis. This paracrine stimulation of tumor stroma is also
observed in carcinomas of the colon, lung, breast, and prostate
(Bhardwaj, B., et al. Clin Cancer Res, 1996, 2(4), 773-82;
Nakanishi, K., et al. Mod Pathol, 1997, 10(4), 341-7; Sundberg, C.,
et al. Am J Pathol, 1997, 151(2), 479-92; Lindmark, G., et al. Lab
Invest, 1993, 69(6), 682-9; Vignaud, J. M., et al, Cancer Res,
1994, 54(20), 5455-63) where the tumors express PDGF, but not the
receptor. The autocrine stimulation of tumor cell growth, where a
large faction of tumors analyzed express both the ligand PDGF and
the receptor, has been reported in glioblastomas (Fleming, T. P.,
et al. Cancer Res, 1992, 52(16), 4550-3), soft tissue sarcomas
(Wang, J., M. D. Coltrera, and A. M. Gown, Cancer Res, 1994, 54(2),
560-4) and cancers of the ovary (Henriksen, R., et al. Cancer Res,
1993, 53(19), 4550-4), prostate (Fudge, K., C. Y. Wang, and M. E.
Steams, Mod Pathol, 1994, 7(5), 549-54), pancreas (Funa, K., et al.
Cancer Res, 1990, 50(3), 748-53) and lung (Antoniades, H. N., et
al., Proc Natl Acad Sci USA, 1992, 89(9), 3942-6). Ligand
independent activation of the receptor is found to a lesser extent
but has been reported in chronic myelomonocytic leukemia (CMML)
where a chromosomal translocation event forms a fusion protein
between the Ets-like transcription factor TEL and the PDGF
receptor. In addition, activating mutations in PDGFR have been
found in gastrointestinal stromal tumors in which c-Kit activation
is not involved (Heinrich; M. C., et al., Science, 2003, 9, 9).
PDGFR inhibitors will interfere with tumor stromal development and
inhibit tumor growth and metastasis without undue side effects.
Additional factors such as VEGF and FGF, secreted by stromal cells
in response to tumor secreted PDGF, play key roles in stromal
formation, angiogenesis and tumor progression.
[0008] Another major regulator of angiogenesis and vasculogenesis
in both embryonic development and some angiogenic-dependent
diseases is vascular endothelial growth factor (VEGF; also called
vascular permeability factor, VPF). VEGF represents a family of
isoforms of mitogens existing in homodimeric forms due to
alternative RNA splicing. The VEGF isoforms are highly specific for
vascular endothelial cells (for reviews, see: Farrara et al.
Endocr. Rev. 1992, 13, 18; Neufield et al. FASEB J. 1999, 13,
9).
[0009] VEGF expression is induced by hypoxia (Shweiki et al. Nature
1992, 359, 843), as well as by a variety of cytokines and growth
factors, such as interleukin-1, interleukin-6, epidermal growth
factor and transforming growth factor. To date, VEGF and the VEGF
family members have been reported to bind to one or more of three
transmembrane receptor tyrosine kinases (Mustonen et al. J. Cell
Biol., 1995, 129, 895), VEGF receptor-1 (also known as flt-1
(fms-like tyrosine kinase-1)), VEGFR-2 (also known as kinase insert
domain containing receptor (KDR); the murine analogue of KDR is
known as fetal liver kinase-1 (flk-1)), and VEGFR-3 (also known as
flt-4). KDR and flt-1 have been shown to have different signal
transduction properties (Waltenberger et al. J. Biol. Chem. 1994,
269, 26988); Park et al. Oncogene 1995, 10, 135). Thus, KDR
undergoes strong ligand-dependant tyrosine phosphorylation in
intact cells, whereas flt-1 displays a weak response. Thus, binding
to KDR is a critical requirement for induction of the full spectrum
of VEGF-mediated biological responses.
[0010] In vivo, VEGF plays a central role in vasculogenesis, and
induces angiogenesis and permeabilization of blood vessels.
Deregulated VEGF expression contributes to the development of a
number of diseases that are characterized by abnormal angiogenesis
and/or hyperpermeability processes. Regulation of the VEGF-mediated
signal transduction cascade will therefore provide a useful mode
for control of abnormal angiogenesis and/or hyperpermeability
processes.
[0011] Angiogenesis is regarded as an absolute prerequisite for
growth of tumors beyond about 1-2 mm. Oxygen and nutrients may be
supplied to cells in tumor smaller than this limit through
diffusion. However, every tumor is dependent on angiogenesis for
continued growth after it has reached a certain size. Tumorigenic
cells within hypoxic regions of tumors respond by stimulation of
VEGF production, which triggers activation of quiescent endothelial
cells to stimulate new blood vessel formation. (Shweiki et al.
Proc. Nat'l. Acad. Sci., 1995, 92, 768). In addition, VEGF
production in tumor regions where there is no angiogenesis may
proceed through the ras signal transduction pathway (Grugel et al.
J. Biol. Chem., 1995, 270, 25915; Rak et al. Cancer Res. 1995, 55,
4575). In situ hybridization studies have demonstrated VEGF mRNA is
strongly upregulated in a wide variety of human tumors, including
lung (Mattern et al. Br. J. Cancer 1996, 73, 931), thyroid
(Viglietto et al. Oncogene 1995, 11, 1569), breast (Brown et al.
Human Pathol. 1995, 26, 86), gastrointestional tract (Brown et al.
Cancer Res. 1993, 53, 4727; Suzuki et al. Cancer Res. 1996, 56,
3004), kidney and bladder (Brown et al. Am. J. Pathol. 1993, 143I,
1255), ovary (Olson et al. Cancer Res. 1994, 54, 1255), and
cervical (Guidi et al. J. Nat'l Cancer Inst. 1995, 87, 12137)
carcinomas, as well as angiosacroma (Hashimoto et al. Lab. Invest.
1995, 73, 859) and several intracranial tumors (Plate et al. Nature
1992, 359, 845; Phillips et al. Int. J. Oncol. 1993, 2, 913;
Berkman et al. J. Clin. Invest., 1993, 91; 153).
Neutralizing-monoclonal antibodies to KDR have been shown to be
efficacious in blocking tumor angiogenesis (Kim et al. Nature 1993,
362, 841; Rockwell et al. Mol. Cell. Differ. 1995, 3, 315).
[0012] Overexpression of VEGF, for example under conditions of
extreme hypoxia, can lead to intraocular angiogenesis, resulting in
hyperproliferation of blood vessels, leading eventually to
blindness. Such a cascade of events has been observed for a number
of retinopathies, including diabetic retinopathy, ischemic
retinal-vein occlusion, and retinopathy of prematurity (Aiello et
al. New Engl. J. Med. 1994, 331, 1480; Peer et al. Lab. Invest.
1995, 72, 638), and age-related macular degeneration (AMD; see,
Lopez et al. Invest Opththalmol. Vis. Sci. 1996, 37, 855).
[0013] In rheumatoid arthritis (RA), the in-growth of vascular
pannus may be mediated by production of angiogenic factors. Levels
of immunoreactive VEGF are high in the synovial fluid of RA
patients, while VEGF levels were low in the synovial fluid of
patients with other forms of arthritis of with degenerative joint
disease (Koch et al. J. Immunol 1994, 152, 4149). The angiogenesis
inhibitor AGM-170 has been shown to prevent neovascularization of
the joint in the rat collagen arthritis model (Peacock et al. J.
Exper. Med. 1992, 175, 1135).
[0014] Increased VEGF expression has also been shown in psoriatic
skin, as well as bullous disorders associated with subepidermal
blister formation, such as bullous pemphigoid, erythema multiforme,
and dermatitis herpetiformis (Brown et al. J. Invest. Dermatol.
1995, 104, 744).
[0015] The vascular endothelial growth factors (VEGF, VEGF-C,
VEGF-D) and their receptors (VEGFR2, VEGFR3) are not only key
regulators of tumor angiogenesis, but also lymphangiogenesis. VEGF,
VEGF-C and VEGF-D are expressed in most tumors, primarily during
periods of tumor growth and, often at substantially increased
levels. VEGF expression is stimulated by hypoxia, cytokines,
oncogenes such as ras, or by inactivation of tumor suppressor genes
(McMahon, G. Oncologist 2000, 5(Suppl. 1), 3-10; McDonald, N. Q.;
Hendrickson, W. A. Cell 1993, 73, 421-424)
[0016] The biological activities of the VEGFs are mediated through
binding to their receptors. VEGFR3 (also called flt-4) is
predominantly expressed on lymphatic endothelium in normal adult
tissues. VEGFR3 function is needed for new lymphatic vessel
formation, but not for maintenance of the pre-existing lymphatics.
VEGFR3 is also upregulated on blood vessel endothelium in tumors.
Recently VEGF-C and VEGF-D, ligands for VEGFR3, have been
identified as regulators of lymphangiogenesis in mammals.
Lymphangiogenesis induced by tumor-associated lymphangiogenic
factors could promote the growth of new vessels into the tumor,
providing tumor cells access to systemic circulation. Cells that
invade the lymphatics could find their way into the bloodstream via
the thoracic duct. Tumor expression studies have allowed a direct
comparison of VEGF-C, VEGF-D and VEGFR3 expression with
clinicopathological factors that relate directly to the ability of
primary tumors to spread (e.g., lymph node involvement, lymphatic
invasion, secondary metastases, and disease-free survival). In many
instances, these studies demonstrate a statistical correlation
between the expression of lymphangiogenic factors and the ability
of a primary solid tumor to metastasize, (Skobe, M. et al. Nature
Med. 2001, 7(2), 192-198; Stacker, S. A. et al. Nature Med. 2001,
7(2), 186-191; Makinen, T. et al. Nature Med. 2001, 7(2), 199-205;
Mandriota, S. J. et al. EMBO J. 2001, 20(4), 672-82; Karpanen, T.
et al. Cancer Res. 2001, 61(5), 1786-90; Kubo, H. et al. Blood
2000, 96(2), 546-53).
[0017] Hypoxia appears to be an important stimulus for VEGF
production in malignant cells. Activation of p38 MAP kinase is
required for VEGF induction by tumor cells in response to hypoxia
(Blaschke, F. et al. Biochem. Biophys. Res. Commun. 2002, 296,
890-896; Shemirani, B. et al. Oral Oncology 2002, 38, 251-257). In
addition to its involvement in angiogenesis through regulation of
VEGF secretion, p38 MAP kinase promotes malignant cell invasion,
and migration of different tumor types through regulation of
collagenase activity and urokinase plasminogen activator expression
(Laferriere, J. et al. J. Biol. Chem. 2001, 276, 33762-33772;
Westermarck, J. et al. Cancer Res. 2000, 60, 7156-7162; Huang, S.
et al. J. Biol. Chem. 2000, 275, 12266-12272; Simon, C. et al. Exp.
Cell Res. 2001, 271, 344-355). Therefore, inhibition of p38 kinase
is expected to impact tumor growth by interfering with signaling
cascades associated with both angiogenesis and malignant cell
invasion.
[0018] Diarylureas are a class of serine-threonine kinase
inhibitors as well as tyrosine kinase inhibitors well known in the
art. The following publications illustrate their utility as active
ingredient in pharmaceutical compositions for the treatment of
cancer, angiogenesis disorders, and inflammatory disorders: [0019]
Redman et al., Bioorg. Med. Chem. Lett. 2001, 11, 9-12. [0020]
Smith et al., Bioorg. Med. Chem. Lett. 2001, 11, 2775-2778. [0021]
Dumas et al., Bioorg. Med. Chem. Lett. 2000, 10, 2047-2050. [0022]
Dumas et al., Bioorg. Med. Chem. Lett. 2000, 10, 2051-2054. [0023]
Ranges et al., Book of Abstracts, 220th ACS National Meeting,
Washington, D.C., USA, MEDI 149. [0024] Dumas et al., Bioorg. Med.
Chem. Lett. 2002, 12, 1559-1562. [0025] Lowinger et al., Clin.
Cancer Res. 2000, 6(suppl), 335. [0026] Lyons et al.,
Endocr.-Relat. Cancer 2001, 8, 219-225. [0027] Riedl et al., Book
of Abstracts, 92.sup.nd AACR Meeting, New Orleans, La., USA,
abstract 4956. [0028] Khire et al., Book of Abstracts, 93.sup.rd
AACR Meeting, San Francisco, Calif., USA, abstract 4211. [0029]
Lowinger et al., Curr. Pharm. Design 2002, 8, 99-110. [0030] Regan
et al., J. Med. Chem. 2002, 45, 2994-3008. [0031] Pargellis et al.,
Nature Struct. Biol. 2002, 9(4), 268-272. [0032] Carter et al.,
Book of Abstracts, 92.sup.nd AACR Meeting, New Orleans, La., USA,
abstract 4954. [0033] Vincent et al., Book of Abstracts, 38.sup.th
ASCO Meeting, Orlando, Fla., USA, abstract 1900. [0034] Hilger et
al., Book of Abstracts, 38.sup.th ASCO Meeting, Orlando, Fla., USA,
abstract 1916. [0035] Moore et al., Book of Abstracts, 38.sup.th
ASCO Meeting, Orlando, Fla., USA, abstract 1816. [0036] Strumberg
et al., Book of Abstracts, 38.sup.th ASCO Meeting, Orlando, Fla.,
USA, abstract 121. [0037] Madwed J B: Book of Abstracts, Protein
Kinases: Novel Target Identification and Validation for Therapeutic
Development, San Diego, Calif., USA, March 2002. [0038] Roberts et
al., Book of Abstracts, 38.sup.th ASCO Meeting, Orlando, Fla., USA,
abstract 473. [0039] Tolcher et al., Book of Abstracts, 38.sup.th
ASCO Meeting, Orlando, Fla., USA, abstract 334. [0040] Karp et al.,
Book of Abstracts, 38.sup.th AACR Meeting, San Francisco, Calif.,
USA, abstract 2753.
BRIEF DESCRIPTION OF THE DRAWINGS
[0041] FIG. 1. Qualitative assessment consisted of assessing the
staining intensity, identifying the positively staining cells and
the intracellular compartments involved in staining, and evaluating
the overall slide quality. Separate evaluations were performed on
the tumor cells and the tumor cell stroma. Intensity of staining
was graded based upon the following scale: Negative, .+-.
(equivocal), 1+ (weak), 2+ (moderate), 3+ (strong), 4+ (intense).
The intensity of staining was determined by evaluation of the
entire specimen. Semi-quantitative evaluation of target antigen
expression was conducted using a grading system based upon the
percentage of cells throughout the entire specimen expressing each
target antigen as follows: 0-5% with positive target antigen
expression=<5%; 6-25%=1st quartile (1Q); 26-50%=2nd quartile
(2Q); 51-75%=3rd quartile (3Q); 76-100%=4th quartile (4Q).
[0042] FIG. 2. Immunohistochemical Staining for phospho-ERK of
Human Melanoma Biopsies pre- and post-treament.
DESCRIPTION OF THE INVENTION
[0043] The present invention provides methods for treating,
ameliorating, preventing, modulating, etc., conditions and diseases
in humans and other mammals which are associated with signal
transduction pathways comprising, but not limited to, raf, VEGFR,
PDGFR, p38, and/or FLT-3. Preferred methods of the present
invention provide for the modulation of diseases and conditions
associated with raf, VEGFR2, VEGFR3, and/or PDGFR-beta. The methods
can comprise, e.g., administering an aryl urea compound as
described below, pharmaceutically-acceptable salts thereof,
derivatives thereof, etc.
[0044] The present invention also provides compositions and methods
for identifying conditions and diseases which can be modulated with
compounds of the present invention. These methods facilitate the
selection of subjects who can be efficiently treated with compounds
of the present invention. Additionally, the invention provides
methods for monitoring subjects who have been administered a
compound of the present invention. This includes, e.g., determining
the efficacy of compounds of the present invention in the treatment
of various diseases and conditions, and for determining treatment
regimens.
[0045] The aryl urea compounds employed in the methods of this
invention comprise compounds of Formula I, pharmaceutically
acceptable salts thereof, esters thereof, stereoisomers thereof
(both isolated and in mixtures), prodrugs thereof, and any active
derivatives thereof, which are collectively referred to herein as
the "compounds, of the invention" and the like.
[0046] Formula I is as follows:
B--NH--C(O)--NH-L-M-L.sup.1-(Q).sub.1-3 (I)
[0047] wherein B is
[0048] (i) phenyl, optionally substituted with 1-3 substituents
independently selected from the group consisting of R.sup.1,
OR.sup.1, NR.sup.1R.sup.2, S(O).sub.qR.sup.1, SO.sub.2NR1R.sup.2,
NR.sup.1SO.sub.2R.sup.2, C(O)R.sup.1, C(O)OR.sup.1,
C(O)NR.sup.1R.sup.2, NR.sup.1C(O)R.sup.2, NR.sup.1C(O)OR.sup.2,
halogen, cyano; and nitro;
[0049] (ii) naphthyl, optionally substituted with 1-3 substituents
independently selected from the group consisting of R.sup.1,
OR.sup.1; NR.sup.1R.sup.2, S(O).sub.qR.sup.1,
SO.sub.2NR.sup.1R.sup.2, NR.sup.1SO.sub.2R.sup.2, C(O)R.sup.1,
C(O)OR.sup.1, C(O)NR.sup.1R.sup.2, NR.sup.1C(O)R.sup.2,
NR.sup.1C(O)OR.sup.2, halogen, cyano, and nitro;
[0050] (iii) a 5 or 6 membered monocyclic heteroaryl group, having
1-3 heteroatoms independently selected from the group consisting of
O, N and S, optionally substituted with 1-3 substituents
independently selected from the group consisting of R.sup.1,
OR.sup.1, NR.sup.1R.sup.2, S(O).sub.qR.sup.1,
SO.sub.2NR.sup.1R.sup.2, NR.sup.1SO.sub.2R.sup.2, C(O)R.sup.1,
C(O)OR.sup.1, C(O)NR.sup.1R.sup.2; NR.sup.1C(O)R.sup.2,
NR.sup.1C(O)OR.sup.2, halogen, cyano, oxo, and nitro; or
[0051] (iv) an 8 to 10 membered bicyclic heteroaryl group in which
the first ring is bonded to the NH of FIG. 1 and contains 1-3
heteroatoms independently selected from the group consisting of O,
N, and S; and the second ring is fused to the first ring using 3 to
4 carbon atoms. The bicyclic heteroaryl group is optionally
substituted with 1-3 substituents independently selected from the
group consisting of R.sup.1, OR.sup.1, NR.sup.1R.sup.2,
S(O).sub.qR.sup.1, SO.sub.2NR.sup.1R.sup.2,
NR.sup.1SO.sub.2R.sup.2, C(O)R.sup.1, C(O)OR.sup.1,
C(O)NR.sup.1R.sup.2, NR.sup.1C(O)R.sup.2, NR.sup.1C(O)OR.sup.2,
halogen, cyano, oxo, and nitro.
[0052] L is
[0053] (i) phenyl, optionally substituted with 1-3 substituents
independently selected from the group consisting of C.sub.1-C.sub.5
linear or branched alkyl, C.sub.1-C.sub.5 linear or branched
haloalkyl, C.sub.1-C.sub.3 alkoxy, hydroxy, amino, C.sub.1-C.sub.3
alkylamino, C.sub.1-C.sub.6 dialkylamino, halogen, cyano, and
nitro;
[0054] (ii) naphthyl, optionally substituted with 1-3 substituents
independently selected from the group consisting of C.sub.1-C.sub.5
linear or branched alkyl, C.sub.1-C.sub.5 linear or branched
haloalkyl, C.sub.1-C.sub.3 alkoxy, hydroxy, amino, C.sub.1-C.sub.3
alkylamino, C.sub.1-C.sub.6 dialkylamino, halogen, cyano, and
nitro;
[0055] (iii) a 5 or 6 membered monocyclic heteroaryl group, having
1-3 heteroatoms independently selected from the group consisting of
O, N and S, optionally substituted with 1-3 substituents
independently selected from the group consisting of C.sub.1-C.sub.5
linear or branched alkyl, C.sub.1-C.sub.5 linear or branched
haloalkyl, C.sub.1-C.sub.3 alkoxy, hydroxy, amino, C.sub.1-C.sub.3
alkylamino, C.sub.1-C.sub.6 dialkylamino, halogen, cyano, and
nitro; or
[0056] (iv) an 8 to 10 membered bicyclic heteroaryl group having
1-6 heteroatoms independently selected from the group consisting of
O, N and S, optionally substituted with 1-3 substituents
independently selected from the group consisting of C.sub.1-C.sub.5
linear or branched alkyl, C.sub.1-C.sub.5 linear or branched
haloalkyl, C.sub.1-C.sub.3 alkoxy, hydroxy, amino, C.sub.1-C.sub.3
alkylamino, C.sub.1-C.sub.6 dialkylamino, halogen, cyano, and
nitro.
[0057] M is
(a) --(CH.sub.2).sub.m--O--(CH.sub.2).sub.l--,
(b) --(CH.sub.2).sub.m--(CH.sub.2).sub.l--, (c)
--(CH.sub.2).sub.m--C(O)--(CH.sub.2).sub.l--,
(d) --(CH.sub.2).sub.m--NR.sup.3--(CH.sub.2).sub.l--,
(e) --(CH.sub.2).sub.m--NR.sup.3C(O)--(CH.sub.2).sub.l--,
(f) --(CH.sub.2).sub.m--S--(CH.sub.2).sub.l--,
(g) --(CH.sub.2).sub.m--C(O)NR.sup.3--(CH.sub.2).sub.l--,
(h) --(CH.sub.2).sub.m--CF.sub.2--(CH.sub.2).sub.l--,
(i) --(CH.sub.2).sub.m--CCl.sub.2--(CH.sub.2).sub.l--,
(j) --(CH.sub.2).sub.m--CHF--(CH.sub.2).sub.l--,
(k) --(CH.sub.2).sub.m--CH(OH)--(CH.sub.2).sub.l--;
(l) --(CH.sub.2).sub.m--C.ident.C--(CH.sub.2).sub.l--;
(m) --(CH.sub.2).sub.m--C.dbd.C--(CH.sub.2).sub.l--;
(n) --(CH.sub.2).sub.m--CR.sup.4R.sup.5--(CH.sub.2).sub.l--;
or
(o) a single bond, where m and l are 0;
wherein the variables m and l are integers independently selected
from 0-4,
[0058] L' is
[0059] (i) phenyl, optionally substituted with 1-2 additional
substituents other than Q, independently selected from the group
consisting of R.sup.1, OR.sup.1, NR.sup.1R.sup.2,
S(O).sub.qR.sup.1, SO.sub.2NR.sup.1R.sup.2,
NR.sup.1SO.sub.2R.sup.2, NR.sup.1C(O)R.sup.2, NR.sup.1C(O)OR.sup.2,
halogen, cyano and nitro;
[0060] (ii) naphthyl, optionally substituted with 1-2 additional
substituents other than Q, independently selected from the group
consisting of R.sup.1, OR.sup.1, NR.sup.1R.sup.2,
S(O).sub.qR.sup.1, SO.sub.2NR.sup.1R.sup.2,
NR.sup.1SO.sub.2R.sup.2, NR.sup.1C(O)R.sup.2, NR.sup.1C(O)OR.sup.2,
halogen, cyano and nitro;
[0061] (ii) a 5 and 6 membered monocyclic heteroaryl group, having
1-3 heteroatoms independently selected from the group consisting of
O, N and S, optionally substituted with 1-2 additional substituents
other than Q, independently selected from the group consisting of
R.sup.1, OR.sup.1, NR.sup.1R.sup.2, S(O).sub.qR.sup.1,
SO.sub.2NR.sup.1R.sup.2, NR.sup.1SO.sub.2R.sup.2,
NR.sup.1C(O)R.sup.2, NR.sup.1C(O)OR.sup.2, halogen, cyano and nitro
and also oxides (e.g. .dbd.O, --O.sup.- or --OH);
[0062] (iv) an 8 to 10 membered bicyclic heteroaryl group; having
1-6 heteroatoms independently selected from the group consisting of
O, N and S, optionally substituted with 1-2 additional substituents
other than Q, independently selected from the group consisting of
R.sup.1, OR.sup.1, NR.sup.1R.sup.2, S(O).sub.qR.sup.1,
SO.sub.2NR.sup.1R.sup.2, NR.sup.1SO.sub.2R.sup.2,
NR.sup.1C(O)R.sup.2, NR.sup.1C(O)OR.sup.2, halogen, cyano and nitro
and also oxides (e.g. .dbd.O, --O.sup.- or --OH).
