U.S. patent application number 12/347412 was filed with the patent office on 2009-07-09 for raf inhibitor compounds and methods.
Invention is credited to Jonas Grina, Josh Hansen, Ellen Laird, Joseph P. Lyssikatos, Brad Newhouse, Alan Olivero, George Topalov, Mike Welch.
Application Number | 20090176809 12/347412 |
Document ID | / |
Family ID | 36571626 |
Filed Date | 2009-07-09 |
United States Patent
Application |
20090176809 |
Kind Code |
A1 |
Laird; Ellen ; et
al. |
July 9, 2009 |
RAF INHIBITOR COMPOUNDS AND METHODS
Abstract
Pyrazolyl compounds of Formulas Ia and Ib are useful for
inhibiting Raf kinase and for treating disorders mediated thereby.
Methods of using pyrazolyl compounds for in vitro, in situ, and in
vivo diagnosis, prevention or treatment of such disorders in
mammalian cells, or associated pathological conditions are
disclosed. ##STR00001##
Inventors: |
Laird; Ellen; (Longmont,
CO) ; Lyssikatos; Joseph P.; (Superior, CO) ;
Welch; Mike; (Westminster, CO) ; Grina; Jonas;
(Superior, CO) ; Hansen; Josh; (Longmont, CO)
; Newhouse; Brad; (Broomfield, CO) ; Olivero;
Alan; (Half Moon Bay, CA) ; Topalov; George;
(Superior, CO) |
Correspondence
Address: |
VIKSNINS HARRIS & PADYS PLLP
P.O. BOX 111098
ST. PAUL
MN
55111-1098
US
|
Family ID: |
36571626 |
Appl. No.: |
12/347412 |
Filed: |
December 31, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11345828 |
Feb 2, 2006 |
7491829 |
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12347412 |
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60650050 |
Feb 4, 2005 |
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Current U.S.
Class: |
514/259.3 ;
514/318; 514/341; 544/281; 546/194; 546/275.4 |
Current CPC
Class: |
A61P 29/00 20180101;
C07D 405/14 20130101; A61P 25/14 20180101; A61P 25/16 20180101;
A61P 31/12 20180101; A61P 25/00 20180101; A61P 7/00 20180101; A61P
17/02 20180101; A61P 35/04 20180101; A61P 9/00 20180101; A61P 35/00
20180101; A61P 19/08 20180101; A61P 43/00 20180101; C07D 401/04
20130101; C07D 403/02 20130101; A61P 9/10 20180101; A61P 5/00
20180101; A61P 7/02 20180101; A61P 3/10 20180101; A61P 37/00
20180101; C07D 401/14 20130101; A61P 37/08 20180101; A61P 1/16
20180101; A61P 17/06 20180101; A61P 37/04 20180101; C07D 487/04
20130101; A61P 25/28 20180101; A61P 17/00 20180101; A61P 31/00
20180101 |
Class at
Publication: |
514/259.3 ;
546/275.4; 546/194; 544/281; 514/341; 514/318 |
International
Class: |
A61K 31/4439 20060101
A61K031/4439; C07D 401/04 20060101 C07D401/04; C07D 401/14 20060101
C07D401/14; C07D 487/04 20060101 C07D487/04; A61K 31/4545 20060101
A61K031/4545; A61K 31/506 20060101 A61K031/506; A61P 35/00 20060101
A61P035/00 |
Claims
1. A compound selected from Formulas Ia and Ib: ##STR00095## and
stereoisomers, tautomers, solvates and pharmaceutically acceptable
salts thereof, wherein: the A-ring is a 5 or 6 membered
heterocyclic ring having one or two heteroatoms independently
selected from O, N, and S, wherein said heterocyclic, ring is
optionally substituted with one or more groups independently
selected from F, Cl, Br, I, --C(.dbd.Y)R.sup.20,
--C(.dbd.Y)OR.sup.20, --C(.dbd.Y)NR.sup.20R.sup.21,
NR.sup.20R.sup.21, --NR.sup.20C(.dbd.Y)R.sup.21,
--NR.sup.20C(.dbd.Y)OR.sup.21,
--NR.sup.20C(.dbd.Y)NR.sup.20R.sup.21, .dbd.NOR.sup.20,
.dbd.NR.sup.20, .dbd.N+(O)OR.sup.20, .dbd.NNR.sup.20R.sup.21,
.dbd.O , --OR.sup.20, --OC(.dbd.Y)R.sup.20, --OC(.dbd.Y)OR.sup.20,
--OC(.dbd.Y)NR.sup.20R.sup.21, --OS(O).sub.2(OR.sup.20),
--OP(.dbd.Y)(OR.sup.20)(OR.sup.21), --OP(OR.sup.20)(OR.sup.21),
--P(.dbd.Y)(OR.sup.20)(OR.sup.21),
--P(.dbd.Y)(OR.sup.20)NR.sup.20R.sup.21, .dbd.S, --SRW,
--S(O)R.sup.20, --S(O).sub.2R.sup.20,
--S(O).sub.2NR.sup.20R.sup.21, --S(O)(OR.sup.20),
--S(O).sub.2(OR.sup.20), --SC(.dbd.Y)R.sup.20,
--SC(.dbd.Y)OR.sup.20, --SC(.dbd.Y)NR.sup.20R.sup.21,
C.sub.1-C.sub.8 alkyl, C.sub.2-C.sub.8 alkenyl, C.sub.2-C.sub.8
alkynyl, C.sub.6-C.sub.20 aryl, C.sub.3-C.sub.12 carbocyclyl,
C.sub.2-C.sub.20 heterocyclyl, and a protecting group, and wherein
said alkyl, alkenyl, alkynyl, aryl, carbocyclyl and heterocyclyl
are optionally and independently substituted with one or more
groups independently selected from F, Cl, Br, I,
--C(.dbd.Y)R.sup.20, --C(.dbd.Y)OR.sup.20,
--C(.dbd.Y)NR.sup.20R.sup.21, NR.sup.20R.sup.21,
--NR.sup.20C(.dbd.Y)R.sup.21, --NR.sup.20C(.dbd.Y)OR.sup.21,
--NR.sup.23C(.dbd.Y)NR.sup.20R.sup.21, --OR.sup.20,
--OC(.dbd.Y)R.sup.20, --OC(.dbd.Y)OR.sup.20,
--OC(.dbd.Y)NR.sup.20R.sup.21, --OS(O).sub.2(OR.sup.20),
--OP(.dbd.Y)(OR.sup.20)(OR.sup.21), --OP(OR.sup.20)(OR.sup.21),
--P(.dbd.Y)(OR.sup.20)(OR.sup.21),
--P(.dbd.Y)(OR)NR.sup.20R.sup.21, --SR.sup.20, --S(O)R.sup.20,
--S(O).sub.2R.sup.20, --S(O).sub.2NR.sup.20R.sup.21,
--S(O)(OR.sup.20), --S(O).sub.2(OR.sup.20), --SC(.dbd.Y)R.sup.20,
--SC(.dbd.Y)OR.sup.20, --SC(.dbd.Y)NR.sup.20R.sup.21,
C.sub.1-C.sub.8 alkyl, C.sub.2-C.sub.8 alkenyl, C.sub.2-C.sub.8
alkynyl, C.sub.6-C.sub.20 aryl, C.sub.3-C.sub.12 carbocyclyl,
C.sub.2-C.sub.20 heterocyclyl; X is a C.sub.2-C.sub.20
heterocyclyl, wherein said heterocycle is optionally substituted
with one or more groups independently selected from F, Cl, Br, I,
--C(.dbd.Y)R.sup.20, --C(.dbd.Y)OR.sup.20,
--C(.dbd.Y)NR.sup.20R.sup.21, --NR.sup.20R.sup.21,
--NR.sup.20C(.dbd.Y)R.sup.21, --NR.sup.20C(.dbd.Y)OR.sup.21,
--NR.sup.23C(.dbd.Y)NR.sup.20R.sup.21, --OR.sup.20,
--OC(.dbd.Y)R.sup.20, --OC(.dbd.Y)OR.sup.20,
--OC(.dbd.Y)NR.sup.20R.sup.21, --OS(O).sub.2(OR.sup.20),
--OP(.dbd.Y)(OR.sup.20)(OR.sup.21), --OP(OR.sup.20)(OR.sup.21),
--P(.dbd.Y)(OR.sup.20)(OR.sup.21),
--P(.dbd.Y)(OR.sup.23)NR.sup.20R.sup.21, --SR.sup.20,
--S(O)R.sup.20, --S(O).sub.2R.sup.20,
--S(O).sub.2NR.sup.20R.sup.21, --S(O)(OR.sup.20),
--S(O).sub.2(OR.sup.20), --SC(.dbd.Y)R.sup.20,
--SC(.dbd.Y)OR.sup.20, --SC(.dbd.Y)NR.sup.20R.sup.21, 5-7 membered
ring lactam, 5-7 membered ring lactone, 5-7 membered ring sultam,
C.sub.1-C.sub.8 alkyl, C.sub.2-C.sub.8 alkenyl, C.sub.2-C.sub.8
alkynyl, C.sub.3-C.sub.12 carbocyclyl, C.sub.6-C.sub.20 aryl, and
C.sub.2-C.sub.20 heterocyclyl, wherein said alkyl, alkenyl,
alkynyl, carbocyclyl, aryl, and heterocyclyl are optionally
substituted with one or more groups independently selected from F,
Cl, Br, I, OR.sup.20, NR.sup.20R.sup.21--SR.sup.20, --S(O)R.sup.20,
--S(O).sub.2R.sup.20, alkyl, alkenyl, alkynyl, carbocyclyl, aryl,
and heterocyclyl; R.sup.1 is selected from H, C.sub.1-C.sub.8
alkyl, C.sub.2-C.sub.8 alkenyl, C.sub.2-C.sub.8 alkynyl,
(C.sub.1-C.sub.8 alkyl)NR.sup.20R.sup.21, a C.sub.2-C.sub.20
heterocyclyl, a C.sub.3-C.sub.12 carbocyclyl, and a
C.sub.6-C.sub.20 aryl, wherein said alkyl, alkenyl, alkynyl,
heterocycle, carbocyclyl and aryl are optionally substituted with
one or more groups independently selected from F, Cl, Br, I,
--C(.dbd.Y)R.sup.20, --C(.dbd.Y)OR.sup.20,
--C(.dbd.Y)NR.sup.20R.sup.21, --NR.sup.20R.sup.21, OR.sup.20, CN,
C(.dbd.O)NR.sup.20R.sup.21, C(.dbd.O)OR.sup.20, alkyl,
(C.sub.1-C.sub.8 alkyl)NR.sup.20R.sup.21, and heterocyclyl; R.sup.2
is selected from H, F, Cl, Br, I, --C(.dbd.Y)R.sup.20,
--C(.dbd.Y)OR.sup.20, --C(.dbd.Y)NR.sup.20R.sup.21, --OR.sup.20,
--OC(.dbd.Y)R.sup.20, --OC(.dbd.Y)OR.sup.20,
--OC(.dbd.Y)NR.sup.20R.sup.21, --OS(O).sub.2(OR.sup.20),
--OP(.dbd.Y)(OR.sup.20)(OR.sup.21), --OP(OR.sup.20)(OR.sup.21),
--P(.dbd.Y)(OR.sup.20)(OR.sup.21),
--P(.dbd.Y)(OR)NR.sup.20R.sup.21, --SR.sup.20, --S(O)R.sup.20,
--S(O).sub.2R.sup.20, S(O).sub.2NR.sup.20R.sup.21,
--S(O)(OR.sup.20), --S(O).sub.2(OR.sup.20), --SC(.dbd.Y)R.sup.20,
--SC(.dbd.Y)OR.sup.20, --SC(.dbd.Y)NR.sup.20R.sup.21, 5-7 membered
ring lactam, 5-7 membered ring lactone, 5-7 membered ring sultam,
C.sub.1-C.sub.8 alkyl, C.sub.2-C.sub.8 alkenyl, C.sub.2-C.sub.8
alkynyl, C.sub.3-C.sub.12 carbocyclyl, C.sub.6-C.sub.20 aryl, and
C.sub.2-C.sub.20 heterocyclyl, wherein said alkyl, alkenyl,
alkynyl, carbocyclyl, aryl, and heterocyclyl are optionally
substituted with one or more groups independently selected from F,
Cl, Br, I, OR.sup.20, NR.sup.20R.sup.21--SR.sup.20, --S(O)R.sup.20,
--S(O).sub.2R.sup.20, alkyl, alkenyl, alkynyl, carbocyclyl, aryl,
and heterocyclyl, R.sup.3, R.sup.4, and R.sup.5 are independently
selected from H, F, Cl, Br, I, --C(.dbd.Y)R.sup.20,
--C(.dbd.Y)OR.sup.20, C(.dbd.Y)NR.sup.20R.sup.21,
--NR.sup.20R.sup.21, --NR.sup.20C(.dbd.Y)R.sup.21,
--NR.sup.20C(.dbd.Y)OR.sup.21,
--NR.sup.23C(.dbd.Y)NR.sup.20R.sup.21, --OR.sup.20,
--OC(.dbd.Y)R.sup.20, --OC(.dbd.Y)OR.sup.20,
--OC(.dbd.Y)NR.sup.20R.sup.21, --OS(O).sub.2(OR.sup.20),
--OP(.dbd.Y)(OR.sup.20)(OR.sup.21), --OP(OR.sup.20)(OR.sup.21),
--P(.dbd.Y)(OR.sup.20)(OR.sup.21),
--P(.dbd.Y)(OR.sup.23)NR.sup.20R.sup.21, --SR.sup.20,
--S(O)R.sup.20, --S(O).sub.2R.sup.20,
--S(O).sub.2NR.sup.20R.sup.21, --S(O)(OR.sup.20),
--S(O).sub.2(OR.sup.20), --SC(.dbd.Y)R.sup.20,
--SC(.dbd.Y)OR.sup.20, --SC(.dbd.Y)NR.sup.20R.sup.21, 5-7 membered
ring lactam, 5-7 membered ring lactone, 5-7 membered ring sultam,
C.sub.1-C.sub.8 alkyl, C.sub.2-C.sub.8 alkenyl, C.sub.2-C.sub.8
alkynyl, C.sub.3-C.sub.12 carbocyclyl, C.sub.6-C.sub.20 aryl, and
C.sub.2-C.sub.20 heterocyclyl, wherein said alkyl, alkenyl,
alkynyl, carbocyclyl, aryl, and heterocyclyl are optionally
substituted with one or more groups independently selected from F,
Cl, Br, I, OR.sup.20, NR.sup.20R.sup.21--SR.sup.20, --S(O)R.sup.20,
--S(O).sub.2R.sup.21, alkyl, alkenyl, alkynyl, carbocyclyl, aryl,
and heterocyclyl; R.sup.20 and R.sup.21 are independently selected
from H, C.sub.1-C.sub.8 alkyl, C.sub.2-C.sub.8 alkenyl,
C.sub.2-C.sub.8 alkynyl, C.sub.6-C.sub.20 aryl, C.sub.2-C.sub.20
heterocyclyl, and a protecting group, wherein said alkyl, alkenyl,
alkynyl, aryl, and heterocyclyl are optionally and independently
substituted with one or more groups independently selected from F,
Cl, Br, I, --C(.dbd.Y)R.sup.a, --C(.dbd.Y)OR.sup.a,
--C(.dbd.Y)NR.sup.aR.sup.b, --OR.sup.a, --OC(.dbd.Y)R.sup.a,
--OC(.dbd.Y)OR.sup.a, --OC(.dbd.Y)NR.sup.aR.sup.b,
--OS(O).sub.2(OR.sup.a), --OP(.dbd.Y)(OR.sup.a)(OR.sup.b),
--OP(OR.sup.a)(OR.sup.b), --P(.dbd.Y)(OR.sup.a)(OR.sup.b),
--P(.dbd.Y)(OR)NR.sup.aR.sup.b, --SR.sup.a, --S(O)R.sup.a,
--S(O).sub.2R.sup.a, S(O).sub.2NR.sup.aR.sup.b, --S(O)(OR.sup.a),
--S(O).sub.2(OR.sup.a), --SC(.dbd.Y)R.sup.a, --SC(.dbd.Y)OR.sup.a,
and --SC(.dbd.Y)NR.sup.aR.sup.b, or R.sup.20 and R.sup.21 together
with the atoms to which they are attached form a heterocyclic ring,
wherein said heterocyclic ring is optionally substituted with one
or more groups independently selected from F, Cl, Br, I, alkyl,
alkenyl and alkynyl; R.sup.23 is H, C.sub.1-C.sub.8 alkyl,
C.sub.2-C.sub.8 alkenyl, C.sub.2-C.sub.8 alkynyl, C.sub.6-C.sub.20
aryl, C.sub.2-C.sub.20 heterocyclyl, or a protecting group; R.sup.a
and R.sup.b are independently H, C.sub.1-C.sub.8 alkyl,
C.sub.2-C.sub.8 alkenyl, C.sub.2-C.sub.8 alkynyl, C.sub.6-C.sub.20
aryl, or C.sub.2-C.sub.20 heterocyclyl; Y is independently O, S,
NR.sup.20, .sup.+N(O)R.sup.20, N(OR.sup.20), .sup.+N(O)(OR.sup.20),
or N--NR.sup.20R.sup.21; and protecting group is selected from
trialkylsilyl, dialkylphenylsilyl, benzoate, benzyl,
benzyloxymethyl, methyl, methoxymethyl, triarylmethyl, phthalimido,
t-butoxycarbonyl (BOC), benzyloxycarbonyl (CBz),
9-fluorenylmethylenoxycarbonyl (Fmoc), and tetrahydropyranyl.
2. The compound of claim 1 wherein R.sup.1 is selected from H,
methyl, CH.sub.2CH.sub.2NH.sub.2, CH.sub.2CH.sub.2CH.sub.2NH.sub.2,
CH.sub.2CH.sub.2CH.sub.2NH.sub.2,
CH.sub.2CH.sub.2CH.sub.2NHCH.sub.2C(.dbd.O)OCH.sub.3,
CH.sub.2CH.sub.2NHCH.sub.2CH.sub.2OH, CH.sub.2CH.sub.2OH,
CH.sub.2CH(OH)CH.sub.2OH, CH.sub.2C(.dbd.O)OH, ##STR00096##
3-22. (canceled)
23. The compound of claim 2, wherein X is an optionally substituted
2-pyridyl, 3-pyridyl, 4-pyridyl, 2-imidazolyl, 4-imidazolyl,
3-pyrazolyl, 4-pyrazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl,
3-pyridazinyl, 4-pyridazinyl, 5-pyridazinyl, 2-pyrimidinyl,
5-pyrimidinyl, 6-pyrimidinyl, 2-pyrazinyl, 2-oxazolyl, 4-oxazolyl,
or 5-oxazolyl.
24. The compound of claim 23 wherein X is optionally substituted
4-pyridyl.
25. The compound of claim 1, wherein the A-ring is an optionally
substituted ring selected from tetrahydrofuranyl,
tetrahydropyranyl, tetrahydropyridyl, piperazinyl, pyrrolidinyl,
pyridyl, pyrimidinyl, dihydrothiophenyl, thiophenyl, imidazolyl,
thiazolyl, oxazolyl, isoxazolyl, and pyrazolyl.
26. (canceled)
27. (canceled)
28. The compound of claim 24, wherein the A-ring is an optionally
substituted ring selected from tetrahydrofuranyl,
tetrahydropyranyl, tetrahydropyridyl, piperazinyl, pyrrolidinyl,
pyridyl, pyrimidinyl, dihydrothiophenyl, thiophenyl, imidazolyl,
thiazolyl, oxazolyl, isoxazolyl, and pyrazolyl.
29. The compound of claim 1 selected from Formulas IIa-h and
IIIa-f: ##STR00097## ##STR00098## ##STR00099## wherein Z is
selected from NR.sup.20, O, and S.
30. (canceled)
31. The compound of claim 29 wherein Z is O.
32. The compound of claim 29 wherein Y is O.
33. The compound of claim 29 wherein Y is N--OR.sup.20.
34. The compound of claim 33 wherein Y is N--OH.
35. The compound of claim 29 wherein Z is O, and Y is
N--OR.sup.2--.
36. (canceled)
37. (canceled)
38. (canceled)
39. The compound of claim 29 selected from Formulas IVa, IVb, Va
and Vb: ##STR00100##
40-57. (canceled)
58. The compound of claim 1 selected from:
5-(1-methyl-3-pyridin-4-yl-1H-pyrazol-4-yl)-benzofuran-3(2H)-one
oxime;
5-(1-methyl-3-pyridin-4-yl-1H-pyrazol-4-yl)-2,3-dihydrochromen-4-one
oxime;
5-(1-methyl-3-pyridin-4-yl-1H-pyrazol-4-yl)-3,4-dihydroisoquinolin-
-1(2H)-one oxime;
5-(1-(2-hydroxyethyl)-3-pyridin-4-yl-1H-pyrazol-4-yl)-indolin-2-one;
5-(1-(2-hydroxyethyl)-3-pyridin-4-yl-1H-pyrazol-4-yl)-isoquinolin-1-ol;
5-(1-methyl-3-pyridin-4-yl-1H-pyrazol-4-yl)-indolin-2-one;
5-(1-(2-hydroxyethyl)-3-pyridin-4-yl-1H-pyrazol-4-yl)-2,3-dihydrophthalaz-
ine-1,4-dione;
5-(1-(2-hydroxyethyl)-3-pyridin-4-yl-1H-pyrazol-4-yl)-6-1H-indole;
5-(1-(2-hydroxyethyl)-3-pyridin-4-yl-1H-pyrazol-4-yl)-2-(2-methoxyethyl)i-
soindoline-1,3-dione;
5-(1-(2-hydroxyethyl)-3-pyridin-4-yl-1H-pyrazol-4-yl)-benzo[d][1,3]dioxol-
e;
5-(1-(2-hydroxyethyl)-3-pyridin-4-yl-1H-pyrazol-4-yl)-quinazolin-4(3H)--
one;
5-(1-(2-hydroxyethyl)-3-pyridin-4-yl-1H-pyrazol-4-yl)-N-tert-butyloxy-
carbonyl-2,3-dihydro-1H-inden-1-amine;
5-(1-(2-hydroxyethyl)-3-pyridin-4-yl-1H-pyrazol-4-yl)-5-1H-indole;
5-(1-(2-hydroxyethyl)-3-pyridin-4-yl-1H-pyrazol-4-yl)-5-isoindoline-1,3-d-
ione;
(Z)-5-(1-methyl-3-(pyridin-4-yl)-1H-pyrazol-4-yl)isoindolin-1-one
oxime;
(Z)-5-(1-(1-methylpiperidin-4-yl)-3-(pyridin-4-yl)-1H-pyrazol-4-yl-
)isoindolin-1-one oxime; and
5-(1-(1-methylpiperidin-4-yl)-3-(pyridin-4-yl)-1H-pyrazol-4-yl)isoindolin-
-1-imine.
59. A pharmaceutical composition comprised of a compound of claim 1
and a pharmaceutically acceptable carrier.
60. The composition according to claim 59, additionally comprising
an additional therapeutic agent selected from an anti-proliferative
agent, an anti-inflammatory agent, an immunomodulatory agent, a
neurotrophic factor, an agent for treating cardiovascular disease,
an agent for treating liver disease, an anti-viral agent, an agent
for treating blood disorders, an agent for treating diabetes, or an
agent for treating immunodeficiency disorders.
61. A composition comprising the compound of claim 1 in an amount
to detectably inhibit Raf kinase activity and a pharmaceutically
acceptable carrier, adjuvant, or vehicle.
62-76. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional application of U.S.
application Ser. No. 11/345,828 filed on Feb. 2, 2006, which
application is a nonprovisional application filed under 37 CFR
.sctn.1.53(b), and claims the benefit under 35 USC .sctn.119(e) of
U.S. Provisional Application Ser. No. 60/650,050 filed on Feb. 4,
2005, which applications are incorporated by reference in their
entireties.
FIELD OF THE INVENTION
[0002] In one aspect, the invention relates to novel pyrazolyl
compounds which are inhibitors of Raf kinase, as well as
compositions containing these compounds and methods of use. The
pyrazolyl compounds are useful for inhibiting Raf kinase and for
treating disorders mediated thereby. The invention also relates to
methods of using pyrazolyl compounds for in vitro, in situ, and in
vivo diagnosis or treatment of mammalian cells, or associated
pathological conditions.
BACKGROUND OF THE INVENTION
[0003] The Raf/MEK/ERK (extracellular signal-regulated kinase)
kinase cascade is pivotal in transmitting signals from membrane
receptors to transcription factors that control gene expression
culminating in the regulation of cell cycle progression (Robinson,
M J and Cobb, M H (1997) Curr. Opin. Cell Biol. 9:180-186). This
cascade can prevent cell death through ERK2 and p90(Rsk) activation
and phosphorylation of apoptotic and cell cycle regulatory proteins
(Shelton J G et al (2003) Oncogene. 22(16):2478-92). The PI3K/Akt
kinase cascade also controls apoptosis and can phosphorylate many
apoptotic and cell cycle regulatory proteins. These pathways are
interwoven as Akt can phosphorylate Raf (Rapidly growing
Fibrosarcoma) and result in its inactivation, and Raf can be
required for the anti-apoptotic effects of Akt. Raf is a key
serine-threonine protein kinase which participates in the
transmission of growth, anti-apoptotic and differentiation
messages. These signals can be initiated after receptor ligation
and are transmitted to members of the MAP kinase cascade that
subsequently activate transcription factors controlling gene
expression. Raf is a multigene family which expresses oncoprotein
kinases: Raf-1, A-Raf and B-Raf (McCubrey J A. et al (1998)
Leukemia. 12(12):1903-1929; Ikawa et al (1988) Mol. and Cell. Biol.