[0063] (v) a saturated and partially saturated C.sub.3-C.sub.6
monocyclic carbocyclic moiety optionally substituted with 1-2
additional substituents other than Q, independently selected from
the group consisting of R.sup.1, OR.sup.1, NR.sup.1R.sup.2,
S(O).sub.qR.sup.1, SO.sub.2NR.sup.1R.sup.2,
NR.sup.1SO.sub.2R.sup.2, NR.sup.1C(O)R.sup.2, NR.sup.1C(O)OR.sup.2,
halogen, cyano and, nitro;
[0064] (vi) a saturated and partially saturated C.sub.8-C.sub.10
bicyclic carbocyclic moiety, optionally substituted with 1-2
additional substituents other than Q, independently selected from
the group consisting of R.sup.1, OR.sup.1, NR.sup.1R.sup.2,
S(O).sub.qR.sup.1, SO.sub.2NR.sup.1R.sup.2,
NR.sup.1SO.sub.2R.sup.2, NR.sup.1C(O)R.sup.2, NR.sup.1C(O)OR.sup.2,
halogen, cyano and nitro;
[0065] (vii) a saturated and partially saturated 5 and 6 membered
monocyclic heterocyclic moiety, having 1-3 heteroatoms
independently selected from the group consisting of O, N and S,
optionally substituted with 1-2 additional substituents other than
Q, independently selected from the group consisting of R.sup.1,
OR.sup.1, NR.sup.1R.sup.2, S(O).sub.qR.sup.1,
SO.sub.2NR.sup.1R.sup.2, NR.sup.1SO.sub.2R.sup.2N,
R.sup.1C(O)R.sup.2, NR.sup.1C(O)OR.sup.2, halogen, cyano and nitro,
and also oxides (e.g. .dbd.O, --O.sup.- or --OH); or
[0066] (viii) a saturated and partially saturated 8 to 10 membered
bicyclic heterocyclic moiety, having 1-6 heteroatoms independently
selected from the group consisting of O, N and S, optionally
substituted with 1-2 additional substituents other than Q,
independently selected from the group consisting of R.sup.1,
OR.sup.1, NR.sup.1R.sup.2, S(O).sub.qR.sup.1,
SO.sub.2NR.sup.1R.sup.2, NR.sup.1SO.sub.2R.sup.2,
NR.sup.1C(O)R.sup.2, NR.sup.1C(O)OR.sup.2, halogen, cyano and
nitro, and also oxides (e.g. .dbd.O, --O.sup.- or --OH);
each Q is independently C(O)R.sup.4, C(O)OR.sup.4 and
C(O)NR.sup.4R.sup.5;
wherein each R.sup.1-R.sup.5 is independently selected from the
group consisting of:
(a) hydrogen,
(b) C.sub.1-C.sub.5 linear, branched, or cyclic alkyl,
(c) phenyl,
(d) C.sub.1-C.sub.3 alkyl-phenyl, wherein the alkyl moiety is
optionally substituted with halogen up to per-halo;
(e) up to per-halo substituted C.sub.1-C.sub.5 linear or branched
alkyl.
[0067] (f) --(CH.sub.2).sub.q--X, where X is a 5 or 6 membered
monocyclic heterocyclic ring, containing 1-4 atoms selected from
oxygen, nitrogen and sulfur, which is saturated, partially
saturated, or aromatic, or a 8-10 membered bicyclic heteroaryl
having 1-4 heteroatoms selected from the group consisting of O, N
and S; and wherein said alkyl moiety is optionally substituted with
halogen up to per-halo,
[0068] wherein each R.sup.1-R.sup.5, other than per-halo
substituted C.sub.1-C.sub.5 linear or branched alkyl, is optionally
substituted with 1-3 substituents independently selected from the
group consisting of C.sub.1-C.sub.5 linear or branched alkyl, up to
perhalo substitiuted C.sub.1-C.sub.5 linear or branched alkyl,
C.sub.1-C.sub.3 alkoxy, hydroxy, carboxy, amino, C.sub.1-C.sub.3
alkylamino, C.sub.1-C.sub.6 dialkylamino, halogen, cyano, and
nitro;
wherein the variable p is an integer selected from 0, 1, or 2 and
the variable q is an integer selected from 0, 1, 2, 3, or 4.
[0069] In formula I, suitable hetaryl groups include, but are not
limited to, 5-10 membered ring systems containing monocyclic and
bicyclic rings, at least one of which is aromatic, in which one or
more, e.g., 1-4 carbon atoms in one or more of the rings can be
replaced by oxygen, nitrogen or sulfur atoms. In bicyclic ring
systems, each ring can have from 3-7 atoms.
[0070] "Monocyclic heteroaryl" means an aromatic monocyclic ring
having 5 to 6 ring atoms, at least one of which is a hetero atom
selected from N, O and S, the remaining atoms being carbon. When
more than one hetero atom is present in the moiety, they are
selected independently from the other(s) so that they may be the
same or different. Monocyclic heteroaryl moieties include, but are
not limited to pyrrole, furan, thiophene, imidazole, pyrazole,
thiazole, oxazole, isoxazole, isothiazole, triazole, tetrazole,
thiadiazole, oxadiazole, pyridine, pyrimidine, pyridazine,
pyrazine, and triazine.
[0071] Bicyclic heteroaryl means fused bicyclic moieties where one
of the rings is chosen from the monocyclic heteroaryl rings
described above and the second ring is either benzene or another
monocyclic heteroaryl ring described above. When both rings in the
bicyclic moiety are heteroaryl rings, they may be the same or
different, as long as they are chemically accessible by means known
in the art. Bicyclic heteroaryl rings include synthetically
accessible 5-5, 5-6, or 6-6 fused bicyclic aromatic structures
including, for example but not by way of limitation, benzoxazole
(fused phenyl and oxazole), quinoline (fused phenyl and pyridine),
imidazopyrimidine (fused imidazole and pyrimidine), and the
like.
[0072] The phrase "5 or 6 membered heterocyclic ring, containing at
least one atom selected from oxygen, nitrogen and sulfur, which is
saturated, partially saturated, or aromatic" includes, by no way of
limitation, tetrahydropyrane, tetrahydrofurane, 1,3-dioxolane,
1,4-dioxane, morpholine, thiomorpholine, piperazine, piperidine,
piperidinone, tetrahydropyrimidone, pentamethylene sulfide,
tetramethylene sulfide, dihydropyrane, dihydrofuran,
dihydrothiophene, pyrrole, furan, thiophene, imidazole, pyrazole,
thiazole, oxazole, isoxazole, isothiazole, triazole, pyridine,
pyrimidine, pyridazine, pyrazine, triazine, and the like.
[0073] The term "C.sub.1-C.sub.3 alkyl-phenyl" includes, by no way
of limitation, 3-phenyl-propyl, 2-phenyl-1-methyl-ethyl.
Substituted examples include 2-[2-chlorophenyl]ethyl,
3,4-dimethylphenyl-methyl, and the like.
[0074] Suitable substituted and unsubstituted heteroaryl groups for
the compounds of this invention, such as those for B, L and L' of
formula I, include, but are not limited to the following monocyclic
heteroaryl groups:
[0075] 2- or 3-furyl,
[0076] 2- or 3-thienyl,
[0077] 2- or 4-triazinyl,
[0078] 1-, 2- or 3-pyrrolyl,
[0079] 1-, 2-, 4- or 5-imidazolyl,
[0080] 1-, 3-, 4- or 5-pyrazolyl,
[0081] 2-, 4- or 5-oxazolyl,
[0082] 3-, 4- or 5-isoxazolyl,
[0083] 2-, 4- or 5-thiazolyl,
[0084] 3-, 4- or 5-isothiazolyl,
[0085] 2-, 3- or 4-pyridyl,
[0086] 2-, 4-, 5- or 6-pyrimidinyl,
[0087] 1,2,3-triazol-1-, 4- or 5-yl,
[0088] 1,2,4-triazol-1-, -3- or -5-yl,
[0089] 1- or 5-tetrazolyl,
[0090] 1,2,3-oxadiazol-4- or -5-yl,
[0091] 1,2,4-oxadiazol-3- or -5-yl,
[0092] 1,3,4-thiadiazol-2- or -5-yl,
[0093] 1,2,4-oxadiazol-3- or -5-yl,
[0094] 1,3,4-thiadiazol-2- or -5-yl,
[0095] 1,3,4-thiadiazol-3- or -5-yl,
[0096] 1,2,3-thiadiazol-4- or -5-yl,
[0097] 2-, 3-, 4-, 5- or 6-2H-thiopyranyl,
[0098] 2-, 3- or 4-4H-thiopyranyl,
[0099] 3- or 4-pyridazinyl, pyrazinyl, and
[0100] the following bicyclic heterocyclic groups:
[0101] benzofuryl, benzothienyl, indolyl, benzimidazolyl,
benzopyrazolyl, benzoxazolyl, benzisoxazolyl, benzothiazolyl,
benzisothiazolyl, benz-1,3-oxadiazolyl, quinolinyl, isoquinolinyl,
quinazolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl,
dihydrobenzofuryl, pyrazolo[3,4-b]pyrimidinyl, purinyl,
benzodiazine, pterindinyl, pyrrolo[2,3-b]pyridinyl,
pyrazolo[3,4-b]pyridinyl, oxazo[4,5-b]pyridinyl,
imidazo[4,5-b]pyridinyl, cyclopentenopyridine, cyclohexanopyridine,
cyclopentanopyrimidine, cyclohexanopyrimidine,
cyclcopentanopyrazine, cyclohexanopyrazine,
cyclopentanopyridiazine, cyclohexanopyridazine,
cyclopentanoimidazole, cyclohexanoimidazole, cyclopentanothiophene
and cyclohexanothiophene.
[0102] Suitable aryl groups which do not contain heteroatoms
include, for example, phenyl and 1- and 2-naphthyl,
tetrahydronaphthyl, indanyl, indenyl, benzocyclobutanyl,
benzocycloheptanyl and benzocycloheptenyl.
[0103] Suitable linear alkyl groups and alkyl portions of groups,
e.g., alkoxy, alkylphenyl and alkylheteroaryl etc. throughout
include methyl, ethyl, propyl, butyl, pentyl, etc. Suitable
branched alkyl groups include all branched isomers such as
isopropyl, isobutyl, sec-butyl, tert-butyl, etc.
[0104] The term "alkoxy" means a straight or branched chain alkoxy
group having saturated carbon atoms which may be linear or branched
with single or multiple branching, and includes such groups as
methoxy, ethoxy, n-propoxy, isopropoxy, and the like. It also
includes halogenated groups such as 2,2-dichloroethoxy,
trifluoromethoxy, and the like.
[0105] C.sub.1-C.sub.3alkylamino means methylamino, ethylamino,
propylamino or isopropylamino. Examples of C.sub.1-C.sub.6
dialkylamino group include but are not limited to diethylamino,
ethyl-isopropylamino, means methylamino, methyl-isobutylamino,
dihexylamino.
[0106] Suitable halogens include F, Cl, Br, and/or I, from one to
per-substitution (i.e. all H atoms on a group replaced by a halogen
atom) being possible where an alkyl group is substituted by
halogen, mixed substitution of halogen atom types also being
possible on a given moiety. Preferred halogens are Cl, Br and
F.
[0107] The term "up to perhalo substituted linear and branched
alkyl," includes alkyl groups having one alkyl hydrogen replaced
with halogen, alkyl groups wherein all hydrogens are replaced with
halogen, alkyl groups wherein more than one but less than all
hydrogens are replaced by halogen and alkyl groups having alkyl
hydrogens replaced by halogen and other substituents. Examples
include chloromethyl, dichloromethyl, trichloromethyl,
fluoromethyl, difluoromethyl, trifluoromethyl, and the like.
[0108] The term "cycloalkyl", as used herein, refers to cyclic
structures having 3-8 members in the ring such as cyclopropyl,
cyclobutyl and cyclopentyl and cyclic structures having 3-8 members
with alkyl substituents such that, for example, "C.sub.3
cycloalkyl" includes methyl substituted cyclopropyl groups.
[0109] The term "saturated carbocyclic moieties" defines only the
cyclic structure, i.e. cyclopentyl, cyclohexyl, etc. Any alkyl
substitution on these cyclic structures is specifically
identified.
[0110] Saturated monocyclic and bicyclic carbocyclic moieties
include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and
decahydronaphthalene.
[0111] Partially saturated monocyclic and bicyclic carbocyclic
moieties include cyclopentenyl, cyclohexenyl, cyclohexadienyl and
tetrahydronaphthalene.
[0112] Saturated monocyclic and bicyclic heterocyclic moieties
include tetrahydropyranyl, tetrahydrofuranyl, 1,3-dioxolane,
1,4-dioxanyl, morpholinyl, thiomorpholinyl, piperazinyl,
piperidinyl, piperidinonyl, tetrahydropyrimidonyl, pentamethylene
sulfide and tetramethylene sulfide.
[0113] Partially saturated monocyclic and bicyclic heterocyclic
moieties include dihydropyranyl, dihydrofuranyl, dihydrothienyl,
dihydropiperidinyl, and dihydropyrimidonyl.
[0114] When any moiety is "substituted", it can have up to the
highest number of indicated substituents, and each substituent can
be located at any available position on the moiety and can be
attached through any available atom on the substituent. "Any
available position" means any position on the moiety that is
chemically accessible through means known in the art or taught
herein and that does not create an unduly unstable molecule. When
there are two or more substituents on any moiety, each substituent
is defined independently of any other substituent and can,
accordingly, be the same or different.
[0115] The term "optionally substituted" means that the moiety so
modified may be either unsubstituted, or substituted with the
identified substituent(s).
[0116] It is understood that where L' is pyridine, the term
"hydroxy" as a pyridine substituent includes 2-, 3-, and
4-hydroxypyridine, but also includes those structures referred to
in the art as 1-oxo-pyridine and 1-hydroxy-pyridine.
[0117] Where the plural form of the word compounds, salts, and the
like, is used herein, this is taken to mean also a single compound,
salt, or the like.
[0118] The substituted structures of B and L' are preferably each,
independently, selected from the group consisting of
[0119] methyl, trifluoromethyl, ethyl, n-propyl, n-butyl, n-pentyl,
isopropyl, tert-butyl, sec-butyl, isobutyl, cyclopropyl,
cyclobutyl, cyclopentyl, methoxy, ethoxy, propoxy, Cl, Br and F,
cyano, nitro, hydroxy, amino, methylamino, dimethylamino,
ethylamino and diethylamino.
[0120] Other substituents for B and L' particularly include:
[0121] phenyl, pyridinyl, pyrimidinyl, chlorophenyl,
dichlorophenyl, bromophenyl, dibromophenyl, chloropyridinyl,
bromopyridinyl, dichloropyridinyl, dibromopyridinyl methylphenyl,
methylpyridinyl quinolinyl, isoquinolinyl, isoindolinyl, pyrazinyl,
pyridazinyl, pyrrolinyl, imidazolinyl, thienyl, furyl,
isoxazolinyl, isothiazolinyl, benzopyridinyl, benzothiazolyl,
[0122] C.sub.1-C.sub.5 acyl;
[0123] NH(C.sub.1-C.sub.5 alkyl, phenyl or pyridinyl), such as
aminophenyl; N(C.sub.1-C.sub.5 alkyl)(C.sub.1-C.sub.5 alkyl, phenyl
or pyridinyl), such as diethylamino and dimethyl amino; [0124]
S(O).sub.q(C.sub.1-C.sub.5 alkyl); such as methanesulfonyl; [0125]
S(O).sub.qH;
[0126] SO.sub.2NH.sub.2; [0127] SO.sub.2NH(C.sub.1-C.sub.5 alkyl);
[0128] SO.sub.2N(C.sub.1-C.sub.5 alkyl)(C.sub.1-C.sub.5 alkyl);
[0129] NHSO.sub.2(C.sub.1-C.sub.5 alkyl); N(C.sub.1-C.sub.3 alkyl)
SO.sub.2(C.sub.1-C.sub.5 alkyl); [0130] CO(C.sub.1-C.sub.6 alkyl or
phenyl); [0131] C(O)H; [0132] C(O)O(C.sub.1-C.sub.6 alkyl or
phenyl), such as C(O)OCH.sub.3, --C(O)OCH.sub.2CH.sub.3,
--C(O)OCH.sub.2CH.sub.2CH.sub.3; [0133] C(O)OH; [0134] C(O)NH.sub.2
(carbamoyl); C(O)NH(C.sub.1-C.sub.6 alkyl or phenyl), such as
N-methylethyl carbamoyl, N-methyl carbamoyl, N-ethylcarbamoyl, or
N-dimethylamino ethyl carbamoyl; [0135] C(O)N(C.sub.1-C.sub.6 alkyl
or phenyl)(C.sub.1-C.sub.6 alkyl, phenyl or pyridinyl), such as
N-dimethylcarbamoyl; [0136] C(N(C.sub.1-C.sub.5 alkyl))
(C.sub.1-C.sub.5 alkyl); [0137] NHC(O)(C.sub.1-C.sub.6 alkyl or
phenyl) and [0138] N(C.sub.1-C.sub.5 alkyl,)C(O)(C.sub.1-C.sub.5
alkyl).
[0139] Each of the above substituents is optionally partially or
fully halogenated, such as difluoromethyl sulfonyl.
[0140] An embodiment of this invention includes the administration
of compounds of this invention wherein in formula I, L, B and L'
follow one of the following of combinations:
[0141] B=phenyl, L=phenyl and L' is phenyl, pyridinyl, quinolinyl,
isoquinolinyl or not present,
[0142] B=phenyl, L=pyridinyl and L' is phenyl, pyridinyl,
quinolinyl, isoquinolinyl or not present,
[0143] B=phenyl, L naphthyl and L' is phenyl, pyridinyl,
quinolinyl, isoquinolinyl or not present,
[0144] B=pyridinyl, L=phenyl and L' is phenyl, pyridinyl,
quinolinyl, isoquinolinyl or not present,
[0145] B=pyridinyl, L=pyridinyl and L' is phenyl, pyridinyl,
quinolinyl, isoquinolinyl or not present,
[0146] B=pyridinyl, L=naphthyl and L' is phenyl, pyridinyl,
quinolinyl, isoquinolinyl or not present,
[0147] B=isoquinolinyl, L=phenyl and L' is phenyl, pyridinyl,
quinolinyl, isoquinolinyl or not present,
[0148] B=isoquinolinyl, L=pyridinyl and L' is phenyl, pyridinyl,
quinolinyl, isoquinolinyl or not present,
[0149] B=isoquinolinyl; L=naphthyl and L' is phenyl, pyridinyl,
quinolinyl, isoquinolinyl or not present,
[0150] B=quinolinyl, L=phenyl and L' is phenyl, pyridinyl,
quinolinyl, isoquinolinyl or not present,
[0151] B=quinolinyl, L=pyridinyl and L' is phenyl, pyridinyl,
quinolinyl, isoquinolinyl or not present,
[0152] B=quinolinyl, L=naphthyl and L' is phenyl, pyridinyl,
quinolinyl, isoquinolinyl or not present.
[0153] The structure M of formula I is preferably --O--, a single
bond, --S--, --NH--, --N(CH.sub.3)--, --NHCH.sub.2--,
--NC.sub.2H.sub.4--, --CH.sub.2--, --C(O)--, --CH(OH)--,
--NHC(O)N(CH.sub.3)CH.sub.2--,
--N(CH.sub.3)C(O)N(CH.sub.3)CH.sub.2--,
--CH.sub.2C(O)N(CH.sub.3)--, --C(O)N(CH.sub.3)CH.sub.2--,
--NHC(O)--, --N(CH.sub.3)C(O)--, --C(O)N(CH.sub.3)--, --C(O)NH--,
--CH.sub.2O--, --CH.sub.2S--, --CH.sub.2N(CH.sub.3)--,
--OCH.sub.2--, --CHF--, --CF.sub.2--, --CCl.sub.2--,
--S--CH.sub.2--, and --N(CH.sub.3)CH.sub.2--.
[0154] Compounds of the invention of particular interest include
those of formula I wherein L', L, M and Q are as defined above and
B is phenyl, optionally substituted with 1-4 halogen.
[0155] Compounds of the invention of particular interest also
include those of formula I wherein L, L' and Q are as defined
above, M is --O-- and B is phenyl, optionally substituted with 1-4
halogen.
[0156] Compounds of the invention of particular interest also
include those of formula I wherein B is phenyl or pyridyl,
optionally substituted with 1-6 substituents independently selected
from the group consisting of R.sup.1 and halogen, L' and Q are as
defined above, M is --O-- and L is phenyl, optionally substituted
with 1-4 halogen.
[0157] Compounds of the invention of particular interest also
include those of formula I wherein B is phenyl, optionally
substituted with 1-6 substituents independently selected from the
group consisting of R.sup.1 and halogen, L' and Q are as defined
above, M is --O-- and L is phenyl, optionally substituted with 1-4
halogen.
[0158] Compounds of the invention of particular interest also
include those of formula I wherein B is
4-chloro(2-trifluoromethyl)phenyl, optionally substituted by the
group consisting of R.sup.1 and halogen, L' and Q are as defined
above, M is --O-- and L is phenyl, optionally substituted with 1-4
halogen.
[0159] One of ordinary skill in the art will recognize that some of
the compounds of Formula (I) can exist in different geometrical
isomeric forms. It is intended that all such configurations
(including enantiomers and diastereomers), are included within the
scope of the present invention. A number of the compounds of
Formula I possess asymmetric centers, depending on the location a
nature of various substituents. and can therefore exist in racemic
and optically active forms as well as in the form of racemic or
non-racemic mixtures thereof, and in the form of diastereomers and
diastereomeric mixtures. Asymmetric carbon atoms may be present in
the (R) or (S) configuration or (R,S) configuration. In certain
instances, asymmetry may also be present due to restricted rotation
about a given bond, for example, the central bond adjoining two
substituted aromatic rings of the specified compounds. All of these
compounds, including cis isomers, trans isomers, diastereomic
mixtures, racemates, non-racemic mixtures of enantiomers,
substantially pure, and pure enantiomers, are considered to be
within the scope of the compounds of this invention and are
collectively referred to when reference is made to compounds of
this invention. Therefore, the methods of the present invention
encompass the use of any isolated racemic or optically, active form
of compounds described in Formula I which possess c-raf, b-raf,
p38, VEGFR, PDGFR, and/or FLT-3 activity.
[0160] Methods of separation of enantiomeric and diastereomeric
mixtures are well known to one skilled in the art. The optical
isomers can be obtained by resolution of the racemic mixtures
according to conventional processes, for example, by the formation
of diastereoisomeric salts using an optically active acid or base.
Examples of appropriate acids are tartaric, diacetyltartaric,
dibenzoyltartaric, ditoluoyltartaric and camphorsulfonic acid.
Mixtures of diastereoisomers can be separated into their individual
diastereomers on the basis of their physical chemical differences
by methods known to those skilled in the art, for example, by
chromatography or fractional crystallization. The optically active
bases or acids are liberated from the separated diastereomeric
salts.
[0161] Another process for separation of optical isomers involves
the use of a chiral chromatography column (e.g., chiral HPLC
columns) optimally chosen to maximize the separation of the
enantiomers. Suitable chiral HPLC columns are manufactured by
Diacel, e.g., Chiracel OD and Chiracel OJ. The optically active
compounds of Formula (I) can likewise be obtained by utilizing
optically active starting materials.
[0162] The present invention encompasses any separated, isolated,
pure or partially purified isomers or racemic mixtures of the
compounds of formula I which possess Raf, VEGFR, PDGFR, p38, and/or
FLT-3 activity, and/or an efficacy in modulating any of the
diseases and/or conditions mentioned herein. The term stereoisomer
is understood to encompass diastereoisomers, enantiomers, geometric
isomers, etc.
[0163] Preferred compounds are those with the absolute
configuration of the compound of Formula I which produce the more
desirable biological activity are also included within the scope of
the present invention. The purification of said isomers and the
separation of said isomeric mixtures can be accomplished by
standard techniques known in the art. Herein, substantially pure
enantiomers is intended to mean that no more than 5% w/w of the
corresponding opposite enantiomer is present.
[0164] Pharmaceutically-acceptable salts of these compounds, as
well as commonly used prodrugs of these compounds, are also within
the scope of the invention. The term "pharmaceutically acceptable
salt" refers to a relatively non-toxic, inorganic, or organic acid
addition salt of a compound of the present invention. For example,
see S. M. Berge, et al. "Pharmaceutical Salts," J. Pharm. Sci.
1977, 66, 1-19.
[0165] Suitable salts are especially the pharmaceutically
acceptable salts of compounds of formula (I) or such as, for
example, organic or inorganic acid addition salts of compounds of
formula (I). Suitable acid addition salts include acetate, adipate,
alginate, ascorbate, aspartate, benzoate, benzenesulfonate,
bisulfate, butyrate, citrate, camphorate, camphorsulfonate,
cinnamate, cyclopentanepropionate, digluconate, dodecylsulfate,
ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate,
hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide,
hydroiodide, 2-hydroxyethanesulfonate, itaconate, lactate, maleate,
mandelate, methanesulfonate, 2-naphthalenesulfonate, nicotinate,
nitrate, oxalate, pamoate, pectinate, persulfate,
3-phenylpropionate, picrate, pivalate, propionate, succinate,
sulfonate, tartrate, thiocyanate, tosylate, and undecanoate.
Suitable inorganic acids include but are not limited to halogen
acids (such as hydrochloric acid and hydrobromic acid), sulfuric
acid, or phosphoric acid. Suitable organic acids include but are
not limited to carboxylic, phosphonic, sulfonic, or sulfamic acids,
with examples including acetic acid, propionic acid, octanoic acid,
decanoic acid, trifluoroacetic acid, dodecanoic acid, glycolic
acid, lactic acid, 2- or 3-hydroxybutyric acid,
.gamma.-aminobutyric acid (GABA), gluconic acid,
glucosemonocarboxylic acid, benzoic acid, salicylic acid,
phenylacetic acid and mandelic acid, fumaric acid, succinic acid,
adipic acid, pimelic acid, suberic acid, azeiaic acid, maleic acid,
tartaric acid, citric, acid, glucaric acid, galactaric acid, amino
acids (such as glutamic acid, aspartic acid, N-methylglycine,
acetytaminoacetic acid, N-acetylasparagine or N-acetylcysteine),
pyruvic acid, acetoacetic acid, methanesulfonic acid,
tri-fluoromethane sulfonic acid, 4-toluene sulfonic acid,
benzenesulfonic acid, 1-naphthalenesulfonic acid,
2-naphthalenesulfonic acid, phosphoserine, and 2- or
3-glycerophosphoric acid.
[0166] In addition, pharmaceutically acceptable salts include acid
salts of inorganic bases, such as salts containing alkaline cations
(e.g., Li.sup.+ Na.sup.+ or K.sup.+), alkaline earth cations (e.g.,
Mg.sup.+2, Ca.sup.+2 or Ba.sup.+2), the ammonium cation, as well as
acid salts of organic bases, including aliphatic and aromatic
substituted ammonium, and quaternary ammonium cations, such as
those arising from protonation or peralkylation of triethylamine,
N,N-diethylamine, N,N-dicyclohexylamine, lysine, pyridine,
N,N-dimethylaminopyridine (DMAP), 1,4-diazabiclo[2.2.2]octane
(DABCO), 1,5-diazabicyclo[4.3.0]non-5-ene (DBN) and
1,8-diazabicyclo[5.4.0]undec-7-ene (DBU).
[0167] Base salts include alkali metal salts such as potassium and
sodium salts, alkaline earth metal salts such as calcium and
magnesium salts, and ammonium salts with organic bases such as
dicyclohexylamine and N-methyl-D-glucamine. Additionally, basic
nitrogen containing groups may be quaternized with such agents as
lower alkyl halides such as methyl, ethyl, propyl, and butyl
chlorides, bromides and iodides; dialkyl sulfates like dimethyl,
diethyl, and dibutyl sulfate; and diamyl sulfates, long chain
halides such as decyl, lauryl, myristyl and strearyl chlorides,
bromides and iodides, aralkyl halides like benzyl and phenethyl
bromides and others.
[0168] The esters of appropriate compounds of this invention are
well-tolerated, pharmaceutically acceptable esters such as alkyl
esters including methyl, ethyl, propyl, isopropyl, butyl, isobutyl
or pentyl esters. Additional esters such as phenyl-C.sub.1-C.sub.5
alkyl may be used, although methyl ester is preferred.
[0169] The formation of prodrugs is well known in the art in order
to enhance the properties of the parent compound; such properties
include solubility, absorption, biostability and release time (see
"Pharmaceutical Dosage Form and Drug Delivery Systems" (Sixth
Edition), edited by Ansel et al., published by Williams &
Wilkins, pages 27-29, (1995) which is hereby incorporated by
reference). Commonly used prodrugs of the disclosed
oxazolyl-phenyl-2,4-diamino-pyrimidine compounds are designed to
take advantage of the major drug biotransformation reactions and
are also to be considered within the scope of the invention. Major
drug biotransformation reactions include N-dealkylation,
O-dealkylation, aliphatic hydroxylation, aromatic hydroxylation,
N-oxidation, S-oxidation, deamination, hydrolysis reactions,
glucuronidation, sulfation and acetylation (see Goodman and
Gilman's The Pharmacological Basis of Therapeutics (Ninth Edition),
editor Molinoff et al., pub. by McGraw-Hill, pages 11-13, (1996),
which is hereby incorporated by reference).
[0170] The present invention provides compounds which are capable
of modulating one or more signal transduction pathways comprising,
but not limited to, raf, VEGFR-2, VEGFR-3, p38, PDGFR-beta, and/or
Flt-3. Raf is an important signaling molecule involved in the
regulation of a number of key cellular processes, including cell
growth, cell survival and invasion. It is a member of the
Ras/Raf/MEK/ERK pathway. This pathway is present in most tumor
cells. VEGFR-2, VEGFR-3, PDGFR-beta, and Flt-3 are transmembrane
receptor molecules which, when stimulated by an appropriate ligand,
trigger the Ras/Raf/MEK/ERK cell signaling pathway, leading to a
cascade of cellular events. Each of these receptor molecules have
tyrosine kinase activity.
[0171] The VEGFR receptors are stimulated by vascular endothelial
growth factors (VEGF), and are important control points in the
regulation of endothelial cell development and function. The
PDGF-beta receptor regulates cell proliferation and survival in a
number of cell types, including mesenchymal cells. Flt-3 is a
receptor for the FL ligand. It is structurally similar to c-kit,
and modulates the growth of pluripotent haemopoietic cells,
influencing the development of T-cells, B-cells, and dendritic
cells.
[0172] Any gene or isoform of raf, VEGFR-2, VEGFR-3, p38,
PDGFR-beta, and/or Flt-3 can be modulated in accordance with
present invention, including both wild-type and mutant forms. Raf
or raf-1 kinase is a family of serine/threonine kinases which
comprise at least three family members, A-Raf, B-Raf, and c-raf or
Raf-1. See, e.g., Dhillon and Kolch, Arch. Biochem. Biophys.,
404:3-9, 2002. C-raf and B-Raf are preferred targets for compounds
of the present invention. Activating B-Raf mutations (e.g., V599E
mutant) have been identified in various cancers, including
melanoma, and the compounds described herein can be utilized to
inhibit their activity.
[0173] By the term "modulate," it is meant that the functional
activity of the pathway (or a component of it) is changed in
comparison to its normal activity in the absence of the compound.
This effect includes any quality or degree of modulation,
including, increasing, agonizing, augmenting, enhancing,
facilitating, stimulating, decreasing, blocking, inhibiting,
reducing, diminishing, antagonizing, etc.
[0174] Tables 8 and 9 show the activity of a compound
N-[4-chloro-3-(trifluoromethyl)phenyl]-N'-{4-[2-N-methylcarbamoyl-4-pyrid-
yloxy]phenyl}urea of the present invention in vitro (biochemical)
and in vivo (cellular) assays, respectively. As indicated by its
biochemical activity profile (Table 8), the compound is a potent
inhibitor of a number of different cellular kinases. Moreover, it
has the ability to affect kinase activity, cell growth, and other
cellular processes in whole cells. Table 9 shows the concentration
of compound which inhibited target phosphorylation in whole cells
by 50% (IC50). While the activity profile is representative of an
active compound of the present invention, not all active compounds
are required to have the same rank order of potency for them to be
useful in the present invention. Assays for biochemical and
cellular activity are conventional and can be carried out using any
suitable format, including as described in the examples below. See,
also, VEGFR-3 kinase assays (e.g., Manley et al., J. Med. Chem.,
45(26):5687-93, 2002; Stahl et al., Angew Chem. Int. Ed. Engl.,
41(7):1174-8, 2002); FLT3 kinase assays (Yee et al., Blood,
100(8):2941-9, 2002; Kelly et al., Cancer Cell., 1(5):421-32,
2002).
[0175] The compounds of the present invention can also modulate one
or more of the following processes, including, but not limited to,
e.g., cell growth (including, e.g., differentiation, cell survival,
and/or proliferation), tumor cell growth (including, e.g.,
differentiation, cell survival, and/or proliferation), tumor
regression, endothelial cell growth (including, e.g.,
differentiation, cell survival, and/or proliferation), angiogenesis
(blood vessel growth), lymphangiogenesis (lymphatic vessel growth),
and/or hematopoiesis (e.g., T- and B-cell development, dendritic
cell development, etc.).
[0176] While not wishing to be bound by any theory or mechanism of
action, it has been found that compounds of the present invention
possess the ability to modulate kinase activity. The methods of the
present invention, however, are not limited to any particular
mechanism or how the compounds achieve their therapeutic effect. By
the phrase "kinase activity," it is meant a catalytic activity in
which a gamma-phosphate from adenosine triphosphate (ATP) is
transferred to an amino acid residue (e.g., serine, threonine, or
tyrosine) in a protein substrate. A compound can modulate kinase
activity, e.g., inhibiting it by directly competing with ATP for
the ATP-binding pocket of the kinase, by producing a conformational
change in the enzyme's structure that affects its activity (e.g.,
by disrupting the biologically-active three-dimensional structure),
etc.
[0177] Kinase activity can be determined routinely using
conventional assay methods. Kinase assays typically comprise the
kinase enzyme, substrates, buffers, and components of a detection
system. A typical kinase assay involves the reaction of a protein
kinase with a peptide substrate and an ATP, such as 32P-ATP, to
produce a phosphorylated end-product (for instance, a
phosphoprotein when a peptide substrate is used. The resulting
end-product can be detected using any suitable method. When
radioactive ATT is utilized, a radioactively labeled phosphoprotein
can be separated from the unreacted gamma-32P-ATP using an affinity
membrane or gel electrophoresis, and then visualized on the gel
using autoradiography or detected with a scintillation counter.
Non-radioactive methods can also be used. Methods can utilize an
antibody which recognizes the phosphorylated substrate, e.g., an
anti-phosphotyrosine antibody. For instance, kinase enzyme can
incubated with a substrate, in the presence of ATP and kinase
buffer under conditions which are effective for the enzyme to
phosphorylate the substrate. The reaction mixture can be separated,
e.g., electrophoretically, and then phosphorylation of the
substrate can be measured, e.g., by Western blotting using an
anti-phosphotyrosine antibody. The antibody can be labeled with a
detectable label, e.g., an enzyme, such as HRP, avidin or biotin,
chemiluminescent reagents, etc. Other methods can utilize ELISA
formats, affinity membrane separation, fluorescence polarization
assays, luminescent assays, etc.
[0178] An alternative to a radioactive format is time-resolved
fluorescence resonance energy transfer (TR-FRET). This method
follows the standard kinase reaction, where a substrate, e.g.,
biotinylated poly(GluTyr), is phosphorylated by a protein kinase in
the presence of ATP. The end-product can then detected with a
europium chelate phosphospecific antibody (anti-phosphotyrosine or
phosphoserine/threonine), and streptavidin-APC, which binds the
biotinylated substrate. These two-components are brought together
spatially upon binding, and energy transfer from the
phosphospecific antibody to the acceptor (SA-APC) produces
fluorescent readout in the homogeneous format.
[0179] The compounds of the present invention can be used to treat
and/or prevent any disease or condition involving one or more
cellular signal transduction pathways comprising raf, VEGFR-2,
VEGFR-3, p38, PDGFR-beta, and/or Flt-3. The term "treating" is used
conventionally, e.g., the management or care of a subject for the
purpose of combating, alleviating, reducing, relieving, improving
the condition of, etc., of a disease or disorder. Diseases and
conditions that can be treated include any of those mentioned above
and below, as well as:
[0180] Raf associated diseases include, e.g., cell-proliferation
disorders, cancer, tumors, etc.;
[0181] VEGFR-2 associated diseases include, e.g., cancer, tumor
growth, inflammatory disease, rheumatoid arthritis, retinopathy,
psoriasis, glomerulonephritis, asthma, chronic bronchitis,
atherosclerosis, transplant rejection, conditions involving
angiogenesis, etc.;
[0182] VEGFR-3 associated diseases include, e.g., cancer, corneal
disease, inflamed cornea (e.g., Hamrah, Am. J. Path., 163:57-68,
2003), corneal transplantation (Cursiefen et al., Cornea,
22:273-81, 2003), lymphatic hyperplasia, conditions involving
lymphangiogenesis, etc.;
[0183] PDGFR-beta associated diseases include, e.g., diseases or
conditions characterized by cell proliferation, cell matrix
production, cell movement, and/or extracellular matrix production.
Specific examples, include, e.g., tumors, malignancies, cancer,
metastasis, chronic myeloid leukemia, inflammation, renal disease,
diabetic nephropathy, mesangial proliferative glomerulonephritis,
fibrotic conditions, atherosclerosis, restenosis,
hypertension-related arterosclerosis, Venous bypass graft
arterosclerosis, scleroderma, interstitial pulmonary diseases,
synovial disorders, arthritis, leukemias, lymphomas, etc;
[0184] Flt-3 associated diseases include, e.g., immune-related
disorders, blood cell disorders, conditions involving hematopoietic
cell development (e.g., T-cells, B-cells, dendritic cells, cancer,
anemia, HIV, acquired immune deficiency syndrome, etc.
[0185] In addition, compounds of the present invention can be used
to treat conditions and disorders disclosed in U.S. Pat. No.
6,316,479, e.g, glomerular sclerosis, interstitial nephritis,
interstitial pulmonary fibrosis, atherosclerosis, wound scarring
and scleroderma.
[0186] The compounds of this invention also have a broad
therapeutic activity to treat or prevent the progression of a broad
array of diseases, such as inflammatory conditions, coronary
restenosis, tumor-associated angiogenesis, atherosclerosis,
autoimmune diseases, inflammation, certain kidney diseases
associated with proliferation of glomerular or mesangial cells, and
ocular diseases associated with retinal vessel proliferation.
psoriasis, hepatic cirrhosis, diabetes, atherosclerosis,
restenosis, vascular graft restenosis, in-stent stenosis,
angiogenesis, ocurlar diseases, pulmonary fibrosis, obliterative
bronchiolitis, glomerular nephritis, rheumatoid arthritis,
[0187] The present invention also provides for treating,
preventing, modulating, etc., one or more of the following
conditions in humans and/or other mammals: retinopathy, including
diabetic retinopathy, ischemic retinal-vein occlusion, retinopathy
of prematurity and age related macular degeneration; rheumatoid
arthritis, psoriasis, or bullous disorder associated with
subepidermal blister formation, including bullous pemphigoid,
erythema multiforme, or dermatitis herpetiformis, rheumatic fever,
bone resorption, postmenopausal osteoperosis, sepsis, gram negative
sepsis, septic shock, endotoxic shock, toxic shock syndrome,
systemic inflammatory response syndrome, inflammatory bowel disease
(Crohn's disease and ulcerative colitis), Jarisch-Herxheimer
reaction, asthma, adult respiratory distress syndrome, acute
pulmonary fibrotic disease, pulmonary sarcoidosis, allergic
respiratory disease, silicosis, coal worker's pneumoconiosis,
alveolar injury, hepatic failure, liver disease during acute
inflammation, severe alcoholic hepatitis, malaria (Plasmodium
falciparum malaria and cerebral malaria), non-insulin-dependent
diabetes mellitus (NIDDM), congestive heart failure, damage
following heart disease, atherosclerosis, Alzheimer's disease,
acute encephalitis, brain injury, multiple sclerosis (demyelation
and oligiodendrocyte loss in multiple sclerosis), advanced cancer,
lymphoid malignancy, pancreatitis, impaired wound healing in
infection, inflammation and cancer, myelodysplastic syndromes,
systemic lupus erythematosus, biliary cirrhosis, bowel necrosis,
radiation injury/toxicity following administration of monoclonal
antibodies, host-versus-graft reaction (ischemia reperfusion injury
and allograft rejections of kidney, liver, heart, and skin), lung
allograft rejection (obliterative bronchitis), or complications due
to total hip replacement, ad an infectious disease selected from
tuberculosis, Helicobacter pylori infection during peptic ulcer
disease, Chaga's disease resulting from Trypanosoma cruzi
infection, effects of Shiga-like toxin resulting from E. coli
infection, effects of enterotoxin A resulting from Staphylococcus
infection, meningococcal infection, and infections from Borrelia
burgdorferi, Treponema pallidum, cytomegalovirus, influenza virus,
Theiler's encephalomyelitis virus, and the human immunodeficiency
virus (HIV), papilloma, blastoglioma, Kaposi's sarcoma, melanoma,
lung cancer, ovarian cancer, prostate cancer, squamous cell
carcinoma, astrocytoma, head cancer, neck cancer, bladder cancer,
breast cancer, colorectal cancer, thyroid cancer, pancreatic
cancer, gastric cancer, hepatocellular carcinoma, leukemia,
lymphoma, Hodgkin's disease, Burkitt's disease, arthritis,
rheumatoid arthritis, diabetic retinopathy, angiogenesis,
restenosis, in-stent restenosis, vascular graft restenosis,
pulmonary fibrosis, hepatic cirrhosis, atherosclerosis,
glomerulonophritis, diabetic nephropathy, thrombic micoangiopathy
syndromes, transplant rejection, psoriasis, diabetes, wound
healing, inflammation, and neurodegenerative diseases. hyperimmune
disorders, hemangioma, myocardial angiogenesis, coronary and
cerebral collateral vascularization, ischemia, corneal disease,
rubeosis, neovascular glaucoma, macular degeneration retinopathy of
prematurity, wound healing, ulcer Helicobacter related diseases,
fractures, endometriosis, a diabetic condition, cat scratch fever,
thyroid hyperplasia, asthma or edema following burns, trauma,
chronic lung disease, stroke, polyps, cysts, synovitis, chronic and
allergic inflammation, ovarian hyperstimulation syndrome, pulmonary
and cerebral edema, keloid, fibrosis, cirrhosis, carpal tunnel
syndrome, adult respiratory distress syndrome, ascites, an ocular
condition, a cardiovascular condition, Crow-Fukase (POEMS) disease,
Crohn's disease, glomerulonophritis, osteoarthritis, multiple
sclerosis, graft rejection, Lyme disease, sepsis, von Hippel Lindau
disease, pemphigoid, Paget's disease, polycystic kidney disease,
sarcoidosis, throiditis, hyperviscosity syndrome, Osler-Weber-Rendu
disease, chronic occlusive pulmonary disease, radiation, hypoxia,
preeclampsia, menometrorrhagia, endometriosis, infection by Herpes
simplex, ischemic retinopathy, corneal angiogenisis, Herpes Zoster,
human immunodeficiency virus, parapoxvirus, protozoa,
toxoplasmosis, and tumor-associated effusions and edema.
[0188] Compounds can possess more than one of the mentioned
activities, and therefore can target a plurality of signal
transduction pathways. Thus, these compounds can achieve
therapeutic and prophylactic effects which normally are only
obtained when using a combination of different compounds. For
instance, the ability to inhibit both new vessel formation (e.g.,
associated with VEGFR-2 and -3 function) (e.g., blood and/or lymph)
and cell-proliferation (e.g., associated with raf and PDGFR-beta
function) using a single compound is especially beneficial in the
treatment of cancer, and other cell-proliferation disorders that
are facilitated by neovascularization. Thus, the present invention
relates specifically to compounds which possess at least anti-cell
proliferation and anti-angiogenic (i.e., inhibits angiogenesis)
activity. Any disorder or condition that would benefit from
inhibiting vessel growth and cell proliferation can be treated in
accordance with the present invention. Using a single compound is
also advantageous because its range of activities can be more
precisely defined.
[0189] As indicated above, the present invention relates to methods
of treating and/or preventing diseases and conditions; and/or
modulating one or more of the pathways, polypeptides, genes,
diseases, conditions, etc., associated with raf, VEGFR-2, VEGFR-3,
PDGFR-beta, and/or Flt-3. These methods generally involve
administering effective amounts of compounds of the present
invention, where an effective amount is the quantity of the
compound which is useful to achieve the desired result. Compounds
can be administered in any effective form by any effective route,
as discussed in more detail below.
[0190] Methods include modulating tumor cell proliferation,
including inhibiting cell proliferation. The latter indicates that
the growth and/or differentiation of tumor cells is reduced,
decreased, diminished, slowed, etc. The term "proliferation"
includes any process which relates to cell growth and division, and
includes differentiation and apoptosis. As discussed above, raf
kinases play a key role in the activation of the cytoplasmic
signaling cascade involved in cell proliferation, differentiation,
and apoptosis. For example, studies have found that inhibiting
c-raf-1 by anti-sense oligonucleotides can block cell proliferation
(see above). Any amount of inhibition is considered
therapeutic.
[0191] Any tumor or cancer can be treated, including, but not
limited to, cancers having one or more mutations in raf, VEGFR-2,
VEGFR-3, PDGFR-beta, Flt-3, and/or ras, as well as any upstream or
downstream member of the signaling pathways of which they are a
part. As discussed earlier, a cancer can be treated with a compound
of the present invention irrespective of the mechanism which is
responsible for it. Cancers of any organ can be treated, including
cancers of, but are not limited to, e.g., colon, pancreas, breast,
prostate, bone, liver, kidney, lung, testes, skin, pancreas,
stomach, colorectal cancer, renal cell carcinoma, hepatocellular
carcinoma, melanoma, etc.
[0192] Examples of breast cancer include, but are not limited to,
invasive ductal carcinoma, invasive lobular carcinoma, ductal
carcinoma in situ, and lobular carcinoma in situ.
[0193] Examples of cancers of the respiratory tract include, but
are not limited to, small-cell and non-small-cell lung carcinoma,
as well as bronchial adenoma and pleuropulmonary blastoma.
[0194] Examples of brain cancers include, but are not limited to,
brain stem and hypophtalmic glioma, cerebellar and cerebral
astrocytoma, medulloblastoma, ependymoma, as well as
neuroectodermal and pineal tumor.
[0195] Tumors of the male reproductive organs include, but are not
limited to, prostate and testicular cancer. Tumors of the female
reproductive organs include, but are not limited to, endometrial,
cervical, ovarian, vaginal, and vulvar cancer, as well as sarcoma
of the uterus.
[0196] Tumors of the digestive tract include, but are not limited
to, anal, colon, colorectal, esophageal, gallbladder, gastric,
pancreatic, rectal, small-intestine, and salivary gland
cancers.
[0197] Tumors of the urinary tract include; but are not limited to,
bladder, penile, kidney, renal pelvis, ureter, and urethral
cancers.
[0198] Eye cancers include, but are not limited to, intraocular
melanoma and retinoblastoma.
[0199] Examples of liver cancers include, but are not limited to,
hepatocellular carcinoma (liver cell carcinomas with or without
fibrolamellar variant), cholangiocarcinoma (intrahepatic bile duct
carcinoma), and mixed hepatocellular cholangiocarcinoma.
[0200] Skin cancers include, but are not limited to, squamous cell
carcinoma, Kaposi's sarcoma, malignant melanoma, Merkel cell skin
cancer, and non-melanoma skin cancer.
[0201] Head-and-neck cancers include, but are not limited to,
laryngeal, hypopharyngeal, nasopharyngeal, and/or oropharyngeal
cancers, and lip and oral cavity cancer.
[0202] Lymphomas include, but are not limited to, AIDS-related
lymphoma, non-Hodgkin's lymphoma, cutaneous T-cell lymphoma,
Hodgkin's disease, and lymphoma of the central nervous system.
[0203] Sarcomas include, but are not limited to, sarcoma of the
soft tissue, osteosarcoma, malignant fibrous histiocytoma,
lymphosarcoma, and rhabdomyosarcoma.
[0204] Leukemias include, but are not limited to, acute myeloid
leukemia, acute lymphoblastic leukemia, chronic lymphocytic
leukemia, chronic myelogenous leukemia, and hairy cell
leukemia.
[0205] In addition to inhibiting the proliferation of tumor cells,
compounds of the present invention can also cause tumor regression,
e.g., a decrease in the size of a tumor, or in the extent of cancer
in the body.
[0206] The present invention also relates to methods of modulating
angiogenesis and/or lymphangiogenesis in a system comprising cells,
comprising administering to the system an effective amount of a
compound described herein. A system comprising cells can be an in
vivo system, such as a tumor in a patient, isolated organs,
tissues, or cells, in vitro assays systems (CAM, BCE, etc), animal
models (e.g., in vivo, subcutaneous, cancer models), hosts in need
of treatment (e.g., hosts suffering from diseases having angiogenic
and/or lymphangiogenic component, such as cancer), etc. Preferred
compounds of the present invention inhibit angiogenesis and/or
lymphangiogenesis, e.g., the formation of new blood vessels.
[0207] Inappropriate and ectopic expression of angiogenesis can be
deleterious to an organism. A number of pathological conditions are
associated with the growth of extraneous blood vessels. These
include, e.g., diabetic retinopathy, neovascular glaucoma,
psoriasis, retrolental fibroplasias, angiofibroma, inflammation,
etc. In addition, the increased blood supply associated with
cancerous and neoplastic tissue, encourages growth, leading to
rapid tumor enlargement and metastasis. Moreover, the growth of new
blood and lymph vessels in a tumor provides an escape route for
renegade cells, encouraging metastasis and the consequence spread
of the cancer.
[0208] Useful systems for modulating angiogenesis, include, e.g.,
neovascularization of tumor explants (e.g., U.S. Pat. Nos.
5,192,744; 6,024,688), chicken chorioallantoic membrane (CAM) assay
(e.g., Taylor and Folkman, Nature, 297:307-312, 1982; Eliceiri et
al., J. Cell Biol., 140, 1255-1263, 1998), bovine capillary
endothelial (BCE) cell assay (e.g., U.S. Pat. No. 6,024,688;
Polverini, P. J. et al., Methods Enzymol., 198: 440-450, 1991),
migration assays, and HUVEC (human umbilical cord vascular
endothelial cell) growth inhibition assay (e.g., U.S. Pat. No.
6,060,449). In addition, useful systems for modulating
lymphangiogenesis, include, e.g., rabbit ear model (e.g., Szuba et
al., FASEB J., 16(14):1985-7, 2002).
[0209] Modulation of angiogenesis can be determined by any suitable
method. For example, the degree of tissue vascularity is typically
determined by assessing the number and density of vesssels present
in a given sample. For example, microvessel density (MVD) can be
estimated by counting the number of endothelial clusters in a
high-power microscopic field, or detecting a marker specific for
microvascular endothelium or other markers of growing or
established blood vessels, such as CD31 (also known as
platelet-endothelial cell adhesion molecule or PECAM). A CD31
antibody can be employed in conventional immunohistological methods
to immunostain tissue sections as described by, e.g., Penfold et
al., Br. J. Oral and Maxill. Surg., 34: 37-41; U.S. Pat. No.
6,017,949; Dellas et al., Gyn. Oncol., 67:27-33, 1997; and others.