8(6):2651-2654; Sithanandam et al (1990) Oncogene 5:1775-1780;
Konishi et al (1995) Biochem. and Biophys. Res. Comm.
216(2):526-534). All three Raf kinases are functionally present in
certain human hematopoietic cells, and their aberrant expression
can result in abrogation of cytokine dependency. Their regulatory
mechanisms differ because C-Raf and A-Raf require additional serine
and tyrosine phosphorylation within the N region of the kinase
domain for full activity (Mason et al (1999) EMBO J. 18:2137-2148),
and B-Raf has a much higher basal kinase activity than either A-Raf
or C-Raf. The three Raf oncoproteins play critical roles in the
transmission of mitogenic and anti-apoptotic signals. B-Raf has
recently been shown to be frequently mutated in various human
cancers (Wan et al (2004) Cell 116:855-867). Development of
specific Raf inhibitors may prove efficacious in cancer therapy.
The cytoplasmic serine/threonine kinase B-Raf and receptor tyrosine
kinases of the platelet-derived growth factor receptor (PDGFR)
family are frequently activated in cancer by mutations of an
equivalent amino acid. Structural studies have provided important
insights into why these very different kinases share similar
oncogenic hot spots and why the PDGFR juxtamembrane region is also
a frequent oncogenic target (Dibb N J (2004) Nature Reviews.
Cancer. 4(9):718-27).
[0004] Transformation of normal melanocytes into melanoma cells is
accomplished by the activation of growth stimulatory pathways,
typically leading to cellular proliferation, and the inactivation
of apoptotic and tumor suppressor pathways. Small molecule
inhibitors of proteins in the growth stimulatory pathways are under
active investigation, and their application to melanoma patients
would represent a new treatment strategy to inhibit cell
proliferation or induce cell death (Polsky D. (2003) Oncogene,
22(20):3087-91; Konopleva, M et al. (2003) Blood,
102(11):625a).
[0005] B-Raf encodes a Ras-regulated kinase that mediates cell
growth and malignant transformation kinase pathway activation that
controls cell growth and survival. Activation of the Ras/Raf/MEK
pathway results in a cascade of events from the cell surface to the
nucleus ultimately affecting cellular proliferation, apoptosis,
differentiation and transformation. Raf is a downstream effector
enzyme of Ras. When activated, Raf goes on to activate MEK1 and
MEK2 kinases which in turn phosphorylate and activate ERK1 and ERK2
which translocate to the nucleus where they stimulate pathways
required for translation initiation and transcription activation
leading to proliferation (Sorbera et al. (2002) Drugs of the Future
27(12):1141-1147). Activating B-Raf mutations have been identified
in 66% of melanomas and a smaller percentage of many other human
cancers. B-Raf mutations also account for the MAP kinase pathway
activation common in non-small cell lung carcinomas (NSCLCs),
including V600E and other mutations identified as novel, altering
residues important in AKT-mediated B-Raf phosphorylation which
suggest that disruption of AKT-induced B-Raf inhibition can play a
role in malignant transformation. Although >90% of B-Raf
mutations in melanoma involve codon 600 (57 of 60), 8 of 9 B-Raf
mutations reported to date in NSCLC are non-V600 (89%;
P<10(-7)), strongly suggesting that B-Raf mutations in NSCLC are
qualitatively different from those in melanoma; thus, there may be
therapeutic differences between lung cancer and melanoma in
response to Raf inhibitors (Karasarides et al. (2004) Oncogene
23(37):6292-6298; Bollag et al. (2003) Current Opinion in Invest.
Drugs 4(12):1436-1441). Although uncommon, B-Raf mutations in human
lung cancers may identify a subset of tumors sensitive to targeted
therapy (Brose M S et al. (2002) Cancer Research 62(23):6997-7000;
US 2005/267060).
[0006] Raf protein kinases are key components of signal
transduction pathways by which specific extracellular stimuli
elicit precise cellular responses in mammalian cells. Activated
cell surface receptors activate ras/rap proteins at the inner
aspect of the plasma membrane which in turn recruit and activate
Raf proteins. Activated Raf proteins phosphorylate and activate the
intracellular protein kinases MEK1 and MEK2. In turn, activated
MEKs catalyze phosphorylation and activation of p42/p44
mitogen-activated protein kinase (MAPK). A variety of cytoplasmic
and nuclear substrates of activated MAPK are known which directly
or indirectly contribute to the cellular response to environmental
change. In fact, B-Raf mutation has been shown to predict
sensitivity to pharmacological MEK inhibition by small molecule
inhibitors by limiting tumor growth in B-Raf mutant xenografts
(Solit et al (2005) Nature Letters to Editor 6 Nov. 2005,
doi:10.1038). Three distinct genes have been identified in mammals
that encode Raf proteins; A-Raf, B-Raf and C-Raf (also known as
Raf-1) and isoformic variants that result from differential
splicing of mRNA are known. In particular, it has been suggested
that B-Raf is the major Raf isoform activated by the neurotrophin,
nerve growth factor (NGF), for NGF induced extracellular signaling
by kinase activation (York et al. (2000) Mol. and Cell. Biol.
20(21):8069-8083).
[0007] Cancer chemotherapy drugs typically have a narrow
therapeutic index, and often the responses produced are only just
palliative as well as unpredictable. In contrast, targeted therapy
that has been introduced in recent years is directed against
cancer-specific molecules and signaling pathways and thus has more
limited nonspecific toxicities. Tyrosine kinases are an esp.
important target because they play an important role in the
modulation of growth factor signaling (Arora et al. (2005) Jour. of
Pharm. and Exp. Ther. 315(3):971-979). Small molecule inhibitors of
tyrosine kinase compete with the ATP binding site of the catalytic
domain of several oncogenic tyrosine kinases (Fabian et al. (2005)
Nature Biotechnology 23(3):329-336). Several tyrosine kinase
inhibitors (TKIs) have been found to have effective antitumor
activity and have been approved or are in clinical trials,
including imatinib mesylate (ST1571; Gleevec), gefitinib (Iressa),
erlotinib (OSI-1774; Tarceva), lapatinib (GW-572016), canertinib
(CI-1033), semaxinib (SU5416), vatalanib (PTK787/ZK222584),
sorafenib (BAY 43-9006), sutent (SUI 1248), and leflunomide
(SUIOI). TKIs are thus an important new class of targeted therapy
that interfere with specific cell signaling pathways and thus allow
target-specific therapy for selected malignancies. The Raf/MEK/ERK
pathway is the focus of intense drug discovery efforts (Thompson et
al. (2005) Current Opinion in Pharmacology 5(4):350-356; Sridhar et
al (2005) Molecular Cancer Therapeutics 4(4):677-685).
[0008] Inhibitors of Raf kinases have been suggested for use in
disruption of tumor cell growth and hence in the treatment of
cancers, e.g. histiocytic lymphoma, lung adenocarcinoma, small cell
lung cancer and pancreatic and breast carcinoma; and also in the
treatment and/or prophylaxis of disorders associated with neuronal
degeneration resulting from ischemic events, including cerebral
ischemia after cardiac arrest, stroke and multi-infarct dementia
and also after cerebral ischemic events such as those resulting
from head injury, surgery and/or during childbirth (Strumberg et
al. (2005) Onkologie 28(2):101-107). Sorafenib (NEXAVAR.TM.;
BAY-43-9006; Bayer and Onyx) is an oral cytostatic pan-kinase
inhibitor, approved by the FDA for advanced renal cell carcinoma,
and is being developed for the potential treatment of additional,
various cancers (Ahmad et al. (2004) Clinical Cancer Res. 10(18,
Pt. 2):6388S-6392S; Lee et al. (2003) Current Opinion in Invest.
Drugs 4(6):757-763). Sorafenib prevents tumor growth by inhibition
of tumor cell proliferation and tumor angiogenesis (Clark et al.
(2005) Clinical Cancer Res. 11(15):5472-5480; Yu et al. (2005)
Oncogene 24(46):6861-6869; Wilhelm et al. (2004) Cancer Res.
64(19):7099-7109).
[0009] Use of these targeted therapies is not without limitations
such as the development of resistance and the lack of tumor
response in the general population. The availability of newer
inhibitors and improved patient selection will help overcome these
problems in the future. There remains a significant need to develop
Raf kinase inhibitors for the treatment of solid tumors
SUMMARY OF THE INVENTION
[0010] In one aspect, the invention relates to a group of novel
compounds that are inhibitors of Raf kinases, in particular
inhibitors of B-Raf kinase. The compounds of the invention can be
used in the treatment of hyperproliferative disorders such as
cancer. Certain hyperproliferative disorders are characterized by
the overactivation of Raf kinase function, for example by mutations
or overexpression of the protein. Compounds of the invention are
useful in the treatment of melanoma and other cancers of the skin,
and at various stages of progression of the disease. In one aspect,
the compounds of the invention include a group of novel pyrazole
compounds having Formulas Ia and Ib.
##STR00002##
[0011] and stereoisomers, tautomers, solvates and pharmaceutically
acceptable salts thereof, wherein:
[0012] the A-ring is: (i) a 5 or 6 membered heterocyclic ring
having one or two heteroatoms independently selected from O, N, and
S, (ii) a 5 or 6 membered carbocyclic ring optionally fused to a 5
or 6 membered heterocycle, or (iii) a phenyl ring, wherein said
heterocyclic, carbocyclic and phenyl rings are optionally
substituted with one or more groups independently selected from F,
Cl, Br, I, --C(.dbd.Y)R.sup.20, --C(.dbd.Y)OR.sup.20,
--C(.dbd.Y)NR.sup.20R.sup.21, NR.sup.20R.sup.21,
--NR.sup.20C(.dbd.Y)R.sup.21, --NR.sup.20C(.dbd.Y)OR.sup.21,
--NR.sup.23C(.dbd.Y)NR.sup.20R.sup.21, .dbd.NOR.sup.20,
.dbd.NR.sup.20, .dbd.N+(O)OR.sup.20, .dbd.NNR.sup.20R.sup.21,
.dbd.O, OR.sup.20, --OC(.dbd.Y)R.sup.20, --OC(.dbd.Y)OR.sup.20,
--OC(.dbd.Y)NR.sup.20R.sup.21, --OS(O).sub.2(OR.sup.20),
--OP(.dbd.Y)(OR.sup.20)(OR.sup.21), --OP(OR.sup.20)(OR.sup.21),
--P(.dbd.Y)(OR.sup.20)(OR.sup.21),
--P(.dbd.Y)(OR)NR.sup.20R.sup.21, .dbd.S, --SR.sup.20,
--S(O)R.sup.20, --S(O).sub.2R, --S(O).sub.2NR.sup.20R.sup.21,
--S(O)(OR.sup.20), --S(O).sub.2(OR.sup.20), --SC(.dbd.Y)R.sup.20,
--SC(.dbd.Y)OR.sup.20, --SC(.dbd.Y)NR.sup.20R.sup.21,
C.sub.1-C.sub.8 alkyl, C.sub.2-C.sub.8 alkenyl, C.sub.2-C.sub.8
alkynyl, C.sub.6-C.sub.20 aryl, C.sub.3-C.sub.12 carbocyclyl,
C.sub.2-C.sub.20 heterocyclyl, and a protecting group, and wherein
said alkyl, alkenyl, alkynyl, aryl, carbocyclyl and heterocyclyl
are optionally and independently substituted with one or more
groups independently selected from F, Cl, Br, I,
--C(.dbd.Y)R.sup.20, --C(.dbd.Y)OR.sup.20,
--C(.dbd.Y)NR.sup.20R.sup.21, NR.sup.20R.sup.21,
--NR.sup.20C(.dbd.Y)R.sup.21, --NR.sup.20C(.dbd.Y)OR.sup.21,
--NR.sup.23C(.dbd.Y)NR.sup.20R.sup.21, --OR.sup.20,
--OC(.dbd.Y)R.sup.20, --OC(.dbd.Y)OR.sup.20,
--OC(.dbd.Y)NR.sup.20R.sup.21, --OS(O).sub.2(OR.sup.20),
--OP(.dbd.Y)(OR.sup.20)(OR.sup.21), --OP(OR.sup.20)(OR.sup.21),
--P(.dbd.Y)(OR.sup.20)(OR.sup.21),
--P(.dbd.Y)(OR)NR.sup.20R.sup.21, --SR.sup.20, --S(O)R.sup.20,
--S(O).sub.2R.sup.20, --S(O).sub.2NR.sup.20R.sup.21,
--S(O)(OR.sup.20), --S(O).sub.2(OR.sup.20), --SC(.dbd.Y)R.sup.20,
--SC(.dbd.Y)OR.sup.20, --SC(.dbd.Y)NR.sup.20R.sup.21,
C.sub.1-C.sub.8 alkyl, C.sub.2-C.sub.8 alkenyl, C.sub.2-C.sub.8
alkynyl, C.sub.6-C.sub.20 aryl, C.sub.3-C.sub.12 carbocyclyl, and
C.sub.2-C.sub.20 heterocyclyl;
[0013] X is selected from a C.sub.2-C.sub.20 heterocyclyl, a
C.sub.3-C.sub.12 carbocyclyl, and a C.sub.6-C.sub.20 aryl, wherein
said heterocycle, carbocyclyl and aryl are optionally substituted
with one or more groups independently selected from F, Cl, Br, I,
--C(.dbd.Y)R.sup.20, --C(.dbd.Y)OR.sup.20,
--C(.dbd.Y)NR.sup.20R.sup.21, --NR.sup.20R.sup.21,
--NR.sup.20C(.dbd.Y)R.sup.21, --NR.sup.20C(.dbd.Y)OR.sup.21,
--NR.sup.23C(.dbd.Y)NR.sup.20R.sup.21, --OR.sup.20,
--OC(.dbd.Y)R.sup.20, --OC(.dbd.Y)OR.sup.20,
--OC(.dbd.Y)NR.sup.20R.sup.21, --OS(O).sub.2(OR.sup.20),
--OP(.dbd.Y)(OR.sup.20)(OR.sup.21), --OP(OR.sup.20)(OR.sup.21),
--P(.dbd.Y)(OR.sup.20)(OR.sup.21),
--P(.dbd.Y)(OR.sup.23)NR.sup.20R.sup.21, --SR.sup.20,
--S(O)R.sup.20, --S(O).sub.2R.sup.20,
--S(O).sub.2NR.sup.20R.sup.21, --S(O)(OR.sup.20),
--S(O).sub.2(OR.sup.21), --SC(.dbd.Y)R.sup.20,
--SC(.dbd.Y)OR.sup.20, --SC(.dbd.Y)NR.sup.20R.sup.21, 5-7 membered
ring lactam, 5-7 membered ring lactone, 5-7 membered ring sultam,
C.sub.1-C.sub.8 alkyl, C.sub.2-C.sub.8 alkenyl, C.sub.2-C.sub.8
alkynyl, C.sub.3-C.sub.12 carbocyclyl, C.sub.6-C.sub.20 aryl, and
C.sub.2-C.sub.20 heterocyclyl, wherein said alkyl, alkenyl,
alkynyl, carbocyclyl, more groups independently selected from F,
Cl, Br, I, OR.sup.20, NR.sup.20R.sup.21--SR.sup.20, -aryl, and
heterocyclyl are optionally substituted with one or S(O)R.sup.20,
--S(O).sub.2R.sup.20, alkyl, alkenyl, alkynyl, carbocyclyl, aryl,
and heterocyclyl;
[0014] R.sup.1 is selected from H, C.sub.1-C.sub.8 alkyl,
C.sub.2-C.sub.8 alkenyl, C.sub.2-C.sub.8 alkynyl,
(C.sub.1-C.sub.8alkyl)NR.sup.20R.sup.21, C.sub.2-C.sub.20
heterocyclyl, C.sub.3-C.sub.12 carbocyclyl, and C.sub.6-C.sub.20
aryl, wherein said alkyl, alkenyl, alkynyl, heterocycle,
carbocyclyl and aryl are optionally substituted with one or more
groups independently selected from F, Cl, Br, I,
--C(.dbd.Y)R.sup.20, --C(.dbd.Y)OR.sup.20,
--C(.dbd.Y)NR.sup.20R.sup.21, --NR.sup.20R.sup.21, OR.sup.20, CN,
C(.dbd.O)NR.sup.20R.sup.21, C(.dbd.O)OR.sup.20, alkyl,
(C.sub.1-C.sub.8alkyl)NR.sup.20R.sup.21, and heterocyclyl;
[0015] R.sup.2 is selected from H, F, Cl, Br, I,
--C(.dbd.Y)R.sup.20, --C(.dbd.Y)OR.sup.20,
--C(.dbd.Y)NR.sup.20R.sup.21, --OR.sup.20, --OC(.dbd.Y)R.sup.20,
--OC(.dbd.Y)OR.sup.20, --OC(.dbd.Y)NR.sup.20R.sup.21,
--OS(O).sub.2(OR.sup.20), --OP(.dbd.Y)(OR.sup.20)(OR.sup.21),
--OP(OR.sup.20)(OR.sup.21), --P(.dbd.Y)(OR.sup.20)(OR.sup.21),
--P(.dbd.Y)(OR)NR.sup.20R.sup.21, --SR.sup.20, --S(O)R.sup.20,
--S(O).sub.2R.sup.20, --S(O).sub.2NR.sup.20R.sup.21,
--S(O)(OR.sup.20), --S(O).sub.2(OR.sup.20), --SC(.dbd.Y)R.sup.20,
--SC(.dbd.Y)OR.sup.20, --SC(.dbd.Y)NR.sup.20R.sup.21, 5-7 membered
ring lactam, 5-7 membered ring lactone, 5-7 membered ring sultam,
C.sub.1-C.sub.8 alkyl, C.sub.2-C.sub.8 alkenyl, C.sub.2-C.sub.8
alkynyl, C.sub.3-C.sub.12 carbocyclyl, C.sub.6-C.sub.20 aryl, and
C.sub.2-C.sub.20 heterocyclyl, wherein said alkyl, alkenyl,
alkynyl, carbocyclyl, aryl, and heterocyclyl are optionally
substituted with one or more groups independently selected from F,
Cl, Br, I, OR.sup.20, NR.sup.20R.sup.21--SR.sup.20, --S(O)R.sup.20,
--S(O).sub.2R.sup.20, alkyl, alkenyl, alkynyl, carbocyclyl, aryl,
and heterocyclyl,
[0016] or R.sup.1 and R.sup.2 of Formula Ia together with the atoms
to which they are attached optionally form a saturated, partially
unsaturated or aromatic 5 or 6 membered fused heterocycle ring
having at least two heteroatoms independently selected from O, N
and S, wherein said heterocycle ring is optionally substituted with
one or more groups independently selected from F, Cl, Br, I,
--C(.dbd.Y)R.sup.20, --C(.dbd.Y)OR.sup.20,
--C(.dbd.Y)NR.sup.20R.sup.21, --NR.sup.20R.sup.21,
--NR.sup.20C(.dbd.Y)R.sup.21, --NR.sup.20C(.dbd.Y)OR.sup.21,
--NR.sup.23C(.dbd.Y)NR.sup.20R.sup.21, --OR.sup.20,
--OC(.dbd.Y)R.sup.20, --OC(.dbd.Y)OR.sup.20,
--OC(.dbd.Y)NR.sup.20R.sup.21, --OS(O).sub.2(OR.sup.20),
--OP(.dbd.Y)(OR.sup.20)(OR.sup.21), --OP(OR.sup.20)(OR.sup.21),
--P(.dbd.Y)(OR.sup.20)(OR.sup.21),
--P(.dbd.Y)(OR.sup.23)NR.sup.20R.sup.21, --SR.sup.20,
--S(O)R.sup.20, --S(O).sub.2R.sup.20,
--S(O).sub.2NR.sup.20R.sup.21, --S(O)(OR.sup.20),
--S(O).sub.2(OR.sup.20), --SC(.dbd.Y)R.sup.20,
--SC(.dbd.Y)OR.sup.20, SC(.dbd.Y)NR.sup.20R.sup.21, 5-7 membered
ring lactam, 5-7 membered ring lactone, 5-7 membered ring sultam,
C.sub.1-C.sub.8 alkyl, C.sub.2-C.sub.8 alkenyl, C.sub.2-C.sub.8
alkynyl, C.sub.3-C.sub.12 carbocyclyl, C.sub.6-C.sub.20 aryl, and
C.sub.2-C.sub.20 heterocyclyl, wherein said alkyl, alkenyl,
alkynyl, carbocyclyl, aryl, and heterocyclyl are optionally
substituted with one or more groups independently selected from F,
Cl, Br, I, OR.sup.20, NR.sup.20R.sup.21--SR.sup.20, --S(O)R.sup.20,
--S(O).sub.2R.sup.20, alkyl, alkenyl, alkynyl, carbocyclyl, aryl,
and heterocyclyl;
[0017] R.sup.3, R.sup.4, and R.sup.5 are independently selected
from H, F, Cl, Br, I, --C(.dbd.Y)R.sup.20, --C(.dbd.Y)OR.sup.20,
--C(.dbd.Y)NR.sup.20R.sup.21, --NR.sup.20R.sup.21,
--NR.sup.20C(.dbd.Y)R.sup.21, --NR.sup.20C(.dbd.Y)OR.sup.21,
--NR.sup.23C(.dbd.Y)NR.sup.20R.sup.21, --OR.sup.20,
--OC(.dbd.Y)R.sup.20, --OC(.dbd.Y)OR.sup.20,
--OC(.dbd.Y)NR.sup.20R.sup.21, --OS(O).sub.2(OR.sup.20),
--OP(.dbd.Y)(OR.sup.20)(OR.sup.21), --OP(OR.sup.20)(OR.sup.21),
--P(.dbd.Y)(OR.sup.20)(OR.sup.21),
--P(.dbd.Y)(OR.sup.23)NR.sup.20R.sup.21, --SR.sup.20,
--S(O)R.sup.20, --S(O).sub.2R.sup.20,
--S(O).sub.2NR.sup.20R.sup.21, --S(O)(OR.sup.21),
--S(O).sub.2(OR.sup.20), --SC(.dbd.Y)R.sup.20,
--SC(.dbd.Y)OR.sup.20, --SC(.dbd.Y)NR.sup.20R.sup.21, 5-7 membered
ring lactam, 5-7 membered ring lactone, 5-7 membered ring sultam,
C.sub.1-C.sub.8 alkyl, C.sub.2-C.sub.8 alkenyl, C.sub.2-C.sub.8
alkynyl, C.sub.3-C.sub.12 carbocyclyl, C.sub.6-C.sub.20 aryl, and
C.sub.2-C.sub.20 heterocyclyl, wherein said alkyl, alkenyl,
alkynyl, carbocyclyl, aryl, and heterocyclyl are optionally
substituted with one or more groups independently selected from F,
Cl, Br, I, OR.sup.20, NR.sup.20R.sup.21--SR.sup.20, --S(O)R.sup.20,
--S(O).sub.2R.sup.20, alkyl, alkenyl, alkynyl, carbocyclyl, aryl,
and heterocyclyl;
[0018] R.sup.20 and R.sup.21 are independently selected from H,
C.sub.1-C.sub.8 alkyl, C.sub.2-C.sub.8 alkenyl, C.sub.2-C.sub.8
alkynyl, C.sub.6-C.sub.20 aryl, C.sub.2-C.sub.20 heterocyclyl, and
a protecting group, wherein said alkyl, alkenyl, alkynyl, aryl, and
heterocyclyl are optionally and independently substituted with one
or more groups independently selected from F, Cl, Br, I,
--C(.dbd.Y)R.sup.a, --C(.dbd.Y)OR.sup.a,
--C(.dbd.Y)NR.sup.aR.sup.b, --OR.sup.a, --OC(.dbd.Y)R.sup.a,
OC(.dbd.Y)OR.sup.a, --OC(.dbd.Y)NR.sup.aR.sup.b,
--OS(O).sub.2(OR.sup.a), --OP(.dbd.Y)(OR.sup.a)(OR.sup.b),
--OP(OR.sup.a)(OR.sup.b), --P(.dbd.Y)(OR.sup.a)(OR.sup.b),
--P(.dbd.Y)(OR)NR.sup.aR.sup.b, --SR.sup.a, --S(O)R.sup.a,
--S(O).sub.2R.sup.a, --S(O).sub.2NR.sup.aR.sup.b, --S(O)(OR.sup.a),
--S(O).sub.2(OR.sup.a), --SC(.dbd.Y)R.sup.a, --SC(.dbd.Y)OR.sup.a,
and SC(.dbd.Y)NR.sup.aR.sup.b,
[0019] or R.sup.20 and R.sup.21 together with the atoms to which
they are attached form a heterocyclic ring, wherein said
heterocyclic ring is optionally substituted with one or more groups
independently selected from F, Cl, Br, I, alkyl, alkenyl and
alkynyl;
[0020] R.sup.23 is H, C.sub.1-C.sub.8 alkyl, C.sub.2-C.sub.8
alkenyl, C.sub.2-C.sub.8 alkynyl, C.sub.6-C.sub.20 aryl,
C.sub.2-C.sub.20 heterocyclyl, or a protecting group;
[0021] R.sup.a and R.sup.b are independently H, C.sub.1-C.sub.8
alkyl, C.sub.2-C.sub.8 alkenyl, C.sub.2-C.sub.8 alkynyl,
C.sub.6-C.sub.20 aryl, or C.sub.2-C.sub.20 heterocyclyl;
[0022] Y is independently O, S, NR.sup.20, .sup.+N(O)R.sup.20,
N(OR.sup.20), .sup.+N(O)(OR.sup.20), or N--NR.sup.20R.sup.21;
and
[0023] protecting group is selected from trialkylsilyl,
dialkylphenylsilyl, benzoate, benzyl, benzyloxymethyl, methyl,
methoxymethyl, triarylmethyl, phthalimido, t-butoxycarbonyl (BOC),
benzyloxycarbonyl (CBz), 9-fluorenylmethylenoxycarbonyl (Fmoc), and
tetrahydropyranyl.