Other markers for angiogenesis, include, e.g., Vezf1 (e.g., Xiang
et al., Dev. Bio., 206:123-141, 1999), angiopoietin, Tie-1, and
Tie-2 (e.g., Sato et al., Nature, 376:70-74, 1995). Additionally,
levels of circulating VEGF, such as VEGF-165, VEGF-C, VEGF-D, can
be measured in an ELISA to determine whether it is above a
threshold value that indicates angiongenic activity in the
body.
[0210] Additionally, the present invention relates to methods of
screening patients to determine their susceptibility to compounds
of the present invention. For example, the presenting invention
relates to methods of selecting subjects having a disease for
treatment with a compound of formula I, comprising, one or more of
the following steps in any effective order, e.g., measuring the
expression or activity of Raf, VEGFR-2, VEGFR-3, p38, PDGFR-beta,
and/or Flt-3, in a sample obtained from a subject having a disease,
and administering said compound of formula I to subjects who are
identified as having high levels of expression or activity, where
said compound is a compound of formula I of claim 1.
[0211] The term "susceptibility" is used broadly to indicate, e.g.,
ability to respond, toxicity or other adverse effects, etc. For
example, the invention relates to methods of determining whether a
condition can be modulated by a compound disclosed herein,
comprising measuring the expression or activity of Raf, VEGFR-2,
VEGFR-3, p38, PDGFR-beta, and/or Flt-3 in cells having said
condition. The results can be used to determine or predict whether
a subject will respond to a compound of the present invention. For
example, where the condition is a tumor, the methods can be used to
predict whether the tumor is susceptible to compounds of the
present invention. By the term "susceptible," it is meant that
tumor can be treated with it, e.g., causing tumor regression or
cell death, inhibiting cell proliferation, inhibiting tumor growth,
inhibiting tumor metastasis, etc.
[0212] Whether a condition, such as a tumor, is susceptible to a
compound of the present invention can be determined routinely. For
instance, cells or tissues (e.g., tumor cells, a biopsy sample,
etc.) that exhibit the condition can be assayed for the presence
and/or activity of Raf, VEGFR-2, VEGFR-3, p38, PDGFR-beta, and/or
Flt-3. When high levels of expression and/or activity are
identified, this can indicate that the subject will respond to, and
benefit from, a compound of the present invention. Levels of gene
expression (e.g., mRNA levels), gene amplification, or gene product
activity (e.g., tyrosine kinase activity) can be utilized to
characterize the state of the cell with respect to the
corresponding gene and signaling pathway. For example, the target
genes of the present invention possess tyrosine kinase activity,
and therefore kinase activity can be used to assess the cell or
tissue state. In the example below, activity was measured by
looking at the levels of substrate phosphorylated by it. This can
be done quantitatively (e.g., using isotopes, spectroscopy, etc.)
or semi-quantitatively as in the example where the levels were
assessed visually and assigned a level of intensity from +1 to +4.
A cell or tissue which has a high level of phosphorylated substrate
(and a high number of cells exihibiting the heightened activity)
can be considered to have a high level of kinase activity, and
therefore be a candidate for therapy with a compound of the present
invention. More than one activity can be assessed, and the results
from several targets can be utilized in deciding whether a
subject's condition (e.g., a tumor) will be responsive to a
compound of the present invention.
[0213] High levels of target activity can be relative to a control
or other standard. For instance, in the example below, high levels
of activity were with reference to a cell type (stromal) in the
tissue section which normally does not express substantial levels
of the target gene. High levels can therefore be where cells
express a statistically higher amount of measured activity or
phosphoryated substrate than the standard or control used as a
comparison. High levels can also be where 25% or more cells express
the target activity (e.g., phospho-ERK).
[0214] The method can further comprise a step of comparing the
expression in a sample with a normal control, or expression in a
sample obtained from normal or unaffected tissue. Comparing can be
done manually, against a standard, in an electronic form (e.g.,
against a database), etc. The normal control can be a standard
sample that is provided with the assay; it can be obtained from
adjacent, but unaffected, tissue from the same patient; or, it can
be pre-determined values, etc. Gene expression, protein expression
(e.g., abundance in a cell), protein activity (e.g., kinase
activity), etc., can be determined.
[0215] For instance, a biopsy from a cancer patient can be assayed
for the presence, quantity, and/or activity of Raf, VEGFR-2,
VEGFR-3, p38, PDGFR-beta, and/or Flt-3. Increased expression or
activity of one or more of these can indicate that the cancer can
be targeted for treatment by a compound of the present invention.
For example, as described in the examples below, raf activity can
be monitored by its ability to initiate the cascade leading to ERK
phosphorylation (i.e., raf/MEK/ERK), resulting in phospho-ERK.
Increased phospho-ERK levels in a cancer shows that its raf
activity is elevated, suggesting the use of compounds of the
present invention to treat it. In addition to biopsy samples,
phospho-ERK (other markers) can also be measured in other body
fluids, such as serum, blood, cerebral spinal fluid, urine, etc.,
such as in peripheral blood lymphocytes (PBLs). For the latter,
inhibition of ERK phosphorylation can be measured following
activation with phorbol myristate acetate using antibodies as
described in the examples below.
[0216] In addition, patients having cancer can be selected and
monitored on the basis of whether the tissue is experiencing
neovacularization, and how much. This can be assessed as discussed
above, e.g., using immunohistochemistry for vessel markers (e.g.,
CD31), circulating levels of a VGFR ligand, etc.
[0217] Patient selection and monitoring can also be made on the
basis of the appearance in a body fluid (such as blood) above
normal levels of the shedded ectodomains derived from the various
receptors, including the extracellular portions of VEGFR-2,
VEGFR-3, p38, PDGFR-beta, and Flt-3. Detection methods can be
carried out routinely, e.g., using antibodies which specifically
bind to the extracellular domain.
[0218] Measuring expression includes determining or detecting the
amount of the polypeptide present in a cell or shed by it, as well
as measuring the underlying mRNA, where the quantity of mRNA
present is considered to reflect the quantity of polypeptide
manufactured by the cell. Furthermore, the genes for Raf, VEGFR-2,
VEGFR-3, p38, PDGFR-beta, and/or Flt-3 can be analyzed to determine
whether there is a gene defect responsible for aberrant expression
or polypeptide activity. Genes sequences are publically available;
e.g., NM.sub.--004333 Homo sapiens v-raf murine sarcoma viral
oncogene homolog B1 (BRAF); NM.sub.--004119 Homo sapiens
fms-related tyrosine kinase 3 (FLT3); NM.sub.--002609 Homo sapiens
platelet-derived growth factor receptor, beta polypeptide (PDGFRB);
NM.sub.--002253 Homo sapiens VEGFR2; NM.sub.--182925 Homo sapiens
fms-related tyrosine kinase 4 (FLT4); L35253 Homo sapiens p38
mitogen activated protein (MAP) kinase.
[0219] Polypeptide detection can be carried out by any available
method, e.g., by Western blots, ELISA, dot blot,
immunoprecipitation, RIA, immunohistochemistry, etc. For instance,
a tissue section can be prepared and labeled with a specific
antibody (indirect or direct and visualized with a microscope.
Amount of a polypeptide can be quantitated without visualization,
e.g., by preparing a lysate of a sample of interest, and then
determining by ELISA or Western the amount of polypeptide per
quantity of tissue. Antibodies and other specific binding agents
can be used. There is no limitation on how detection is
performed.
[0220] Assays can be utilized which permit quantification and/or
presence/absence detection of a target nucleic acid (e.g., genes,
mRNA, etc., for raf, VEGFR, PDGFR, Flt-3, etc) in a sample. Assays
can be performed at the single-cell level, or in a sample
comprising many cells, where the assay is "averaging" expression
over the entire collection of cells and tissue present in the
sample. Any suitable assay format can be used, including, but not
limited to, e.g., Southern blot analysis, Northern blot analysis,
polymerase chain reaction ("PCR") (e.g., Saiki et al., Science,
241:53, 1988; U.S. Pat. Nos. 4,683,195, 4,683,202, and 6,040,166;
PCR Protocols: A Guide to Methods and Applications, Innis et al.,
eds., Academic Press, New York, 1990), reverse transcriptase
polymerase chain reaction ("RT-PCR"), anchored PCR, rapid
amplification of cDNA ends ("RACE") (e.g., Schaefer in Gene Cloning
and Analysis: Current Innovations, Pages 99-115, 1997), ligase
chain reaction ("LCR") (EP 320 308), one-sided PCR (Ohara et al.,
Proc. Natl. Acad. Sci., 86:5673-5677, 1989), indexing methods
(e.g., U.S. Pat. No. 5,508,169), in situ hybridization,
differential display (e.g., Liang et al., Nucl. Acid. Res., 21:3269
3275, 1993; U.S. Pat. Nos. 5,262,311, 5,599,672 and 5,965,409;
WO97/18454; Prashar and Weissman, Proc. Natl. Acad. Sci.,
93:659-663, and U.S. Pat. Nos. 6,010,850 and 5,712,126; Welsh et
al., Nucleic Acid Res., 20:4965-4970, 1992, and U.S. Pat. No.
5,487,985) and other RNA fingerprinting techniques, nucleic acid
sequence based amplification ("NASBA") and other transcription
based amplification systems (e.g., U.S. Pat. Nos. 5,409,818 and
5,554,527; WO 88/10315), polynucleotide arrays (e.g., U.S. Pat.
Nos. 5,143,854, 5,424,186; 5,700,637, 5,874,219, and 6,054,270; PCT
WO 92/10092; PCT WO 90/15070), Qbeta Replicase (PCT/US87/00880),
Strand Displacement Amplification ("SDA"), Repair Chain Reaction
("RCR"), nuclease protection assays, subtraction-based methods,
Rapid-Scan, etc. Additional useful methods include, but are not
limited to, e.g., template-based amplification methods, competitive
PCR (e.g., U.S. Pat. No. 5,747,251), redox-based assays (e.g., U.S.
Pat. No. 5,871,918), Taqman-based assays (e.g., Holland et al.,
Proc. Natl. Acad, Sci., 88:7276-7280, 1991; U.S. Pat. Nos.
5,210,015 and 5,994,063), real-time fluorescence-based monitoring
(e.g., U.S. Pat. No. 5,928,907), molecular energy transfer labels
(e.g., U.S. Pat. Nos. 5,348,853, 5,532,129, 5,565,322, 6,030,787,
and 6,117,635; Tyagi and Kramer, Nature Biotech., 14:303-309,
1996). Any method suitable for single cell analysis of gene or
protein expression can be used, including in situ hybridization,
immunocytochemistry, MACS, FACS, flow cytometry, etc. For single
cell assays, expression products can be measured using antibodies,
PCR, or other types of nucleic acid amplification (e.g., Brady et
al., Methods Mol. & Cell. Biol. 2, 17-25, 1990; Eberwine et
al., 1992, Proc. Natl. Acad. Sci., 89, 3010-3014, 1992, U.S. Pat.
No. 5,723,290). These and other methods can be carried out
conventionally, e.g., as described in the mentioned
publications.
[0221] Activity of raf, VEGFR-2, VEGFR-3, p38, PDGFR-beta, and
Flt-3 can be assessed routinely, e.g., as described in the examples
below, or using standard assays for kinase activity (see,
above).
[0222] Measuring expression includes evaluating the all aspects of
the transcriptional and translational machinery, of the gene. For
instance, if a promoter defect causes, or is suspected of causing,
the disorder, then a sample can be evaluated (i.e., "assessed") by
looking (e.g., sequencing or restriction mapping) at the promoter
sequence in the gene, by detecting transcription products (e.g.,
RNA), by detecting translation product (e.g., polypeptide). Any
measure of whether the gene is functional can be used, including,
polypeptide, polynucleotide, and functional assays for the gene's
biological activity.
[0223] In making the assessment, it can be useful to compare the
results to a gene which is not associated with the disorder, or to
the same gene but in a unaffected tissue or region of the same
tissue. The nature of the comparison can be determined routinely,
depending upon how the assessing is accomplished. If, for example,
the mRNA levels of a sample is detected, then the mRNA levels of a
normal can serve as a comparison, or a gene which is known not to
be affected by the disorder. Methods of detecting mRNA are well
known, and discussed above, e.g., but not limited to, Northern blot
analysis, polymerase chain reaction (PCR), reverse transcriptase
PCR, RACE PCR, etc. Similarly, if polypeptide production is used to
evaluate the gene, then the polypeptide in a normal tissue sample
can be used as a comparison, or, polypeptide from a different gene
whose expression is known not to be affected by the disorder. These
are only examples of how such a method could be carried out.
[0224] Patients can also be selected for treatment if they have a
particular genotype which is known to be associated with a cancer,
especially genotypes which affect the Raf/Mek/Erk pathway, such as
mutations in the BRAF, KRAS, or MEK genes. Along these lines, the
the present invention relates to methods for selecting patients for
treatment involving determining the the presence of a Raf, VEGFR-2,
VEGFR-3, p38, PDGFR-beta, and/or Flt-3 gene mutation in a sample
obtained from a subject, wherein said mutation is associated with a
disease, and administering said compound of formula I to subjects
who are identified as having said mutation.
[0225] The presence of the mutation can be determined
conventionally, e.g., obtaining cells or a tissue sample from a
subject, extracting nucleic acid from it, determining the gene
sequence or structure of a target gene (using, e.g., mRNA, cDNA,
genomic DNA, etc), comparing the sequence or structure of the
target gene to the structure of the normal gene, whereby a
difference in sequence or structure indicates a mutation in the
gene in the subject. Mutations can be determined using any
effective method, e.g., comparing restriction maps, nucleotide
sequences, amino acid sequences, RFLPs, DNAse sites, DNA
methylation fingerprints (e.g., U.S. Pat. No. 6,214,556), protein
cleavage sites, molecular weights, electrophoretic mobilities,
charges, ion mobility, etc., between a standard gene and the
subject's gene. Proteins can also be compared. To carry out such
methods, all or part of the gene or polypeptide can be compared.
For example, if nucleotide sequencing is utilized, the entire gene
can be sequenced, including promoter, introns, and exons, or only
parts of it can be sequenced and compared, e.g., exon 1, exon 2,
etc.
[0226] The present invention also provides methods of assessing the
efficacy of a compound of the present invention in treating a
disease, comprising one or more of the following steps in any
effective order, e.g., measuring the expression or activity of Raf,
VEGFR-2, VEGFR-3, p38, PDGFR-beta, and/or Flt-3 in a sample
obtained from said subject who has been treated with a compound of
the present invention, and determining the effects of said compound
on said expression or activity. The measuring step can be carried
out as described already.
[0227] For instance, biopsy samples can be removed from patients
who have been treated with a compound of the present invention, and
then assayed for the presence and/or activity of the mentioned
signaling molecules. As discussed above, decreased levels of
phospho-ERK in the cancer tissue (e.g., compared to a normal tissue
or before treatment) indicate that the compound is exerting in vivo
efficacy and a therapeutic effect.
[0228] Determining the effects of the compound on expression or
activity includes performing a comparison step between a tissue
sample and a control, or other type of standard. Examples of
standards that can be used, include, but are not limited to, a
tissue sample prior to treatment, a tissue sample from an
unaffected tissue or from an unaffected region of the affected
tissue (e.g., from a region of the tissue which is not transformed,
cancerous, etc.), etc. A standard can also be a value, or range of
values, that is representative of normal levels of expression that
have been established for that marker. The comparison can also be
made between samples collected from at least two different
timepoints during the treatment regimen with a compound of the
present invention. For example, samples can be collected from
various times after initiation of the drug treatment, and analysis
of expression and/or activity levels can be used to monitor the
progress/prognosis of the subject, e.g., how the subject is
responding to the drug regimen. Any timepoint can be used, e.g.,
daily, twice a week, weekly, every two weeks, every month, yearly,
a plurality of timepoints (at least 2, 3, 4, 8, 12, etc.).
[0229] The phrase "determining the effect" indicates that the
result produced by the compound is analyzed and/or identified. For
instance, in Example 3, data is shown that compound reduced the
levels of phospho-ERK (i.e., the effect of the compound on raf
activity was determined by measuring phospho-ERK). Any type of
effect can be identified, e.g., where the expression and/or
activity is reduced, decreased, down-regulated, inhibited, blocked,
increased, up-regulated, unchanged, etc.
[0230] The method can be used to determine appropriate dosages and
dosing regimens, e.g., how much compound to administer and at what
frequency to administer it. By monitoring its effect on the
signaling molecules in the tissue, the clinician can determine the
appropriate treatment protocol and whether it is achieving the
desired effect, e.g., on modulating or inhibiting the signal
transduction pathway. For instance, if the compound is not
effective in knocking down the amounts of a marker, such as
phospho-ERK, the dosage can be increased in the patient or given
more frequently. Similarly, dosages and/or frequency can be reduced
when it is shown that the compound is effective in knocking down
the levels of phospho-ERK or other marker for the disease state.
Since the compounds can be administered in combination with others
treatments, e.g., radiation, chemotherapy, and other agents, the
monitoring of the subject can be used to assess the combined
effects of the treatment regimen on the progress of the
disease.
[0231] Examples of mutations, include mutations in K-RAS; mutations
in the BRAF gene, such as mutations at position 599, such as V599E,
and positions 461, 462, 463, 465, 468, 593, 596, 60, etc., which
are associated with cancers, such as melanoma.
[0232] Compounds of the present invention also can be used as
markers to determine the presence and quantity of raf, VEGFR-2,
VEGFR-3, PDGFR-beta, and/or Flt-3. Methods can involve the presence
of Raf, VEGFR-2, VEGFR-3, PDGFR-beta, and/or Flt-3 in a sample
comprising a biological material, comprising one or more of the
following steps in any effective order, e.g., contacting said
sample comprising a biological material with a compound of the
present invention, and determining whether said compound binds to
said material. The compound can be labeled, or it can be used as a
competitor to a labeled compound, such as labeled-ATP.
[0233] The invention also provides methods for treating,
preventing, modulating, etc., diseases and conditions in mammals
comprising administering a compound of this invention with another
modulator of the signal transduction pathway comprising, but not
limited to raf, VEGFR, PDGFR, and/or FLT-3. These can be present in
the same composition or in separate formulations or dosage units.
Administration can be the same or different routes, and can be
simultaneous, sequential, etc.
[0234] The invention also relates to methods for treating,
preventing, modulating, etc., diseases and conditions, comprising
administering a compound of this invention with another active
agent, e.g., once or more per day for up to 28 consecutive days
with the concurrent or intermittent administration of another
active agent over the same total time period.
[0235] Optional anti-hyper-proliferative agents which can be added
to the composition include but are not limited to compounds listed
on the cancer chemotherapy drug regimens in the 11.sup.th Edition
of the Merck Index, (1996), which is hereby incorporated by
reference, such as asparaginase, bleomycin, carboplatin,
carmustine, chlorambucil, cisplatin, colaspase, cyclophosphamide,
cytarabine, dacarbazine, dactinomycin, daunorubicin, doxorubicin
(adriamycine), epirubicin, etoposide, 5-fluorouracil,
hexamethylmelamine, hydroxyurea, ifosfamide, irinotecan,
leucovorin, lomustine, mechlorethamine, 6-mercaptopurine, mesna,
methotrexate, mitomycin C, mitoxantrone, prednisolone, prednisone,
procarbazine, raloxifen, streptozocin, tamoxifen, thioguanine,
topotecan, vinblastine, vincristine, and vindesine.
[0236] Other anti-hyper-proliferative agents suitable for use with
the composition of the invention include, but are not limited to,
those compounds acknowledged to be used in the treatment of
neoplastic diseases in Goodman and Gilman's The Pharmacological
Basis of Therapeutics (Ninth Edition), editor Molinoff et al.,
publ. by McGraw-Hill, pages 1225-1287, (1996), which is hereby
incorporated by reference, such as aminoglutethimide,
L-asparaginase, azathioprine, 5-azacytidine cladribine, busulfan,
diethylstilbestrol, 2',2'-difluorodeoxycytidine, docetaxel,
erythrohydroxynonyladenine, ethinyl estradiol,
5-fluorodeoxyuridine, 5-fluorodeoxyuridine monophosphate,
fludarabine phosphate, fluoxymesterone, flutamide,
hydroxyprogesterone caproate, idarubicin, interferon,
medroxyprogesterone acetate, megestrol acetate, melphalan,
mitotane, paclitaxel, pentostatin, N-phosphonoacetyl-L-aspartate
(PALA), plicamycin, semustine, teniposide, testosterone propionate,
thiotepa, trimethylmelamine, uridine, and vinorelbine.
[0237] Compounds of the present invention can be administered in
any form by any effective route, including, e.g., oral, parenteral,
enteral, intravenous, intraperitoneal, topical, transdermal (e.g.,
using any standard patch), ophthalmic, nasally, local, non-oral,
such as aerosal, inhalation, subcutaneous, intramuscular, buccal,
sublingual, rectal, vaginal, intra-arterial, and intrathecal, etc.
They can be administered alone, or in combination with any
ingredient(s), active or inactive. They can be administered in any
effective dosage, e.g., from about 0.1 to about 200 mg/kg of total
body weight.
[0238] The present invention relates to a method for using the
compounds described above (Compounds of Formula I), including salts
and esters thereof and compositions thereof, to treat mammalian
hyper-proliferative disorders. This method comprises administering
to a mammal in need thereof, including a human, an amount of a
compound of this invention, or a pharmaceutically acceptable salt
or ester thereof, which is effective to treat the disorder.
Hyper-proliferative disorders include but are not limited to solid
tumors, such as cancers of the breast, respiratory tract, brain,
reproductive organs, digestive tract, urinary tract, eye, liver,
skin, head and neck, thyroid, parathyroid and their distant
metastases. Those disorders also include lymphomas, sarcomas, and
leukemias.
[0239] Synthetic transformations that may be employed in the
synthesis of compounds of Formula I and in the synthesis of
intermediates involved in the synthesis of compounds of Formula I
are known by or accessible to one skilled in the art. Collections
of synthetic transformations may be found in compilations, such as:
[0240] J. March. Advanced Organic Chemistry, 4th ed.; John Wiley:
New York (1992) [0241] R. C. Larock. Comprehensive Organic
Transformations, 2nd ed.; Wiley-VCH: New York (1999) [0242] F. A.
Carey; R. J. Sundberg. Advanced Organic Chemistry, 2nd ed.; Plenum
Press: New York (1984) [0243] T. W. Greene; P. G. M. Wuts.
Protective Groups in Organic Synthesis, 3rd ed.; John Wiley: New
York (1999) [0244] L. S. Hegedus. Transition Metals in the
Synthesis of Complex Organic Molecules, 2nd ed.; University Science
Books: Mill Valley, Calif. (1994) [0245] L. A. Paquette, Ed. The
Encyclopedia of Reagents for Organic Synthesis; John Wiley: New
York (1994) [0246] A. R. Katritzky; O. Meth-Cohn; C. W. Rees, Eds.
Comprehensive Organic Functional Group Transformations; Pergamon
Press: Oxford, UK (1995) [0247] G. Wilkinson; F. G A. Stone; E. W.
Abel, Eds. Comprehensive Organometallic Chemistry; Pergamon Press:
Oxford, UK (1982) [0248] B. M. Trost; I. Fleming. Comprehensive
Organic Synthesis; Pergamon Press: Oxford, UK (1991) [0249] A. R
Katritzky; C. W. Rees Eds. Comprehensive Heterocylic Chemistry;
Pergamon Press: Oxford, UK (1984) [0250] A. R. Katritzky; C. W.
Rees; E. F. V. Scriven, Eds. Comprehensive Heterocylic Chemistry
II; Pergamon Press: Oxford, UK (1996) [0251] C. Hansch; P. G.
Sammes; J. B. Taylor, Eds. Comprehensive Medicinal Chemistry:
Pergamon Press: Oxford, UK (1990).
[0252] In addition, recurring reviews of synthetic methodology and
related topics include Organic Reactions; John Wiley: New York;
Organic Syntheses; John Wiley: New York; Reagents for Organic
Synthesis: John Wiley: New York; The Total Synthesis of Natural
Products; John Wiley: New York; The Organic Chemistry of Drug
Synthesis; John Wiley: New York; Annual Reports in Organic
Synthesis; Academic Press: San Diego Calif.; and Methtoden der
Organischen Chemie (Houben-Weyl); Thieme: Stuttgart Germany.
Furthermore, databases of synthetic transformations include
Chemical Abstracts, which may be searched using either CAS OnLine
or SciFinder, Handbuch der Organischen Chemie (Beilstein), which
may be searched using SpotFire, and REACCS.
General Preparative Methods
[0253] The diaryl ureas of Formula I may be prepared by the use of
known chemical reactions and procedures, some from starting
materials which are commercially available. Nevertheless, general
preparative methods are provided below to aid one skilled in the
art in synthesizing these compounds, with more detailed examples
being provided in the Experimental section which follows.
[0254] Substituted anilines may be generated using standard methods
(March. Advanced Organic Chemistry, 3.sup.rd Ed.; John Wiley: New
York (1985). Larock. Comprehensive Organic Transformations; VCH
Publishers: New York (1989)). As shown in Scheme I, aryl amines are
commonly synthesized by reduction of nitroaryls using a metal
catalyst, such as Ni, Pd, or Pt, and H.sub.2 or a hydride transfer
agent, such as formate, cyclohexadiene, or a borohydride (Rylander.
Hydrogenation Methods; Academic Press: London, UK (1985)).