[0024] One aspect of the invention is to provide methods of
inhibiting Raf kinase activity by contacting these enzymes with an
effective inhibitory amount of the novel inhibitors of the present
invention, or a composition containing these compounds.
[0025] Another aspect of the invention are methods of treating such
as, but not limited to, a hyperproliferative disorder (e.g.,
cancer), cardiovascular disease, neurodegenerative disease, or
inflammatory disease, , by administering to a mammal in need of
such treatment an effective amount of one or more compounds of the
invention, or a composition containing the compound and a carrier
or excipient.
[0026] Another aspect of the invention are methods of treating
cancer by administering to a mammal in need of such treatment an
effective amount of one or more compounds of the invention in
combination with one or more additional compounds having
anti-cancer properties.
[0027] This invention also provides a compound of this invention
for use in treating a disease or disorder such as, but not limited
to, a hyperproliferative disorder (e.g., cancer), cardiovascular
disease, neurodegenerative disease, or inflammatory disease.
[0028] An additional aspect of the invention is the use of a
compound of this invention in the preparation of a medicament for
the treatment or prevention of a disease or disorder such as, but
not limited to, a hyperproliferative disorder (e.g., cancer),
cardiovascular disease, neurodegenerative disease, or inflammatory
disease.
[0029] Another aspect of the invention includes articles of
manufacture, i.e. kits, comprising a pyrazolyl compound of Formulas
Ia or Ib, a container, and a package insert or label indicating a
treatment.
[0030] Another aspect of the invention includes methods of
preparing, methods of synthesis, methods of separation, and methods
of purification of the pyrazolyl compounds of Formula Ia and Ib. In
addition, the invention includes novel intermediates for preparing
the pyrazolyl compounds of Formula Ia and Ib.
[0031] Additional advantages and novel features of this invention
shall be set forth in part in the description that follows, and in
part will become apparent to those skilled in the art upon
examination of the following specification or may be learned by the
practice of the invention. The advantages of the invention may be
realized and attained by means of the instrumentalities,
combinations, compositions, and methods particularly pointed out in
the appended claims.
DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS
[0032] Reference will now be made in detail to certain embodiments
of the invention, examples of which are illustrated in the
accompanying structures and formulas. While the invention will be
described in conjunction with the enumerated embodiments, it will
be understood that they are not intended to limit the invention to
those embodiments. On the contrary, the invention is intended to
cover all alternatives, modifications, and equivalents, which may
be included within the scope of the present invention as defined by
the claims. One skilled in the art will recognize many methods and
materials similar or equivalent to those described herein, which
could be used in the practice of the present invention. The present
invention is in no way limited to the methods and materials
described. In the event that one or more of the incorporated
literature, patents, and similar materials differs from or
contradicts this application, including but not limited to defined
terms, term usage, described techniques, or the like, this
application controls.
DEFINITIONS
[0033] Unless stated otherwise, the following terms and phrases as
used herein are intended to have the following meanings:
[0034] The term "alkyl" as used herein refers to a saturated linear
or branched-chain monovalent hydrocarbon radical of one to eighteen
carbon atoms, wherein the alkyl radical may be optionally
substituted independently with one or more substituents described
below. Examples of alkyl groups include C.sub.1-C.sub.8 hydrocarbon
moieties such as methyl (Me, --CH.sub.3), ethyl (Et,
--CH.sub.2CH.sub.3), 1-propyl (n-Pr, n-propyl,
--CH.sub.2CH.sub.2CH.sub.3), 2-propyl (i-Pr, i-propyl,
--CH(CH.sub.3).sub.2), 1-butyl (n-Bu, n-butyl,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.3), 2-methyl-1-propyl (i-Bu,
i-butyl, --CH.sub.2CH(CH.sub.3).sub.2), 2-butyl (s-Bu, s-butyl,
--CH(CH.sub.3)CH.sub.2CH.sub.3), 2-methyl-2-propyl (t-Bu, t-butyl,
--C(CH.sub.3).sub.3), 1-pentyl (n-pentyl,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.3), 2-pentyl
(--CH(CH.sub.3)CH.sub.2CH.sub.2CH.sub.3), 3-pentyl
(--CH(CH.sub.2CH.sub.3).sub.2), 2-methyl-2-butyl
(--C(CH.sub.3).sub.2CH.sub.2CH.sub.3), 3-methyl-2-butyl
(--CH(CH.sub.3)CH(CH.sub.3).sub.2), 3-methyl-1-butyl
(--CH.sub.2CH.sub.2CH(CH.sub.3).sub.2), 2-methyl-1-butyl
(--CH.sub.2CH(CH.sub.3)CH.sub.2CH.sub.3), 1-hexyl
(--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.3), 2-hexyl
(--CH(CH.sub.3)CH.sub.2CH.sub.2CH.sub.2CH.sub.3), 3-hexyl
(--CH(CH.sub.2CH.sub.3)(CH.sub.2CH.sub.2CH.sub.3)),
2-methyl-2-pentyl (--C(CH.sub.3).sub.2CH.sub.2CH.sub.2CH.sub.3),
3-methyl-2-pentyl (--CH(CH.sub.3)CH(CH.sub.3)CH.sub.2CH.sub.3),
4-methyl-2-pentyl (--CH(CH.sub.3)CH.sub.2CH(CH.sub.3).sub.2),
3-methyl-3-pentyl (--C(CH.sub.3)(CH.sub.2CH.sub.3).sub.2),
2-methyl-3-pentyl (--CH(CH.sub.2CH.sub.3)CH(CH.sub.3).sub.2),
2,3-dimethyl-2-butyl (--C(CH.sub.3).sub.2CH(CH.sub.3).sub.2),
3,3-dimethyl-2-butyl (--CH(CH.sub.3)C(CH.sub.3).sub.3, 1-heptyl,
1-octyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,
cycloheptyl, and cyclooctyl. Alkyl groups are optionally
substituted independently with one or more substituents described
herein.
[0035] The term "alkenyl" refers to linear or branched-chain
monovalent hydrocarbon radical of two to eighteen carbon atoms with
at least one site of unsaturation, i.e., a carbon-carbon, sp.sup.2
double bond, wherein the alkenyl radical may be optionally
substituted independently with one or more substituents described
herein, and includes radicals having "cis" and "trans"
orientations, or alternatively, "E" and "Z" orientations. Examples
include, but are not limited to, ethylenyl or vinyl
(--CH.dbd.CH.sub.2), allyl (--CH.sub.2CH.dbd.CH.sub.2),
1-cyclopent-1-enyl, 1-cyclopent-2-enyl, 1-cyclopent-3-enyl,
5-hexenyl, 1-cyclohex-1-enyl, 1-cyclohex-2-enyl, and
1-cyclohex-3-enyl. Alkenyl groups are optionally substituted
independently with one or more substituents described herein.
[0036] The term "alkynyl" refers to a linear or branched monovalent
hydrocarbon radical of two to eighteen carbon atoms with at least
one site of unsaturation, i.e., a carbon-carbon, sp triple bond,
wherein the alkynyl radical may be optionally substituted
independently with one or more substituents described herein.
Examples include, but are not limited to, ethynyl (--C.ident.CH)
and propynyl (propargyl, --CH.sub.2C.ident.CH). Alkynyl groups are
optionally substituted independently with one or more substituents
described herein.
[0037] "Alkylene" refers to a saturated, branched or straight chain
or cyclic hydrocarbon radical of 1-18 carbon atoms, and having two
monovalent radical centers derived by the removal of two hydrogen
atoms from the same or two different carbon atoms of a parent
alkane. Typical alkylene radicals include, but are not limited to,
methylene (--CH.sub.2--) 1,2-ethyl (--CH.sub.2CH.sub.2--),
1,3-propyl (--CH.sub.2CH.sub.2CH.sub.2--), 1,4-butyl
(--CH.sub.2CH.sub.2CH.sub.2CH.sub.2--), and the like. Alkylene
groups are optionally substituted independently with one or more
substituents described herein.
[0038] "Alkenylene" refers to an unsaturated, branched or straight
chain or cyclic hydrocarbon radical of 2-18 carbon atoms, and
having two monovalent radical centers derived by the removal of two
hydrogen atoms from the same or two different carbon atoms of a
parent alkene. Typical alkenylene radicals include, but are not
limited to, 1,2-ethylene (--CH.dbd.CH--). Alkenylene groups are
optionally substituted independently with one or more substituents
described herein.
[0039] "Alkynylene" refers to an unsaturated, branched or straight
chain or cyclic hydrocarbon radical of 2-18 carbon atoms, and
having two monovalent radical centers derived by the removal of two
hydrogen atoms from the same or two different carbon atoms of a
parent alkyne. Typical alkynylene radicals include, but are not
limited to, acetylene (--C.ident.C--), propargyl
(--CH.sub.2C.ident.C--), and 4-pentynyl
(--CH.sub.2CH.sub.2CH.sub.2C.ident.C--). Alkynylene groups are
optionally substituted independently with one or more substituents
described herein.
[0040] "Aryl" means a monovalent aromatic hydrocarbon radical of
6-20 carbon atoms derived by the removal of one hydrogen atom from
a single carbon atom of a parent aromatic ring system. Some aryl
groups are represented in the exemplary structures as "Ar". Aryl
includes a bicyclic radical comprising an aromatic ring with a
fused non-aromatic or partially saturated ring. Typical aryl groups
include, but are not limited to, radicals derived from benzene,
substituted benzene, naphthalene, anthracene, biphenyl, indenyl,
indanyl, 1,2-dihydronapthalene, 1,2,3,4-tetrahydronapthyl, and the
like. Aryl groups are optionally substituted independently with one
or more substituents described herein.
[0041] The terms "heterocycle," "heterocyclyl", "heterocyclic ring"
and "heteroaryl" refer to a saturated, a partially unsaturated
(i.e., having one or more double and/or triple bonds within the
ring), or aromatic carbocyclic radical of 3 to 20 ring atoms in
which at least one ring atom is a heteroatom independently selected
from nitrogen, oxygen and sulfur, the remaining ring atoms being
carbon, where one or more ring atoms is optionally substituted
independently with one or more substituents described below. A
heterocycle may be a monocycle having 3 to 7 ring members (2 to 6
carbon atoms and 1 to 3 heteroatoms selected from N, O, P, and S)
or a bicycle having 7 to 10 ring members (4 to 9 carbon atoms and 1
to 3 heteroatoms selected from N, O, P, and S), for example: a
bicyclo[4,5], [5,5], [5,6], or [6,6] system. Heterocycles are
described in Paquette, Leo A.; "Principles of Modern Heterocyclic
Chemistry" (W. A. Benjamin, New York, 1968), particularly Chapters
1, 3, 4, 6, 7, and 9; "The Chemistry of Heterocyclic Compounds, A
series of Monographs" (John Wiley & Sons, New York, 1950 to
present), in particular Volumes 13, 14, 16, 19, and 28; and J. Am.
Chem. Soc. (1960) 82:5566. The heterocyclyl may be a carbon-linked
radical or heteroatom-linked radical. The term "heterocycle"
includes heterocycloalkoxy. "Heterocyclyl" also includes radicals
where heterocycle radicals are fused with a carbocyclic,
heterocyclic, aromatic or heteroaromatic ring. Examples of
heterocyclic radicals include, but are not limited to,
pyrrolidinyl, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothienyl,
tetrahydropyranyl, dihydropyranyl, tetrahydrothiopyranyl,
piperidino, morpholino, thiomorpholino, thioxanyl, piperazinyl,
homopiperazinyl, azetidinyl, oxetanyl, thietanyl, homopiperidinyl,
oxepanyl, thiepanyl, oxazepinyl, diazepinyl, thiazepinyl,
2-pyrrolinyl, 3-pyrrolinyl, indolinyl, 2H-pyranyl, 4H-pyranyl,
dioxanyl, 1,3-dioxolanyl, pyrazolinyl, dithianyl, dithiolanyl,
dihydropyranyl, dihydrothienyl, dihydrofuranyl,
pyrazolidinylimidazolinyl, imidazolidinyl,
3-azabicyco[3.1.0]hexanyl, 3-azabicyclo[4.1.0]heptanyl,
azabicyclo[2.2.2]hexanyl, 3H-indolyl quinolizinyl and N-pyridyl
ureas. Spiro moieties are also included within the scope of this
definition. Examples of a heterocyclic group wherein 2 ring carbon
atoms are substituted with oxo (.dbd.O) moieties are pyrimidinonyl
and 1,1-dioxo-thiomorpholinyl. The heterocycle groups herein are
optionally substituted independently with one or more substituents
described herein.
[0042] The term "heteroaryl" includes 1) monocyclic aromatic 5-,
6-, and 7-membered rings containing one or more heteroatoms
independently selected from nitrogen, oxygen, and sulfur, and 2)
fused ring systems of 8 to 20 atoms wherein at least one aromatic
ring contains one or more heteroatoms independently selected from
nitrogen, oxygen, and sulfur. Examples of heteroaryl groups are
pyridinyl (including, for example, 2-hydroxypyridinyl), imidazolyl,
imidazopyridinyl, pyrimidinyl (including, for example,
4-hydroxypyrimidinyl), pyrazolyl, triazolyl, pyrazinyl, tetrazolyl,
furyl, thienyl, isoxazolyl, thiazolyl, oxazolyl, isothiazolyl,
pyrrolyl, quinolinyl, isoquinolinyl, indolyl, benzimidazolyl,
benzofuranyl, cinnolinyl, indazolyl, indolizinyl, phthalazinyl,
pyridazinyl, triazinyl, isoindolyl, pteridinyl, purinyl,
oxadiazolyl, triazolyl, thiadiazolyl, thiadiazolyl, furazanyl,
benzofurazanyl, benzothiophenyl, benzothiazolyl, benzoxazolyl,
quinazolinyl, quinoxalinyl, naphthyridinyl, and furopyridinyl.
Heteroaryl groups are optionally substituted independently with one
or more substituents described herein.
[0043] The heterocycle may be C-attached or N-attached where such
is possible. By way of example and not limitation, carbon bonded
heterocycles are bonded at position 2, 3, 4, 5, or 6 of a pyridine,
position 3, 4, 5, or 6 of a pyridazine, position 2, 4, 5, or 6 of a
pyrimidine, position 2, 3, 5, or 6 of a pyrazine, position 2, 3, 4,
or 5 of a furan, tetrahydrofuran, thiofuran, thiophene, pyrrole or
tetrahydropyrrole, position 2, 4, or 5 of an oxazole, imidazole or
thiazole, position 3, 4, or 5 of an isoxazole, pyrazole, or
isothiazole, position 2 or 3 of an aziridine, position 2, 3, or 4
of an azetidine, position 2, 3, 4, 5, 6, 7, or 8 of a quinoline or
position 1, 3, 4, 5, 6, 7, or 8 of an isoquinoline. (2-pyridyl,
3-pyridyl, 4-pyridyl, 5-pyridyl, 6-pyridyl).
[0044] By way of example and not limitation, nitrogen bonded
heterocycles are bonded at position 1 of an aziridine, azetidine,
pyrrole, pyrrolidine, 2-pyrroline, 3-pyrroline, imidazole,
imidazolidine, 2-imidazoline, 3-imidazoline, pyrazole, pyrazoline,
2-pyrazoline, 3-pyrazoline, piperidine, piperazine, indole,
indoline, 1H-indazole, position 2 of a isoindole, or isoindoline,
position 4 of a morpholine, and position 9 of a carbazole, or
.beta.-carboline.
[0045] "Carbocycle", "carbocyclyl" and "cycloalkyl" mean a
non-aromatic, saturated or unsaturated ring having 3 to 12 carbon
atoms as a monocycle or 7 to 12 carbon atoms as a bicycle.
Monocyclic carbocycles have 3 to 6 ring atoms, still more typically
5 or 6 ring atoms. Bicyclic carbocycles have 7 to 12 ring atoms,
e.g. arranged as a bicyclo[4,5], [5,5], [5,6] or [6,6] system, or 9
or 10 ring atoms arranged as a bicyclo[5,6] or [6,6] system, or as
bridged systems such as bicyclo[2.2.1]heptane,
bicyclo[2.2.2]octane, and bicyclo[3.2.2]nonane. Examples of
monocyclic carbocycles include cyclopropyl, cyclobutyl,
cyclopentyl, 1-cyclopent-1-enyl, 1-cyclopent-2-enyl,
1-cyclopent-3-enyl, cyclohexyl, 1-cyclohex-1-enyl,
1-cyclohex-2-enyl, 1-cyclohex-3-enyl, cyclohexadienyl, cycloheptyl,
cyclooctyl, cyclononyl, cyclodecyl, cycloundecyl, and
cyclododecyl.
[0046] "Substituted alkyl", "substituted aryl", "substituted
heterocyclyl", and "substituted carbocyclyl" mean alkyl, aryl,
heterocyclyl and carbocyclyl respectively, in which one or more
hydrogen atoms are each independently replaced with a substituent.
Typical substituents include, but are not limited to, X, R,
O.sup.-, --OR, --SR, --NR.sub.2, --NR.sub.3, .dbd.NR, .dbd.N--OR,
.dbd.O, --CX.sub.3, --CN, --OCN, --SCN, --N.dbd.C.dbd.O, --NCS,
--NO, --NO.sub.2, .dbd.N.sub.2, --N.sub.3, NC(.dbd.O)R,
--C(.dbd.O)R, --C(.dbd.O)NR.sub.2, --SO.sub.3.sup.-, --SO.sub.3H,
--S(.dbd.O).sub.2R, --OS(.dbd.O).sub.2OR, --S(.dbd.O).sub.2NR,
--S(.dbd.O)R, --OP(.dbd.O)(OR).sub.2, --P(.dbd.O)(OR).sub.2,
--PO.sub.3, --PO.sub.3H.sub.2, --C(.dbd.O)R, --C(.dbd.O)X,
--C(.dbd.S)R, --CO.sub.2R, --CO.sub.2--, --C(.dbd.S)OR,
--C(.dbd.O)SR, --C(.dbd.S)SR, --C(.dbd.O)NR.sub.2,
--C(.dbd.S)NR.sub.2, and --C(.dbd.NR)NR.sub.2, where each X is
independently a halogen (F, Cl, Br, or I), and each R is
independently H, C.sub.1-C.sub.18 alkyl, C.sub.6-C.sub.20 aryl,
C.sub.3-C.sub.14 heterocycle, protecting group or prodrug moiety.
Alkylene, alkenylene, and alkynylene groups as described above may
also be similarly substituted.
[0047] The terms "treat" or "treatment" refer to both therapeutic
treatment and prophylactic or preventative measures, wherein the
object is to prevent or slow down (lessen) an undesired
physiological change or disorder, such as the development or spread
of cancer. For purposes of this invention, beneficial or desired
clinical results include, but are not limited to, alleviation of
symptoms, diminishment of extent of disease, stabilized (i.e., not
worsening) state of disease, delay or slowing of disease
progression, amelioration or palliation of the disease state, and
remission (whether partial or total), whether detectable or
undetectable. "Treatment" can also mean prolonging survival as
compared to expected survival if not receiving treatment. Those in
need of treatment include those already with the condition or
disorder as well as those prone to have the condition or disorder
or those in which the condition or disorder is to be prevented.
[0048] The phrase "therapeutically effective amount" means an
amount of a compound of the present invention that (i) treats or
prevents the particular disease, condition, or disorder, (ii)
attenuates, ameliorates, or eliminates one or more symptoms of the
particular disease, condition, or disorder, or (iii) prevents or
delays the onset of one or more symptoms of the particular disease,
condition, or disorder described herein. In the case of cancer, the
therapeutically effective amount of the drug may reduce the number
of cancer cells; reduce the tumor size; inhibit (i.e., slow to some
extent and preferably stop) cancer cell infiltration into
peripheral organs; inhibit (i.e., slow to some extent and
preferably stop) tumor metastasis; inhibit, to some extent, tumor
growth; and/or relieve to some extent one or more of the symptoms
associated with the cancer. To the extent the drug may prevent
growth and/or kill existing cancer cells, it may be cytostatic
and/or cytotoxic. For cancer therapy, efficacy can, for example, be
measured by assessing the time to disease progression (TTP) and/or
determining the response rate (RR).
[0049] The term "bioavailability" refers to the systemic
availability (i.e., blood/plasma levels) of a given amount of drug
administered to a patient. Bioavailability is an absolute term that
indicates measurement of both the time (rate) and total amount
(extent) of drug that reaches the general circulation from an
administered dosage form.
[0050] The terms "cancer" and "cancerous" refer to or describe the
physiological condition in mammals that is typically characterized
by unregulated cell growth. A "tumor" comprises one or more
cancerous cells. Examples of cancer include, but are not limited
to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or
lymphoid malignancies. More particular examples of such cancers
include squamous cell cancer (e.g., epithelial squamous cell
cancer), lung cancer including small-cell lung cancer, non-small
cell lung cancer ("NSCLC"), adenocarcinoma of the lung and squamous
carcinoma of the lung, cancer of the peritoneum, hepatocellular
cancer, gastric or stomach cancer including gastrointestinal
cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian
cancer, liver cancer, bladder cancer, hepatoma, breast cancer,
colon cancer, rectal cancer, colorectal cancer, endometrial or
uterine carcinoma, salivary gland carcinoma, kidney or renal
cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic
carcinoma, anal carcinoma, penile carcinoma, as well as head and
neck cancer.
[0051] The term "prodrug" as used in this application refers to a
precursor or derivative form of a pharmaceutically active substance
that is less cytotoxic to tumor cells compared to the parent drug
and is capable of being enzymatically or hydrolytically activated
or converted into the more active parent form. See, e.g., Wilman,
"Prodrugs in Cancer Chemotherapy" Biochemical Society Transactions,
14, pp. 375-382, 615th Meeting Belfast (1986) and Stella et al.,
"Prodrugs: A Chemical Approach to Targeted Drug Delivery," Directed
Drug Delivery, Borchardt et al., (ed.), pp. 247-267, Humana Press
(1985). The prodrugs of this invention include, but are not limited
to, phosphate-containing prodrugs, thiophosphate-containing
prodrugs, sulfate-containing prodrugs, peptide-containing prodrugs,
D-amino acid-modified prodrugs, glycosylated prodrugs,
.beta.-lactam-containing prodrugs, optionally substituted
phenoxyacetamide-containing prodrugs or optionally substituted
phenylacetamide-containing prodrugs, 5-fluorocytosine and other
5-fluorouridine prodrugs which can be converted into the more
active cytotoxic free drug. Examples of cytotoxic drugs that can be
derivatized into a prodrug form for use in this invention include,
but are not limited to, those chemotherapeutic agents described
above.
[0052] A "liposome" is a small vesicle composed of various types of
lipids, phospholipids and/or surfactant which is useful for
delivery of a drug (such as the Raf inhibitors disclosed herein
and, optionally, a chemotherapeutic agent) to a mammal. The
components of the liposome are commonly arranged in a bilayer
formation, similar to the lipid arrangement of biological
membranes.
[0053] The term "package insert" is used to refer to instructions
customarily included in commercial packages of therapeutic
products, that contain information about the indications, usage,
dosage, administration, contraindications and/or warnings
concerning the use of such therapeutic products.
[0054] The term "chiral" refers to molecules which have the
property of non-superimposability of the mirror image partner,
while the term "achiral" refers to molecules which are
superimposable on their mirror image partner.
[0055] The term "stereoisomers" refers to compounds which have
identical chemical constitution, but differ with regard to the
arrangement of the atoms or groups in space.
[0056] "Diastereomer" refers to a stereoisomer with two or more
centers of chirality and whose molecules are not mirror images of
one another. Diastereomers have different physical properties, e.g.
melting points, boiling points, spectral properties, and
reactivities. Mixtures of diastereomers may separate under high
resolution analytical procedures such as electrophoresis and
chromatography.
[0057] "Enantiomers" refer to two stereoisomers of a compound which
are non-superimposable mirror images of one another.
[0058] Stereochemical definitions and conventions used herein
generally follow S. P. Parker, Ed., McGraw-Hill Dictionary of
Chemical Terms (1984) McGraw-Hill Book Company, New York; and
Eliel, E. and Wilen, S., "Stereochemistry of Organic Compounds",
John Wiley & Sons, Inc., New York, 1994. Many organic compounds
exist in optically active forms, i.e., they have the ability to
rotate the plane of plane-polarized light. In describing an
optically active compound, the prefixes D and L, or R and S, are
used to denote the absolute configuration of the molecule about its
chiral center(s). The prefixes d and l or (+) and (-) are employed
to designate the sign of rotation of plane-polarized light by the
compound, with (-) or d meaning that the compound is levorotatory.
A compound prefixed with (+) or d is dextrorotatory. For a given
chemical structure, these stereoisomers are identical except that
they are mirror images of one another. A specific stereoisomer may
also be referred to as an enantiomer, and a mixture of such isomers
is often called an enantiomeric mixture. A 50:50 mixture of
enantiomers is referred to as a racemic mixture or a racemate,
which may occur where there has been no stereoselection or
stereospecificity in a chemical reaction or process. The terms
"racemic mixture" and "racemate" refer to an equimolar mixture of
two enantiomeric species, devoid of optical activity.
[0059] The phrase "pharmaceutically acceptable salt," as used
herein, refers to pharmaceutically acceptable organic or inorganic
salts of a compound of the invention. Exemplary salts include, but
are not limited, to sulfate, citrate, acetate, oxalate, chloride,
bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate,
isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate,
tannate, pantothenate, bitartrate, ascorbate, succinate, maleate,
gentisinate, fumarate, gluconate, glucuronate, saccharate, formate,
benzoate, glutamate, methanesulfonate, ethanesulfonate,
benzenesulfonate, p-toluenesulfonate, and pamoate (i.e.,
1,1'-methylene-bis -(2-hydroxy-3-naphthoate)) salts. A
pharmaceutically acceptable salt may involve the inclusion of
another molecule such as an acetate ion, a succinate ion or other
counter ion. The counter ion may be any organic or inorganic moiety
that stabilizes the charge on the parent compound. Furthermore, a
pharmaceutically acceptable salt may have more than one charged
atom in its structure. Instances where multiple charged atoms are
part of the pharmaceutically acceptable salt can have multiple
counter ions. Hence, a pharmaceutically acceptable salt can have
one or more charged atoms and/or one or more counter ion.
[0060] A "solvate" refers to an association or complex of one or
more solvent molecules and a compound of the invention. Examples of
solvents that form solvates include, but are not limited to, water,
isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid,
and ethanolamine. The term "hydrate" refers to the complex where
the solvent molecule is water.
[0061] The term "protecting group" or "Pg" refers to a substituent
that is commonly employed to block or protect a particular
functionality while reacting other functional groups on the
compound. For example, an "amino-protecting group" is a substituent
attached to an amino group that blocks or protects the amino
functionality in the compound. Suitable amino-protecting groups
include acetyl, trifluoroacetyl, phthalimido, t-butoxycarbonyl
(BOC), benzyloxycarbonyl (CBz) and 9-fluorenylmethylenoxycarbonyl
(Fmoc). Similarly, a "hydroxy-protecting group" refers to a
substituent of a hydroxy group that blocks or protects the hydroxy
functionality. Suitable hydroxy-protecting groups include acetyl,
trialkylsilyl, dialkylphenylsilyl, benzoyl, benzyl,
benzyloxymethyl, methyl, methoxymethyl, triarylmethyl, and
tetrahydropyranyl. A "carboxy-protecting group" refers to a
substituent of the carboxy group that blocks or protects the
carboxy functionality. Common carboxy-protecting groups include
--CH.sub.2CH.sub.2SO.sub.2Ph, cyanoethyl, 2-(trimethylsilyl)ethyl,
2-(trimethylsilyl)ethoxymethyl, 2-(p-toluenesulfonyl)ethyl,
2-(p-nitrophenylsulfenyl)ethyl, 2-(diphenylphosphino)-ethyl,
nitroethyl and the like. For a general description of protecting
groups and their use, see T. W. Greene and P. Wuts, Protective
Groups in Organic Synthesis, Third Ed., John Wiley & Sons, New
York, 1999; and P. Kocienski, Protecting Groups, Third Ed., Verlag,
2003.
[0062] The term "animal" refers to humans (male or female),
companion animals (e.g., dogs, cats and horses), food-source
animals, zoo animals, marine animals, birds and other similar
animal species.
[0063] The phrase "pharmaceutically acceptable" indicates that the
substance or composition must be compatible chemically and/or
toxicologically, with the other ingredients comprising a
formulation, and/or the mammal being treated therewith.
Pyrazolyl Raf Inhibitor Compounds
[0064] The present invention provides compounds having Formulas Ia
and Ib, and pharmaceutical formulations thereof, that are
potentially useful in the treatment of diseases, conditions and/or
disorders modulated by Raf kinases. Formula Ia and Ib compounds
include:
##STR00003##
[0065] and stereoisomers, tautomers, solvates and pharmaceutically
acceptable salts thereof, wherein:
[0066] the A-ring is: (i) a 5 or 6 membered heterocyclic ring
having one or two heteroatoms independently selected from O, N, and
S, (ii) a 5 or 6 membered carbocyclic ring optionally fused to a 5
or 6 membered heterocycle, or (iii) a phenyl ring, wherein said
heterocyclic, carbocyclic and phenyl rings are optionally
substituted with one or more groups independently selected from F,
Cl, Br, I, --C(.dbd.Y)R.sup.20, --C(.dbd.Y)OR.sup.20,
--C(.dbd.Y)NR.sup.20R.sup.21, NR.sup.20R.sup.21,
--NR.sup.20C(.dbd.Y)R.sup.21, --NR.sup.20C(.dbd.Y)OR.sup.21,
--NR.sup.23C(.dbd.Y)NR.sup.20R.sup.21, --NOR.sup.20,
.dbd.NR.sup.20, .dbd.N+O)OR.sup.20, .dbd.NNR.sup.20R.sup.21,
.dbd.O, --OR.sup.20, --OC(.dbd.Y)R.sup.20, --OC(.dbd.Y).sup.20,
--C(.dbd.Y)NR.sup.20R.sup.21, --OS(O).sub.2(OR.sup.20),
--OP(.dbd.Y)(OR.sup.20)(OR.sup.21), --OP(OR.sup.20)(OR.sup.21),
--P(.dbd.Y)(OR.sup.20)(OR.sup.21),
--P(.dbd.Y)(OR)NR.sup.20R.sup.21, .dbd.S, --SR.sup.20,
--S(O)R.sup.20, --S(O).sub.2R.sup.20,
--S(O).sub.2NR.sup.20R.sup.21, --S(O)(OR.sup.20),
--S(O).sub.2(OR.sup.20), --SC(.dbd.Y)R.sup.21,
--SC(.dbd.Y)OR.sup.2, --SC(.dbd.Y)NR.sup.20R.sup.21,
C.sub.1-C.sub.8 alkyl, C.sub.2-C.sub.8 alkenyl, C.sub.2-C.sub.8
alkynyl, C.sub.6-C.sub.20 aryl, C.sub.3-C.sub.12 carbocyclyl,
C.sub.2-C.sub.20 heterocyclyl, and a protecting group, and wherein
said alkyl, alkenyl, alkynyl, aryl, carbocyclyl and heterocyclyl
are optionally and independently substituted with one or more
groups independently selected from F, Cl, Br, I,
--C(.dbd.Y)R.sup.20, --C(.dbd.Y)OR.sup.20,
--C(.dbd.Y)NR.sup.20R.sup.21, NR.sup.20R.sup.21,
--NR.sup.20C(.dbd.Y)R.sup.21, --NR.sup.20C(.dbd.Y)OR.sup.21,
--NR.sup.23C(.dbd.Y)NR.sup.20R.sup.21, --OR.sup.20,
--OC(.dbd.Y)R.sup.20, --OC(.dbd.Y)OR.sup.20,
--OC(.dbd.Y)NR.sup.20R.sup.21, --OS(O).sub.2(OR.sup.20),
--OP(.dbd.Y)(OR.sup.20)(OR.sup.21), --OP(OR.sup.20)(OR.sup.21),
--P(.dbd.Y)(OR.sup.20)(OR.sup.21),
--P(.dbd.Y)(OR.sup.20)NR.sup.20R.sup.21, --SR.sup.20,
--S(O)R.sup.20, --S(O).sub.2R.sup.20,
--S(O).sub.2NR.sup.20R.sup.21, --S(O)(OR.sup.20),
--S(O).sub.2(OR.sup.20), --SC(.dbd.Y)R.sup.20,
--SC(.dbd.Y)OR.sup.20, --SC(.dbd.Y)NR.sup.20R.sup.21,
C.sub.1-C.sub.8 alkyl, C.sub.2-C.sub.8 alkenyl, C.sub.2-C.sub.8
alkynyl, C.sub.6-C.sub.20 aryl, C.sub.3-C.sub.12 carbocyclyl and
C.sub.2-C.sub.20 heterocyclyl;
[0067] X is selected from a C.sub.2-C.sub.20 heterocyclyl, a
C.sub.3-C.sub.12 carbocyclyl, and a C.sub.6-C.sub.20 aryl, wherein
said heterocycle, carbocyclyl and aryl are optionally substituted
with one or more groups independently selected from F, Cl, Br, I,
--C(.dbd.Y)R.sup.20, --C(.dbd.Y)OR.sup.20,
--C(.dbd.Y)NR.sup.20R.sup.21, --NR.sup.20R.sup.21,
--NR.sup.20C(.dbd.Y)R.sup.21, --NR.sup.20C(.dbd.Y)OR.sup.21,
--NR.sup.23C(.dbd.Y)NR.sup.20R.sup.21, --OR.sup.20,
--OC(.dbd.Y)R.sup.20, --OC(.dbd.Y)OR.sup.20,
--OC(.dbd.Y)NR.sup.20R.sup.21, --OS(O).sub.2(OR.sup.20),
--OP(.dbd.Y)(OR.sup.20)(OR.sup.21), --OP(OR.sup.20)(OR.sup.21),
--P(.dbd.Y)(OR.sup.20)(OR.sup.21),
--P(.dbd.Y)(OR.sup.23)NR.sup.20R.sup.21, --SR.sup.20,
--S(O)R.sup.20, --S(O).sub.2R.sup.20,
--S(O).sub.2NR.sup.20R.sup.21, --S(O)(OR.sup.20),
--S(O).sub.2(OR.sup.20), --SC(.dbd.Y)R.sup.20,
--SC(.dbd.Y)OR.sup.20, --SC(.dbd.Y)NR.sup.20R.sup.21, 5-7 membered
ring lactam, 5-7 membered ring lactone, 5-7 membered ring sultam,
C.sub.1-C.sub.8 alkyl, C.sub.2-C.sub.8 alkenyl, C.sub.2-C.sub.8
alkynyl, C.sub.3-C.sub.12 carbocyclyl, C.sub.6-C.sub.20 aryl, and
C.sub.2-C.sub.20 heterocyclyl, wherein said alkyl, alkenyl,
alkynyl, carbocyclyl, aryl, and heterocyclyl are optionally
substituted with one or more groups independently selected from F,
Cl, Br, I, OR.sup.20, NR.sup.20R.sup.21--SR.sup.20, --S(O)R.sup.20,
--S(O).sub.2R.sup.20, alkyl, alkenyl, alkynyl, carbocyclyl, aryl,
and heterocyclyl;
[0068] R.sup.1 is selected from H, C.sub.1-C.sub.8 alkyl,
C.sub.2-C.sub.8 alkenyl, C.sub.2-C.sub.8 alkynyl,
(C.sub.1-C.sub.8-alkyl)NR.sup.20R.sup.21, C.sub.2-C.sub.20
heterocyclyl, C.sub.3-C.sub.12 carbocyclyl, and C.sub.6-C.sub.20
aryl, wherein said alkyl, alkenyl, alkynyl, heterocycle,
carbocyclyl and aryl are optionally substituted with one or more
groups independently selected from F, Cl, Br, I,
--C(.dbd.Y)R.sup.20, --C(.dbd.Y)OR.sup.20,
--C(.dbd.Y)NR.sup.20R.sup.21, --NR.sup.20R.sup.21, OR.sup.20, CN,
C(.dbd.O)NR.sup.20R.sup.21, C(.dbd.O)OR.sup.20, alkyl,
(C.sub.1-C.sub.8-alkyl)NR.sup.20R.sup.21, and heterocyclyl;
[0069] R.sup.2 is selected from H, F, Cl, Br, I,
--C(.dbd.Y)R.sup.20, --C(.dbd.Y)OR.sup.20,
--C(.dbd.Y)NR.sup.20R.sup.21, --OR.sup.20, --OC(.dbd.Y)R.sup.20,
--OC(.dbd.Y)OR.sup.20, --OC(.dbd.Y)NR.sup.20R.sup.21,
--OS(O).sub.2(OR.sup.20), --OP(.dbd.Y)(OR.sup.20)(OR.sup.21),
--OP(OR.sup.20)(OR.sup.21), --P(.dbd.Y)(OR.sup.20)(OR.sup.21),
--P(.dbd.Y)(OR)NR.sup.20R.sup.21, --SR.sup.20, --S(O)R.sup.20,
--S(O).sub.2R.sup.20, --S(O).sub.2NR.sup.20R.sup.21,
--S(O)(OR.sup.20), --S(O).sub.2(OR.sup.20), --SC(.dbd.Y)R.sup.20,
--SC(.dbd.Y)OR.sup.20, --SC(.dbd.Y)NR.sup.20R.sup.21, 5-7 membered
ring lactam, 5-7 membered ring lactone, 5-7 membered ring sultam,
C.sub.1-C.sub.8 alkyl, C.sub.2-C.sub.8 alkenyl, C.sub.2-C.sub.8
alkynyl, C.sub.3-C.sub.12 carbocyclyl, C.sub.6-C.sub.20 aryl, and
C.sub.2-C.sub.20 heterocyclyl, wherein said alkyl, alkenyl,
alkynyl, carbocyclyl, aryl, and heterocyclyl are optionally
substituted with one or more groups independently selected from F,
Cl, Br, I, OR.sup.20, NR.sup.20R.sup.21--SR.sup.20, --S(O)R.sup.20,
--S(O).sub.2R.sup.20, alkyl, alkenyl, alkynyl, carbocyclyl, aryl,
and heterocyclyl,
[0070] or R.sup.1 and R.sup.2 of Formula Ia together with the atoms
to which they are attached optionally form a saturated, partially
unsaturated or aromatic 5 or 6 membered fused heterocycle ring
having at least two heteroatoms independently selected from O, N
and S, wherein said heterocycle ring is optionally substituted with
one or more groups independently selected from F, Cl, Br, I,
--C(.dbd.Y)R.sup.20, --C(.dbd.Y)OR.sup.20,
--C(.dbd.Y)NR.sup.20R.sup.21, --NR.sup.20R.sup.21,
--NR.sup.20C(.dbd.Y)R.sup.21, --NR.sup.20C(.dbd.Y)OR.sup.21,
--NR.sup.20C(.dbd.Y)NR.sup.20R.sup.21, --OR.sup.20,
--OC(.dbd.Y)R.sup.20, --OC(.dbd.Y)OR.sup.20,
--OC(.dbd.Y)NR.sup.20R.sup.21, --OS(O).sub.2(OR.sup.20),
--OP(.dbd.Y)(OR.sup.20)(OR.sup.21), --OP(OR.sup.20)(OR.sup.21),
--P(.dbd.Y)(OR.sup.20)(OR.sup.21),
--P(.dbd.Y)(OR.sup.20)NR.sup.20R.sup.21, --SR.sup.20,
--S(O)R.sup.20, --S(O).sub.2R.sup.20,
--S(O).sub.2NR.sup.20R.sup.21, --S(O)(OR.sup.20),
--S(O).sub.2(OR.sup.20), --SC(.dbd.Y)R.sup.20,
--SC(.dbd.Y)OR.sup.20, --SC(.dbd.Y)NR.sup.20R.sup.21, 5-7 membered
ring lactam, 5-7 membered ring lactone, 5-7 membered ring sultam,
C.sub.1-C.sub.8 alkyl, C.sub.2-C.sub.8 alkenyl, C.sub.2-C.sub.8
alkynyl, C.sub.3-C.sub.12 carbocyclyl, C.sub.6-C.sub.20 aryl, and
C.sub.2-C.sub.20 heterocyclyl, wherein said alkyl, alkenyl,
alkynyl, carbocyclyl, aryl, and heterocyclyl are optionally
substituted with one or more groups independently selected from F,
Cl, Br, I, OR.sup.20, NR.sup.20R.sup.21--SR.sup.20, --S(O)R.sup.20,
--S(O).sub.2R.sup.20, alkyl, alkenyl, alkynyl, carbocyclyl, aryl,
and heterocyclyl;
[0071] R.sup.3, R.sup.4, and R.sup.5 are independently selected
from H, F, Cl, Br, I, --C(.dbd.Y)R.sup.20, --C(.dbd.Y)OR.sup.20,
--C(.dbd.Y)NR.sup.20R.sup.21, --NR.sup.20R.sup.21,
--NR.sup.20C(.dbd.Y)R.sup.21, --NR.sup.20C(.dbd.Y)OR.sup.21,
NR.sup.23C(.dbd.Y)NR.sup.20R.sup.21, --OR.sup.20,
--OC(.dbd.Y)R.sup.20, --OC(.dbd.Y)OR.sup.20,
--OC(.dbd.Y)NR.sup.20R.sup.21, --OS(O).sub.2(OR.sup.20),
--OP(.dbd.Y)(OR.sup.20)(OR.sup.21), --OP(OR.sup.20)(OR.sup.21),
--P(.dbd.Y)(OR.sup.20)(OR.sup.21),
--P(.dbd.Y)(OR.sup.23)NR.sup.20R.sup.21, --SR.sup.20,
--S(O)R.sup.20, --S(O).sub.2NR.sup.20R.sup.21,
--S(O)NR.sup.20R.sup.21, --S(O)(OR.sup.20),
--S(O).sub.2(OR.sup.20), --SC(.dbd.Y)R.sup.20,
--SC(.dbd.Y)OR.sup.20, --SC(.dbd.Y)NR.sup.20R.sup.21, 5-7 membered
ring lactam, 5-7 membered ring lactone, 5-7 membered ring sultam,
C.sub.1-C.sub.8 alkyl, C.sub.2-C.sub.8 alkenyl, C.sub.2-C.sub.8
alkynyl, C.sub.3-C.sub.12 carbocyclyl, C.sub.6-C.sub.20 aryl, and
C.sub.2-C.sub.20 heterocyclyl, wherein said alkyl, alkenyl,
alkynyl, carbocyclyl, aryl, and heterocyclyl are optionally
substituted with one or more groups independently selected from F,
Cl, Br, I, OR.sup.20, NR.sup.20R.sup.21--SR.sup.20, --S(O)R.sup.20,
--S(O).sub.2R.sup.20, alkyl, alkenyl, alkynyl, carbocyclyl, aryl,
and heterocyclyl;
[0072] R.sup.20 and R.sup.21 are independently selected from H,
C.sub.1-C.sub.8 alkyl, C.sub.2-C.sub.8 alkenyl, C.sub.2-C.sub.8
alkynyl, C.sub.6-C.sub.20 aryl, C.sub.2-C.sub.20 heterocyclyl, and
a protecting group, wherein said alkyl, alkenyl, alkynyl, aryl, and
heterocyclyl are optionally and independently substituted with one
or more groups independently selected from F, Cl, Br, I,
--C(.dbd.Y)R.sup.a, --C(.dbd.Y)OR.sup.a,
--C(.dbd.Y)NR.sup.aR.sup.b, --OR.sup.a, --OC(.dbd.Y)R.sup.a,
--OC(.dbd.Y)OR.sup.a, --OC(.dbd.Y)NR.sup.aR.sup.e,
--OS(O).sub.2(OR.sup.a), --OP(.dbd.Y)(OR.sup.a)(OR.sup.b),
--OP(OR.sup.a)(OR.sup.b), --P(.dbd.Y)(OR.sup.a)(OR.sup.b),
--P(.dbd.Y)(OR)NR.sup.aR.sup.b, --SR.sup.a, --S(O)R.sup.a,
--S(O).sub.2R.sup.a, --S(O).sub.2NR.sup.aR.sup.b, --S(O)(OR.sup.a),
--S(O).sub.2(OR.sup.a), --SC(.dbd.Y)R.sup.a, --SC(.dbd.Y)OR.sup.a,
and --SC(.dbd.Y)NR.sup.aR.sup.b,
[0073] or R.sup.20 and R.sup.21 together with the atoms to which
they are attached form a saturated or partially unsaturated
heterocyclic ring, wherein said heterocyclic ring is optionally
substituted with one or more groups independently selected from F,
Cl, Br, I, alkyl, alkenyl and alkynyl;
[0074] R.sup.23 is H, C.sub.1-C.sub.8 alkyl, C.sub.2-C.sub.8
alkenyl, C.sub.2-C.sub.8 alkynyl, C.sub.6-C.sub.20 aryl,
C.sub.2-C.sub.20 heterocyclyl, or a protecting group;
[0075] R.sup.a and R.sup.b are independently H, C.sub.1-C.sub.8
alkyl, C.sub.2-C.sub.8 alkenyl, C.sub.2-C.sub.8 alkynyl,
C.sub.6-C.sub.20 aryl, or C.sub.2-C.sub.20 heterocyclyl;
[0076] Y is independently O, S, NR.sup.20, .sup.+N(O)R.sup.20,
.sup.+N(OR.sup.20), .sup.+N(O)(OR.sup.20), or N--NR.sup.20R.sup.21;
and
[0077] protecting group is selected from trialkylsilyl,
dialkylphenylsilyl, benzoate, benzyl, benzyloxymethyl, methyl,
methoxymethyl, triarylmethyl, phthalimido, t-butoxycarbonyl (BOC),
benzyloxycarbonyl (CBz), 9-fluorenylmethylenoxycarbonyl (Fmoc), and
tetrahydropyranyl.
[0078] In another embodiment, Formula Ia and Ib compounds include
where:
[0079] A forms: (i) a 5 or 6 membered fused heterocyclic ring
having one or two heteroatoms independently selected from O, N, and
SI (ii) a 5 or 6 membered carbocyclic ring, or (iii) a phenyl
ring;
[0080] X is selected from a C.sub.2-C.sub.20 heterocycle, a
C.sub.3-C.sub.12 carbocycle, and a C.sub.6-C.sub.20 aryl;
[0081] R.sup.1 is selected from H, C.sub.1-C.sub.8 alkyl,
C.sub.1-C.sub.8 alkenyl, C.sub.1-C.sub.8 alkynyl, a
C.sub.2-C.sub.20 heterocycle, a C.sub.3-C.sub.12 carbocycle, and a
C.sub.6-C.sub.20 aryl;
[0082] R.sup.2 is selected from H, F, Cl, Br, I, --C(.dbd.Y)R,
--C(.dbd.Y)OR, --C(.dbd.Y)NR.sub.2, --OR, --OC(.dbd.Y)R,
--OC(.dbd.Y)OR, --OC(.dbd.Y)(N(R).sub.2), --OS(O).sub.2(OR),
--OP(.dbd.Y)(OR).sub.2, --OP(OR).sub.2, --P(.dbd.Y)(OR).sub.2,
--P(.dbd.Y)(OR)NR.sub.2, --SR, --S(O)R, --S(O).sub.2R,
--S(O).sub.2NR, --S(O)(OR), --S(O).sub.2(OR), --SC(.dbd.Y)R,
--SC(.dbd.Y)OR, --SC(.dbd.Y)NR.sub.2, C.sub.1-C.sub.8 alkylhalide,
C.sub.1-C.sub.8 alkylsulfonate, C.sub.1-C.sub.8 alkylamino,
C.sub.1-C.sub.8 alkylhydroxyl, C.sub.1-C.sub.8 alkylthiol, 5-7
membered ring lactam, 5-7 membered ring lactone, 5-7 membered ring
sultam, C.sub.1-C.sub.8 alkyl, C.sub.1-C.sub.8 alkenyl,
C.sub.1-C.sub.8 alkynyl, C.sub.6-C.sub.20 aryl, and
C.sub.2-C.sub.20 heterocycle;
[0083] R.sup.1 and R.sup.2 of Formula Ia optionally form a 5 or 6
membered fused heterocycle ring having at least two heteroatoms
selected from O, N and S;
[0084] R.sup.3, R.sup.4, and R.sup.5 are independently selected
from H, F, Cl, Br, I, --C(.dbd.Y)R, --C(.dbd.Y)OR,
--C(.dbd.Y)NR.sub.2, --NR.sub.2, --.sup.+NR.sub.3,
--N(R)C(.dbd.Y)R, --N(R)C(.dbd.Y)OR, --N(R)C(.dbd.Y)NR.sub.2, --OR,
--OC(.dbd.Y)R, --OC(.dbd.Y)OR, --OC(.dbd.Y)(N(R).sub.2),
--OS(O).sub.2(OR), --OP(.dbd.Y)(OR).sub.2, --OP(OR).sub.2,
--P(.dbd.Y)(OR).sub.2, --P(.dbd.Y)(OR)NR.sub.2, --SR, --S(O)R,
--S(O).sub.2R, --S(O).sub.2NR, --S(O)(OR), --S(O).sub.2(OR),
--SC(.dbd.Y)R, --SC(.dbd.Y)OR, --SC(.dbd.Y)NR.sub.2,
C.sub.1-C.sub.8 alkylhalide, C.sub.1-C.sub.8 alkylsulfonate,
C.sub.1-C.sub.8 alkylamino, C.sub.1-C.sub.8 alkylhydroxyl,
C.sub.1-C.sub.8 alkylthiol, 5-7 membered ring lactam, 5-7 membered
ring lactone, 5-7 membered ring sultam, C.sub.1-C.sub.8 alkyl,
C.sub.1-C.sub.8 alkenyl, C.sub.1-C.sub.8 alkynyl, C.sub.6-C.sub.20
aryl, and C.sub.2-C.sub.20 heterocycle;
[0085] R is H, C.sub.1-C.sub.8 alkyl, C.sub.1-C.sub.8 alkenyl,
C.sub.1-C.sub.8 alkynyl, C.sub.6-C.sub.20 aryl, C.sub.2-C.sub.20
heterocycle, or a protecting group; and
[0086] Y is independently O, S, NR, .sup.+N(O)(R), N(OR),
.sup.+N(O)(OR), or N--NR.sub.2;
[0087] each alkyl, alkenyl, alkynyl, aryl, carbocycle, and
heterocycle is optionally and independently substituted with one or
more substituents selected from F, Cl, Br, I, OH, OR, R,
--C(.dbd.Y)R, --C(.dbd.Y)OR, --C(.dbd.Y)N(R).sub.2, --N(R).sub.2,
--.sup.+N(R).sub.3, --N(R)C(.dbd.Y), --N(R)C(.dbd.Y)O,
--N(R)C(.dbd.Y)N(R).sub.2, --S, --OC(.dbd.Y), --OC(.dbd.Y)O,
--OC(.dbd.Y)(N(R).sub.2), --OS(O).sub.2(OR),
--OP(.dbd.Y)(OR).sub.2, --OP(OR).sub.2, --P(.dbd.Y)(OR).sub.2,
--P(.dbd.Y)(OR)NR.sub.2, --S(O), --S(O).sub.2, --S(O).sub.2N,
--S(O)(OR), --S(O).sub.2(OR), --SC(.dbd.Y), --SC(.dbd.Y)O, .dbd.Y,
and --SC(.dbd.Y)(N(R).sub.2).