Nitroaryls may also be directly reduced using a strong hydride
source, such as LiAlH.sub.4 (Seyden-Penne. Reductions by the
Alumino- and Borohydrides in Organic Synthesis; VCH Publishers: New
York (1991)), or using a zero valent metal, such as Fe, Sn or Ca,
often in acidic media. Many methods exist for the synthesis of
nitroaryls (March. Advanced Organic Chemistry, 3.sup.rd Ed.; John
Wiley: New York (1985). Larock. Comprehensive Organic
Transformations; VCH Publishers: New York (1989)). ##STR1##
[0255] Nitroaryls are commonly formed by electrophilic aromatic
nitration using HNO.sub.3, or an alternative NO.sub.2.sup.+ source.
Nitroaryls may be further elaborated prior to reduction. Thus,
nitroaryls substituted with ##STR2## potential leaving groups (e.g.
F, Cl, Br, etc.) may undergo substitution reactions on treatment
with nucleophiles, such as thiolate (exemplified in Scheme II) or
phenoxide. Nitroaryls may also undergo Ullman-type coupling
reactions (Scheme II). ##STR3##
[0256] Nitroaryls may also undergo transition metal mediated cross
coupling reactions. For example, nitroaryl electrophiles, such as
nitroaryl bromides, iodides or triflates, undergo palladium
mediated cross coupling reactions with aryl nucleophiles, such as
arylboronic acids (Suzuki reactions, exemplified below), aryltins
(Stille reactions) or arylzincs (Negishi reaction) to afford the
biaryl (5). ##STR4##
[0257] As shown in Scheme III, non-symmetrical urea formation may
involve reaction of an aryl isocyanate (14) with an aryl amine
(13). The heteroaryl isocyanate may be synthesized from a
heteroaryl amine by treatment with phosgene or a phosgene
equivalent, such as trichloromethyl chloroformate (diphosgene),
bis(trichloromethyl) carbonate (triphosgene), or
N,N'-carbonyldiimidazole (CDI). The isocyanate may also be derived
from a heterocyclic carboxylic acid derivative, such as an ester,
an acid halide or an anhydride by a Curtius-type rearrangement.
Thus, reaction of acid derivative 16 with an azide source, followed
by rearrangement affords the isocyanate. The corresponding
carboxylic acid (17) may also be subjected to Curtius-type
rearrangements using diphenylphosphoryl azide (DPPA) or a similar
reagent. ##STR5##
[0258] Finally, ureas may be further manipulated using methods
familiar to those skilled in the art.
[0259] The compounds may be administered orally, topically,
parenterally, by inhalation or spray or rectally in dosage unit
formulations. The term `administration by injection` includes
intravenous, intramuscular, subcutaneous and parenteral injections,
as well as use of infusion techniques. One or more compounds may be
present in association with one or more non-toxic pharmaceutically
acceptable carriers and if desired other active ingredients.
[0260] Compositions intended for oral use may be prepared according
to any suitable method known to the art for the manufacture of
pharmaceutical compositions. Such compositions may contain one or
more agents selected from the group consisting of diluents,
sweetening agents, flavoring agents, coloring agents and preserving
agents in order to provide palatable preparations. Tablets contain
the active ingredient in admixture with non-toxic pharmaceutically
acceptable excipients which are suitable for the manufacture of
tablets. These excipients may be, for example, inert diluents, such
as calcium carbonate, sodium carbonate, lactose, calcium phosphate
or sodium phosphate; granulating and disintegrating agents, for
example, corn starch, or alginic acid; and binding agents, for
example magnesium stearate, stearic acid or talc. The tablets may
be uncoated or they may be coated by known techniques to delay
disintegration and adsorption in the gastrointestinal tract and
thereby provide a sustained action over a longer period. For
example, a time delay material such as glyceryl monostearate or
glyceryl distearate may be employed. These compounds may also be
prepared in solid, rapidly released form.
[0261] Formulations for oral use may also be presented as hard
gelatin capsules wherein the active ingredient is mixed with an
inert solid diluent, for example, calcium carbonate, calcium
phosphate or kaolin, or as soft gelatin capsules wherein the active
ingredient is mixed with water or an oil medium, for example peanut
oil, liquid paraffin or olive oil.
[0262] Aqueous suspensions contain the active materials in
admixture with excipients suitable for the manufacture of aqueous
suspensions. Such excipients are suspending agents, for example
sodium carboxymethylcellulose, methylcellulose, hydroxypropyl
methylcellulose, sodium alginate, polyvinylpyrrolidone, gum
tragacanth and gum acacia; dispersing or wetting agents may be a
naturally occurring phosphatide, for example, lecithin, or
condensation products or an alkylene oxide with fatty acids, for
example polyoxyethylene stearate, or condensation products of
ethylene oxide with long chain aliphatic alcohols, for example
heptadecaethylene oxycetanol, or condensation products of ethylene
oxide with partial esters derived from fatty acids and hexitol such
as polyoxyethylene sorbitol monooleate, or condensation products of
ethylene oxide with partial esters derived from fatty acids and
hexitol anhydrides, for example polyethylene sorbitan monooleate.
The aqueous suspensions may also contain one or more preservatives,
for example ethyl, or n-propyl p-hydroxybenzoate, one or more
coloring agents, one or more flavoring agents, and one or more
sweetening agents, such as sucrose or saccharin.
[0263] Dispersible powders and granules suitable for preparation of
an aqueous suspension by the addition of water provide the active
ingredient in admixture with a dispersing or wetting agent,
suspending agent and one or more preservatives. Suitable dispersing
or wetting agents and suspending agents are exemplified by those
already mentioned above. Additional excipients, for example,
sweetening, flavoring and coloring agents, may also be present.
[0264] The compounds may also be in the form of non-aqueous liquid
formulations, e.g., oily suspensions which may be formulated by
suspending the active ingredients in a vegetable oil, for example
arachis oil, olive oil, sesame oil or peanut oil, or in a mineral
oil such as liquid paraffin. The oily suspensions may contain a
thickening agent, for example beeswax, hard paraffin or cetyl
alcohol. Sweetening agents such as those set forth above, and
flavoring agents may be added to provide palatable oral
preparations. These compositions may be preserved by the addition
of an anti-oxidant such as ascorbic acid.
[0265] Pharmaceutical compositions of the invention may also be in
the form of oil-in-water emulsions. The oily phase may be a
vegetable oil, for example olive oil or arachis oil, or a mineral
oil, for example liquid paraffin or mixtures of these. Suitable
emulsifying agents may be naturally-occurring gums, for example gum
acacia or gum tragacanth, naturally-occurring phosphatides, for
example soy bean, lecithin, and esters or partial esters derived
from fatty acids and hexitol anhydrides, for example sorbitan
monooleate, and condensation products of the said partial esters
with ethylene oxide, for example polyoxyethylene sorbitan
monooleate. The emulsions may also contain sweetening and flavoring
agents.
[0266] Syrups and elixirs may be formulated with sweetening agents,
for example glycerol, propylene glycol, sorbitol or sucrose. Such
formulations may also contain a demulcent, a preservative and
flavoring and coloring agents.
[0267] The compounds may also be administered in the form of
suppositories for rectal administration of the drug. These
compositions can be prepared by mixing the drug with a suitable
non-irritating excipient which is solid at ordinary temperatures
but liquid at the rectal temperature and will therefore melt in the
rectum to release the drug. Such materials include cocoa butter and
polyethylene glycols.
[0268] The compounds of this invention may also be administered
parenterally, that is, subcutaneously, intravenously,
intraocularly, intrasynovially, intramuscularly, or
interperitoneally, as injectable dosages of the compound in a
physiologically acceptable diluent with a pharmaceutical carrier
which can be a sterile liquid or mixture of liquids such as water,
saline, aqueous dextrose and related sugar solutions, an alcohol
such as ethanol, isopropanol, or hexadecyl alcohol, glycols such as
propylene glycol or polyethylene glycol, glycerol ketals such as
2,2-dimethyl-1,1-dioxolane-4-methanol, ethers such as poly(ethylene
glycol) 400, an oil, a fatty acid, a fatty acid ester or, a fatty
acid glyceride, or an acetylated fatty acid glyceride, with or
without the addition of a pharmaceutically acceptable surfactant
such as a soap or a detergent, suspending agent such as pectin,
carbomers, methycellulose, hydroxypropylmethylcellulose, or
carboxymethylcellulose, or emulsifying agent and other
pharmaceutical adjuvants.
[0269] Illustrative of oils which can be used in the parenteral
formulations of this invention are those of petroleum, animal,
vegetable, or synthetic origin, for example, peanut oil, soybean
oil, sesame oil, cottonseed oil, corn oil, olive oil, petrolatum
and mineral oil. Suitable fatty acids include oleic acid, stearic
acid, isostearic acid and myristic acid. Suitable fatty acid esters
are, for example, ethyl oleate and isopropyl myristate. Suitable
soaps include fatty acid alkali metal, ammonium, and
triethanolamine salts and suitable detergents include cationic
detergents, for example dimethyl dialkyl ammonium halides, alkyl
pyridinium halides, and alkylamine acetates; anionic detergents,
for example, alkyl, aryl, and olefin sulfonates, alkyl, olefin,
ether, and monoglyceride sulfates, and sulfosuccinates; non-ionic
detergents, for example, fatty amine oxides, fatty acid
alkanolamides, and poly(oxyethylene-oxypropylene)s or ethylene
oxide or propylene oxide copolymers; and amphoteric detergents, for
example, alkyl-beta-aminopropionates, and 2-alkylimidazoline
quarternary ammonium salts, as well as mixtures.
[0270] The parenteral compositions of this invention will typically
contain from about 0.5% to about 25% by weight of the active
ingredient in solution. Preservatives and buffers may also be used
advantageously. In order to minimize or eliminate irritation at the
site of injection, such compositions may contain a non-ionic
surfactant having a hydrophile-lipophile balance (HLB) of from
about 12 to about 17. The quantity of surfactant in such
formulation ranges from about 5% to about 15% by weight. The
surfactant can be a single component having the above HLB or can be
a mixture of two or more components having the desired HLB.
[0271] Illustrative of surfactants used in parenteral formulations
are the class of polyethylene sorbitan fatty acid esters, for
example, sorbitan monooleate and the high molecular weight adducts
of ethylene oxide with a hydrophobic base, formed by the
condensation of propylene oxide with propylene glycol.
[0272] The pharmaceutical compositions may be in the form of
sterile injectable aqueous suspensions. Such suspensions may be
formulated according to known methods using suitable dispersing or
wetting agents and suspending agents such as, for example, sodium
carboxymethylcellulose, methylcellulose,
hydroxypropylmethyl-cellulose, sodium alginate,
polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or
wetting agents which may be a naturally occurring phosphatide such
as lecithin, a condensation product of an alkylene oxide with a
fatty acid, for example, polyoxyethylene stearate, a condensation
product of ethylene oxide with a long chain aliphatic alcohol, for
example, heptadeca-ethyleneoxycetanol, a condensation product of
ethylene oxide with a partial ester derived form a fatty acid and a
hexitol such as polyoxyethylene sorbitol monooleate, or a
condensation product of an ethylene oxide with a partial ester
derived from a fatty acid and a hexitol anhydride, for example
polyoxyethylene sorbitan monooleate.
[0273] The sterile injectable preparation may also be a sterile
injectable solution or suspension in a non-toxic parenterally
acceptable diluent or solvent. Diluents and solvents that may be
employed are, for example, water, Ringer's solution, isotonic
sodium chloride solutions and isotonic glucose solutions. In
addition, sterile fixed oils are conventionally employed as
solvents or suspending media. For this purpose, any bland, fixed
oil may be employed including synthetic mono- or diglycerides. In
addition, fatty acids such as oleic acid can be used in the
preparation of injectables.
[0274] Compounds of the invention may also be administrated
transdermally using methods ("patches") known to those skilled in
the art (see, for example: Chien; "Transdermal Controlled Systemic
Medications"; Marcel Dekker, Inc.; 1987. Lipp et al. WO94/04157 3
Mar. 94). Such transdermal patches may be used to provide
continuous or discontinuous infusion of the compounds of the
present invention in controlled amounts. The construction and use
of transdermal patches for the delivery of pharmaceutical agents is
well known in the art (see, e.g., U.S. Pat. No. 5,023,252, issued
Jun. 11, 1991, incorporated herein by reference). Such patches may
be constructed for continuous, pulsatile, or on demand delivery of
pharmaceutical agents. For example, a solution or suspension of a
compound of Formula I in a suitable volatile solvent optionally
containing penetration enhancing agents can be combined with
additional additives known to those skilled in the art, such as
matrix materials and bacteriocides. After sterilization, the
resulting mixture can be formulated following known procedures into
dosage forms. In addition, on treatment with emulsifying agents and
water, a solution or suspension of a compound of Formula I may be
formulated into a lotion or salve.
[0275] Suitable solvents for processing transdermal delivery
systems are known to those skilled in the art, and include lower
alcohols such as ethanol or isopropyl alcohol, lower ketones such
as acetone, lower carboxylic acid esters such as ethyl acetate,
polar ethers such as tetrahydrofuran, lower hydrocarbons such as
hexane, cyclohexane or benzene, or halogenated hydrocarbons such as
dichloromethane, chloroform, trichlorotrifluoroethane, or
trichlorofluoroethane. Suitable solvents may also include mixtures
of one or more materials selected from lower alcohols, lower
ketones, lower carboxylic acid esters, polar ethers, lower
hydrocarbons, halogenated hydrocarbons.
[0276] Suitable penetration enhancing materials for transdermal
delivery system are known to those skilled in the art, and include,
for example, monohydroxy or polyhydroxy alcohols such as ethanol,
propylene glycol or benzyl alcohol, saturated or unsaturated
C.sub.8-C.sub.18 fatty alcohols such as lauryl alcohol or cetyl
alcohol, saturated or unsaturated C.sub.8-C.sub.18 fatty acids such
as stearic acid, saturated or unsaturated fatty esters with up to
24 carbons such as methyl, ethyl, propyl, isopropyl, n-butyl,
sec-butyl isobutyl tertbutyl or monoglycerin esters of acetic acid,
capronic acid, lauric acid, myristinic acid, stearic acid, or
palmitic acid, or diesters of saturated or unsaturated dicarboxylic
acids with a total of up to 24 carbons such as diisopropyl adipate,
diisobutyl adipate, diisopropyl sebacate, diisopropyl maleate, or
diisopropyl fumarate. Additional penetration enhancing materials
include phosphatidyl derivatives such as lecithin or cephalin,
terpenes, amides, ketones, ureas and their derivatives, and ethers
such as dimethyl isosorbid and diethyleneglycol monoethyl ether.
Suitable penetration enhancing formulations may also include
mixtures of one or more materials selected from monohydroxy or
polyhydroxy alcohols, saturated or unsaturated C.sub.8-C.sub.18
fatty alcohols, saturated or unsaturated C.sub.8-C.sub.18 fatty
acids, saturated or unsaturated fatty esters with up to 24 carbons,
diesters of saturated or unsaturated discarboxylic acids with a
total of up to 24 carbons, phosphatidyl derivatives, terpenes,
amides, ketones, ureas and their derivatives, and ethers.
[0277] Suitable binding materials for transdermal delivery systems
are known to those skilled in the art and include polyacrylates,
silicones, polyurethanes, block polymers, styrenebutadiene
copolymers, and natural and synthetic rubbers. Cellulose ethers,
derivatized polyethylenes, and silicates may also be used as matrix
components. Additional additives, such as viscous resins or oils
may be added to increase the viscosity of the matrix.
[0278] Controlled release formulations for parenteral
administration include liposomal, polymeric microsphere and
polymeric gel formulations which are known in the art.
[0279] It may be desirable or necessary to introduce the
pharmaceutical composition to the patient via a mechanical delivery
device. The construction and use of mechanical delivery devices for
the delivery of pharmaceutical agents is well known in the art.
Direct techniques for, for example, administering a drug directly
to the brain usually involve placement of a drug delivery catheter
into the patient's ventricular system to bypass the blood-brain
barrier. One such implantable delivery system, used for the
transport of agents to specific anatomical regions of the body, is
described in U.S. Pat. No. 5,011,472, issued Apr. 30, 1991.
[0280] The compositions of the invention can also contain other
conventional pharmaceutically acceptable compounding ingredients,
generally referred to as carriers or diluents, as necessary or
desired. Conventional procedures for preparing such compositions in
appropriate dosage forms can be utilized. Such ingredients and
procedures include those described in the following references,
each of which is incorporated herein by reference: Powell, M. F. et
al, "Compendium of Excipients for Parenteral Formulations" PDA
Journal of Pharmaceutical Science & Technology 1998, 52(5),
238-311; Strickley, R. G "Parenteral Formulations of Small Molecule
Therapeutics Marketed in the United States (1999)-Part-1" PDA
Journal of Pharmaceutical Science & Technology 1999, 53(6),
324-349; and Nema, S. et al, "Excipients and Their Use in
Injectable Products" PDA Journal of Pharmaceutical Science &
Technology 1997, 51(4), 166-171.
[0281] This invention also relates to administering pharmaceutical
compositions containing one or more compounds of the present
invention. These compositions can be utilized to achieve the
desired pharmacological effect by administration to a patient in
need thereof. A patient, for the purpose of this invention, is a
mammal, including a human, in need of treatment for the particular
condition or disease. Therefore, the present invention includes
pharmaceutical compositions which are comprised of a
pharmaceutically acceptable carrier and a pharmaceutically
effective amount of a compound, or salt thereof, of the present
invention. A pharmaceutically acceptable carrier is any carrier
which is relatively non-toxic and innocuous to a patient at
concentrations consistent with effective activity of the active
ingredient so that any side effects ascribable to the carrier do
not vitiate the beneficial effects of the active ingredient. A
pharmaceutically effective amount of compound is that amount which
produces a result or exerts an influence on the particular
condition being treated. The compounds of the present invention can
be administered with pharmaceutically-acceptable carriers well
known in the art using any effective conventional dosage unit
forms, including immediate, slow and timed release preparations,
orally, parenterally, topically, nasally, ophthalmically, otically,
sublingually, rectally, vaginally, and the like.
[0282] For oral administration, the compounds can be formulated
into solid or liquid preparations such as capsules, pills, tablets,
troches, lozenges, melts, powders, solutions, suspensions, or
emulsions, and may be prepared according to methods known to the
art for the manufacture of pharmaceutical compositions. The solid
unit dosage forms can be a capsule which can be of the ordinary
hard- or soft-shelled gelatin type containing, for example,
surfactants, lubricants, and inert fillers such as lactose,
sucrose, calcium phosphate, and corn starch.
[0283] In another embodiment, the compounds of this invention may
be tableted with conventional tablet bases such as lactose, sucrose
and cornstarch in combination with binders such as acacia, corn
starch or gelatin, disintegrating agents intended to assist the
break-up and dissolution of the tablet following administration
such as potato starch, alginic acid, corn starch, and guar gum, gum
tragacanth, acacia, lubricants intended to improve the flow of
tablet granulation and to prevent the adhesion of tablet material
to the surfaces of the tablet dies and punches, for example talc,
stearic acid, or magnesium, calcium or zinc stearate, dyes,
coloring agents, and flavoring agents such as peppermint, oil of
wintergreen, or cherry flavoring, intended to enhance the aesthetic
qualities of the tablets and make them more acceptable to the
patient. Suitable excipients for use in oral liquid dosage forms
include dicalcium phosphate and diluents such as water and
alcohols, for example, ethanol, benzyl alcohol, and polyethylene
alcohols, either with or without the addition of a pharmaceutically
acceptable surfactant, suspending agent or emulsifying agent.
Various other materials may be present as coatings or to otherwise
modify the physical form of the dosage unit. For instance tablets,
pills or capsules may be coated with shellac, sugar or both.
[0284] The compositions of the invention can also contain other
conventional pharmaceutically acceptable compounding ingredients,
generally referred to as carriers or diluents, as necessary or
desired. Conventional procedures for preparing such compositions in
appropriate dosage forms can be utilized. Such ingredients and
procedures include those described in the following references,
each of which is incorporated herein by reference: Powell, M. F. et
al, "Compendium of Excipients for Parenteral Formulations" PDA
Journal of Pharmaceutical Science & Technology 1998, 52(5),
238-311; Strickley, R. G "Parenteral Formulations of Small Molecule
Therapeutics Marketed in the United States (1999)-Part-1" PDA
Journal of Pharmaceutical Science & Technology 1999, 53(6),
324-349; and Nema, S. et al, "Excipients and Their Use in
Injectable Products" PDA Journal of Pharmaceutical Science &
Technology 1997, 51(4), 166-171.
[0285] Commonly used pharmaceutical ingredients which can be used
as appropriate to formulate the composition for its intended route
of administration include:
[0286] acidifying agents (examples include but are not limited to
acetic acid, citric acid, fumaric acid, hydrochloric acid, nitric
acid);
[0287] alkalinizing agents (examples include but are not limited to
ammonia solution, ammonium carbonate, diethanolamine,
monoethanolamine, potassium hydroxide, sodium borate, sodium
carbonate, sodium hydroxide, triethanolamine, trolamine);
[0288] adsorbents (examples include but are not limited to powdered
cellulose and activated charcoal);
[0289] aerosol propellants (examples include but are not limited to
carbon dioxide, CCl.sub.2F.sub.2, F.sub.2ClC--CClF.sub.2 and
CClF.sub.3)
[0290] air displacement agents (examples include but are not
limited to nitrogen and argon);
[0291] antifungal preservatives (examples include but are not
limited to benzoic acid, butylparaben, ethylparaben, methylparaben,
propylparaben, sodium benzoate);
[0292] antimicrobial preservatives (examples include but are not
limited to benzalkonium chloride, benzethonium chloride, benzyl
alcohol, cetylpyridinium chloride, chlorobutanol, phenol,
phenylethyl alcohol, phenylmercuric nitrate and thimerosal);
[0293] antioxidants (examples include but are not limited to
ascorbic acid, ascorbyl palmitate, butylated hydroxyanisole,
butylated hydroxytoluene, hypophosphorus acid, monothioglycerol,
propyl gallate, sodium ascorbate, sodium bisulfite, sodium
formaldehyde sulfoxylate, sodium metabisulfite);
[0294] binding materials (examples include but are not limited to
block polymers, natural and synthetic rubber, polyacrylates,
polyurethanes, silicones, polysiloxanes and styrene-butadiene
copolymers);
[0295] buffering agents (examples include but are not limited to
potassium metaphosphate, dipotassium phosphate, sodium acetate,
sodium citrate anhydrous and sodium citrate dihydrate)
[0296] carrying agents (examples include but are not limited to
acacia syrup, aromatic syrup, aromatic elixir, cherry syrup, cocoa
syrup, orange syrup, syrup, corn oil, mineral oil, peanut oil,
sesame oil, bacteriostatic sodium chloride injection and
bacteriostatic water for injection)
[0297] chelating agents (examples include but are not limited to
edetate disodium and edetic acid)
[0298] colorants (examples include but are not limited to FD&C
Red No. 3, FD&C Red No. 20, FD&C Yellow No. 6, FD&C
Blue No. 2, D&C Green No. 5, D&C Orange No. 5, D&C Red
No. 8, caramel and ferric oxide red);
[0299] clarifying agents (examples include but are not limited to
bentonite);
[0300] emulsifying agents (examples include but are not limited to
acacia, cetomacrogol, cetyl alcohol, glyceryl monostearate,
lecithin, sorbitan monooleate, polyoxyethylene 50
monostearate);
[0301] encapsulating agents (examples include but are not limited
to gelatin and cellulose acetate phthalate)
[0302] flavorants (examples include but are not limited to anise
oil, cinnamon oil, cocoa, menthol, orange oil, peppermint oil and
vanillin);
[0303] humectants (examples include but are not limited to
glycerol, propylene glycol and sorbitol);
[0304] levigating agents (examples include but are not limited to
mineral oil and glycerin);
[0305] oils (examples include but are not limited to arachis oil,
mineral oil, olive oil, peanut oil, sesame oil and vegetable
oil);
[0306] ointment bases (examples include but are not limited to
lanolin, hydrophilic ointment, polyethylene glycol ointment,
petrolatum, hydrophilic petrolatum, white ointment, yellow
ointment, and rose water ointment);
[0307] penetration enhancers (transdermal delivery) (examples
include but are not limited to monohydroxy or polyhydroxy alcohols,
mono- or polyvalent alcohols, saturated or unsaturated fatty
alcohols, saturated or unsaturated fatty esters, saturated or
unsaturated dicarboxylic acids, essential oils, phosphatidyl
derivatives, cephalin, terpenes, amides, ethers, ketones and
ureas)
[0308] plasticizers (examples include but are not limited to
diethyl phthalate and glycerol);
[0309] solvents (examples include but are not limited to ethanol,
corn oil, cottonseed oil, glycerol, isopropanol, mineral oil, oleic
acid, peanut oil, purified water, water for injection, sterile
water for injection and sterile water for irrigation);
[0310] stiffening agents (examples include but are not limited to
cetyl alcohol, cetyl esters wax, microcrystalline wax, paraffin,
stearyl alcohol, white wax and yellow wax);
[0311] suppository bases (examples include but are not limited to
cocoa butter and polyethylene glycols (mixtures));
[0312] surfactants (examples include but are not limited to
benzalkonium chloride, nonoxynol 10, oxtoxynol 9, polysorbate 80,
sodium lauryl sulfate and sorbitan mono-palmitate);
[0313] suspending agents (examples include but are not limited to
agar, bentonite, carbomers, carboxymethylcellulose sodium,
hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl
methylcellulose, kaolin, methylcellulose, tragacanth and
veegum);
[0314] sweetening agents (examples include but are not limited to
aspartame, dextrose, glycerol, mannitol, propylene glycol,
saccharin sodium, sorbitol and sucrose);
[0315] tablet anti-adherents (examples include but are not limited
to magnesium stearate and talc);
[0316] tablet binders (examples include but are not limited to
acacia, alginic acid, carboxymethylcellulose sodium, compressible
sugar, ethylcellulose, gelatin, liquid glucose, methylcellulose,
non-crosslinked polyvinyl pyrrolidone, and pregelatinized
starch);
[0317] tablet and capsule diluents (examples include but are not
limited to dibasic calcium phosphate, kaolin, lactose, mannitol,
microcrystalline cellulose, powdered cellulose, precipitated
calcium carbonate, sodium carbonate, sodium phosphate, sorbitol and
starch);
[0318] tablet coating agents (examples include but are not limited
to liquid glucose, hydroxyethyl cellulose, hydroxypropyl cellulose,
hydroxypropyl methylcellulose, methylcellulose, ethylcellulose,
cellulose acetate phthalate and shellac);
[0319] tablet direct compression excipients (examples include but
are not limited to dibasic calcium phosphate);
[0320] tablet disintegrants (examples include but are not limited
to alginic acid, carboxymethylcellulose calcium, microcrystalline
cellulose, polacrillin potassium, cross-linked
polyvinylpyrrolidone, sodium alginate, sodium starch glycollate and
starch);
[0321] tablet glidants (examples include but are not limited to
colloidal silica, corn starch and talc);
[0322] tablet lubricants (examples include but are not limited to
calcium stearate, magnesium stearate, mineral oil, stearic acid and
zinc stearate);
[0323] tablet/capsule opaquants (examples include but are not
limited to titanium dioxide);
[0324] tablet polishing agents (examples include but are not
limited to carnauba wax and white wax);
[0325] thickening agents (examples include but are not limited to
beeswax, cetyl alcohol and paraffin);
[0326] tonicity agents (examples include but are not limited to
dextrose and sodium chloride);
[0327] viscosity increasing agents (examples include but are not
limited to alginic acid, bentonite, carbomers,
carboxymethylcellulose sodium, methylcellulose, polyvinyl
pyrrolidone, sodium alginate and tragacanth); and
[0328] wetting agents (examples include but are not limited to
heptadecaethylene oxycetanol, lecithins, sorbitol monooleate,
polyoxyethylene sorbitol monooleate, and polyoxyethylene
stearate).