[0088] Formula Ia and Ib compounds are regioisomers, differing by
the attachment of R1 at the non-equivalent nitrogen atoms of the
pyrazole ring. Formula Ia and Ib compounds include stereoisomers,
tautomers, solvates and pharmaceutically acceptable salts thereof,
wherein:
[0089] In certain embodiments, R.sup.20 and R.sup.21 are optionally
and independently a protecting group such as, but not limited to,
trialkylsilyl, dialkylphenylsilyl, benzoate, benzyl,
benzyloxymethyl, methyl, methoxymethyl, a triarylmethyl,
phthalimido, t-butoxycarbonyl (BOC), benzyloxycarbonyl (CBz) and
9-fluorenylmethylenoxycarbonyl (Fmoc), or tetrahydropyranyl.
[0090] Exemplary embodiments of the A-ring include an optionally
substituted a 5 or 6 membered fused heterocyclic ring having one or
two heteroatoms independently selected from O, N, and S, such as
tetrahydrofuranyl, dioxolanyl, tetrahydropyranyl,
tetrahydropyridyl, piperazinyl, pyrrolidinyl, pyridyl, pyrimidinyl,
dihydrothiophenyl, thiophenyl, imidazolyl, thiazolyl, oxazolyl,
isoxazolyl, and pyrazolyl, including exemplary structures:
##STR00004##
[0091] The A-ring may be an optionally substituted, fused saturated
or partially unsaturated 5 or 6 membered carbocyclic ring, such as
cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, and
substituted forms thereof, including exemplary structures:
##STR00005##
[0092] The A-ring may be an optionally substituted ring selected
from phenyl. For example, the A-ring may be:
##STR00006##
[0093] and substituted forms thereof.
[0094] Exemplary embodiments of X include 2-pyridyl, 3-pyridyl,
4-pyridyl, 2-imidazolyl, 4-imidazolyl, 3-pyrazolyl, 4-pyrazolyl,
2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 3-pyridazinyl,
4-pyridazinyl, 5-pyridazinyl, 2-pyrimidinyl, 5-pyrimidinyl,
6-pyrimidinyl, 2-pyrazinyl, 2-oxazolyl, 4-oxazolyl, 5-oxazolyl, and
substituted forms thereof, and shown as:
##STR00007##
[0095] Exemplary embodiments of 5-7 membered ring lactams, 5-7
membered ring lactones, and 5-7 membered ring sultams include the
structures:
##STR00008##
[0096] Exemplary embodiments of Formulas Ia and Ib compounds
include compounds of Formulas IIa-h and IIIa-f:
##STR00009## ##STR00010## ##STR00011##
[0097] wherein Z is selected from CR.sup.20R.sup.21, C(.dbd.Y),
NR.sup.20, O, and S.
[0098] Exemplary embodiments of Formulas Ia and Ib compounds also
include compounds of Formulas IVa, IVb, Va and Vb:
##STR00012##
[0099] Exemplary embodiments of Formulas Ia and Ib compounds also
include compounds of Formulas VIIa, VIIb, VIIa and VIIb:
##STR00013##
[0100] Exemplary embodiments of Formulas Ia and Ib compounds also
include Formulas VIIIa, VIIb, IXa and IXb:
##STR00014##
[0101] wherein R.sup.6, R.sup.7, R.sup.8, and R.sup.9 are
independently selected from H, F, Cl, Br, I, --OR.sup.20,
--C(.dbd.Y)R.sup.20, --C(.dbd.Y)OR.sup.20,
--C(.dbd.Y)NR.sup.20R.sup.21, --NR.sup.20R.sup.21,
--NR.sup.20C(.dbd.Y)R.sup.21, --NR.sup.20C(.dbd.Y)OR.sup.21,
--NR.sup.23C(.dbd.Y)NR.sup.20R.sup.21, --OC(.dbd.Y)R.sup.20,
--OC(.dbd.Y)OR.sup.20, --OC(.dbd.Y)NR.sup.20R.sup.21,
--OS(O).sub.2(OR.sup.20), --OP(.dbd.Y)(OR.sup.20)(OR.sup.21),
--OP(OR.sup.20)(OR.sup.21), --P(.dbd.Y)(OR.sup.20)(OR.sup.21),
--P(.dbd.Y)(OR.sup.20)NR.sup.20R.sup.21, --SR.sup.20,
--S(O)R.sup.20, --S(O).sub.2R.sup.20,
--S(O).sub.2NR.sup.20R.sup.21, --S(O)(OR.sup.20),
--S(O).sub.2(OR.sup.20), --SC(.dbd.Y)R.sup.20,
--SC(.dbd.Y)OR.sup.20, --SC(.dbd.Y)NR.sup.20R.sup.21, 5-7 membered
ring lactam, 5-7 membered ring lactone, 5-7 membered ring sultam,
C.sub.1-C.sub.8 alkyl, C.sub.2-C.sub.8 alkenyl, C.sub.2-C.sub.8
alkynyl, C.sub.3-C.sub.12 carbocyclyl, C.sub.6-C.sub.20 aryl, and
C.sub.2-C.sub.20 heterocyclyl, wherein said alkyl, alkenyl,
alkynyl, carbocyclyl, aryl, and heterocycle are optionally
substituted with one or more groups independently selected from F,
Cl, Br, I, OR.sup.20, NR.sup.20R.sup.21--SR.sup.20, S(O)R.sup.20,
--S(O).sub.2R.sup.20, alkyl, alkenyl, alkynyl, carbocyclyl, aryl,
and heterocyclyl.
[0102] Exemplary embodiments of Formulas Ia and Ib compounds also
include compounds of Formulas Xa, Xb, XMa and XMb:
##STR00015##
[0103] wherein R.sup.6, R.sup.7, R.sup.8, R.sup.9, and R.sup.10 are
independently selected from H, F, Cl, Br, I, --OR.sup.20,
--C(.dbd.Y)R.sup.20, --C(.dbd.Y)OR.sup.20,
--C(.dbd.Y)NR.sup.20R.sup.21, --NR.sup.20R.sup.21,
--NR.sup.20C(.dbd.Y)R.sup.21, --NR.sup.20C(Y)OR.sup.21,
--NR.sup.23C(.dbd.Y)NR.sup.20R.sup.21, --OC(.dbd.Y)R.sup.20,
--OC(.dbd.Y)OR.sup.20, --OC(.dbd.Y)NR.sup.20R.sup.21,
--OS(O).sub.2(OR.sup.20),
--OP(.dbd.Y)(OR.sup.20)(OR.sup.21)--OP(OR.sup.20)(OR.sup.21),
--P(.dbd.Y)(OR.sup.20)(OR.sup.21),
--P(.dbd.Y)(OR.sup.23)NR.sup.20R.sup.21, --SR.sup.20,
--S(O)R.sup.20, --S(O).sub.2R.sup.20,
--S(O).sub.2NR.sup.20R.sup.21, --S(O)(OR.sup.20),
--S(O).sub.2(OR.sup.20), --SC(.dbd.Y)R.sup.20,
--SC(.dbd.Y)OR.sup.20, --SC(.dbd.Y)NR.sup.20R.sup.21, 5-7 membered
ring lactam, 5-7 membered ring lactone, 5-7 membered ring sultam,
C.sub.1-C.sub.8 alkyl, C.sub.2-C.sub.8 alkenyl, C.sub.2-C.sub.8
alkynyl, C.sub.3-C.sub.12 carbocyclyl, C.sub.6-C.sub.20 aryl, and
C.sub.2-C.sub.20 heterocyclyl, wherein said alkyl, alkenyl,
alkynyl, carbocyclyl, aryl, and heterocyclyl are optionally
substituted with one or more groups independently selected from F,
Cl, Br, I, OR.sup.20, NR.sup.20R.sup.21--SR.sup.20, --S(O)R.sup.20,
--S(O).sub.2R.sup.20, alkyl, alkenyl, alkynyl, carbocyclyl, aryl,
and heterocyclyl.
[0104] Exemplary embodiments of Formulas Ia and Ib compounds also
include Formulas XIa-d and XIIIa-j:
##STR00016## ##STR00017##
[0105] wherein R.sup.11, R.sup.12, and R.sup.13 are independently
selected from H, F, Cl, Br, I, --OR.sup.20, --C(.dbd.Y)R.sup.20,
--C(.dbd.Y)OR.sup.20, --C(.dbd.Y)NR.sup.20R.sup.21,
--NR.sup.20R.sup.21, --NR.sup.20C(.dbd.Y)R.sup.21,
--NR.sup.20C(.dbd.Y)OR.sup.21,
--NR.sup.20C(.dbd.Y)NR.sup.20R.sup.21, --OC(.dbd.Y)R.sup.20,
--OC(.dbd.Y)OR.sup.20, --OC(.dbd.Y)NR.sup.20R.sup.21,
--OS(O).sub.2(OR.sup.20), --OP(.dbd.Y)(OR.sup.20)(OR.sup.21),
--OP(OR.sup.20)(OR.sup.21), --P(.dbd.Y)(OR.sup.20)(OR.sup.21),
--P(.dbd.Y)(OR.sup.23)NR.sup.20R.sup.21, --SR.sup.20,
--S(O)R.sup.20, --S(O).sub.2R.sup.20,
--S(O).sub.2NR.sup.20R.sup.21, --S(O)(OR.sup.20),
--S(O).sub.2(OR.sup.20), --SC(.dbd.Y)R.sup.20,
--SC(.dbd.Y)OR.sup.20, --SC(.dbd.Y)NR.sup.20R.sup.21, 5-7 membered
ring lactam, 5-7 membered ring lactone, 5-7 membered ring sultam,
C.sub.1-C.sub.8 alkyl, C.sub.2-C.sub.8 alkenyl, C.sub.2-C.sub.8
alkynyl, C.sub.3-C.sub.12 carbocyclyl, C.sub.6-C.sub.20 aryl, and
C.sub.2-C.sub.20 heterocycle, wherein said alkyl, alkenyl, alkynyl,
carbocyclyl, aryl, and heterocyclyl are optionally substituted with
one or more groups independently selected from F, Cl, Br, I,
OR.sup.20, NR.sup.20R.sup.21--SR.sup.20, --S(O)R.sup.20,
--S(O).sub.2R.sup.20, alkyl, alkenyl, alkynyl, carbocyclyl, aryl,
and heterocyclyl.
[0106] Exemplary embodiments of Formulas Ia and Ib compounds also
include Formulas XIVa-d:
##STR00018##
[0107] Exemplary embodiments of Formulas Ia and Ib compounds also
include Formulas XVa-d:
##STR00019##
[0108] Compounds 101-160 listed in Table 1 were prepared,
characterized, and assayed for B-Raf binding activity and in vitro
activity against tumor cells.
TABLE-US-00001 TABLE 1 No. Structure Name 101 ##STR00020##
5-(1-(2-aminoethyl)-3-pyridin-4-yl-1H-
pyrazol-4-yl)-2,3-dihydroinden-1-one oxime 102 ##STR00021##
5-(1-(3-aminopropyl)-3-pyridin-4-yl-1H-
pyrazol-4-yl)-2,3-dihydroinden-1-one oxime 103 ##STR00022##
5-(1-(4-aminobutyl)-3-pyridin-4-yl-1H-
pyrazol-4-yl)-2,3-dihydroinden-1-one oxime 104 ##STR00023##
5-(1-(pyrrolidin-3-yl)-3-pyridin-4-yl-1H-
pyrazol-4-yl)-2,3-dihydroinden-1-one oxime 105 ##STR00024##
5-(1-(piperidin-4-yl)-3-pyridin-4-yl-1H-
pyrazol-4-yl)-2,3-dihydroinden-1-one oxime 106 ##STR00025##
5-(1-(4-methylpiperidinyl)-3-pyridin-4-yl-1H-
pyrazol-4-yl)-2,3-dihydroinden-1-one oxime 107 ##STR00026##
5-(1-(3R-tetrahydrofuranyl)-3-pyridin-4-yl-
1H-pyrazol-4-yl)-2,3-dihydroinden-1-one oxime 108 ##STR00027##
5-(1-(3S-tetrahydrofuranyl)-3-pyridin-4-yl-
1H-pyrazol-4-yl)-2,3-dihydroinden-1-one oxime 109 ##STR00028##
5-(1-(4-tetrahydropyranyl)-3-pyridin-4-yl-1H-
pyrazol-4-yl)-2,3-dihydroinden-1-one oxime 110 ##STR00029##
5-(1-(2-hydroxyethyl)-3-pyridin-4-yl-1H-
pyrazol-4-yl)-2,3-dihydroinden-1-one oxime 111 ##STR00030##
5-(1-(3S-(1R-hydroxy)cyclohexyl)-3-pyridin-
4-yl-1H-pyrazol-4-yl)-2,3-dihydroinden-1-one oxime 112 ##STR00031##
5-(1-(2,3 dihydroxypropyl)-3-pyridin-4-yl-
1H-pyrazol-4-yl)-2,3-dihydroinden-1-one oxime 113 ##STR00032##
5-(1-methyl-3-pyridin-4-yl-1H-pyrazol-4-yl)- 2,3-dihydroinden-1-one
oxime 114 ##STR00033## 5-(1-(3-cyano-pyridin-2-yl)-3-pyridin-4-yl-
1H-pyrazol-4-yl)-2,3-dihydroinden-1-one oxime 115 ##STR00034##
5-(1-(3S-(1S-hydroxy)cyclohexyl)-3-pyridin-
4-yl-1H-pyrazol-4-yl)-2,3-dihydroinden-1-one oxime 116 ##STR00035##
5-(1-(3-aminomethyl-pyridin-2-yl)-3-pyridin-
4-yl-1H-pyrazol-4-yl)-2,3-dihydroinden-1-one oxime 117 ##STR00036##
5-(1-(5-(aminomethyl)pyridin-2-yl)-3-pyridin-
4-yl-1H-pyrazol-4-yl)-2,3-dihydroinden-1-one oxime 118 ##STR00037##
5-(1-(3S-(1S, 2R-dihydroxy)cyclohexyl)-3-
pyridin-4-yl-1H-pyrazol-4-yl)-2,3- dihydroinden-1-one oxime 119
##STR00038## 5-(1-(3-methoxyacetamidopropyl)-3-pyridin-
4-yl-1H-pyrazol-4-yl)-2,3-dihydroinden-1-one oxime 120 ##STR00039##
5-(1-(4-aminomethylphenyl)-3-pyridin-4-yl-
1H-pyrazol-4-yl)-2,3-dihydroinden-1-one oxime 121 ##STR00040##
5-(1-(5-(acetamido)pyridin-2-yl)-3-pyridin-4-
yl-1H-pyrazol-4-yl)-2,3-dihydroinden-1-one oxime 122 ##STR00041##
5-(1-Methyl-5-pyridin-4-yl-1H-pyrazol-4-yl)- 2,3-dihydroinden-1-one
oxime 123 ##STR00042## 5-(1-(2-hydroxyethyl)-5-pyridin-4-yl-1H-
pyrazol-4-yl)-2,3-dihydroinden-1-one oxime 124 ##STR00043##
5-(2-(pyridin-4-yl)pyrazolo[1.5-.alpha.]pyrimidin-
3-yl)-2,3-dihydroinden-1-one oxime 125 ##STR00044##
5-(1-(2S-(1S-hydroxy)cyclopentyl)-3-pyridin-
4-yl-1H-pyrazol-4-yl)-2,3-dihydroinden-1-one oxime 126 ##STR00045##
5-(3-pyridin-4-yl-1H-pyrazol-4-yl)-2,3- dihydroinden-1-one oxime
127 ##STR00046## 5-(1-cyclopentyl-3-pyridin-4-yl-1H-pyrazol-
4-yl)-2,3-dihydroinden-1-one oxime 128 ##STR00047##
5-(1-(2-(4-methylpiperazinyl)ethyl)-3-pyridin-
4-yl-1H-pyrazol-4-yl)-2,3-dihydroinden-1-one oxime 129 ##STR00048##
5-(1-methyl-3-pyridin-4-yl-1H-pyrazol-4-yl)- benzofuran-3(2H)-one
oxime 130 ##STR00049## 5-(1-methyl-3-pyridin-4-yl-1H-pyrazol-4-yl)-
2,3-dihydrochromen-4-one oxime 131 ##STR00050##
5-(1-methyl-3-pyridin-4-yl-1H-pyrazol-4-yl)- 2,3-dihydroinden-1-one
132 ##STR00051## 5-(1-(2-(2-hydroxyethyl)aminoethyl)-3-
pyridin-4-yl-1H-pyrazol-4-yl)-2,3- dihydroinden-1-one oxime 133
##STR00052## 5-(1-(2-(4-methylpiperazinyl)ethyl)-3-pyridin-
4-yl-1H-pyrazol-4-yl)-2,3-dihydro-1H-inden- 1-amine 134
##STR00053## 5-(2-hydroxyethyl-5-pyridin-4-yl-1H-pyrazol-
4-yl)-2,3-dihydro-1H-inden-1-amine 135 ##STR00054##
5-(1-(2S-(1S-hydroxy)cyclopentyl)-3-pyridin-
4-yl-1H-pyrazol-4-yl)-2,3-dihydro-1H-inden- 1-amine 136
##STR00055## 5-(1-(2-(N-piperidyl)ethyl)-3-pyridin-4-yl-
1H-pyrazol-4-yl)-2,3-dihydroinden-1-one oxime 137 ##STR00056##
5-(1-methyl-3-pyridin-4-yl-1H-pyrazol-4-yl)-
3,4-dihydroisoquinolin-1(2H)-one oxime 138 ##STR00057##
5-(1-(4-piperidyl)-3-pyridin-4-yl-1H-pyrazol-
4-yl)-2,4,5,6-tetrahydrocyclopenta[c]phenyl pyrazole 139
##STR00058## 5-(1-(2-hydroxyethyl)-3-pyridin-4-yl-1H-
pyrazol-4-yl)-2,3-dihydroinden-1-one 140 ##STR00059##
5-(1-(4-(N-methylpiperidyl))-3-pyridin-4-yl-
1H-pyrazol-4-yl)-2,3-dihydroinden-1-one oxime 141 ##STR00060##
5-(3-pyridin-4-yl-1H-pyrazol-4-yl)-2,3- dihydroinden-1-one 142
##STR00061## 5-(1-(2-hydroxyethyl)-3-pyridin-4-yl-1H-
pyrazol-4-yl)-indolin-2-one 143 ##STR00062##
5-(1-(2-hydroxyethyl)-3-pyridin-4-yl-1H-
pyrazol-4-yl)-isoquinolin-1-ol 144 ##STR00063##
5-(1-methyl-3-pyridin-4-yl-1H-pyrazol-4-yl)- indolin-2-one 145
##STR00064## 5-(1-acetic acid-3-pyridin-4-yl-1H-pyrazol-4-
yl)-2,3-dihydroinden-1-one oxime 146 ##STR00065##
5-(1-(2-hydroxyethyl)-3-pyridin-4-yl-1H-
pyrazol-4-yl)-2,3-dihydrophthalazine-1,4- dione 147 ##STR00066##
5-(1-(2-hydroxyethyl)-3-pyridin-4-yl-1H- pyrazol-4-yl)-6-1H-indole
148 ##STR00067## 5-(1-(2-hydroxyethyl)-3-pyridin-4-yl-1H-
pyrazol-4-yl)-2-(2-methoxyethyl)isoindoline- 1,3-dione 149
##STR00068## 5-(1-(2-hydroxyethyl)-3-pyridin-4-yl-1H-
pyrazol-4-yl)-benzo[d][1,3]dioxole 150 ##STR00069##
5-(1-(2-hydroxyethyl)-3-pyridin-4-yl-1H-
pyrazol-4-yl)-quinazolin-4(3H)-one 151 ##STR00070##
5-(1-(2-hydroxyethyl)-3-pyridin-4-yl-1H-
pyrazol-4-yl)-2,3-dihydro-1H-inden-1-amine 152 ##STR00071##
5-(1-(2-hydroxyethyl)-3-pyridin-4-yl-1H-
pyrazol-4-yl)-N-tert-butyloxycarbonyl-2,3- dihydro-1H-inden-1-amine
153 ##STR00072## 5-(1-(2-hydroxyethyl)-3-pyridin-4-yl-1H-
pyrazol-4-yl)-5-1H-indole 154 ##STR00073##
5-(1-(2-hydroxyethyl)-3-pyridin-4-yl-1H-
pyrazol-4-yl)-5-isoindoline-1,3-dione 155 ##STR00074##
(Z)-5-(1-methyl-3-(pyridin-4-yl)-1H-pyrazol- 4-yl)isoindolin-1-one
oxime 156 ##STR00075##
(Z)-5-(1-(1-methylpiperidin-4-yl)-3-(pyridin-
4-yl)-1H-pyrazol-4-yl)isoindolin-1-one oxime 157 ##STR00076##
5-(1-(1-methylpiperidin-4-yl)-3-(pyridin-4-
yl)-1H-pyrazol-4-yl)isoindolin-1-imine 158 ##STR00077##
6-(1-methyl-3-(pyridin-4-yl)-1H-pyrazol-4- yl)naphthalen-1-ol 159
##STR00078## 6-(1-((1S,2S)-2-hydroxycyclohexyl)-3-
(pyridin-4-yl)-1H-pyrazol-4-yl)naphthalen-1- ol 160 ##STR00079##
6-(1-methyl-3-(pyridin-4-yl)-1H-pyrazol-4- yl)naphthalen-2-ol
[0109] The compounds of the invention may contain asymmetric or
chiral centers, and, therefore, exist in different stereoisomeric
forms. It is intended that all stereoisomeric forms of the
compounds of the invention, including but not limited to:
diastereomers, enantiomers, and atropisomers as well as mixtures
thereof such as racemic mixtures, form part of the present
invention. In addition, the present invention embraces all
geometric and positional isomers. For example, if a compound of the
present invention incorporates a double bond or a fused ring, both
the cis- and trans-forms, as well as mixtures, are embraced within
the scope of the invention. Both the single positional isomers and
mixture of positional isomers, e.g., resulting from the N-oxidation
of the pyrimidine and pyrazine rings, or the E and Z forms of oxime
moieties, are also within the scope of the present invention.
[0110] In the structures shown herein, where the stereochemistry of
any particular chiral atom is not specified, then all stereoisomers
are contemplated and included as the compounds of the invention.
Where stereochemistry is specified by a solid wedge or dashed line
representing a particular configuration, then that stereoisomer is
so specified and defined.
[0111] The compounds of the present invention may exist in
unsolvated as well as solvated forms with pharmaceutically
acceptable solvents such as water, ethanol, and the like, and it is
intended that the invention embrace both solvated and unsolvated
forms.
[0112] It is also possible that the compounds of the present
invention may exist in different tautomeric forms, and all such
forms are embraced within the scope of the invention. The term
"tautomer" or "tautomeric form" refers to structural isomers of
different energies which are interconvertible via a low energy
barrier. For example, proton tautomers (also known as prototropic
tautomers) include interconversions via migration of a proton, such
as keto-enol and imine-enamine isomerizations. Valence tautomers
include interconversions by reorganization of some of the bonding
electrons.
[0113] Hydroxyimino or alkoxyimino (oxime) moieties of the
compounds of the invention, e.g. Formula I-XIX compounds, can be
positioned on any of carbon atoms of ring A. The oxime moiety can
exist as either the E or Z isomer, or as a mixture of both.
[0114] The present invention also embraces isotopically-labeled
compounds of the present invention which are identical to those
recited herein, but for the fact that one or more atoms are
replaced by an atom having an atomic mass or mass number different
from the atomic mass or mass number usually found in nature. All
isotopes of any particular atom or element as specified are
contemplated within the scope of the compounds of the invention,
and their uses. Exemplary isotopes that can be incorporated into
compounds of the invention include isotopes of hydrogen, carbon,
nitrogen, oxygen, phosphorus, sulfur, fluorine, chlorine, and
iodine, such as .sup.2H, .sup.3H, .sup.11C, .sup.13C, .sup.14C,
.sup.13N, .sup.15N, .sup.15O, .sup.17O, .sup.18O, .sup.32P,
.sup.33P, .sup.35S, .sup.18F .sup.36Cl, .sup.123I, and .sup.125I
respectively. Certain isotopically-labeled compounds of the present
invention (e.g., those labeled with .sup.3H and .sup.14C) are
useful in compound and/or substrate tissue distribution assays.
Tritiated (i.e., .sup.3H) and carbon-14 (i.e., .sup.14C) isotopes
are useful for their ease of preparation and detectability.