[0329] The total amount of the active ingredient to be administered
will generally range from about 0.001 mg/kg to about 200 mg/kg, and
preferably from about 0.01 mg/kg to about 20 mg/kg body weight per
day. A unit dosage may contain from about 0.5 mg to about 1500 mg
of active ingredient, and can be administered one or more times per
day. For all regimens of use disclosed herein for compounds of
Formula I, the daily oral dosage regimen will preferably be from
0.01 to 200 mg/Kg of total body weight. The daily dosage for
administration by injection, including intravenous, intramuscular,
subcutaneous and parenteral injections, and use of infusion
techniques will preferably be from 0.01 to 200 mg/Kg of total body
weight. The daily rectal dosage regime will preferably be from 0.01
to 200 mg/Kg of total body weight. The daily vaginal dosage regime
will preferably be from 0.01 to 200 mg/Kg of total body weight. The
daily topical dosage regime will preferably be from 0.1 to 200 mg
administered between one to four times daily. The transdermal
concentration will preferably be that required to maintain a daily
dose of from 0.01 to 200 mg/Kg. The daily inhalation dosage regime
will preferably be from 0.01 to 100 mg/Kg of total body weight.
These dosages regimes can be achieved with multiple dosages within
a single day or extended dosages, such as those given on a weekly
or monthly basis.
[0330] Based upon standard laboratory techniques known to evaluate
compounds useful for the treatment of hyper-proliferative
disorders, by standard toxicity tests and by standard
pharmacological assays for the determination of treatment of the
conditions identified above in mammals, and by comparison of these
results with the results of known medicaments that are used to
treat these conditions, the effective dosage of the compounds of
this invention can readily be determined for treatment of each
desired indication. The amount of the active ingredient to be
administered in the treatment of one of these conditions can vary
widely according to such considerations as the particular compound
and dosage unit employed, the mode of administration, the period of
treatment, the age and gender of the patient treated, and the
nature and extent of the condition treated.
[0331] It will be appreciated by those skilled in the art that the
particular method of administration will depend on a variety of
factors, all of which are considered routinely when administering
therapeutics. It will also be appreciated by one skilled in the art
that the specific dose level for a given patient depends on a
variety of factors, including specific activity of the compound
administered, age, body weight, health, sex, diet, time and route
of administration, rate of excretion, etc. It will be further
appreciated by one skilled in the art that the optimal course of
treatment, i.e., the mode of treatment and the daily number of
doses of a compound of Formula I or a pharmaceutically acceptable
salt thereof given for a defined number of days, can be ascertained
by those skilled in the art using conventional treatment tests.
[0332] It will be understood, however, that the specific dose level
for any particular patient will depend upon a variety of factors,
including the activity of the specific compound employed, the age,
body weight, general health, sex, diet, time of administration,
route of administration, and rate of excretion, drug combination
and the severity of the condition undergoing therapy.
[0333] It will be further appreciated by one skilled in the art
that the optimal course of treatment, i.e., the mode of treatment
and the daily number of doses of a compound of this invention given
for a defined number of days, can be ascertained by those skilled
in the art using conventional treatment tests.
[0334] Specific preparations of the compounds of this invention are
already described in the patent literature, and can be adapted to
the compounds of the present invention. For example, Riedl, B., et
al., "O-Carboxy Aryl Substituted Diphenyl Ureas as raf Kinase
Inhibitors" PCT Int. Appl., WO 00 42012, Riedl, B., et al.,
"O-Carboxy Aryl Substituted Diphenyl Ureas as p38 Kinase
Inhibitors" PCT Int. Appl., WO 00 41698, incorporated herein by
reference.
[0335] Pharmaceutical compositions according to the present
invention can be illustrated as follows:
[0336] Sterile IV Solution: A 5 mg/ml solution of the desired
compound of this invention is made using sterile, injectable water,
and the pH is adjusted if necessary. The solution is diluted for
administration to 1-2 mg/ml with sterile 5% dextrose and is
administered as an IV infusion over 60 minutes.
[0337] Lyophilized powder for IV administration: A sterile
preparation can be prepared with (i) 100-1000 mg of the desired
compound of this invention as a lyophilized powder, (ii) 32-327
mg/ml sodium citrate, and (iii) 300-3000 mg Dextran 40. The
formulation is reconstituted with sterile, injectable saline or
dextrose 5% to a concentration of 10 to 20 mg/ml, which is further
diluted with saline or dextrose 5% to 0.2-0.4 mg/ml, and is
administered either IV bolus or by IV infusion over 15-60
minutes.
Intramuscular suspension: The following solution or suspension can
be prepared, for intramuscular injection:
[0338] 50 mg/ml of the desired, water-insoluble compound of this
invention [0339] 5 mg/ml sodium carboxymethylcellulose [0340] 4
mg/ml TWEEN 80 [0341] 9 mg/ml sodium chloride [0342] 9 mg/ml benzyl
alcohol Hard Shell Capsules: A large number of unit capsules are
prepared by filling standard two-piece hard galantine capsules each
with 100 mg of powdered active ingredient, 150 mg of lactose, 50 mg
of cellulose and 6 mg of magnesium stearate. Soft Gelatin Capsules:
A mixture of active ingredient in a digestible oil such as soybean
oil, cottonseed oil or olive oil is prepared and injected by means
of a positive displacement pump into molten gelatin to form soft
gelatin capsules containing 100 mg of the active ingredient. The
capsules are washed and dried. The active ingredient can be
dissolved in a mixture of polyethylene glycol, glycerin and
sorbitol to prepare a water miscible medicine mix. Tablets: A large
number of tablets are prepared by conventional procedures so that
the dosage unit was 100 mg of active ingredient, 0.2 mg. of
colloidal silicon dioxide, 5 mg of magnesium stearate, 275 mg of
microcrystalline cellulose, 11 mg. of starch, and 98.8 mg of
lactose. Appropriate aqueous and non-aqueous coatings may be
applied to increase palatability, improve elegance and stability or
delay absorption. Immediate Release Tablets/Capsules: These are
solid oral dosage forms made by conventional and novel processes.
These units are taken orally without water for immediate
dissolution and delivery of the medication. The active ingredient
is mixed in a liquid containing ingredient such as sugar, gelatin,
pectin and sweeteners. These liquids are solidified into solid
tablets or caplets by freeze drying and solid state extraction
techniques. The drug compounds may be compressed with viscoelastic
and thermoelastic sugars and polymers or effervescent components to
produce porous matrices intended for immediate release, without the
need of water.
[0343] Methods for preparing the compounds of this invention are
also described in the following U.S. applications:
[0344] Ser. No. 09/425,228, filed Oct. 22, 1999;
[0345] Ser. No. 09/722,418 filed Nov. 28, 2000
[0346] Ser. No. 09/758,547, filed Jan. 12, 2001;
[0347] Ser. No. 09/838,285, filed Apr. 20, 2001;
[0348] Ser. No. 09/838,286, filed Apr. 20, 2001; and
[0349] The entire disclosure of all applications, patents and
publications cited above and below are hereby incorporated by
reference.
[0350] The compounds can be produced from known compounds (or from
starting materials which, in turn, can be produced from known
compounds), e.g., through the general preparative methods shown
below. The activity of a given compound to inhibit raf kinase can
be routinely assayed, e.g., according to procedures disclosed
below. The following examples are for illustrative purposes only
and are not intended, nor should they be construed to limit the
invention in any way.
EXAMPLES
[0351] All reactions were performed in flame-dried or oven-dried
glassware under a positive pressure of dry argon or dry nitrogen,
and were stirred magnetically unless otherwise indicated. Sensitive
liquids and solutions were transferred via syringe or cannula, and
introduced into reaction vessels through rubber septa. Unless
otherwise stated, the term `concentration under reduced pressure`
refers to use of a Buchi rotary evaporator at approximately 15
mmHg. Unless otherwise stated, the term `under high vacuum` refers
to a vacuum of 0.4-1.0 mmHg.
[0352] All temperatures are reported uncorrected in degrees Celsius
(.degree. C.). Unless otherwise indicated, all parts and
percentages are by weight.
[0353] Commercial grade reagents and solvents were used without
further purification.
N-cyclohexyl-N'-(methylpolystyrene)carbodiimide was purchased from
Calbiochem-Novabiochem Corp. 3-tert-Butylaniline,
5-tert-butyl-2-methoxyamine, 4-bromo-3-(trifluoromethyl)aniline,
4-chloro-3-(trifluoromethyl)aniline
2-methoxy-5-(trifluoromethyl)aniline, 4-tert-butyl-2-nitroaniline,
3-amino-2-naphthol, ethyl 4-isocyanatobenzoate,
N-acetyl-4-chloro-2-methoxy-5-(trifluoromethyl)aniline and
4-chloro-3-(trifluoromethyl)phenyl isocyanate were purchased and
used without further purification. Syntheses of
3-amino-2-methoxyquinoline (E. Cho et al. WO 98/00402; A. Cordi et
al. EP 542,609; IBID Bioorg. Med. Chem. 3, 1995, 129),
4-(3-carbamoylphenoxy)-1-nitrobenzene (K Ikawa Yakugaku Zasshi 79,
1959, 760; Chem. Abstr. 53, 1959, 12761b), 3-tert-butylphenyl
isocyanate (O. Rohr et al. DE 2,436,108) and
2-methoxy-5-(trifluoromethyl)phenyl isocyanate (K. Inukai et al. JP
42,025,067; IBID Kogyo Kagaku Zasshi 70, 1967, 491) have previously
been described.
[0354] Thin-layer chromatography (TLC) was performed using
Whatman.RTM. pre-coated glass-backed silica gel 60A F-254 250 .mu.m
plates. Visualization of plates was effected by one or more of the
following techniques: (a) ultraviolet illumination, (b) exposure to
iodine vapor, (c) immersion of the plate in a 10% solution of
phosphomolybdic acid in ethanol followed by heating, (d) immersion
of the plate in a cerium sulfate solution followed by heating,
and/or (e) immersion of the plate in an acidic ethanol solution of
2,4-dinitrophenylhydrazine followed by heating. Column
chromatography (flash chromatography) was performed using 230400
mesh EM Science.RTM. silica gel.
[0355] Melting points (mp) were determined using a Thomas-Hoover
melting point apparatus or a Mettler FP66 automated melting point
apparatus and are uncorrected. Fourier transform infrared spectra
were obtained using a Mattson 4020 Galaxy Series spectrophotometer.
Proton (.sup.1H) nuclear magnetic resonance (NMR) spectra were
measured with a General Electric GN-Omega 300 (300 MHz)
spectrometer with either Me.sub.4Si (.delta. 0.00) or residual
protonated solvent (CHCl.sub.3 .delta. 7.26; MeOH .delta. 3.30;
DMSO .delta. 2.49) as standard. Carbon (.sup.13C) NMR spectra were
measured with a General Electric GN-Omega 300 (75 MHz) spectrometer
with solvent (CDCl.sub.3 .delta. 77.0; MeOD-d.sub.3; .delta. 49.0;
DMSO-d.sub.6 .delta. 39.5) as standard. Low-resolution mass spectra
(MS) and high resolution mass spectra (HRMS) were either obtained
as electron impact (EI) mass spectra or as fast atom bombardment
(FAB) mass spectra. Electron impact mass spectra (EI-MS) were
obtained with a Hewlett Packard 5989A mass spectrometer equipped
with a Vacumetrics Desorption Chemical Ionization Probe for sample
introduction. The ion source was maintained at 250.degree. C.
Electron impact ionization was performed with electron energy of 70
eV and a trap current of 300 .mu.A. Liquid-cesium secondary ion
mass spectra (FAB-MS), an updated version of fast atom bombardment
were obtained using a Kratos Concept 1-H spectrometer. Chemical
ionization mass spectra (CI-MS) were obtained using a Hewlett
Packard MS-Engine (5989A) with methane or ammonia as the reagent
gas (1.times.10.sup.-4 torr to 2.5.times.10.sup.-4 torr). The
direct insertion desorption chemical ionization (DCI) probe
(Vaccumetrics, Inc.) was ramped from 0-1.5 amps in 10 sec and held
at 10 amps until all traces of the sample disappeared (.about.1-2
min). Spectra were scanned from 50-800 amu at 2 sec per scan.
HPLC-electrospray mass spectra (HPLC ES-MS) were obtained using a
Hewlett-Packard 1100 HPLC equipped with a quaternary pump, a
variable wavelength detector, a C-18 column, and a Finnigan LCQ ion
trap mass spectrometer with electrospray ionization. Spectra were
scanned from 120-800 amu using a variable ion time according to the
number of ions in the source. Gas chromatography-ion selective mass
spectra (GC-MS) were obtained with a Hewlett Packard 5890 gas
chromatograph equipped with an HP-1 methyl silicone column (0.33 mM
coating; 25 m.times.0.2 mm) and a Hewlett Packard 5971 Mass
Selective Detector (ionization energy 70 eV). Elemental analyses
are conducted by Robertson Microlit Labs, Madison N.J.
[0356] All compounds displayed NMR spectra, LRMS and either
elemental analysis or HRMS consistent with assigned structures.
List of Abbreviations and Acronyms:
AcOH acetic acid
anh anhydrous
atm atmosphere(s)
BOC tert-butoxycarbonyl
CDI 1,1'-carbonyl diimidazole
conc concentrated
d day(s)
dec decomposition
DMAC N,N-dimethylacetamide
DMPU 1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone
DMF N,N-dimethylformamide
DMSO dimethylsulfoxide
DPPA diphenylphosphoryl azide
EDCI 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide
EtOAc ethyl acetate
EtOH ethanol (100%)
Et.sub.2O diethyl ether
Et.sub.3N triethylamine
h hour(s)
HOBT 1-hydroxybenzotriazole
m-CPBA 3-chloroperoxybenzoic acid
MeOH methanol
pet. ether petroleum ether (boiling range 30-60.degree. C.)
temp. temperature
THF tetrahydrofaran
TFA trifluoroAcOH
Tf trifluoromethanesulfonyl
[0357] The following general methods are described in copending
application Ser. No. 09/948,915, filed Sep. 10, 2001, and are
hereby incorporated by reference.
A. General Methods for Synthesis of Substituted Anilines, pages
18-43
B. Synthesis of Urea Precursors, page 43,
C. Methods of Urea Formation, pages 44-51 and
D. Interconversion of Ureas, pages 52-56.
Example A
N-[4-chloro-3-(trifluoromethyl)phenyl]-N'-{4-[2-carbamoyl-(4-yridyloxy)]ph-
enyl}urea
Step 1: Preparation of 4-chloro-2-pyridinecarboxamide
[0358] ##STR6##
[0359] To a stirred mixture of methyl
4-chloro-2-pyridinecarboxylate hydrochloride (1.0 g, 4.81 mmol)
dissolved in conc. aqueous ammonia (32 mL) was added ammonium
chloride (96.2 mg, 1.8 mmol, 0.37 equiv.), and the heterogeneous
reaction mixture was stirred at ambient temperature for 16 h. The
reaction mixture was poured into EtOAc (500 mL) and water (300 mL).
The organic layer was washed with water (2.times.300 mL) and a
saturated NaCl solution (1.times.300 mL), dried (MgSO.sub.4),
concentrated in vacuo to give 4-chloro-2-pyridinecarboxamide as a
beige solid (604.3 mg, 80.3%): TLC (50% EtOAc/hexane) R.sub.f 0.20;
.sup.1H-NMR (DMSO-d.sub.6) .delta. 8.61 (d, J=5.4 Hz, 1H), 8.20
(broad s, 1H), 8.02 (d, J=1.8 Hz, 1H), 7.81 (broad s, 1H), 7.76 to
7.73 (m, 1H).
Step 2: Preparation of 4-(4-aminophenoxy)-2-pyridinecarboxamide
[0360] ##STR7##
[0361] To 4-aminophenol (418 mg, 3.83 mmol) in anh DMF (7.7 mL) was
added potassium tert-butoxide (447 mg, 3.98 mmol, 1.04 equiv.) in
one portion. The reaction mixture was stirred at room temperature
for 2 h, and a solution of 4-chloro-2-pyridinecarboxamide (600 mg,
3.83 mmol, 1.0 equiv.) in anh DMF (4 mL) was then added. The
reaction mixture was stirred at 80.degree. C. for 3 days and poured
into a mixture of EtOAc and a saturated NaCl solution. The organic
layer was sequentially washed with a saturated NH.sub.4Cl solution
then a saturated NaCl solution, dried (MgSO.sub.4), and
concentrated under reduced pressure. The crude product was purified
using MPLC chromatography (Biotage.RTM.; gradient from 100% EtOAc
to followed by 10% MeOH/50% EtOAc/40% hexane) to give the
4-chloro-5-trifluoromethylaniline as a brown solid (510 mg, 58%).
.sup.1H-NMR (DMSO-d.sub.6) .delta. 8.43 (d, J=5.7 Hz, 1H), 8.07 (br
s, 1H), 7.66 (br s, 1H), 7.31 (d, J=2.7 Hz, 1H), 7.07 (dd, J=5.7
Hz, 2.7 Hz, 1H), 6.85 (d, J=9.0 Hz, 2H), 6.62 (d, J=8.7 Hz, 2H),
5.17 (broad s, 2H); HPLC EI-MS ink 230 ((M+H).sup.+.
Step 3: Preparation of
N-[4-chloro-3-(trifluoromethyl)phenyl]-N'-{4-[2-carbamoyl-(4-pyridyloxy)]-
phenyl}urea
[0362] ##STR8##
[0363] A mixture of 4-chloro-5-trifluoromethylaniline (451 mg, 2.31
mmol, 1.1 equiv.) and 1,1'-carbonyl diimidazole (419 mg, 2.54 mmol,
1.2 equiv.) in anh dichloroethane (5.5 mL) was stirred under argon
at 65.degree. C. for 16 h. Once cooled to room temperature, a
solution of 4-(4-aminophenoxy)-2-pyridinecarboxamide (480 mg, 2.09
mmol) in anh THF (4.0 mL) was added, and the reaction mixture was
stirred at 60.degree. C. for 4 h. The reaction mixture was poured
into EtOAc, and the organic layer was washed with water (2.times.)
and a saturated NaCl solution (1.times.), dried (MgSO.sub.4),
filtered, and evaporated in vacuo. Purification using MPLC
chromatography (Biotage.RTM.; gradient from 100% EtOAc to 2%
MeOH/EtOAc) gave
N-[4-chloro-3-(trifluoromethyl)phenyl]-N'-{4-[2-carbamoyl-(4-pyridyloxy)]-
phenyl}urea as a white solid (770 mg, 82%): TLC (EtOAc) R.sub.f
0.11, 100% ethyl acetate .sup.1H-NMR (DMSO-d.sub.6) .delta. 9.21
(s, 1H), 8.99 (s, 1H), 8.50 (d, J=5.6 Hz, 1H), 8.11 (s, 1H), 8.10
(s, 1H), 7.69 (broad s, 1H), 7.64 (dd, J=8.2 Hz, 2.1 Hz, 1H), 7.61
(s, 1H), 7.59 (d, J=8.8 Hz, 2H), 7.39 (d, J=2.5 Hz, 1H), 7.15 (d,
J=8.9 Hz, 2H), 7.14 (m, 1H); MS LC-MS (MH.sup.+=451). Anal. calcd
for C.sub.20H.sub.14ClF.sub.3N.sub.4O.sub.3: C, 53.29%; H, 3.13%;
N, 12.43%. Found: C, 53.33%; H, 3.21%; N, 12.60%.
Example B
N-[4-chloro-3-(trifluoromethyl)phenyl]-N'-{4-[2-N-methylcarbamoyl-4-pyridy-
loxy]phenyl}urea
[0364] ##STR9##
[0365] Step 1: 4-Chloro-N-methyl-2-pyridinecarboxamide is first
synthesized from 4-chloropyridine-2-carbonyl chloride by adding
4-chloropyridine-2-carbonyl chloride HCl salt (7.0 g, 32.95 mmol)
in portions to a mixture of a 2.0 M methylamine solution in THF
(100 mL) and MeOH (20 mL) at 0.degree. C. The resulting mixture is
stored at 3.degree. C. for 4 h, then concentrated under reduced
pressure. The resulting nearly dry solids are suspended in EtOAc
(100 mL) and filtered. The filtrate is washed with a saturated NaCl
solution (2.times.100 mL), dried Na.sub.2SO.sub.4) and concentrated
under reduced pressure to provide
4-chloro-N-methyl-2-pyridinecarboxaxnide as a yellow, crystalline
solid.
[0366] Step 2: A solution of 4-aminophenol (9.60 g, 88.0 mmol) in
anh. DMF (150 mL) is treated with potassium tert-butoxide (10.29 g,
91.7 mmol), and the reddish-brown mixture is stirred at room temp.
for 2 h. The contents are treated with
4-chloro-N-methyl-2-pyridinecarboxamide (15.0 g, 87.9 mmol) from
Step 1 and K.sub.2CO.sub.3 (6.50 g, 47.0 mmol) and then heated at
80.degree. C. for 8 h. The mixture is cooled to room temp. and
separated between EtOAc (500 mL) and a saturated NaCl solution (500
mL). The aqueous phase is back-extracted with EtOAc (300 mL). The
combined organic layers are washed with a saturated NaCl solution
(4.times.1000 mL), dried (Na.sub.2SO.sub.4) and concentrated under
reduced pressure. The resulting solids are dried under reduced
pressure at 35.degree. C. for 3 h to afford
4-(2-(N-methylcarbamoyl)-4-pyridyloxy)aniline as a light-brown
solid. .sup.1H-NMR (DMSO-d.sub.6) .delta. 2.77 (d, J=4.8 Hz, 3H),
5.17 (br s, 2H), 6.64, 6.86 (AA'BB' quartet, J=8.4 Hz, 4H), 7.06
(dd, J=5.5, 2.5 Hz, 1H), 7.33 (d, J=2.5 Hz, 1H), 8.44 (d, J=5.5 Hz,
1H), 8.73 (br d, 1H); HPLC ES-MS m/z 244 ((M+H).sup.+).
[0367] Step 3: A solution of 4-chloro-3-(trifluoromethyl)phenyl
isocyanate (14.60 g, 65.90 mmol) in CH.sub.2Cl.sub.2 (35 mL) is
added dropwise to a suspension of
4-(2-(N-methylcarbamoyl)-4-pyridyloxy)aniline from Step 2; (16.0 g,
65.77 mmol) in CH.sub.2Cl.sub.2 (35 mL) at 0.degree. C. The
resulting mixture is stirred at room temp. for 22 h. The resulting
yellow solids are removed by filtration, then washed with
CH.sub.2Cl.sub.2 (2.times.30 mL) and dried under reduced pressure
(approximately 1 mmHg) to afford
N-(4-chloro-3-(trifluoromethyl)phenyl)-N'-(4-(2-(N-methylcarbamoyl)-4-pyr-
idyloxy)phenyl)urea as an off-white solid: mp 207-209.degree. C.;
.sup.1H-NMR (DMSO-d.sub.6) .delta. 2.77 (d, J=4.8 Hz, 3H), 7.16 (m,
3H), 7.37 (d, J=2.5 Hz, 1H), 7.62 (m, 4H), 8.11 (d, J=2.5 Hz, 1H),
8.49 (d, J=5.5 Hz, 1H), 8.77 (br d, 1H), 8.99 (s, 1H), 9.21 (s,
1H); HPLC ES-MS m/z 465 ((M+H).sup.+).
Example C
N-[2-methoxy-5-(trifluoromethyl)phenyl]-N'-{4-[2-N-methylcarbamoyl-4-pyrid-
yloxy]phenyl}urea
[0368] ##STR10##
[0369] Step 1: 4-Chloro-N-methyl-2-pyridinecarboxamide is first
synthesized from 4-chloropyridine-2-carbonyl chloride by adding
4-chloropyridine-2-carbonyl chloride HCl salt (7.0 g, 32.95 mmol)
in portions to a mixture of a 2.0 M methylamine solution in THF
(100 mL) and MeOH (20 mL) at 0.degree. C. The resulting mixture is
stored at 3.degree. C. for 4 h, then concentrated under reduced
pressure. The resulting nearly dry solids are suspended in EtOAc
(100 mL) and filtered. The filtrate is washed with a saturated NaCl
solution (2.times.100 mL), dried (Na.sub.2SO.sub.4) and
concentrated under reduced pressure to provide
4-chloro-N-methyl-2-pyridinecarboxamide as a yellow, crystalline
solid.
[0370] Step 2: A solution of 4-aminophenol (9.60 g, 88.0 mmol) in
anh. DMF (150 mL) is treated with potassium tert-butoxide (10.29 g,
91.7 mmol), and the reddish-brown mixture is stirred at room temp.
for 2 h. The contents are treated with
4-chloro-N-methyl-2-pyridinecarboxamide (15.0 g, 87.9 mmol) from
Step 1 and K.sub.2CO.sub.3 (6.50 g, 47.0 mmol) and then heated at
80.degree. C. for 8 h. The mixture is cooled to room temp. and
separated between EtOAc (500 mL) and a saturated NaCl solution (500
mL). The aqueous phase is back-extracted with EtOAc (300 mL). The
combined organic layers are washed with a saturated NaCl solution
(4.times.1000 mL), dried (Na.sub.2SO.sub.4) and concentrated under
reduced pressure. The resulting solids are dried under reduced
pressure at 35.degree. C. for 3 h to afford
4-(2-(N-methylcarbamoyl)-4-pyridyloxy)aniline as a light-brown
solid. .sup.1H-NMR (DMSO-d.sub.6) .delta. 2.77 (d, J=4.8 Hz, 3H),
5.17 (br s, 2H), 6.64, 6.86 (AA'BB' quartet, J=8.4 Hz, 4H), 7.06
(dd, J=5.5, 2.5 Hz, 1H), 7.33 (d, J=2.5 Hz, 1H), 8.44 (d, J=5.5 Hz,
1H), 8.73 (br d, 1H); HPLC ES-MS m/z 244 ((M+H).sup.+).
[0371] Step 3: To a solution of
2-methoxy-5-(trifluoromethyl)aniline (0.15 g) in anh
CH.sub.2Cl.sub.2 (15 mL) at 0.degree. C. is added CDI (0.13 g). The
resulting solution is allowed to warm to room temp. over 1 h, is
stirred at room temp. for 16 h, then is treated with
4-(2-(N-methylcarbamoyl)-4-pyridyloxy)aniline (0.18 g) from Step 2.
The resulting yellow solution is stirred at room temp. for 72 h,
then is treated with H.sub.2O (125 mL). The resulting aqueous
mixture is extracted with EtOAc (2.times.150 mL). The combined
organics are washed with a saturated NaCl solution (100 mL), dried
(MgSO.sub.4) and concentrated under reduced pressure. The residue
is triturated (90% EtOAc/10% hexane). The resulting white solids
are collected by filtration and washed with EtOAc. The filtrate is
concentrated under reduced pressure and the residual oil purified
by column chromatography (gradient from 33% EtOAc/67% hexane to 50%
EtOAc/50% hexane to 100% EtOAc) to give
N-(2-methoxy-5-(trifluoromethyl)phenyl)-N'-(4-(2-(N-methylcarbamoyl)-4-py-
ridyloxy)phenyl)urea as a light tan solid: TLC (100% EtOAc) R.sub.f
0.62; .sup.1H NMR (DMSO-d.sub.6) .delta. 2.76 (d, J=4.8 Hz, 3H),
3.96 (s, 3H), 7.1-7.6 and 8.4-8.6 (m, 1H), 8.75 (d, J=4.8 Hz, 1H),
9.55 (s, 1H); FAB-MS m/z 461 ((M+H).sup.+).
[0372] Listed below are compounds listed in the Tables below which
have been synthesized according to the Detailed Experimental
Procedures given above:
Syntheses of Exemplified Compounds
[0373] The synthesis of the exemplified compounds is more
particularly described in U.S. Patent Application No. 20020042517,
published Apr. 11, 2002.
Tables
[0374] The compounds listed in Tables 1-6 below were synthesized
according to the general methods shown above, and the more detailed
exemplary procedures described in U.S. Patent Application No.
20020042517, published Apr. 11, 2002. TABLE-US-00001 TABLE 1
3-tert-Butylphenyl Ureas ##STR11## Entry R mp (.degree. C.) HPLC
(min.) TLC R.sub.f TLC Solvent System Mass Spec. [Source] 1
##STR12## 0.22 50% EtOAc/50% hexane 418 (M + H)+(HPLC ES-MS) 2
##STR13## 0.58 50% EtOAc/50% hexane 403 (M + H)+(HPLC ES-MS) 3
##STR14## 133-135 0.68 100% EtOAc 448 (M + H)+(FAB)
[0375] TABLE-US-00002 2. 5-tert-Butyl-2-methoxyphenyl Ureas
##STR15## Entry R mp (.degree. C.) HPLC (min.) TLC R.sub.f TLC
Solvent System Mass Spec. [Source] 4 ##STR16## 5.93 448 (M +
H)+(HPLC ES-MS) 5 ##STR17## 120-122 0.67 100% EtOAc 478 (M +
H)+(FAB) 6 ##STR18## 0.40 50% EtOAc/50% hexane 460 (M + H)+(HPLC
ES-MS) 7 ##STR19## 0.79 50% EtOAc/50% hexane 446 (M + H)+(HPLC
ES-MS)
[0376] TABLE-US-00003 TABLE 3 5-(Trifluoromethyl)-2-methoxyphenyl
Ureas TLC Mass Entry R mp (.degree. C.) HPLC (min.) TLC R.sub.f
Solvent System Spec. [Source] 8 ##STR20## 250 (dec) 460 (M +
H)+(FAB) 9 ##STR21## 206-208 0.54 10% MeOH/ 90% CH2Cl2 446 (M +
H)+(HPLC ES-MS) 10 ##STR22## 0.33 50% EtOAc/ 50% pet ether 445 (M +
H)+(HPLC ES-MS) 11 ##STR23## 0.20 2% Et3N/ 98% EtOAc 461 (M +
H)+(HPLC ES-MS) 12 ##STR24## 0.27 1% Et3N/ 99% EtOAc 447 (M +
H)+(HPLC ES-MS) 13 ##STR25## 0.62 100% EtOAc 461 (M + H)+(FAB) 14
##STR26## 114-117 0.40 1% Et3N/ 99% EtOAc 447 (M + H)+(FAB) 15
##STR27## 232-235 0.54 100% EtOAc 490(M + H)+(FAB) 16 ##STR28##
210-213 0.29 5% MeOH/ 45% EtOAc/ 50% pet ether 475 (M + H)+(HPLC
ES-MS) 17 ##STR29## 187-188 0.17 50% EtOAc/ 50% pet ether 495 (M +
H)+(HPLC ES-MS) 18 ##STR30## 0.48 100% EtOAc 475 (M + H)+(HPLC
ES-MS) 19 ##STR31## 194-196 0.31 5% MeOH/ 45% EtOAc/ 50% pet ether
475 (M + H)+(HPLC ES-MS) 20 ##STR32## 214-216 0.25 5% MeOH/ 45%
EtOAc/ 50% pet ether 495 (M + H)+(HPLC ES-MS) 21 ##STR33## 208-210
0.30 50% EtOAc/ 50% hexane 481 (M + H)+(HPLC ES-MS) 22 ##STR34##
188-190 0.30 70% EtOAc/ 50% hexane 447 (M + H)+(HPLC ES-MS) 23
##STR35## 0.50 70% EtOAc/ 30% hexane 472 (M + H)+(FAB) 24 ##STR36##
203-205 0.13 100% EtOAc 479 (M + H)+(HPLC ES-MS) 25 ##STR37## 0.09
75% EtOAc/ 25% hexane 458 (M + H)+(HPLC ES-MS) 26 ##STR38## 169-171
0.67 50% EtOAc/ 50% pet ether 474 (M + H)+(HPLC ES-MS) 27 ##STR39##
218-219 0.40 50% EtOAc/ 50% pet ether 477 (M + H)+(HPLC ES-MS) 28
##STR40## 212-214 0.30 40% EtOAc/ 60% hexane 29 ##STR41## 0.33 50%
EtOAc/ 50% pet ether 474 (M + H)+(HPLC ES-MS) 30 ##STR42## 210-211
31 ##STR43## 210-204 0.43 10% MeOH/ CH2Cl2 32 ##STR44## 247-249
0.57 10% MeOH/ CH2Cl2 33 ##STR45## 217-219 0.07 10% MeOH/ CH2Cl2 34
##STR46## 0.11 70% EtOAc/ 30% hexane 35 ##STR47## 0.38 70% EtOAc/
30% hexane 36 ##STR48## 0.77 70% EtOAc/ 30% hexane 37 ##STR49##
0.58 70% EtOAc/ 30% hexane 38 ##STR50## 0.58 70% EtOAc/ 30% hexane
39 ##STR51## 0.17 70% EtOAc/ 30% hexane 40 ##STR52## 0.21 70%
EtOAc/ 30% hexane
[0377] TABLE-US-00004 TABLE 4 3-(Trifluoromethyl)-4-chlorophenyl
Ureas ##STR53## Mass Entry R mp (.degree. C.) HPLC (min.) TLC
R.sub.f TLC Solvent System Spec. [Source] 41 ##STR54## 163-165 0.08
50% EtOAc/ 50% pet ether 464 (M + H)+(HPLC ES-MS) 42 ##STR55## 215
0.06 50% EtOAc/ 50% pet ether 465 (M + H)+(HPLC ES-MS) 43 ##STR56##
0.10 50% EtOAc/ 50% pet ether 451 (M + H)+(HPLC ES-MS) 44 ##STR57##
0.25 30% EtOAc/ 70% pet ether 451 (M + H)+(HPLC ES-MS) 45 ##STR58##
0.31 30% EtOAc/ 70% pet ether 465 (M + H)+(HPLC ES-MS) 46 ##STR59##
176-179 0.23 40% EtOAc/ 60% hexane 476 (M + H)+(FAB) 47 ##STR60##
0.29 5% MeOH/ 45% EtOAc/ 50% pet ether 478 (M + H)+(HPLC ES-MS) 48
##STR61## 206-209 49 ##STR62## 147-151 0.22 50% EtOAc/ 50% pet
ether 499 (M + H)+(HPLC ES-MS) 50 ##STR63## 0.54 100% EtOAc 479 (M
+ H)+(HPLC ES-MS) 51 ##STR64## 187-189 0.33 5% MeOH/ 45% EtOAc/ 50%
pet ether 479 (M + H)+(HPLC ES-MS) 52 ##STR65## 219 0.18 5% MeOH/
45% EtOAc/ 50% pet ether 499 (M + H)+(HPLC ES-MS) 53 ##STR66##
246-248 0.30 50% EtOAc/ 50% hexane 485 (M + H)+(HPLC ES-MS) 54
##STR67## 196-200 0.30 70% EtOAc/ 30% hexane) 502 (M + H)+(HPLC
ES-MS) 55 ##STR68## 228-230 0.30 30% EtOAc/ 70% CH2Cl2 466 (M +
H)+(HPLC ES-MS) 56 ##STR69## 238-245 57 ##STR70## 221-222 0.75 80%
EtOAc/ 20% hexane 492 (M + H)+(FAB) 58 ##STR71## 247 0.35 100%
EtOAc 59 ##STR72## 198-200 0.09 100% EtOAc 479 (M + H)+(HPLC ES-MS)
60 ##STR73## 158-160 0.64 50% EtOAc/ 50% pet ether 61 ##STR74##
195-197 0.39 10% MeOH/ CH2Cl2 62 ##STR75## 170-172 0.52 10% MeOH/
CH2Cl2 63 ##STR76## 168-171 0.39 10% MeOH/ CH2Cl2 64 ##STR77##
176-177 0.35 10% MeOH/ CH2Cl2 65 ##STR78## 130-133 487 (M +
H)+(HPLC ES-MS) 66 ##STR79## 155 67 ##STR80## 225-229 0.23 100%
EtOAc 68 ##STR81## 234-236 0.29 40% EtOAc/ 60% hexane 69 ##STR82##
0.48 50% EtOAc/ 50% pet ether 481 (M + H)+(HPLC ES-MS) 70 ##STR83##
0.46 5% MeOH/ 95% CH2Cl2 564 (M + H)+(HPLC ES-MS) 71 ##STR84##
199-201 0.50 10% MeOH/ CH2Cl2 72 ##STR85## 235-237 0.55 10% MeOH/
CH2Cl2 73 ##STR86## 200-201 0.21 50% MeOH/ CH2Cl2 74 ##STR87##
145-148 75 ##STR88## 0.12 70% EtOAc/ 30% hexane 527 (M + H)+(HPLC
ES-MS) 76 ##STR89## 0.18 70% EtOAc/ 30% hexane 77 ##STR90## 0.74
70% EtOAc/ 30% hexane 78 ##STR91## 0.58 70% EtOAc/ 30% hexane 79
##STR92## 0.47 70% EtOAc/ 30% hexane 569 (M + H)+(HPLC ES-MS) 80
##STR93## 0.18 70% EtOAc/ 30% hexane 508 (M + H)+(HPLC ES-MS) 81
##STR94## 0.58 70% EtOAc/ 30% hexane 557 (M + H)+(HPLC ES-MS) 82
##STR95## 0.37 70% EtOAc/ 30% hexane 611 (M + H)+(HPLC ES-MS) 83
##STR96## 0.19 70% EtOAc/ 30% hexane 84 ##STR97## 179-183
[0378] TABLE-US-00005 TABLE 5 3-(Trifluoromethyl)-4-bromophenyl
Ureas ##STR98## Mass Entry R mp (.degree. C.) HPLC (min.) TLC
R.sub.f TLC Solvent System Spec. [Source] 85 ##STR99## 186-187 0.13
50% EtOAc/ 50% pet ether 509 (M + H)+(HPLC ES-MS) 86 ##STR100##
150-152 0.31 50% EtOAc/ 50% pet ether 545 (M + H)+(HPLC ES-MS) 87
##STR101## 217-219 0.16 50% EtOAc/ 50% pet ether 545 (M + H)+(HPLC
ES-MS) 88 ##STR102## 183-184 0.31 50% EtOAc/ 50% pet ether 525 (M +
H)+(HPLC ES-MS) 89 ##STR103## 0.21 50% EtOAc/ 50% pet ether 511 (M
+ H)+(HPLC ES-MS) 90 ##STR104## 0.28 50% EtOAc/ 50% pet ether 525
(M + H)+(HPLC ES-MS) 91 ##STR105## 214-216 0.28 50% EtOAc/ 50% pet
ether 522 (M + H)+(HPLC ES-MS) 92 ##STR106## 0.47 50% EtOAc/ 50%
pet ether 527 (M + H)+(HPLC ES-MS) 93 ##STR107## 0.46 50% EtOAc/
50% pet ether 527 (M + H)+(HPLC ES-MS) 94 ##STR108## 145-150 0.41
5% MeOH/ 95% CH2Cl2
[0379] TABLE-US-00006 TABLE 6
5-(Trifluoromethyl)-4-chloro-2-methoxyphenyl Ureas ##STR109## Entry
R mp (.degree. C.) HPLC (min.) TLC R.sub.f TLC Solvent System Mass
Spec. [Source] 95 ##STR110## 140-144 0.29 5% MeOH/ 45% EtOAc/50%
pet ether 495 (M + H)+(HPLC ES-MS) 96 ##STR111## 244-245 0.39 5%
MeOH/ 45% EtOAc/50% pet ether 529 (M + H)+(HPLC ES-MS) 97
##STR112## 220-221 0.25 5% MeOH/ 45% EtOAc/50% pet ether 529 (M +
H)+(HPLC ES-MS) 98 ##STR113## 0.27 5% MeOH/ 45% EtOAc/50% pet ether
495 (M + H)+(HPLC ES-MS) 99 ##STR114## 180-181 0.52 5% MeOH/ 45%
EtOAc/50% pet ether 509 (M + H)+(HPLC ES-MS) 100 ##STR115##
162-165
[0380] TABLE-US-00007 TABLE 7 Additional Ureas mp HPLC TLC TLC
Entry R (.degree. C.) (min.) R.sub.f Solvent System Mass Spec.
[Source] 101 ##STR116## 162-165 102 ##STR117## 0.10 50% EtOAc/ 50%
hexane 442 (M + H)+(HPLC ES-MS) 103 ##STR118## 125-130 0.24 40%
EtOAc/ 60% hexane 512 (M + H)+(FAB)
Selected Compounds are Named Below
[0381] From WO 2000/41698 TABLE-US-00008 Entry No Name 1
{3-[4-({[3-(tert-
butyl)phenyl]amino}carbonylamino)phenoxy]phenyl}-N-
methylcarboxamide 11
N-[2-methoxy-5-(trifluoromethyl)phenyl]({3-[2-(N-
methylcarbamoyl)(4-pyridyloxy)]phenyl}amino)carboxamide 12
4-[3-({N-[2-methoxy-5-
(trifluoromethyl)phenyl]carbamoyl}amino)phenoxy]pyridine-2-
carboxamide 13 N-[2-methoxy-5-(trifluoromethyl)phenyl]({4-[2-(N-
methylcarbamoyl)(4-pyridyloxy)]phenyl}amino)carboxamide 14
4-[4-({N-[2-methoxy-5-
(trifluoromethyl)phenyl]carbamoyl}amino)phenoxy]pyridine-2-
carboxamide 16 {4-[4-({N-[2-methoxy-5-
(trifluoromethyl)phenyl]carbamoyl}amino)-3-
methylphenoxy](2-pyridyl)}-N-methylcarboxamide 17
({2-chloro-4-[2-(N-methylcarbamoyl)(4-
pyridyloxy)]phenyl}amino)-N-[2-methoxy-5-
(trifluoromethyl)phenyl]carboxamide 19
({4-[2-(N-ethylcarbamoyl)(4-pyridyloxy)]phenyl}amino)-N-[2-
methoxy-5-(trifluoromethyl)phenyl]carboxamide 20
({3-chloro-4-[2-(N-methylcarbamoyl)(4-
pyridyloxy)]phenyl}amino)-N-[2-methoxy-5-
(trifluoromethyl)phenyl]carboxamide 22 3-[4-({N-[2-methoxy-5-
(trifluoromethyl)phenyl]carbamoyl}amino)phenoxy]benzamide 24
({4-[2-(N,N-dimethylcarbamoyl)(4-
pyridyloxy)]phenyl}amino)-N-[2-methoxy-5-
(trifluoromethyl)phenyl]carboxamide 27
N-[2-methoxy-5-(trifluoromethyl)phenyl]({4-[2-(N-
methylcarbamoyl)(4-pyridylthio)]phenyl}amino)carboxamide 29
N-[2-methoxy-5-(trifluoromethyl)phenyl]({3-[2-(N-
methylcarbamoyl)(4-pyridylthio)]phenyl}amino)carboxamide 31
N-[2-methoxy-5-(trifluoromethyl)phenyl][(4-{5-[N-(2-
morpholin-4-ylethyl)carbamoyl](3-
pyridyloxy)}phenyl)amino]carboxamide 32
N-[2-methoxy-5-(trifluoromethyl)phenyl]({4-[5-(N-
methylcarbamoyl)(3-pyridyloxy)]phenyl}amino)carboxamide 34
N-[2-methoxy-5-(trifluoromethyl)phenyl]({4-[3-(N-(3-
pyridyl)carbamoyl)phenoxy]phenyl}amino)carboxamide 42
{4-[4-({[4-chloro-3-
(trifluoromethyl)phenyl]amino}carbonylamino)phenoxy](2-
pyridyl)}-N-methylcarboxamide 43 4-[4-({[4-chloro-3-
(trifluoromethyl)phenyl]amino}carbonylamino)phenoxy]pyridine-
2-carboxamide 44 4-[3-({[4-chloro-3-
(trifluoromethyl)phenyl]amino}carbonylamino)phenoxy]pyridine-
2-carboxamide 45
{[4-chloro-3-(trifluoromethyl)phenyl]amino}-N-{3-[2-(N-
methylcarbamoyl)(4-pyridyloxy)]phenyl}carboxamide 47
{[4-chloro-3-(trifluoromethyl)phenyl]amino}-N-{2-methyl-4-
[2-(N-methylcarbamoyl)(4-pyridyloxy)]phenyl}carboxamide 49
{4-[3-chloro-4-({[4-chloro-3-
(trifluoromethyl)phenyl]amino}carbonylamino)phenoxy](2-
pyridyl)}-N-methylcarboxamide 51
N-[4-chloro-3-(trifluoromethyl)phenyl]({4-[2-(N-
ethylcarbamoyl)(4-pyridyloxy)]phenyl}amino)carboxamide 61
{3-[4-({[4-chloro-3-
(trifluoromethyl)phenyl]amino}carbonylamino)phenoxy]phenyl}-
N-(2-morpholin-4-ylethyl)carboxamide 62 {3-[4-({[4-chloro-3-
(trifluoromethyl)phenyl]amino}carbonylamino)phenoxy]phenyl}-
N-(2-piperidylethyl)carboxamide 65 {4-[4-({[4-chloro-3-
(trifluoromethyl)phenyl]amino}carbonylamino)phenylthio](2-
pyridyl)}-N-methylcarboxamide 69
{[4-chloro-3-(trifluoromethyl)phenyl]amino}-N-{3-[2-(N-
methylcarbamoyl)(4-pyridylthio)]phenyl}carboxamide 70
{4-[4-({[4-chloro-3-
(trifluoromethyl)phenyl]amino}carbonylamino)phenoxy](2-
pyridyl)}-N-(2-morpholin-4-ylethyl)carboxamide 72
{5-[4-({[4-chloro-3-
(trifluoromethyl)phenyl]amino}carbonylamino)phenoxy](3-
pyridyl)}-N-methylcarboxamide 75
N-[4-chloro-3-(trifluoromethyl)phenyl]({4-[3-(N-(3-
pyridyl)carbamoyl)phenoxy]phenyl}amino)carboxamide 84
{4-[4-({[4-chloro-3-
(trifluoromethyl)phenyl]amino}carbonylamino)phenoxy](2-
pyridyl)}-N-(2-hydroxyethyl)carboxamide 87 {4-[4-({[4-bromo-3-
(trifluoromethyl)phenyl]amino}carbonylamino)-2-
chlorophenoxy](2-pyridyl)}-N-methylcarboxamide 88
N-[4-bromo-3-(trifluoromethyl)phenyl]({4-[2-(N-
ethylcarbamoyl)(4-pyridyloxy)]phenyl}amino)carboxamide 89
{[4-bromo-3-(trifluoromethyl)phenyl]amino}-N-{3-[2-(N-
methylcarbamoyl)(4-pyridyloxy)]phenyl}carboxamide 90
{[4-bromo-3-(trifluoromethyl)phenyl]amino}-N-{4-methyl-3-
[2-(N-methylcarbamoyl)(4-pyridyloxy)]phenyl}carboxamide 93
{[4-bromo-3-(trifluoromethyl)phenyl]amino}-N-{3-[2-(N-
methylcarbamoyl)(4-pyridylthio)]phenyl}carboxamide 94
{4-[4-({[4-bromo-3-
(trifluoromethyl)phenyl]amino}carbonylamino)phenoxy](2-
pyridyl)}-N-(2-morpholin-4-ylethyl)carboxamide 95
N-[4-chloro-2-methoxy-5-(trifluoromethyl)phenyl]({4-[2-(N-
methylcarbamoyl)(4-pyridyloxy)]phenyl}amino)carboxamide 96
N-[4-chloro-2-methoxy-5-(trifluoromethyl)phenyl]({2-chloro-
4-[2-(N-methylcarbamoyl)(4- pyridyloxy)]phenyl}amino)carboxamide 97
N-[4-chloro-2-methoxy-5-(trifluoromethyl)phenyl]({3-chloro-
4-[2-(N-methylcarbamoyl)(4- pyridyloxy)]phenyl}amino)carboxamide 98
N-[4-chloro-2-methoxy-5-(trifluoromethyl)phenyl]({3-[2-(N-
methylcarbamoyl)(4-pyridyloxy)]phenyl}amino)carboxamide 99
N-[4-chloro-2-methoxy-5-(trifluoromethyl)phenyl]({4-[2-(N-
ethylcarbamoyl)(4-pyridyloxy)]phenyl}amino)carboxamide
[0382] The compounds listed below are suitable for use in this
invention and their synthesis is described with greater
particularity in WO 2002/85859 TABLE-US-00009 Entry No Name 16
[(4-fluorophenyl)amino]-N-(3-isoquinolyl)carboxamide 25
N-(2-methoxy(3-quinolyl))[(4-(4-
pyridyloxy)phenyl)amino]carboxamide 27
N-(2-methoxy(3-quinolyl))[(3-(4-
pyridylthio)phenyl)amino]carboxamide 28
N-[1-(4-methylpiperazinyl)(3-isoquinolyl)][(4-(4-
pyridyloxy)phenyl)amino]carboxamide
[0383] and WO 2002/85857 TABLE-US-00010 Entry No. Name 25
N-(2-methoxy(3-quinolyl))[(4-(4-
pyridyloxy)phenyl)amino]carboxamide 27
N-(2-methoxy(3-quinolyl))[(3-(4-
pyridylthio)phenyl)amino]carboxamide 28
N-[1-(4-methylpiperazinyl)(3-isoquinolyl)][(4-(4-
pyridyloxy)phenyl)amino]carboxamide
[0384] The following publications relate to VEGFR-3 inhibition and
are incorporated herein for their description of disease states
mediated by VEGFR-3 and assays to determine such activity.