Further, substitution with heavier isotopes such as deuterium
(i.e., .sup.2H) may afford certain therapeutic advantages resulting
from greater metabolic stability (e.g., increased in vivo half-life
or reduced dosage requirements) and hence may be preferred in some
circumstances. Positron emitting isotopes such as .sup.15O,
.sup.13N, .sup.11C, and .sup.18F are useful for positron emission
tomography (PET) studies to examine substrate receptor occupancy.
Isotopically labeled compounds of the present invention can
generally be prepared by following procedures analogous to those
disclosed in the Schemes and/or in the Examples herein below, by
substituting an isotopically labeled reagent for a non-isotopically
labeled reagent.
[0115] Synthesis of Pyrazolyl Raf Inhibitor Compounds
[0116] Compounds of the present invention may be synthesized by
synthetic routes that include processes analogous to those
well-known in the chemical arts, particularly in light of the
description contained herein. The starting materials are generally
available from commercial sources such as Aldrich Chemicals
(Milwaukee, Wis.) or are readily prepared using methods well known
to those skilled in the art (e.g., prepared by methods generally
described in Louis F. Fieser and Mary Fieser, Reagents for Organic
Synthesis, v. 1-19, Wiley, N.Y. (1967-1999 ed.), or Beilsteins
Handbuch der organischen Chemie, 4, Aufl. ed. Springer-Verlag,
Berlin, including supplements (also available via the Beilstein
online database)).
[0117] Compounds of Formulas Ia and Ib are pyrazolyl compounds
(pyrazolo), which may be readily prepared using procedures
well-known to prepare other heterocycles, which are described for
instance in "Comprehensive Heterocyclic Chemistry, Editors Katrizky
and Rees, Pergamon Press, 1984. Methods for pyrazole synthesis are
also disclosed in: U.S. Pat. No. 5,008,363; Penning, et al (1997)
Bioorganic & Med. Chem. Letters 7(16):2121-2124; Dombroski et
al (2004) Bioorganic & Med. Chem. Letters 14:919-923; Almansa
et al (2001) J. Med. Chem. 44:350-361; Fray et al (1995) J. Med.
Chem. 38:3524-3535).
[0118] Compounds of Formulas Ia and Ib may be prepared singly or as
compound libraries comprising at least 2, for example 5 to 1,000
compounds, or 10 to 100 compounds of Formulas Ia and Ib. Libraries
of compounds of the invention may be prepared by a combinatorial
split and mix approach or by multiple parallel syntheses using
either solution phase or solid phase chemistry, by procedures known
to those skilled in the art. Thus according to a further aspect of
the invention there is provided a compound library comprising at
least 2 compounds of Formulas Ia and Ib, or pharmaceutically
acceptable salts thereof.
[0119] For illustrative purposes, the reaction schemes depicted
below provide potential routes for synthesizing the compounds of
the present invention as well as key intermediates. For a more
detailed description of the individual reaction steps, see the
Examples section below. Those skilled in the art will appreciate
that other synthetic routes may be used to synthesize the inventive
compounds. Although specific starting materials and reagents are
depicted in the Schemes and discussed below, other starting
materials and reagents can be easily substituted to provide a
variety of derivatives and/or reaction conditions. In addition,
many of the compounds prepared by the methods described below can
be further modified in light of this disclosure using conventional
chemistry well known to those skilled in the art.
[0120] In the preparation of compounds of the present invention,
protection of remote functionality (e.g., primary or secondary
amine) of intermediates may be necessary. The need for such
protection will vary depending on the nature of the remote
functionality and the conditions of the preparation methods.
Suitable amino-protecting groups (NH-Pg) include acetyl,
trifluoroacetyl, t-butoxycarbonyl (BOC), benzyloxycarbonyl (CBz)
and 9-fluorenylmethyleneoxycarbonyl (Fmoc). The need for such
protection is readily determined by one skilled in the art. For a
general description of protecting groups and their use, see T. W.
Greene, Protective Groups in Organic Synthesis, John Wiley &
Sons, New York, 1991.
[0121] The following general synthetic schemes can be used to
prepare compounds of the present invention, wherein the
R.sup.1-R.sup.5 substituents, the A-ring, and X are as defined for
Formulas Ia and Ib.
##STR00080##
##STR00081##
[0122] Formation of the pyrazoleboronic acids 3 and 6 is carried
out by treating bromopyrazoles 1 or 5 with n-butyllithium in
tetrahydrofuran at -78.degree. C., quenching the lithiated pyrazole
intermediate with trimethylborate, and isolating the hydrolyzed
product by aqueous acidic workup. Coupling of the fused A-ring with
the pyrazole can be done via a Suzuki reaction. This is
accomplished either by coupling of the bromopyrazole 1 or 5 with
the fused A-ring boronic acid 2, or by coupling the pyrazole
boronic acid 3 or 6 with the bromo-substituted fused A-ring 4. The
Suzuki reactions are typically conducted in a solvent mixture of
acetonitrile and water, sometimes with N,N-dimethylformamide (DMF)
as a co-solvent. (For reviews see: Miyaura et al. (1995) Chem. Rev.
95:2457-2483; Suzuki, A. (1999) J. Organomet. Chem. 576:147-168;
Suzuki, A. in Metal-Catalyzed Cross-Coupling Reactions, Diederich,
F., Stang, P. J., Eds., VCH, Weinheim, Del. (1998), pp 49-97). The
coupling reactions are carried out at an elevated temperature of
about 80.degree. C. in the presence of several equivalents of a
base, such as potassium carbonate, and a catalytic amount of a
source of Pd(0), such as tetrakis(triphenylphosphine)palladium(0).
Other catalysts, bases, solvent systems, and temperatures can be
successfully employed in the Suzuki reaction (Owens et al (2003)
Bioorganic & Med. Chem. Letters 13:4143-4145; Molander et al
(2002) Organic Letters 4(11):1867-1870; U.S. Pat. No. 6,448,433).
In particular, the catalysts Pd (PPh.sub.3).sub.4, Pd(OAc).sub.2,
PdCl.sub.2(dppf)-DCM, Pd.sub.2(dba).sub.3/Pt--Bu).sub.3 may be
employed, depending on reaction variables.
##STR00082##
[0123] Scheme III shows a synthetic route to the intermediate
bromopyrazoles 1 and 5, where R.sup.2 is H. The enaminoketone 8 is
prepared by the reaction of the acetyl starting material 7 with
N,N-dimethylformamide dimethylacetal in a suitable solvent, such as
toluene, at reflux (e.g. Example 1, where X is 4-pyridyl).
Enaminoketone 8 is then cyclized to the tautomeric pyrazole 9 by
heating with excess hydrazine monohydrate in ethanol. In the
presence of base and an electrophile, pyrazole 9 can be substituted
with R.sup.1. Suitable electrophiles include, but are not limited
to alkyl halides, cycloalkyl halides, heterocyclyl halides, alkyl
tosylates and mesylates, cycloalkyl tosylates and mesylates,
heterocyclyl tosylates and mesylates, acid chlorides, sulfonyl
chlorides, activated alkyl esters, activated aryl esters, epoxides,
.alpha.,.beta.-unsaturated ketones, and activated aryl halides.
Bromination of pyrazoles 10 and 11 provides the intermediate
bromopyrazoles 1 and 5, where R.sup.2 is H. Bromination reactions
can be carried out using bromine in a suitable solvent such as
chloroform, chloroform and methanol mixture, or an acetic acid and
sodium acetate mixture. Typically, the bromination can be conducted
between -40.degree. C. and room temperature, preferably at
0.degree. C. As an alternative to bromine, N-bromosuccinimide can
be used to accomplish the bromination of pyrazoles 10 and 11.
##STR00083##
[0124] An alternative route to the intermediate bromopyrazoles 1
and 5, where R.sup.2 is H, is shown in Scheme IV. The steps are
essentially the same as in Scheme III, but in a different order.
The previously described substituted pyrazole 9 is brominated in a
similar fashion as described above in Scheme III. The resulting
bromopyrazole tautomeric mixture 12 is treated with a base and an
electrophile to afford the R.sup.1-substituted intermediate
bromopyrazoles 1 and 5, where R.sup.2 is H.
##STR00084##
[0125] Another synthetic route to the intermediate pyrazoles 1 and
5, where R.sup.2 is H, is shown in Scheme V. The previously
described enaminoketone 8 can be cyclized with monosubstituted
hydrazines by heating in a suitable solvent, such as ethanol. The
resulting mixture of pyrazoles 13 and 14 is separated by
chromatography and/or recrystallization. Bromination by the methods
described above affords the intermediate bromopyrazoles 1 and 5,
where R.sup.2 is H.
##STR00085##
[0126] Synthetic Scheme VI shows the synthesis of the intermediate
bromopyrazolylpyrimidine 20, which represents an example of
structure 1a in Scheme I, where R.sup.1 and R.sup.2 form a fused
heterocycle ring. Ester 15 is converted to ketonitrile 16 by
treatment with sodium methoxide and acetonitrile in refluxing
toluene. The ketonitrile 16 is cyclized to aminopyrazole 17 by
heating with hydrazine in a suitable solvent, such as ethanol.
Pyrazolylpyrimidine 18 is prepared by treating aminopyrazole 17
with malonaldehyde bis(dimethyl acetal), catalytic zinc chloride,
and hydrochloric acid in refluxing ethanol. Pyrazolylpyrimidine 18
is brominated with bromine in chloroform solution to yield the
intermediate bromopyrazolylpyrimidine 20.
##STR00086##
[0127] Scheme VII shows the synthesis of
3,4-dihydroisoquinolin-1(2H)-one oxime intermediate 24. Indanone
intermediate 21 may be ring expanded to
3,4-dihydroisoquinolin-1(2H)-one lactam intermediate 22 with azide
reagents. Vilsemeier-Hack type activation with phosphorus
pentachloride and hydroxylamine displacement of chloride from
intermediate 23 gives N-hydroxyacetamidine intermediate 24. Where X
is B(OH).sub.2 or Br, intermediate 24 can be coupled by Suzuki
reaction with intermediates 1, 3, 5, or 6 as in Schemes I and
II.
##STR00087##
[0128] Scheme VIII shows the synthesis of a benzyl-protected
isoindolin-1-one oxime intermediate 27. Isoindolinone 25 is
alkylated with triethyloxonium tetrafluoroborate in chloroform to
afford the 3-ethoxy-1H-isoindole 26, which is reacted with
O-benzylhydroxylamine hydrochloride and sodium carbonate in
ethanol, providing intermediate 27. Intermediate 27 can be coupled
by Suzuki reaction with intermediates 3 or 6 as in Schemes I and
II. Alternatively, intermediate 27 can be converted to the boronate
ester intermediate 28 by reaction with bis(pinacolato)diboron in
dioxane, using [1,1'-bis(diphenylphosphino)ferrocene]palladium(II)
chloride dichloromethane complex. The boronate ester intermediate
28 can be coupled by Suzuki reaction with intermediates 1 or 5 as
in Schemes I and II.
[0129] Methods of Separation
[0130] In each of the exemplary Schemes it may be advantageous to
separate reaction products from one another and/or from starting
materials. The desired products of each step or series of steps is
separated and/or purified (hereinafter separated) to the desired
degree of homogeneity by the techniques common in the art.
Typically such separations involve multiphase extraction,
crystallization from a solvent or solvent mixture, distillation,
sublimation, or chromatography. Chromatography can involve any
number of methods including, for example: reverse-phase and normal
phase; size exclusion; ion exchange; high, medium, and low pressure
liquid chromatography methods and apparatus; small scale
analytical; simulated moving bed (SMB) and preparative thin or
thick layer chromatography, as well as techniques of small scale
thin layer and flash chromatography.
[0131] Another class of separation methods involves treatment of a
mixture with a reagent selected to bind to or render otherwise
separable a desired product, unreacted starting material, reaction
by product, or the like. Such reagents include adsorbents or
absorbents such as activated carbon, molecular sieves, ion exchange
media, or the like. Alternatively, the reagents can be acids in the
case of a basic material, bases in the case of an acidic material,
binding reagents such as antibodies, binding proteins, selective
chelators such as crown ethers, liquid/liquid ion extraction
reagents (LIX), or the like.
[0132] Selection of appropriate methods of separation depends on
the nature of the materials involved. For example, boiling point,
and molecular weight in distillation and sublimation, presence or
absence of polar functional groups in chromatography, stability of
materials in acidic and basic media in multiphase extraction, and
the like. One skilled in the art will apply techniques most likely
to achieve the desired separation.
[0133] Diastereomeric mixtures can be separated into their
individual diastereoisomers on the basis of their physical chemical
differences by methods well known to those skilled in the art, such
as by chromatography and/or fractional crystallization. Enantiomers
can be separated by converting the enantiomeric mixture into a
diastereomeric mixture by reaction with an appropriate optically
active compound (e.g., chiral auxiliary such as a chiral alcohol or
Mosher's acid chloride), separating the diastereoisomers and
converting (e.g., hydrolyzing) the individual diastereoisomers to
the corresponding pure enantiomers. Also, some of the compounds of
the present invention may be atropisomers (e.g., substituted
biaryls) and are considered as part of this invention. Enantiomers
can also be separated by use of a chiral HPLC column.
[0134] A single stereoisomer, e.g. an enantiomer, substantially
free of its stereoisomer may be obtained by resolution of the
racemic mixture using a method such as formation of diastereomers
using optically active resolving agents (Eliel, E. and Wilen, S.
"Stereochemistry of Organic Compounds," John Wiley & Sons,
Inc., New York, 1994; Lochmuller, C. H., (1975) J. Chromatogr.,
113(3):283-302). Racemic mixtures of chiral compounds of the
invention can be separated and isolated by any suitable method,
including: (1) formation of ionic, diastereomeric salts with chiral
compounds and separation by fractional crystallization or other
methods, (2) formation of diastereomeric compounds with chiral
derivatizing reagents, separation of the diastereomers, and
conversion to the pure stereoisomers, and (3) separation of the
substantially pure or enriched stereoisomers directly under chiral
conditions. See: "Drug Stereochemistry, Analytical Methods and
Pharmacology," Irving W. Wainer, Ed., Marcel Dekker, Inc., New York
(1993).
[0135] Under method (1), diastereomeric salts can be formed by
reaction of enantiomerically pure chiral bases such as brucine,
quinine, ephedrine, strychnine,
.alpha.-methyl-.beta.-phenylethylamine (amphetamine), and the like
with asymmetric compounds bearing acidic functionality, such as
carboxylic acid and sulfonic acid. The diastereomeric salts may be
induced to separate by fractional crystallization or ionic
chromatography. For separation of the optical isomers of amino
compounds, addition of chiral carboxylic or sulfonic acids, such as
camphorsulfonic acid, tartaric acid, mandelic acid, or lactic acid
can result in formation of the diastereomeric salts.
[0136] Alternatively, by method (2), the substrate to be resolved
is reacted with one enantiomer of a chiral compound to form a
diastereomeric pair (E. and Wilen, S. "Stereochemistry of Organic
Compounds", John Wiley & Sons, Inc., 1994, p. 322).
Diastereomeric compounds can be formed by reacting asymmetric
compounds with enantiomerically pure chiral derivatizing reagents,
such as menthyl derivatives, followed by separation of the
diastereomers and hydrolysis to yield the pure or enriched
enantiomer. A method of determining optical purity involves making
chiral esters, such as a menthyl ester, e.g. (-) menthyl
chloroformate in the presence of base, or Mosher ester,
.alpha.-methoxy-.alpha.-(trifluoromethyl)phenyl acetate (Jacob III.
(1982) J. Org. Chem. 47:4165), of the racemic mixture, and
analyzing the NMR spectrum for the presence of the two
atropisomeric enantiomers or diastereomers. Stable diastereomers of
atropisomeric compounds can be separated and isolated by normal-
and reverse-phase chromatography following methods for separation
of atropisomeric naphthyl-isoquinolines (WO 96/15111). By method
(3), a racemic mixture of two enantiomers can be separated by
chromatography using a chiral stationary phase ("Chiral Liquid
Chromatography" (1989) W. J. Lough, Ed., Chapman and Hall, New
York; Okamoto, (1990) J. of Chromatogr. 513:375-378). Enriched or
purified enantiomers can be distinguished by methods used to
distinguish other chiral molecules with asymmetric carbon atoms,
such as optical rotation and circular dichroism.
[0137] Positional isomers, such as the E and Z forms of
oxime-containing compounds of Formula Ia and Ib compounds, and
intermediates for their synthesis, may be observed by
characterization methods such as NMR and analytical HPLC. For
certain compounds where the energy barrier for interconversion is
sufficiently high, the E and Z oxime isomers may be separated, for
example by preparatory HPLC.
[0138] Biological Evaluation
[0139] B-Raf mutant protein 447-717 (V600E) was co-expressed with
the chaperone protein Cdc37, complexed with Hsp90 (Roe et al (2004)
Cell 116:87-98; Stancato et al (1993) J. Biol. Chem.
268:21711-21716).
[0140] Determining the activity of Raf in the sample is possible by
a number of direct and indirect detection methods (US 2004/082014).
Activity of human recombinant B-Raf protein may be assessed in
vitro by assay of the incorporation of radiolabelled phosphate to
recombinant MAP kinase (MEK), a known physiologic substrate of
B-Raf, according to US 2004/127496 and WO 03/022840. The
activity/inhibition of V600E full-length B-Raf was estimated by
measuring the incorporation of radiolabeled phosphate from
[.gamma.-.sup.33P]ATP into FSBA-modified wild-type MEK (Example
8).
[0141] Suitable methods of Raf activity depend on the nature of the
sample. In cells, the activity of Raf is on the one hand determined
by the amount of the Raf expressed in the cell, and on the other
hand by the amount of the activated Raf. The activation of the
transcription of the genes coding for Raf protein, in particular
B-Raf protein, may for instance be made by determining the amount
of the Raf mRNA. Prior art standard methods comprise for instance
the DNA chip hybridization, RT-PCR, primer extension and RNA
protection. Furthermore, the determination of the Raf activity
based on the induction or repression of the transcription of the
respective Raf gene(s), may also take place by the coupling of the
Raf promoter to suitable reporter gene constructs. Examples for
suitable reporter genes are the chloramphenicol transferase gene,
the green fluorescent protein (GFP) and variants thereof, the
luciferase gene and the Renilla gene. The detection of the increase
of expression of Raf proteins may however also be made on the
protein level, in this case the amount of protein being detected
for instance by antibodies directed against Raf protein. The change
of the activity of the Raf protein can however also be put down to
increased or reduced phosphorylation or dephosphorylation of the
protein. For instance, the B-Raf kinase is regulated by the
phosphorylation of the 599Thr and 602Ser remainders (Zhang B. H.
and Guan K. L. (2000) EMBO J. 19:5429). The change of the
phosphorylation of B-Raf proteins may for instance be detected by
antibodies directed against phosphorylated threonine or serine.
[0142] Since Raf proteins are threonine/serine kinases, the
activity of the Raf proteins can also be determined by their
enzymatic activity. The protein MEK is for instance a substrate of
B-Raf and the degree of the phosphorylation of MEK permits the
determination of the B-Raf activity in the sample. In the same way,
the phosphorylation of other substrates, as for instance MBP and
peptides which are specifically phosphorylated by Raf (Salh et al.
(1999) Anticancer Res. 19:731-740; Bondzi et al. (2000) Oncogene
19:5030-5033), of the Raf proteins can be used for determining the
respective activity. Since Raf is part of a signal cascade where a
series of kinases are respectively phosphorylated and activated by
a superordinated kinase, the activity of Raf can also be determined
by evaluating the phosphorylation degree of each kinase
subordinated to Raf. This so-called map kinase pathway leads, among
other features, also to a specific activation of transcription
factors and thus to a transcriptional activation of genes, such
that the activity of Raf can indirectly be determined by measuring
the activity of these target genes.
[0143] The activity of test compounds (Formulas Ia and Ib) as B-Raf
inhibitors may be determined by the in vitro fluorescence
anisotropy kinase binding assay as described in Example 9, and
according to US 2004/127496 and WO 03/022840.
[0144] The neuroprotective properties of B-Raf inhibitors may be
determined by the in vitro assay in Rat Hippocampal Slice Cultures,
according to US 2004/127496; US 2004/082014; WO 03/022840 and as
described in Example 10.
[0145] The ERK inhibition properties of the compounds of the
invention may also be determined by the spectrophotometric
coupled-enzyme assay as described in Example 11 (Fox et al. (1998)
Protein Sci. 7:2249).
[0146] Inhibition of basal ERK1/2 phosphorylation was determined by
incubating Malme 3-M cells with compound for 1 hour and quantifying
the fluorescent pERK signal on fixed cells and normalizing to total
ERK signal according to the protocol of Example 12. The ERK
inhibition properties of the compounds of the invention may also be
determined by the in vitro cellular proliferation assay as
described in Example 12, and according to US 2003/0139452.
[0147] Viable cells after a 3 day incubation with compound were
quantified using the MTS/PMS colorimetric assay as described in
Example 13.
[0148] Exemplary compounds 101-160 listed in Table 1 were prepared,
characterized, and assayed for their B-Raf binding activity and in
vitro activity against tumor cells. The range of B-Raf binding
activities was less than 1 nM to about 10 .mu.M. Certain exemplary
compounds of the invention had B-Raf binding activity IC.sub.50
values less than 10 nM. Certain compounds of the invention had
cell-based activity, i.e. cells expressing activated mutants of the
B-Raf target kinase, IC.sub.50 values less than 100 nM.
[0149] Administration of Pyrazolyl Compounds
[0150] The pyrazolyl compounds of the invention may be administered
by any route appropriate to the condition to be treated. Suitable
routes include oral, parenteral (including subcutaneous,
intramuscular, intravenous, intraarterial, intradermal, intrathecal
and epidural), transdermal, rectal, nasal, topical (including
buccal and sublingual), vaginal, intraperitoneal, intrapulmonary,
and intranasal. For local immunosuppressive treatment, the
compounds may be administered by intralesional administration,
including perfusing or otherwise contacting the graft with the
inhibitor before transplantation. It will be appreciated that the
preferred route may vary with for example the condition of the
recipient. Where the pyrazolyl compound is administered orally, it
may be formulated as a pill, capsule, tablet, etc. with a
pharmaceutically acceptable carrier or excipient. Where the
pyrazolyl compound is administered parenterally, it may be
formulated with a pharmaceutically acceptable parenteral vehicle
and in a unit dosage injectable form, as detailed below.
[0151] A dose to treat human patients may range from about 10 mg to
about 1000 mg of pyrazolyl compound. A typical dose may be about
100 mg to about 300 mg of pyrazolyl compound. A dose may be
administered once a day (QID), twice per day (BID), or more
frequently, depending on the pharmacokinetic and pharmacodynamic
properties, including absorption, distribution, metabolism, and
excretion of the particular compound. In addition, toxicity factors
may influence the dosage and administration regimen. When
administered orally, the pill, capsule, or tablet may be ingested
daily or less frequently for a specified period of time. The
regimen may be repeated for a number of cycles of therapy.
[0152] Another aspect of this invention provides a compound of this
invention for use as a medicament in the treatment of the diseases
or conditions described herein in a mammal, for example, a human,
suffering from such disease or condition. Also provided is the use
of a compound of this invention in the preparation of a medicament
for the treatment of the diseases and conditions described herein
in a warm-blooded animal, such as a mammal, for example a human,
suffering from such disorder.
[0153] Pharmaceutical Formulations of Pyrazolyl Compounds
[0154] Compounds of the present invention are useful for treating
diseases, conditions and/or disorders characterized by over
expression of Raf kinases, e.g. B-Raf kinase; therefore, another
embodiment of the present invention is a pharmaceutical
composition, i.e. formulation, comprising a therapeutically
effective amount of a compound of the present invention and a
pharmaceutically acceptable excipient, diluent or carrier.
[0155] A typical formulation is prepared by mixing a compound of
the present invention and a carrier, diluent or excipient. Suitable
carriers, diluents and excipients are well known to those skilled
in the art and include materials such as carbohydrates, waxes,
water soluble and/or swellable polymers, hydrophilic or hydrophobic
materials, gelatin, oils, solvents, water, and the like. The
particular carrier, diluent or excipient used will depend upon the
means and purpose for which the compound of the present invention
is being applied. Solvents are generally selected based on solvents
recognized by persons skilled in the art as safe (GRAS) to be
administered to a mammal. In general, safe solvents are non-toxic
aqueous solvents such as water and other non-toxic solvents that
are soluble or miscible in water. Suitable aqueous solvents include
water, ethanol, propylene glycol, polyethylene glycols (e.g.,
PEG400, PEG300), etc. and mixtures thereof. The formulations may
also include one or more buffers, stabilizing agents, surfactants,
wetting agents, lubricating agents, emulsifiers, suspending agents,
preservatives, antioxidants, opaquing agents, glidants, processing
aids, colorants, sweeteners, perfuming agents, flavoring agents and
other known additives to provide an elegant presentation of the
drug (i.e., a compound of the present invention or pharmaceutical
composition thereof) or aid in the manufacturing of the
pharmaceutical product (i.e., medicament).
[0156] The formulations may be prepared using conventional
dissolution and mixing procedures. For example, the bulk drug
substance (i.e., compound of the present invention or stabilized
form of the compound, such as a complex with a cyclodextrin
derivative or other known complexation agent) is dissolved in a
suitable solvent in the presence of one or more of the excipients
described above. The compound of the present invention is typically
formulated into pharmaceutical dosage forms to provide an easily
controllable dosage of the drug and to enable patient compliance
with the prescribed regimen.
[0157] The pharmaceutical composition (or formulation) for
application may be packaged in a variety of ways depending upon the
method used for administering the drug. Generally, an article for
distribution includes a container having deposited therein the
pharmaceutical formulation in an appropriate form. Suitable
containers are well-known to those skilled in the art and include
materials such as bottles (plastic and glass), sachets, ampoules,
plastic bags, metal cylinders, and the like. The container may also
include a tamper-proof assemblage to prevent indiscreet access to
the contents of the package. In addition, the container has
deposited thereon a label that describes the contents of the
container. The label may also include appropriate warnings.
[0158] Pharmaceutical, formulations of therapeutic pyrazolyl
compounds of the invention may be prepared for various routes and
types of administration. A pyrazolyl compound having the desired
degree of purity is optionally mixed with pharmaceutically
acceptable diluents, carriers, excipients or stabilizers
(Remington's Pharmaceutical Sciences (1980) 16th edition, Osol, A.
Ed.), in the form of a lyophilized formulation, milled powder, or
an aqueous solution. Formulation may be conducted by mixing at
ambient temperature at the appropriate pH, and at the desired
degree of purity, with physiologically acceptable carriers, i.e.,
carriers that are non-toxic to recipients at the dosages and
concentrations employed. The pH of the formulation depends mainly
on the particular use and the concentration of compound, but may
range from about 3 to about 8. Formulation in an acetate buffer at
pH 5 is a suitable embodiment.
[0159] The inhibitory compound for use herein is preferably
sterile. The compound ordinarily will be stored as a solid
composition, although lyophilized formulations or aqueous solutions
are acceptable.
[0160] The pharmaceutical compositions of the invention will be
formulated, dosed, and administered in a fashion, i.e. amounts,
concentrations, schedules, course, vehicles, and route of
administration, consistent with good medical practice. Factors for
consideration in this context include the particular disorder being
treated, the particular mammal being treated, the clinical
condition of the individual patient, the cause of the disorder, the
site of delivery of the agent, the method of administration, the
scheduling of administration, and other factors known to medical
practitioners. The "therapeutically effective amount" of the
compound to be administered will be governed by such
considerations, and is the minimum amount necessary to prevent,
ameliorate, or treat the coagulation factor mediated disorder. Such
amount is preferably below the amount that is toxic to the host or
renders the host significantly more susceptible to bleeding.
[0161] As a general proposition, the initial pharmaceutically
effective amount of the inhibitor administered parenterally per
dose will be in the range of about 0.01-100 mg/kg, namely about 0.1
to 20 mg/kg of patient body weight per day, with the typical
initial range of compound used being 0.3 to 15 mg/kg/day.
[0162] Acceptable diluents, carriers, excipients, and stabilizers
are nontoxic to recipients at the dosages and concentrations
employed, and include buffers such as phosphate, citrate, and other
organic acids; antioxidants including ascorbic acid and methionine;
preservatives (such as octadecyldimethylbenzyl ammonium chloride;
hexamethonium chloride; benzalkonium chloride, benzethonium
chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as
methyl or propyl paraben; catechol; resorcinol; cyclohexanol;
3-pentanol; and m-cresol); low molecular weight (less than about 10
residues) polypeptides; proteins, such as serum albumin, gelatin,
or immunoglobulins; hydrophilic polymers such as
polyvinylpyrrolidone; amino acids such as glycine, glutamine,
asparagine, histidine, arginine, or lysine; monosaccharides,
disaccharides, and other carbohydrates including glucose, mannose,
or dextrins; chelating agents such as EDTA; sugars such as sucrose,
mannitol, trehalose or sorbitol; salt-forming counter-ions such as
sodium; metal complexes (e.g. Zn-protein complexes); and/or
non-ionic surfactants such as TWEEN.TM., PLURONICS.TM. or
polyethylene glycol (PEG). The active pharmaceutical ingredients
may also be entrapped in microcapsules prepared, for example, by
coacervation techniques or by interfacial polymerization, for
example, hydroxymethylcellulose or gelatin-microcapsules and
poly-(methylmethacylate) microcapsules, respectively, in colloidal
drug delivery systems (for example, liposomes, albumin
microspheres, microemulsions, nano-particles and nanocapsules) or
in macroemulsions. Such techniques are disclosed in Remington's
Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).
[0163] Sustained-release preparations may be prepared. Suitable
examples of sustained-release preparations include semipermeable
matrices of solid hydrophobic polymers containing the pyrazolyl
compound, which matrices are in the form of shaped articles, e.g.
films, or microcapsules. Examples of sustained-release matrices
include polyesters, hydrogels (for example,
poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)),
polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic
acid and gamma-ethyl-L-glutamate, non-degradable ethylene-vinyl
acetate, degradable lactic acid-glycolic acid copolymers such as
the LUPRON DEPOT.TM. (injectable microspheres composed of lactic
acid-glycolic acid copolymer and leuprolide acetate), and
poly-D-(-)-3-hydroxybutyric acid.
[0164] The formulations to be used for in vivo administration must
be sterile, which is readily accomplished by filtration through
sterile filtration membranes.
[0165] The formulations include those suitable for the
administration routes detailed herein. The formulations may
conveniently be presented in unit dosage form and may be prepared
by any of the methods well known in the art of pharmacy. Techniques
and formulations generally are found in Remington's Pharmaceutical
Sciences (Mack Publishing Co., Easton, Pa.). Such methods include
the step of bringing into association the active ingredient with
the carrier which constitutes one or more accessory ingredients. In
general the formulations are prepared by uniformly and intimately
bringing into association the active ingredient with liquid
carriers or finely divided solid carriers or both, and then, if
necessary, shaping the product.
[0166] Formulations of pyrazolyl compound suitable for oral
administration may be prepared as discrete units such as pills,
capsules, cachets or tablets each containing a predetermined amount
of the pyrazolyl compound.
[0167] Compressed tablets may be prepared by compressing in a
suitable machine the active ingredient in a free-flowing form such
as a powder or granules, optionally mixed with a binder, lubricant,
inert diluent, preservative, surface active or dispersing agent.
Molded tablets may be made by molding in a suitable machine a
mixture of the powdered active ingredient moistened with an inert
liquid diluent. The tablets may optionally be coated or scored and
optionally are formulated so as to provide slow or controlled
release of the active ingredient therefrom.
[0168] Tablets, troches, lozenges, aqueous or oil suspensions,
dispersible powders or granules, emulsions, hard or soft capsules,
e.g. gelatin capsules, syrups or elixirs may be prepared for oral
use. Formulations of a pyrazolyl compound intended for oral use may
be prepared according to any method known to the art for the
manufacture of pharmaceutical compositions and such compositions
may contain one or more agents including sweetening agents,
flavoring agents, coloring agents and preserving agents, in order
to provide a palatable preparation. Tablets containing the active
ingredient in admixture with non-toxic pharmaceutically acceptable
excipient which are suitable for manufacture of tablets are
acceptable. These excipients may be, for example, inert diluents,
such as calcium or sodium carbonate, lactose, calcium or sodium
phosphate; granulating and disintegrating agents, such as maize
starch, or alginic acid; binding agents, such as starch, gelatin or
acacia; and lubricating agents, such as magnesium stearate, stearic
acid or talc. Tablets may be uncoated or may be coated by known
techniques including microencapsulation 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 alone
or with a wax may be employed.
[0169] For infections of the eye or other external tissues e.g.
mouth and skin, the formulations are preferably applied as a
topical ointment or cream containing the active ingredient(s) in an
amount of, for example, 0.075 to 20% w/w. When formulated in an
ointment, the active ingredients may be employed with either a
paraffinic or a water-miscible ointment base. Alternatively, the
active ingredients may be formulated in a cream with an
oil-in-water cream base.
[0170] If desired, the aqueous phase of the cream base may include
a polyhydric alcohol, i.e. an alcohol having two or more hydroxyl
groups such as propylene glycol, butane 1,3-diol, mannitol,
sorbitol, glycerol and polyethylene glycol (including PEG 400) and
mixtures thereof. The topical formulations may desirably include a
compound which enhances absorption or penetration of the active
ingredient through the skin or other affected areas. Examples of
such dermal penetration enhancers include dimethyl sulfoxide and
related analogs.
[0171] The oily phase of the emulsions of this invention may be
constituted from known ingredients in a known manner. While the
phase may comprise merely an emulsifier (otherwise known as an
emulgent), it desirably comprises a mixture of at least one
emulsifier with a fat or an oil or with both a fat and an oil.
Preferably, a hydrophilic emulsifier is included together with a
lipophilic emulsifier which acts as a stabilizer. It is also
preferred to include both an oil and a fat. Together, the
emulsifier(s) with or without stabilizer(s) make up the so-called
emulsifying wax, and the wax together with the oil and fat make up
the so-called emulsifying ointment base which forms the oily
dispersed phase of the cream formulations. Emulgents and emulsion
stabilizers suitable for use in the formulation of the invention
include Tween.RTM. 60, Span.RTM. 80, cetostearyl alcohol, benzyl
alcohol, myristyl alcohol, glyceryl mono-stearate and sodium lauryl
sulfate.
[0172] Aqueous suspensions of the invention contain the active
materials in admixture with excipients suitable for the manufacture
of aqueous suspensions. Such excipients include a suspending agent,
such as sodium carboxymethylcellulose, croscarmellose, povidone,
methylcellulose, hydroxypropyl methylcelluose, sodium alginate,
polyvinylpyrrolidone, gum tragacanth and gum acacia, and dispersing
or wetting agents such as a naturally occurring phosphatide (e.g.,
lecithin), a condensation product of an alkylene oxide with a fatty
acid (e.g., polyoxyethylene stearate), a condensation product of
ethylene oxide with a long chain aliphatic alcohol (e.g.,
heptadecaethyleneoxycetanol), a condensation product of ethylene
oxide with a partial ester derived from a fatty acid and a hexitol
anhydride (e.g., polyoxyethylene sorbitan monooleate). The aqueous
suspension may also contain one or more preservatives such as ethyl
or n-propyl p-hydroxy-benzoate, one or more coloring agents, one or
more flavoring agents and one or more sweetening agents, such as
sucrose or saccharin.
[0173] The pharmaceutical composition of a pyrazolyl compound may
be in the form of a sterile injectable preparation, such as a
sterile injectable aqueous or oleaginous suspension. This
suspension may be formulated according to the known art using those
suitable dispersing or wetting agents and suspending agents which
have been mentioned above. The sterile injectable preparation may
also be a sterile injectable solution or suspension in a non-toxic
parenterally acceptable diluent or solvent, such as a solution in
1,3-butane-diol or prepared as a lyophilized powder. Among the
acceptable vehicles and solvents that may be employed are water,
Ringer's solution and isotonic sodium chloride solution. In
addition, sterile fixed oils may conventionally be employed as a
solvent or suspending medium. For this purpose any bland fixed oil
may be employed including synthetic mono- or diglycerides. In
addition, fatty acids such as oleic acid may likewise be used in
the preparation of injectables.
[0174] The amount of active ingredient that may be combined with
the carrier material to produce a single dosage form will vary
depending upon the host treated and the particular mode of
administration. For example, a time-release formulation intended
for oral administration to humans may contain approximately 1 to
1000 mg of active material compounded with an appropriate and
convenient amount of carrier material which may vary from about 5
to about 95% of the total compositions (weight:weight). The
pharmaceutical composition can be prepared to provide easily
measurable amounts for administration. For example, an aqueous
solution intended for intravenous infusion may contain from about 3
to 500 .mu.g of the active ingredient per milliliter of solution in
order that infusion of a suitable volume at a rate of about 30
mL/hr can occur.
[0175] Formulations suitable for parenteral administration include
aqueous and non-aqueous sterile injection solutions which may
contain anti-oxidants, buffers, bacteriostats and solutes which
render the formulation isotonic with the blood of the intended
recipient; and aqueous and non-aqueous sterile suspensions which
may include suspending agents and thickening agents.
[0176] Formulations suitable for topical administration to the eye
also include eye drops wherein the active ingredient is dissolved
or suspended in a suitable carrier, especially an aqueous solvent
for the active ingredient. The active ingredient is preferably
present in such formulations in a concentration of 0.5 to 20%,
advantageously 0.5 to 10% particularly about 1.5% w/w.
[0177] Formulations suitable for topical administration in the
mouth include lozenges comprising the active ingredient in a
flavored basis, usually sucrose and acacia or tragacanth; pastilles
comprising the active ingredient in an inert basis such as gelatin
and glycerin, or sucrose and acacia; and mouthwashes comprising the
active ingredient in a suitable liquid carrier.
[0178] Formulations for rectal administration may be presented as a
suppository with a suitable base comprising for example cocoa
butter or a salicylate.
[0179] Formulations suitable for intrapulmonary or nasal
administration have a particle size for example in the range of 0.1
to 500 microns (including particle sizes in a range between 0.1 and
500 microns in increments microns such as 0.5, 1, 30 microns, 35
microns, etc.), which is administered by rapid inhalation through
the nasal passage or by inhalation through the mouth so as to reach
the alveolar sacs. Suitable formulations include aqueous or oily
solutions of the active ingredient. Formulations suitable for
aerosol or dry powder administration may be prepared according to
conventional methods and may be delivered with other therapeutic
agents such as compounds heretofore used in the treatment or
prophylaxis of HIV infections as described below.
[0180] Formulations suitable for vaginal administration may be
presented as pessaries, tampons, creams, gels, pastes, foams or
spray formulations containing in addition to the active ingredient
such carriers as are known in the art to be appropriate.
[0181] The formulations may be packaged in unit-dose or multi-dose
containers, for example sealed ampoules and vials, and may be
stored in a freeze-dried (lyophilized) condition requiring only the
addition of the sterile liquid carrier, for example water, for
injection immediately prior to use. Extemporaneous injection
solutions and suspensions are prepared from sterile powders,
granules and tablets of the kind previously described. Preferred
unit dosage formulations are those containing a daily dose or unit
daily sub-dose, as herein above recited, or an appropriate fraction
thereof, of the active ingredient.
[0182] The invention further provides veterinary compositions
comprising at least one active ingredient as above defined together
with a veterinary carrier therefore. Veterinary carriers are
materials useful for the purpose of administering the composition
and may be solid, liquid or gaseous materials which are otherwise
inert or acceptable in the veterinary art and are compatible with
the active ingredient. These veterinary compositions may be
administered parenterally, orally or by any other desired
route.
[0183] Combination Therapy
[0184] A pyrazolyl compound of the invention may be combined in a
pharmaceutical combination formulation, or dosing regimen as
combination therapy, with a second compound that has
anti-hyperproliferative properties or that is useful for treating a
hyperproliferative disorder (e.g. cancer). The second compound of
the pharmaceutical combination formulation or dosing regimen
preferably has complementary activities to the pyrazolyl compound
of the combination such that they do not adversely affect each
other. Such molecules are suitably present in combination in
amounts that are effective for the purpose intended.
[0185] The combination therapy may be administered as a
simultaneous or sequential regimen. When administered sequentially,
the combination may be administered in two or more administrations.
The combined administration includes coadministration, using
separate formulations or a single pharmaceutical formulation, and
consecutive administration in either order, wherein preferably
there is a time period while both (or all) active agents
simultaneously exert their biological activities.
[0186] Suitable dosages for any of the above coadministered agents
are those presently used and may be lowered due to the combined
action (synergy) of the newly identified agent and other
chemotherapeutic agents or treatments.
[0187] The combination therapy may provide "synergy" and prove
"synergistic", i.e. the effect achieved when the active ingredients
used together is greater than the sum of the effects that results
from using the compounds separately. A synergistic effect may be
attained when the active ingredients are: (1) co-formulated and
administered or delivered simultaneously in a combined, unit dosage
formulation; (2) delivered by alternation or in parallel as
separate formulations; or (3) by some other regimen. When delivered
in alternation therapy, a synergistic effect may be attained when
the compounds are administered or delivered sequentially, e.g. by
different injections in separate syringes. In general, during
alternation therapy, an effective dosage of each active ingredient
is administered sequentially, i.e. serially, whereas in combination
therapy, effective dosages of two or more active ingredients are
administered together.
[0188] Metabolites of the Pyrazolyl Compounds
[0189] Also falling within the scope of this invention are the in
vivo metabolic products of the pyrazolyl compounds described
herein, to the extent such products are novel and unobvious over
the prior art. Such products may result for example from the
oxidation, reduction, hydrolysis, amidation, deamidation,
esterification, deesterification, enzymatic cleavage, and the like,
of the administered compound. Accordingly, the invention includes
novel and unobvious compounds produced by a process comprising
contacting a compound of this invention with a mammal for a period
of time sufficient to yield a metabolic product thereof.
[0190] Metabolite products typically are identified by preparing a
radiolabelled (e.g. .sup.14C or .sup.3H) isotope of a compound of
the invention, administering it parenterally in a detectable dose
(e.g. greater than about 0.5 mg/kg) to an animal such as rat,
mouse, guinea pig, monkey, or to man, allowing sufficient time for
metabolism to occur (typically about 30 seconds to 30 hours) and
isolating its conversion products from the urine, blood or other
biological samples. These products are easily isolated since they
are labeled (others are isolated by the use of antibodies capable
of binding epitopes surviving in the metabolite). The metabolite
structures are determined in conventional fashion, e.g. by MS,
LC/MS or NMR analysis. In general, analysis of metabolites is done
in the same way as conventional drug metabolism studies well-known
to those skilled in the art. The conversion products, so long as
they are not otherwise found in vivo, are useful in diagnostic
assays for therapeutic dosing of the pyrazolyl compounds of the
invention.
[0191] Articles of Manufacture
[0192] In another embodiment of the invention, an article of
manufacture, or "kit", containing materials useful for the
treatment of the disorders described above is provided. The article
of manufacture comprises a container and a label or package insert
on or associated with the container. Suitable containers include,
for example, bottles, vials, syringes, blister pack, etc. The
containers may be formed from a variety of materials such as glass
or plastic. The container holds a pyrazolyl compound or formulation
thereof which is effective for treating the condition and may have
a sterile access port (for example the container may be an
intravenous solution bag or a vial having a stopper pierceable by a
hypodermic injection needle). At least one active agent in the
composition is a pyrazolyl compound of the invention. The label or
package insert indicates that the composition is used for treating
the condition of choice, such as cancer. In one embodiment, the
label or package inserts indicates that the composition comprising
the pyrazolyl compound can be used to treat a thromboembolic
disorder. In addition, the label or package insert may indicate
that the patient to be treated is one having a thromboembolic
disorder characterized by excessive bleeding. The label or package
insert may also indicate that the composition can be used to treat
other disorders.
[0193] The article of manufacture may comprise (a) a first
container with a pyrazolyl compound contained therein; and (b) a
second container with a second pharmaceutical formulation contained
therein, wherein the second pharmaceutical formulation comprises a
second compound with anti-hyperproliferative activity. The article
of manufacture in this embodiment of the invention may further
comprise a package insert indicating that the first and second
compounds can be used to treat patients at risk of stroke, thrombus
or thrombosis disorder. Alternatively, or additionally, the article
of manufacture may further comprise a second (or third) container
comprising a pharmaceutically-acceptable buffer, such as
bacteriostatic water for injection (BWFI), phosphate-buffered
saline, Ringer's solution and dextrose solution. It may further
include other materials desirable from a commercial and user
standpoint, including other buffers, diluents, filters, needles,
and syringes.
EXAMPLES
[0194] In order to illustrate the invention, the following examples
are included. However, it is to be understood that these examples
do not limit the invention and are only meant to suggest a method
of practicing the invention. Persons skilled in the art will
recognize that the chemical reactions described may be readily
adapted to prepare a number of other Raf inhibitors of the
invention, and alternative methods for preparing the compounds of
this invention are deemed to be within the scope of this invention.
For example, the synthesis of non-exemplified compounds according
to the invention may be successfully performed by modifications
apparent to those skilled in the art, e.g., by appropriately
protecting interfering groups, by utilizing other suitable reagents
known in the art other than those described, and/or by making
routine modifications of reaction conditions. Alternatively, other
reactions disclosed herein or known in the art will be recognized
as having applicability for preparing other compounds of the
invention.
[0195] In the examples described below, unless otherwise indicated
all temperatures are set forth in degrees Celsius. Reagents were
purchased from commercial suppliers such as Aldrich Chemical
Company, Lancaster, TCI or Maybridge, and were used without further
purification unless otherwise indicated.
[0196] The reactions set forth below were done generally under a
positive pressure of nitrogen or argon or with a drying tube
(unless otherwise stated) in anhydrous solvents, and the reaction
flasks were typically fitted with rubber septa for the introduction
of substrates and reagents via syringe. Glassware was oven dried
and/or heat dried.
[0197] Column chromatography was conducted on a Biotage system
(Manufacturer: Dyax Corporation) having a silica gel column or on a
silica SEP PAK.RTM. cartridge (Waters). .sup.1H NMR spectra were
recorded on a Varian instrument operating at 400 MHz. .sup.1H NMR
spectra were obtained as CDCl.sub.3, d.sub.6-DMSO or d.sub.4 MeOH
solutions (reported in ppm), using chloroform as the reference
standard (7.25 ppm). When peak multiplicities are reported, the
following abbreviations are used: s (singlet), d (doublet), t
(triplet), m (multiplet), br (broadened), dd (doublet of doublets),
dt (doublet of triplets). Coupling constants, when given, are
reported in Hertz (Hz).
Example 1
5-(1-Methyl-5-pyridin-4-yl-1H-pyrazol-4-yl)-2,3-dihydroinden-1-one
oxime 122
##STR00088##
[0199] Step 1: Preparation of
(E)-3-(dimethylamino)-1-(pyridin-4-yl)prop-2-en-1-one: To
4-acetylpyridine (23 mL) in 350 mL toluene was added 55 mL
N,N-dimethylformamide dimethyl acetal. The solution was refluxed
for 3 hours and allowed to stir at ambient temperature overnight.
An additional 33 mL N,N-dimethylformamide dimethyl acetal of was
added. After refluxing for 7 hours, the mixture was concentrated to
leave an orange solid. This material was treated with diethyl
ether. The resulting yellow solid was collected by vacuum
filtration. A total of 25.85 g (71% yield) yellow solid was
obtained. .sup.1H NMR (CDCl.sub.3) was consistent with the desired
structure. LC/MS indicated a single peak with a M+1 molecular ion
of m/z 177.1 by positive ESI (electrospray ionization mass
spectrometry).
[0200] Step 2: Preparation of
4-(1-methyl-1H-pyrazole-5-yl)pyridine: To
(E)-3-(dimethylamino)-1-(pyridin-4-yl)prop-2-en-1-one (1.00 g) in
50 mL ethanol was added 0.32 mL methylhydrazine and the resulting
mixture heated to reflux for 1 hour. The solvent was removed by
evaporation and partitioned between ethyl acetate and water. The
aqueous layer was extracted with another portion of ethyl acetate.
The combined ethyl acetate extracts were washed with brine, dried
over magnesium sulfate, filtered, and evaporated to afford 0.62 g
crude product as a yellow oil. The material was purified by
chromatography, eluting with a 10:1 mixture of
dichloromethane-methanol to yield 0.30 g (64% yield) product as a
yellow oil. .sup.1H NMR (CDCl.sub.3) was consistent with a 3.8:1
mixture of desired product and undesired isomer
(4-(1-methyl-1-H-pyrazole-3-yl)pyridine). LC/MS of the mixture
showed 2 peaks, each with a M+1 molecular ion of m/z 160.2 by
positive ESI.
[0201] Step 3: Preparation of
4-(4-bromo-1-methyl-1H-pyrazol-5-yl)pyridine: To a 3.8:1 isomer mix
of 4-(1-methyl-1H-pyrazole-5-yl)pyridine and
4-(1-methyl-1-H-pyrazole-3-yl)pyridine (0.30 g) in 5 mL chloroform
cooled in ice, was added a solution of 104 .mu.L bromine in 3 mL
chloroform. After 5 h, the mixture was treated with excess
saturated aqueous sodium bicarbonate and diluted with
dichloromethane. The dichloromethane was washed with 3 portions of
saturated aqueous sodium bicarbonate, dried over magnesium sulfate,
filtered, and evaporated to yield 0.39 g crude product as a brown
oil. The crude product was purified by chromatography, eluting with
15:1 ethyl acetate-methanol. Fractions containing the major
component were combined to afford 0.17 g (73% yield) of a colorless
oil. .sup.1H NMR (CDCl.sub.3) was consistent with the desired
structure. LC/MS indicated one major product with a M+1 molecular
ion of m/z 240.3 by positive ESI.
[0202] Step 4: Preparation of (E)-5-bromo-2,3-dihydroinden-1-one
O-methyl oxime: To 5-bromoindanone (6.03 g) in 40 mL ethanol was
added 3.58 g methoxylamine hydrochloride and 3.5 mL pyridine. The
mixture was stirred at ambient temperature for 1 hour and heated at
80.degree. C. for 3 hours. The volatiles were removed under vacuum
and the solid residue partitioned between ethyl acetate and
saturated aqueous sodium bicarbonate. The ethyl acetate was washed
with brine, dried over magnesium sulfate, filtered, and evaporated
under vacuum to yield 6.74 g (98% yield) of a tan solid. .sup.1H
NMR (CDCl.sub.3) was consistent with an isomeric mixture of the
desired structure, with the E oxime isomer as the major component.
LC/MS indicated 2 close peaks, both with a M+1 molecular ion of m/z
242.0 by positive ESI.