TABLE-US-00011 WO95/33772 Alitalo, et. al. WO95/33050
Charnock-Jones, et. al.. WO96/39421 Hu, et. al. WO98/33917 Alitalo,
et. al. WO02/057299 Alitalo, et. al. WO02/060950 Alitalo, et. al.
WO02/081520 Boesen, et. al.
[0385] The following publications relate to VEGFR-2 inhibition and
are incorporated herein for their description of disease states
mediated by VEGFR-2 and assays to determine such activity.
TABLE-US-00012 EP0882799 Hanai, et. al. EP1167384 Ferraram, et, al.
EP1086705 Sato, et. al. EP11300032 Tesar, et. al. EP1166798
Haberey, et. al. EP1166799 Haberey, et. al. EP1170017 Maini, et.
al. EP1203827 Smith WO02/083850 Rosen, et. al.
[0386] The following publications relate to FLT-3 inhibition and
are incorporated herein for their description of disease states
mediated by FLT-3 and assays to determine such activity.
TABLE-US-00013 2002/0034517 Brasel, et. al. 2002/0107365 Lyman, et.
al. 2002/0111475 Graddis, et. al. EP0627487 Beckermann, et. al.
WO9846750 Bauer, et. al. WO9818923 McWherter, et. al. WO9428391
Beckermann, et al. WO9426891 Birnbaum, et. al.
[0387] The following patents and publication relate to PDGF/PDGFR
inhibition and are incoropated herein for their description of the
disease states mediated by PDGFR-beta and assays to determine such
activity. TABLE-US-00014 5,094,941 Hart, et. al. 5,371,205 Kelly,
et. al. 5,418,135 Pang 5,444,151 Vassbotn, et. al. 5,468,468
LaRochelle, et. al. 5,567,584 Sledziewski, et. al. 5,618,678 Kelly,
et. al. 5,620,687 Hart, et. al. 5,648,076 Ross, et. al. 5,668,264
Janjic, et. al. 5,686,572 Wolf, et. al. 5,817,310 Ramakrishnan, et.
al. 5,833,986 LaRochelle, et. al. 5,863,739 LaRochelle, et. al.
5,872,218 Wolf, et. al. 5,882,644 Chang, et. al. 5,891,652 Wolf,
et. al. 5,976,534 Hart, et. al. 5,990,141 Hirth, et. al. 6,022,854
Shuman 6,043,211 Williams, et. al. 6,110,737 Escobedo, et. al.
6,207,816B1 Gold, et. al. 6,228,600B1 Matsui, et. al. 6,229,002B1
Janjic, et. al. 6,316,603B1 McTigue, et. al. 6,372,438B1 Williams,
et. al. 6,403,769B1 La Rochelle, et. al. 6,440,445B1 Nowak, et. al.
6,475,782B1 Escobedo, et. al. WO02/083849 Rosen, et. al.
WO02/083704 Rosen, et. al. WO02/081520 Boesen, et. al. WO02/079498
Thomas, et. al. WO02/070008 Rockwell, et. al. WO09959636 Sato, et.
al. WO09946364 Cao, et. al. WO09940118 Hanai, et. al. WO9931238
Yabana, et. al. WO9929861 Klagsbrun, et. al. WO9858053 Kendall, et.
al. WO9851344 Maini, et. al. WO9833917 Alitalo, et. al. WO9831794
Matsumoto, et. al. WO9816551 Ferrara, et. al. WO9813071 Kendall, et
al. WO9811223 Martiny-Baron, et. al. WO9744453 Chen, et. al.
WO9723510 Plouet, et. al. WO9715662 Stinchcomb, et. al. WO9708313
Ferrara, et. al. WO9639515 Cao, et. al. WO9623065 Smith, et. al.
WO9606641 Fleurbaaij, et. al. WO9524473 Cao, et. al. WO9822316
Kyowa WO9521868 Rockwell, et. al. WO02/060489 Xia, et. al.
[0388] PDGFR-Beta TABLE-US-00015 EP0869177 Matsui, et. al.
WO09010013 Matsui, et. al. WO9737029 Matsui, et. al.
[0389] PDGFR-Alpha TABLE-US-00016 EP1000617 Lammers, et. al.
EP0869177 Matsui, et. al. EP0811685 Escobedo, et. al.
[0390] The following patents and publication relate to PDGF/PDGFR
inhibition and are incoropated herein for their description of the
disease states mediated by FGFR and assays to determine such
activity. TABLE-US-00017 5,191,067 Lappi, et. al. 5,288,855
Bergonzoni, et. al. 5,459,015 Janjic, et. al. 5,478,804 Calabresi,
et. al. 5,576,288 Lappi, et. al. 5,639,868 Janjic, et. al.
5,648,076 Ross, et. al. 5,670,323 Nova, et. al. 5,676,637 Lappi,
et. al. 5,707,632 Williams, et. al. 5,744,313 Williams, et. al.
5,789,182 Yayon, et. al. 5,789,382 Wellstein 5,843,893 Courtois
5,891,655 Ornitz 5,965,132 Thorpe, et. al. 6,051,230 Thorpe, et.
al. 6,350,593B1 Williams, et. al.
Biochemistry and Cellular Examples 1. c-Raf (Raf-1) Biochemical
Assay
[0391] The c-Raf biochemical assay was performed with a c-Raf
enzyme that was activated (phosphorylated) by Lck kinase.
Lck-activated c-Raf (Lck/c-Raf) was produced in Sf9 insect cells by
co-infecting cells with baculoviruses expressing, under the control
of the polyhedrin promoter, GST-c-Raf (from amino acid 302 to amino
acid 648) and Lck (full-length). Both baculoviruses were used at
the multiplicity of infection of 2.5 and the cells were harvested
48 hours post infection.
[0392] MEK-1 protein was produced in Sf9 insect cells by infecting
cells with the baculovirus expressing GST-MEK-1 (full-length)
fusion protein at the multiplicity of infection of 5 and harvesting
the cells 48 hours post infection. Similar purification procedure
was used for GST-c-Raf 302-648 and GST-MEK-1.
[0393] Transfected cells were suspended at 100 mg of wet cell
biomass per mL in a buffer containing 10 mM sodium phosphate, 140
mM sodium chloride pH 7.3, 0.5% Triton X-100 and the protease
inhibitor cocktail. The cells were disrupted with Polytron
homogenizer and centrifuged 30,000 g for 30 minutes. The 30,000 g
supernatant was applied onto GSH-Sepharose. The resin was washed
with a buffer containing 50 mM Tris, pH 8.0, 150 M NaCl and 0.01%
Triton X-100. The GST-tagged proteins were eluted with a solution
containing 100 mM Glutathione, 50 mM Tris, pH 8.0, 150 mM NaCl and
0.01% Triton X-100. The purified proteins were dialyzed into a
buffer containing 20 mM Tris, pH 7.5, 150 mM NaCl and 20%
Glycerol.
[0394] Test compounds were serially diluted in DMSO using
three-fold dilutions to stock concentrations ranging typically from
50 .mu.M to 20 nM (final concentrations in the assay range from 1
.mu.M to 0.4 nM). The c-Raf biochemical assay was performed as a
radioactive filtermat assay in 96-well Costar polypropylene plates
(Costar 3365). The plates were loaded with 75 .mu.L solution
containing 50 mM HEPES pH 7.5, 70 mM NaCl, 80 ng of Lck/c-Raf and 1
.mu.g MEK-1. Subsequently, 2 .mu.L of the serially diluted
individual compounds were added to the reaction, prior to the
addition of ATP. The reaction was initiated with 25 .mu.L ATP
solution containing 5 .mu.M ATP and 0.3 .mu.Ci [33P]-ATP. The
plates were sealed and incubated at 32.degree. C. for 1 hour. The
reaction was quenched with the addition of 50 .mu.l of 4%
Phosphoric Acid and harvested onto P30 filtermats (PerkinElmer)
using a Wallac Tomtec Harvester. Filtermats were washed with 1%
Phosphoric Acid first and deinonized H.sub.2O second. The filters
were dried in a microwave, soaked in scintillation fluid and read
in a Wallac 1205 Betaplate Counter (Wallac Inc., Atlanta, Ga.,
U.S.A.). The results were expressed as percent inhibition. %
Inhibition=[100-(T.sub.ib/T.sub.i)].times.100 where
T.sub.ib=(counts per minute with inhibitor)-(background)
T.sub.i=(counts per minute without inhibitor)-(background) 2.
Bio-Plex Phospho-ERK1/2 Immunoassay
[0395] A 96-well phospho-ERK (PERK) immunoassay, using laser flow
cytometry platform has been established to measure inhibition of
basal pERK in cell lines. MDA-MB-231 cells were plated at 50,000
cells per well in 96-well microtitre plates in complete growth
media. For effects of test compounds on basal pERK1/2 inhibition,
the next day after plating, MDA-MB-231 cells were transferred to
DMEM with 0.1% BSA and incubated with test compounds diluted 1:3 to
a final concentration of 3 .mu.M to 12 nM in 0.1% DMSO. Cells were
incubated with test compounds for 2 h, washed, and lysed in
Bio-Plex whole cell lysis buffer A. Samples are diluted with buffer
B 1:1 (v/v) and directly transferred to assay plate or frozen at
-80 C degrees until processed. 50 .mu.L of diluted MDA-MB-231 cell
lysates were incubated with about 2000 of 5 micron Bio-Plex beads
conjugated with an anti-ERK1/2 antibody overnight on a shaker at
room temperature. The next day, biotinylated phospho-ERK1/2
sandwich immunoassay was performed, beads are washed 3 times during
each incubation and then 50 .mu.L of PE-strepavidin was used as a
developing reagent. The relative fluorescence units of pERK1/2 were
detected by counting 25 beads with Bio-Plex flow cell (probe) at
high sensitivity. The IC.sub.50 was calculated by taking untreated
cells as maximum and no cells (beads only) as background.
3. Immunohistochemical Staining of Tumor Sections with Anti-pERK
Antibodies
[0396] Activated Raf kinase phosphorylates and activates MEK
(mitogen-activated protein kinase kinase), which in turn
phosphorylates and activates ERK (extracellular signal-regulated
kinase), which translocates to the nucleus and modifies gene
expression. This pathway is often aberrantly activated in tumor
cells due to the presence of activated ras, mutant BRAF, or
elevation of growth factor receptors. A semi-quantitative
immunohistochemical method was developed to detect phosphorylated
ERK (pERK) in human tumor biopsies as a patient selection biomarker
for determining whether a patient will be responsive to a compound
of the present invention, and also to assess its efficacy. The
antibody used was a polyclonal antibody (rabbit) that detects the
phosphorylation status of p44 and p42 (phospho-p44/42 MAPK
(Thr202/Tyr204)) MAP kinases (ERK1 and ERK2). The procedure was
accomplished as follows:
1. Tumor samples were obtained from patients. Samples were embedded
in paraffin and sectioned.
2. Tumor section slides were deparaffinized, and slides were
incubated in 950 C citrate buffer for 35 min.
3. Slides (in buffer) were placed in a preheated steamer for 30
min.
4. When complete, slides in the heated buffer were allowed to
acclimate to room temperature (RT) for 30 minutes.
5. Slides were washed in Wash Buffer and loaded onto staining racks
in stainer, ensuring that all slides were moist with buffer at all
times.
6. Slides were blocked in a 1.5% Hydrogen Peroxide solution for 10
min and then with, normal blocking serum (goat) for 20 min.
7. Slides were washed in buffer.
8. Slides were incubated with primary anti-pERK antibodies
[phospho-p44/42 MAPK (Thr202/Tyr204)] at a dilution of 1:50 for 30
min. Negative control slides remained in blocking serum.
9. Slides were rinsed with buffer.
10. Slides were incubated in a biotinylated secondary antibody
solution for 30 min.
11. Slides were rinsed with buffer.
12. Slides were exposed to preformed complex comprising horseradish
peroxidase conjugated to avidin for 30 min.
13. Slides were rinsed with buffer.
14. A DAB substrate chromagen was applied to the slides for 1
min.
15. Slides were rinsed with distilled water.
16. Slides were counterstained slides with hematoxylin for 1
min.
17. Slides were rinsed with buffer.
18. Slides were rinsed with distilled water.
19. Slides were rehydrated, and coverslipped with permount.
[0397] 20. Each tissue section was assessed using the Bioquant
TCW98 image analysis system. Five non-overlapping fields/tissue
were selected and captured electronically using an Olympus BX-60
microscope and an Optronics DEI-750 color digital camera connected
to a PC-based computer running the Bioquant software.
[0398] A subject having a melanoma was treated with a compound in
accordance with the present invention, and then a biopsy sample was
assessed using the assay described above. FIG. 1 shows the
semi-quantitative procedure utilized to analyze the tissue
sections.
[0399] FIG. 2 shows data establishing that, prior to the
administration of the compound, a subject having melanoma had high
levels of phospho-ERK in tumor tissue as compared to stromal cells,
indicating that the subject was a candidate for treatment with a
compound of the present invention. The subject was then
administered a 600 mg BID dose of the compound.
[0400] A response to the compound was observed by comparing PET
scans taken at baseline vs. the second cycle. The PET scan, which
had shown significant metabolic activity at baseline, was reported
to be "completely silent" after treatment. This response
corresponds with a decrease in the percent of nuclei expressing
pERK in the post-treatment biopsy. This subject continued to have
stable disease based on CT scan measurements by RECIST through
cycle 6 of treatment. Thus, not only did phospho-ERK (raf activity)
predict that the subject would respond to the compound by showing
measurable improvement, but the compound also reduced the levels of
raf activity in the cells (as measured by phosphor-ERK). The
compound utilized was
N-[4-chloro-3-(trifluoromethyl)phenyl]-N'-{4-[2-N-methylcarbamoyl-4-pyrid-
yloxy]phenyl}urea.
4. Flk-1 (Murine VEGFR-2) Biochemical Assay
[0401] This assay was performed in 96-well opaque plates (Costar
3915) in the TR-FRET format. Reaction conditions are as follows: 10
.mu.M ATP, 25 nM poly GT-biotin, 2 nM Eu-labelled phospho-Tyr Ab,
10 nM APC, 7 nM Flk-1 (kinase domain), 1% DMSO, 50 mM HEPES pH 7.5,
10 mM MgCl.sub.2, 0.1 mM EDTA, 0.015% BRIJ, 0.1 mg/mL BSA, 0.1%
mercapto-ethanol). Reaction is initiated upon addition of enzyme.
Final reaction volume in each well is 100 .mu.L. Plates are read at
both 615 and 665 nM on a Perkin Elmer Victor V Multilabel counter
at about 1.5-2.0 hours after reaction initiation. Signal is
calculated as a ratio: (665 nm/615 nm)*10000 for each well.
5. Murine PDGFR FRET Biochemical Assay
[0402] This assay was formatted in a 96-well black plate (Costar
3915). The following reagents (and their sources) are used:
Europium-labeled anti-phosphotyrosine antibody pY20 and
streptavidin-APC; poly GT-biotin, and mouse PDGFR within DRT. The
reaction conditions are as follows: 1 nM mouse PDGFR is combined
with 20 .mu.M ATP, 7 nM poly GT-biotin, 1 nM pY20 antibody, 5 nM
streptavidin-APC, and 1% DMSO in assay buffer (50 mM HEPES pH 7.5,
10 mM MgCl.sub.2, 0.1 mM EDTA, 0.015% BRIJ 35, 0.1 mg/mL BSA, 0.1%
mercaptoethanol). Reaction is initiated upon addition of enzyme.
Final reaction volume in each well is 100 .mu.L. After 90 minutes,
the reaction is stopped by addition of 10 .mu.L/well of 5 .mu.M
staurosporine. Plates are read at both 615 and 665 nm on a Perkin
Elmer VictorV Multilabel counter at about 1 hour after the reaction
is stopped. Signal is calculated as a ratio: (665 nm/615 nm)*10000
for each well.
[0403] For IC.sub.50 generation for both PDGFR and Flk-1, compounds
were added prior to the enzyme initiation. A 50-fold stock plate
was made with compounds serially diluted 1:3 in a 50% DMSO/50%
dH.sub.2O solution. A 2 .mu.L addition of the stock to the assay
gave final compound concentrations ranging from 10 .mu.M-4.56 nM in
1% DMSO. The data were expressed as percent inhibition: %
inhibition=100-((Signal with inhibitor-background)/(Signal without
inhibitor-background))*100
6. pPDGFR-b Sandwich ELISA in AoSMC Cells
[0404] 100K P3-P6 Aortic SMC were plated in each well of 12-well
cluster in 1000 uL volume/well of SGM-2 using standard cell culture
techniques. Next day, cells were rinsed with 1000 uL D-PBS (Gibco)
once, then serum starved in 500 uL SBM (smooth muscle cell basal
media) with 0.1% BSA (Sigma, Cat A9576) overnight. Compounds were
diluted at a dose range from (10 uM to 1-nM in 10-fold dilution
steps in DMSO. Final DMSO concentration 0.1%). Remove old media by
inversion into the sink quickly then add 100 ul of each dilution to
corresponding well of cells for 1 hr at 37.degree. C. Cells were
then stimulated with 10 ng/mL PDGF BB ligand for 7 minutes at
37.degree. C. The media is decanted and 150 uL of isotonic lysis
buffer with protease inhibitor tablet (Complete; EDTA-free) and 0.2
mM Na vanadate is added. Cells are lysed for 15 min at 4.degree. C.
on shaker in cold room. Lysates are put in eppendorf tubes to which
15 uL of agarose-conjugated anti-PDGFR-b antibody is added (Santa
Cruz, sc-339) and incubated at 4.degree. C. overnight. Next day,
beads are rinsed in 50-volumes of PBS three times and boiled in
1.times.LDS sample buffer (Invitrogen) for 5 minutes. Samples were
run on 3-8% gradient Tris-Acetate gels (Invitrogen) and transferred
onto Nitrocellulose. Membranes were blocked in 1% BSA/TBS-T for 1
hr. before incubation in anti-phospho-PDGFR-b (Tyr-857) antibody in
blocking buffer (1:1000 dilution) for 1 hour. After three washes in
TBS-T, membranes were incubated in Goat anti-rabbit HRP IgG
(Amersham, 1:25000 dilution) for 1 hr. Three more washes followed
before addition of ECL substrate. Membranes were exposed to
Hyperfilm-ECL. Subsequently, membranes were stripped and reprobed
with anti-PDGFR-b antibody (Santa Cruz, SC-339) for total
PDGFR-b.
7. MDA-MB231 Proliferation Assay
[0405] Human breast carcinoma cells (MDA MB-231, NCI) were cultured
in standard growth medium (DMEM) supplemented with 10%
heat-inactivated FBS at 37.degree. C. in 5%. CO.sub.2 (vol/vol) in
a humidified incubator. Cells were plated at a density of 3000
cells per well in 90 .mu.L growth medium in a 96 well culture dish.
In order to determine T.sub.0h CTG values, 24 hours after plating,
100 .mu.L of CellTiter-Glo Luminescent Reagent (Promega) was added
to each well and incubated at room temperature for 30 minutes.
Luminescence was recorded on a Wallac Victor II instrument. The
CellTiter-Glo reagent results in cell lysis and generation of a
luminescent signal proportional to the amount of ATP present,
which, in turn is directly proportional to the number of cells
present.
[0406] Test compounds are dissolved in 100% DMSO to prepare 10 mM
stocks. Stocks were further diluted 1:400 in growth medium to yield
working stocks of 25 .mu.M test compound in 0.25% DMSO. Test
compounds were serially diluted in growth medium containing 0.25%
DMSO to maintain constant DMSO concentrations for all wells. 60
.mu.L of diluted test compound were added to each culture well to
give a final volume of 180 .mu.L. The cells with and without
individual test compounds were incubated for 72 hours at which time
ATP dependent luminescence was measured, as described previously,
to yield T.sub.72h values. Optionally, the IC.sub.50 values can be
determined with a least squares analysis program using compound
concentration versus percent inhibition. % Inhibition=[1-(T.sub.72h
test-T.sub.0h)/(T.sub.72h ctrl-T.sub.0h)].times.100, where
T.sub.72h test=ATP dependent luminescence at 72 hours in the
presence of test compound T.sub.72h ctrl=ATP dependent luminescence
at 72 hours in the absence of test compound T.sub.0h=ATP dependent
luminescence at Time Zero. 8. Tumor Treatment
[0407] To test the efficacy of a compound of the present invention
against a cancer, an
N-[4-chloro-3-(trifluoromethyl)phenyl]-N'-{4-[2-N-methylcarbamoyl-4-pyrid-
yloxy]phenyl}urea salt was administered to staged HCT 116 colon
(mutant k-Ras), MIA PaCa-2 pancreatic (mutant k-Ras), NCI-H460 lung
(mutant k-Ras) and SK-OV-3 ovarian (wt k-Ras) xenograft models. All
treatments were administered p.o. on a qd.times.14 schedule.
Dosages of 10, 30 and 100 mg/kg/dose produced dose-dependent
inhibition of HCT-116 tumor growth that ranged between 45% and 68%.
Similarly, the growth of MIA PaCa-2 tumors was inhibited 44%, 66%,
and 73% at dosages of 10, 30, and 100 mg/kg/dose, respectively. The
growth of NCI-H460 tumors was inhibited 27% and 56% at dosages of
10 and 30 mg/kg/dose of this Raf kinase inhibitor. The SK-OV-3
model was slightly more sensitive, generating 45%-81% tumor growth
inhibitions at dosages of 3 to 100 mg/kg/dose.
[0408] The anti-tumor efficacy of longer duration was also
evaluated in the HCT 116 model. The compound produced net tumor
stasis at dosages of 30 and 100 mg/kg/dose when treatment was
extended to 30 days duration.
[0409] HCT 116, SK-OV-3, and MIA PaCa-2 stock tumors were
maintained as serial s.c. passages of fragments in CD-1 Nu/Nu
female mice (HCT 116 and SK-OV-3) or CB17 SCID female mice
(MIA-PaCa-2). Treatments were administered orally and were
initiated against established tumors. Tumor dimensions were
measured via calipers 2-3 times per week and were converted into
tumor mass by the formula [L.times.(W2)]2, where L and W refer to
the largest and smallest dimensions, respectively.
9. P38 Kinase Assay
[0410] The in vitro inhibitory properties of compounds were
determined using a p38 kinase inhibition assay. P38 activity was
detected using an in vitro kinase assay run in 96-well microtiter
plates. Recombinant human p38 (0.5 .quadrature.g/mL) was mixed with
substrate (myelin basic protein, 5 .quadrature.g/mL) in kinase
buffer (25 mM Hepes, 20 mM MgCl.sub.2 and 150 mM NaCl) and
compound. One .mu.Ci/well of .sup.33P-labeled ATP (10 .mu.M) was
added to a final volume of 100 .mu.L. The reaction was run at
32.degree. C. for 30 min. and stopped with a 1M HCl solution. The
amount of radioactivity incorporated into the substrate was
determined by trapping the labeled substrate onto negatively
charged glass fiber filter paper using a 1% phosphoric acid
solution and read with a scintillation counter. Negative controls
include substrate plus ATP alone.
[0411] Without further elaboration, it is believed that one skilled
in the art can, using the preceding description, utilize the
present invention to its fullest extent. The following preferred
specific embodiments are, therefore, to be construed as merely
illustrative, and not limitative of the remainder of the disclosure
in any way whatsoever. The entire disclosure of all applications,
patents and publications, cited above and in the figures are hereby
incorporated by reference in their entirety, including U.S.
Provisional Application Nos. 60/556,062, filed Mar. 25, 2004,
60/520,399, filed Nov. 17, 2003, and 60/471,735, filed May 20,
2003. TABLE-US-00018 TABLE 8 BIOCHEMICAL ASSAY SIGNALING MOLECULE
IC50 (nM) CRAF 2 BRAF-WT 25 BRAF V599E 38 HER1 INACTIVE >10,000
HER2 INACTIVE >10,000 mPDGFR 57 VEGFR2 4 mVEGFR 1 FGFR 580 p38
38 c-KIT 68 FLT3 58 LCK INACTIVE 2200 BCR-ABL INACTIVE 1350
[0412] TABLE-US-00019 TABLE 9 CELLULAR ASSAY Pathway/Signaling
molecule IC50 (nM) Raf Pathway phospho-ERK Breast 80 Melanoma 880
HER 1 & 2 Pathway HER1 Receptor INACTIVE HER2 Receptor INACTIVE
PDGFR Pathway PDGFR-beta receptor 80 VEGFR- 2 and -3 Pathway
VEGFR-2 receptor 30 mVEGFR-3 receptor 102 c-KIT Pathway c-KIT
phosphor-AKT 402 FLT3 Pathway FLT3 ITD Receptor 20 (internal tandem
duplication mutation)
* * * * *