[0203] Step 5: Preparation of
(E)-1-(methoxyimino)-2,3-dihydro-1H-inden-5-ylboronic acid: To
(E)-5-bromo-2,3-dihydroinden-1-one O-methyl oxime (3.00 g) in 100
mL tetrahydrofuran cooled in dry ice-acetone, was added dropwise,
5.5 mL of a 2.5 M n-butyllithium solution in hexane. A tan
precipitate formed. After 30 minutes at -78.degree. C., 3.1 mL
trimethylborate was added and the mixture allowed to warm to
ambient temperature and allowed to stir overnight. The reaction
mixture was evaporated under vacuum and acidified to pH 1 with 6M
hydrochloric acid, stirred 15 min, basified with 2M sodium
hydroxide, washed with 3 portions diethyl ether, acidified to pH 1
with 6M hydrochloric acid and extracted with 3 portions of ethyl
acetate. The combined ethyl acetate extracts were washed with
brine, dried over magnesium sulfate, filtered, and evaporated under
vacuum to yield 1.95 g pale yellow solid. The crude solid was
triturated with a hexane/diethyl ether mixture to afford 1.46 g
(57% yield) product as a tan solid. .sup.1H NMR (d.sub.6 DMSO) was
consistent with the desired product. LC/MS showed one peak with a
M+1 molecular ion of m/z 206.1 by positive ESI.
[0204] Step 6: Preparation of
(E)-5-(1-methyl-5-(pyridin-4-yl)-1H-pyrazol-4-yl)-2,3-dihydroinden-1-one
O-methyl oxime: To a solution of
4-(4-bromo-1-methyl-1H-pyrazol-5-yl)pyridine (0.17 g) in 7 mL
dimethoxyethane and 3 mL water, was added 0.146 g
(E)-1-(methoxyimino)-2,3-dihydro-1H-inden-5-ylboronic acid (product
of Example 1, Step 5), and 0.79 g K.sub.2CO.sub.3. To this mixture
was added approximately 20 mg
tetrakis(triphenyphosphine)palladium(0) and the suspension heated
to reflux. After 4 hours, the cooled reaction mixture was filtered
through celite and the filter cake was washed with ethyl acetate.
The filtrate was washed with water, brine, dried over magnesium
sulfate, filtered, and evaporated under vacuum to yield 0.63 g
crude product as a tan semisolid. The material was suspended in
ethyl acetate and filtered. The filtrate was evaporated under
vacuum, and the residue purified by chromatography, eluting with
ethyl acetate to provide 82.6 mg (36% yield) product as a colorless
glass. .sup.1H NMR (CDCl.sub.3) was consistent with the desired
product. LC/MS indicated one peak with a M+1 molecular ion of m/z
319.2 by positive ESI.
[0205] Step 7: Preparation of
5-(1-methyl-5-(pyridin-4-yl)-1H-pyrazol-4-yl)-2,3-dihydroinden-1-one:
To
(E)-5-(1-methyl-5-(pyridin-4-yl)-1H-pyrazol-4-yl)-2,3-dihydroinden-1-one
O-methyl oxime (82.6 mg) in 2 mL dioxane and 5 mL acetone was added
0.5 mL 5M hydrochloric acid. The mixture was heated at reflux for 2
hours. The cooled reaction mixture was evaporated under vacuum and
partitioned between saturated aqueous sodium bicarbonate and ethyl
acetate. The ethyl acetate was washed with brine, dried over
magnesium sulfate, filtered, and evaporated under vacuum to yield
0.05 g crude product as an oil. The material was purified by
chromatography, eluting with a 15:1 mixture of ethyl
acetate-methanol to yield 50.9 mg (68% yield) of a colorless oil.
.sup.1H NMR (CDCl.sub.3) was consistent with the desired product.
LC/MS indicated one peak with a M+1 molecular ion of m/z 290.4 by
positive ESI.
[0206] Step 8: Preparation of
5-(1-methyl-5-pyridin-4-yl-1H-pyrazol-4-yl)-indan-1-one oxime 122:
To
5-(1-methyl-5-(pyridin-4-yl)-1H-pyrazol-4-yl)-2,3-dihydroinden-1-one
(0.051 g) in 3 mL ethanol was added 1 mL water and hydroxylamine
hydrochloride (37 mg). The mixture was heated to reflux. After 2
hours, the mixture was cooled and evaporated under vacuum. The
residue was partitioned between saturated aqueous sodium
bicarbonate and ethyl acetate. The ethyl acetate was washed with
brine, dried over magnesium sulfate, filtered, and evaporated under
vacuum to afford crude product. The material was triturated with a
small portion of methanol and dried to yield 40 mg (74% yield) of a
white solid. .sup.1H NMR (CDCl.sub.3) was consistent with
5-(1-methyl-5-pyridin-4-yl-1H-pyrazol-4-yl)-indan-1-one oxime 122.
LC/MS showed a single peak with a M+1 molecular ion of m/z 305.2 by
positive ESI.
Example 2
5-(1-(2-hydroxyethyl)-5-pyridin-4-yl-1H-pyrazol-4-yl)-2,3-dihydroinden-1-o-
ne oxime 123
##STR00089##
[0208] Step 1: Preparation of
2-(5-(pyridin-4-yl)-1H-pyrazol-1-yl)ethanol: To
(E)-3-(dimethylamino)-1-(pyridin-4-yl)prop-2-en-1-one (1.00 g;
Example 1, Step 1) in 50 mL ethanol, was added
2-hydroxyethylhydrazine (0.404 mL) and the mixture heated to
reflux. After 4 hours, the reaction was evaporated under vacuum to
yield a yellow oil. The crude product was purified by
chromatography, eluting with a mixture of 4:1 ethyl
acetate-methanol to afford 0.69 g (64% yield) of product as a white
semisolid. .sup.1H NMR (CDCl.sub.3) indicated a 3.4:1 mixture of
isomers consistent with the desired product as the major component
and 2-(3-(pyridin-4-yl)-1H-pyrazol-1-yl)ethanol as the minor
component. LC/MS showed 2 close peaks, both with a M+1 molecular
ion of m/z 190.2 by positive ESI.
[0209] Step 2: Preparation of
2-(4-bromo-5-(pyridin-4-yl)-1H-pyrazol-1-yl)ethanol: To of a 3.4:1
isomer mixture of 2-(5-(pyridin-4-yl)-1H-pyrazol-1-yl)ethanol and
2-(3-(pyridin-4-yl)-1H-pyrazol-1-yl)ethanol (0.69 g) in 10 mL
chloroform cooled in ice, was added a solution of 200 .mu.L bromine
in 5 mL chloroform. After 1 hour, the mixture was treated with
saturated aqueous sodium bicarbonate and diluted with
dichloromethane. The dichloromethane layer was washed with 3
portions of saturated aqueous sodium bicarbonate, dried over
magnesium sulfate, filtered, and evaporated to yield 0.96 g crude
product as a brown oil. The crude material was purified by
chromatography, eluting with a mixture of 8:1 ethyl
acetate-methanol to afford 0.23 g (23% yield) of product as a
colorless oil. .sup.1H NMR (CDCl.sub.3) was consistent with the
desired product. LC/MS indicated one peak with a M+1 molecular ion
of m/z 270.2 by positive ESI.
[0210] Step 3: Preparation of (E)-5-bromo-2,3-dihydroinden-1-one
O-benzyl oxime: To 5-bromoindanone (5.66 g) in 60 mL ethanol was
added 5.57 g O-benzylhydroxylamine hydrochloride and 3.2 mL
pyridine. The mixture was stirred at ambient temperature for 5
hours. The volatiles were removed under vacuum and the solid
residue partitioned between saturated aqueous sodium bicarbonate
and ethyl acetate. The ethyl acetate was washed with brine, dried
over magnesium sulfate, filtered, and evaporated under vacuum to
afford crude product as an amber oil. The crude material was
crystallized from hexane to afford 5.43 g (84% yield) light tan
solid. .sup.1H NMR (CDCl.sub.3) was consistent with the desired
product. LC/MS indicated a single peak with a M+1 molecular ion of
m/z 318.1 by positive ESI.
[0211] Step 4: Preparation of
(E)-1-(benzyloxyimino)-2,3-dihydro-1H-inden-5-ylboronic acid: To
(E)-5-bromo-2,3-dihydroinden-1-one O-benzyl oxime (5.00 g; Example
2, step 3) in 200 mL tetrahydrofuran cooled in dry ice/acetone was
added 7.0 mL of a 2.5 M solution of n-butyllithium in hexane
dropwise. After 20 minutes, 3.9 mL trimethylborate was added at
-78.degree. C. to the clear brown solution and the reaction mixture
was allowed to warm to ambient temperature for 2 hours. The solvent
was evaporated under vacuum and the residue treated with water and
acidified with 1M hydrochloric acid until pH 3. After stirring 5
minutes, the mixture was basified with 2M sodium hydroxide and
extracted with 3 portions of diethyl ether. The diethyl ether
extracts were extracted with 2 portions of 0.5M sodium hydroxide.
The combined aqueous extracts were acidified to pH 1 with 1M
hydrochloric acid and extracted with 2 portions of ethyl acetate.
The combined ethyl acetate extracts were dried over magnesium
sulfate, filtered, and evaporated to yield 3.46 g (78% yield)
product as a tan solid. .sup.1H NMR (d.sub.6 DMSO) was consistent
with the desired product. LC/MS indicated a peak with a M+1
molecular ion of m/z 282.1 by positive ESI.
[0212] Step 5: Preparation of
(E)-5-(1-(2-hydroxyethyl)-5-(pyridin-4-yl)-1H-pyrazol-4-yl)-2,3-dihydroin-
den-1-one O-benzyl oxime: To
2-(4-bromo-5-(pyridin-4-yl)-1H-pyrazol-1-yl)ethanol (0.050 g;
Example 2, Step 2) dissolved in 5 mL acetonitrile was added 2 mL
water, (E)-1-(benzyloxyimino)-2,3-dihydro-1H-inden-5-ylboronic acid
(0.058 g; Example 2, Step 4), and 0.155 g potassium carbonate. The
mixture was purged with nitrogen and about 20 mg
tetrakis(triphenyphosphine)palladium(0) catalyst was added. The
mixture was heated at 75.degree. C. After 1 hour, the reaction
mixture was filtered through celite, the filter cake washed with
ethyl acetate, and the filtrate evaporated under vacuum. The
residue was partitioned between ethyl acetate and water. The ethyl
acetate was washed with saturated aqueous sodium carbonate, brine,
dried over magnesium sulfate, filtered, and evaporated under vacuum
to yield 0.05 g crude product as a colorless glass. The crude
material was purified by chromatography, eluting with a mixture of
8:1 dichloromethane-methanol to afford 18.6 mg (23% yield) product
as a colorless glass. .sup.1H NMR (CDCl.sub.3) was consistent with
the desired structure. LC/MS showed one peak with a M+1 molecular
ion of m/z 425.2 by positive ESI.
[0213] Step 6: Preparation of
5-(1-(2-hydroxyethyl)-5-pyridin-4-yl-1H-pyrazol-4-yl)-2,3-dihydroinden-1--
one oxime 123: To
(E)-5-(1-(2-hydroxyethyl)-5-(pyridin-4-yl)-1H-pyrazol-4-yl)-2,3-dihydroin-
den-1-one O-benzyl oxime (15.8 mg; Example 2, Step 5) in 5 mL
methanol was added 1 drop 6M hydrochloric acid and about 5 mg
palladium hydroxide (20% by weight on carbon). A hydrogen-filled
balloon was attached to the reaction vessel and the mixture was
stirred at ambient temperature for 1 hour. The mixture was filtered
through celite and the celite was washed with methanol. The
filtrate was concentrated under vacuum and the residue was
partitioned between saturated aqueous sodium carbonate and ethyl
acetate. The ethyl acetate was washed with brine, dried over
magnesium sulfate, filtered, and evaporated to yield 9.1 mg (73%
yield) of a white solid. .sup.1H NMR (d4 MeOH) was consistent with
the desired structure 123. LC/MS showed one peak with a M+1
molecular ion of m/z 335.3 by positive ESI.
Example 3
5-(1-Methyl-3-pyridin-4-yl-1H-pyrazol-4-yl)-2,3-dihydroinden-1-one
oxime 113
##STR00090##
[0215] Step 1: Preparation of 4-(1H-pyrazol-3-yl)pyridine: To 50 mL
absolute ethanol was added
(E)-3-(dimethylamino)-1-(pyridin-4-yl)prop-2-en-1-one (20 g;
Example 1, Step 1). When all solids dissolved, 6.33 mL hydrazine
monohydrate was added dropwise with stirring. After 30 minutes an
additional 1.1 mL hydrazine hydrate was added. After 2 hours the
reaction was treated with water and the yellow solution was
concentrated under vacuum until a slurry formed. The resulting
solid product was collected by vacuum filtration, washed with
water, and dried under high vacuum overnight to yield 15.27 g (93%
yield) of product as off-white crystals. .sup.1H NMR (CDCl.sub.3)
was consistent with a tautomeric mixture of the desired structure.
LC/MS showed one peak with a M+1 molecular ion of m/z 146.4 by
positive ESI.
[0216] Step 2: Preparation of 4-(1-methyl-1H-pyrazol-3-yl)pyridine:
To 150 mL tetrahydrofuran was added 4-(1H-pyrazol-3-yl)pyridine
(4.96 g; Example 3, Step 1) and the solution cooled in ice. To this
was added slowly 1.64 g of a 60% suspension of sodium hydride in
mineral oil. After gas evolution ceased, the mixture was stirred at
ambient temperature for 30 min, cooled back down in ice, and 3.4 mL
dimethylsulfate was added. The mixture was allowed to slowly warm
to ambient temperature. After 1 hour the reaction mixture was
evaporated and the dark brown residue partitioned between saturated
aqueous sodium bicarbonate and ethyl acetate. The ethyl acetate was
washed with saturated aqueous sodium bicarbonate, brine, dried over
magnesium sulfate, filtered, and evaporated under vacuum to afford
4.6 g crude product as a pale yellow solid. The crude material was
taken up in a diethyl ether and hexane mixture, filtered through
celite, and allowed to crystallize. A total of 2.98 g (55% yield)
pale yellow crystals were obtained. .sup.1H NMR (CDCl.sub.3) was
consistent with a single isomer of the desired structure. LC/MS
showed one peak with a M+1 molecular ion of m/z 160.5 by positive
ESI.
[0217] Step 3: Preparation of
4-(4-bromo-1-methyl-1H-pyrazol-3-yl)pyridine: To a solution of
4-(1-methyl-1H-pyrazol-3-yl)pyridine 2.98 g; Example 3, Step 2) in
50 mL chloroform cooled in ice, was added dropwise, a solution of
1.15 mL bromine in 30 mL chloroform. About 15 mL methanol was added
to help dissolution of the yellow precipitate that formed. After 4
hours, the reaction mixture was quenched with saturated aqueous
sodium bicarbonate and diluted with dichloromethane. The
dichloromethane layer was washed with saturated aqueous sodium
bicarbonate, brine, dried over magnesium sulfate, filtered, and
evaporated to yield the crude product as a yellow semisolid. The
crude material was slurried in ethyl acetate and filtered through
celite. The filtrate was evaporated to yield 4.18 g product as a
yellow solid. .sup.1H NMR (CDCl.sub.3) was consistent with the
desired structure. LC/MS showed one peak with a M+1 molecular ion
of m/z 240.5 by positive ESI.
[0218] Step 4: Preparation of
5-(1-methyl-3-(pyridin-4-yl)-1H-pyrazol-4-yl)-2,3-dihydroinden-1-one
O-benzyl oxime: To
(E)-1-(benzyloxyimino)-2,3-dihydro-1H-inden-5-ylboronic acid (3.00
g; Example 2, Step 4) in 32 mL acetonitrile and 8.0 mL water was
added 4-(4-bromo-1-methyl-1H-pyrazol-3-yl)pyridine (2.00 g; Example
3, Step 3). The solution was deoxygenated by bubbling nitrogen
through the solution for 10 min. To the resulting mixture was added
5.62 g potassium carbonate, 10 mL N,N-dimethylformamide, and 290 mg
tetrakis(triphenyphosphine)palladium(0). The mixture was then
refluxed for 8 hours (over the course of this time a small portion
of catalyst was added twice). The cooled reaction mixture was
filtered through celite and the filter cake was washed with ethyl
acetate. The filtrate was washed with water, brine, dried over
sodium sulfate, filtered, and evaporated under vacuum to yield 5.1
g crude product as an oil. The crude material was purified by
chromatography, eluting with ethyl acetate to afford 2.50 g (80%
yield) product as a light yellow foam. .sup.1H NMR (CDCl.sub.3) was
consistent with the desired structure. LC/MS showed one peak with a
M+1 molecular ion of m/z 395.2 by positive ESI.
[0219] Step 5: Preparation of
5-(1-Methyl-3-pyridin-4-yl-1H-pyrazol-4-yl)-2,3-dihydroinden-1-one
oxime 113: To
5-(1-methyl-3-(pyridin-4-yl)-1H-pyrazol-4-yl)-2,3-dihydroinden-1--
one O-benzyl oxime (2.50 g; Example 3, Step 4) in 60 mL methanol
was added 0.89 g palladium hydroxide (20% by weight on carbon) and
6.3 mL 6M hydrochloric acid. A hydrogen-filled balloon was attached
to the reaction vessel and the mixture was stirred at ambient
temperature for 2 hours. The reaction mixture was filtered through
celite and the celite was washed with methanol. The filtrate was
concentrated under vacuum and the residue was partitioned between
saturated aqueous sodium bicarbonate and ethyl acetate. The ethyl
acetate was washed with brine and evaporated to yield 1.0 g of
crude product as a white solid. The aqueous layer was reextracted
with two portions of n-butanol. The n-butanol was evaporated under
vacuum to afford an additional 0.4 g crude product as a white
solid. The solids were combined and washed with two 15 mL portions
of water, dissolved in a 1:1 mixture of dichloromethane-methanol,
filtered, and the filtrate evaporated under vacuum. The residue was
triturated with ethyl acetate and dried to afford 0.98 g (51%
yield) of product as a white solid. .sup.1H NMR (CDCl.sub.3) was
consistent with the desired structure 113. LC/MS showed a peak with
a M+1 molecular ion of m/z 305.4 by positive ESI.
Example 4
5-(1-(2-hydroxyethyl)-3-pyridin-4-yl-1H-pyrazol-4-yl)-2,3-dihydroinden-1-o-
ne oxime
##STR00091##
[0221] Step 1: Preparation of 4-(4-bromo-1H-pyrazol-3-yl)pyridine:
To 4-(1H-pyrazol-3-yl)pyridine (36.1 g; Example 3, Step 1) in 600
mL chloroform and 600 mL acetic acid, was added 20.4 g sodium
acetate and the mixture was stirred for 1 hour until homogeneous.
The solution was then cooled in an ice bath and a solution of 12 mL
bromine in 150 mL acetic acid was added over a 30 minute period. A
precipitate formed during bromine addition. After 2 hours, the
reaction mixture was diluted with dichloromethane and basified with
4M sodium hydroxide to pH 4. The layers were separated and the
aqueous layer was extracted with dichloromethane. The combined
dichloromethane extracts were washed with water, then brine, dried
over magnesium sulfate and applied to a silica gel plug. The plug
was washed with dichloromethane and product was eluted with ethyl
acetate and then a 19:1 mixture of ethyl acetate/methanol. The
eluent contained 33.2 g (60% yield) product as a white solid.
.sup.1H NMR (CDCl.sub.3) was consistent with a tautomeric mixture
of the desired structure. LC/MS showed one peak with a M+1
molecular ion of m/z 226.2 by positive ESI.
[0222] Step 2: Preparation of
2-(4-bromo-3-(pyridin-4-yl)-1H-pyrazol-1-yl)ethyl acetate: To
4-(4-bromo-1H-pyrazol-3-yl)-pyridine (0.50 g; Example 4, Step 1) in
7 mL N,N-dimethylformamide was added 1.09 g cesium carbonate and
0.27 mL 2-bromoethyl acetate. The resulting suspension was stirred
at 60.degree. C. After 1 hour the reaction mixture was diluted with
water and extracted with 2 portions of ethyl acetate. The combined
ethyl acetate extracts were washed with brine, dried over magnesium
sulfate, filtered, and evaporated to yield 0.69 g (100% yield)
product as a yellow oil. .sup.1H NMR (CDCl.sub.3) was consistent
with a 9:1 isomer ratio of the desired structure and the
regioisomer 2-(4-bromo-5-(pyridin-4-yl)-1H-pyrazol-1-yl)ethyl
acetate. LC/MS showed two close peaks, each with a M+1 molecular
ion of m/z 312.2 by positive ESI.
[0223] Step 3: Preparation of
2-(4-bromo-3-(pyridin-4-yl)-1H-pyrazol-1-yl)ethanol: To a 9:1
isomer mixture of 2-(4-bromo-3-(pyridin-4-yl)-1H-pyrazol-1-yl)ethyl
acetate and regioisomer
2-(4-bromo-5-(pyridin-4-yl)-1H-pyrazol-1-yl)ethyl acetate (0.69 g;
Example 4, Step 1) in 20 mL methanol was added 5 mL water and 3 mL
2M sodium hydroxide. After 10 minutes the reaction mixture was
concentrated under vacuum and the aqueous residue was extracted
twice with ethyl acetate. The combined ethyl acetate extracts were
washed with brine, dried over magnesium sulfate, filtered, and
evaporated under vacuum to yield 0.54 g crude product as a white
solid. The crude material was recrystallized from ethyl acetate to
yield 0.39 g (60% yield) white solid in 2 crops. .sup.1H NMR
(CDCl.sub.3) was consistent with 1 isomer of the desired
product.
[0224] Step 4: Preparation of
(E)-5-(1-(2-hydroxyethyl)-3-(pyridin-4-yl)-1H-pyrazol-4-yl)-2,3-dihydroin-
den-1-one O-benzyl oxime: To
2-(4-bromo-3-(pyridin-4-yl)-1H-pyrazol-1-yl)ethanol (100 mg;
Example 4, step 3) in 7 mL acetonitrile and 3 mL water, was added
136 mg (E)-1-(benzyloxyimino)-2,3-dihydro-1H-inden-5-ylboronic acid
(product of Example 2, Step 4), and 155 mg potassium carbonate. The
mixture was purged with nitrogen and about 43 mg
tetrakis(triphenyphosphine)palladium(0) catalyst was added. The
mixture was heated at 80.degree. C. After 1 hour, the reaction
mixture was filtered through celite, the filter cake washed with
ethyl acetate, and filtrate evaporated under vacuum. The residue
was purified by chromatography, eluting with a mixture of 8:1 ethyl
acetate-methanol to afford 110 mg (69% yield) product as a white
solid. .sup.1H NMR (CDCl.sub.3) was consistent with the desired
structure. LC/MS showed one peak with a M+1 molecular ion of m/z
425.2 by positive ESI.
[0225] Step 5: Preparation of
5-(1-(2-hydroxyethyl)-3-pyridin-4-yl-1H-pyrazol-4-yl)-2,3-dihydroinden-1--
one oxime 110: To
(E)-5-(1-(2-hydroxyethyl)-3-(pyridin-4-yl)-1H-pyrazol-4-yl)-2,3-dihydroin-
den-1-one O-benzyl oxime (31.9 mg; Example 4, Step 4) in 5 mL
methanol was added 38 .mu.L 6M hydrochloric acid and about 5 mg
palladium hydroxide (20% by weight on carbon). A hydrogen-filled
balloon was attached to the reaction vessel and the mixture was
stirred at ambient temperature for 1 hour. The mixture was filtered
through celite, the celite was washed with methanol, and the
filtrate treated with a small amount of saturated aqueous sodium
bicarbonate. The filtrate was concentrated under vacuum and the
residue was purified by chromatography, eluting with a 10:1 mixture
of dichloromethane-methanol to yield 13.2 mg (53% yield) of a white
solid. .sup.1H NMR (CDCl.sub.3) was consistent with the desired
structure 110. LC/MS showed one peak with a M+1 molecular ion of
m/z 335.4 by positive ESI.
Example 5
5-(1-(piperidin-4-yl)-3-pyridin-4-yl-1H-pyrazol-4-yl)-2,3-dihydroinden-1-o-
ne oxime
##STR00092##
[0227] Step 1: Preparation of benzyl
4-(4-bromo-3-(pyridin-4-yl)-1H-pyrazol-1-yl)piperidine-1-carboxylate:
To 4-(4-bromo-1H-pyrazol-3-yl)-pyridine (1.00 g; Example 4, Step 1)
in 13 mL N,N-dimethylformamide was added 2.18 g cesium carbonate
and then dropwise, 1.16 mL 4-bromo-N-Z-piperidine. The resulting
suspension was stirred at ambient temperature for 10 minutes then
heated at 55.degree. C. overnight. The cooled reaction mixture was
diluted with water and extracted with 2 portions of ethyl acetate.
The ethyl acetate was washed with water, brine, dried over
magnesium sulfate, filtered, and evaporated under vacuum to yield
2.48 g crude product as a colorless oil. The crude material was
purified by chromatography, eluting with a 9:9:2 mixture of
dichloromethane-ethyl acetate-methanol to afford 0.86 g (44% yield)
product as a colorless oil. .sup.1H NMR (CDCl.sub.3) was consistent
with a 15:1 mixture of the desired structure and regioisomer benzyl
4-(4-bromo-5-(pyridin-4-yl)-1H-pyrazol-1-yl)piperidine-1-carboxylate.
LC/MS showed two peaks, each with a M+1 molecular ion of m/z 444.3
by positive ESI.
[0228] Step 2: Preparation of (E)-benzyl
4-(4-(1-(benzyloxyimino)-2,3-dihydro-1H-inden-5-yl)-5-(pyridin-4-yl)-1H-p-
yrazol-1-yl)piperidine-1-carboxylate: To benzyl
4-(4-bromo-3-(pyridin-4-yl)-1H-pyrazol-1-yl)piperidine-1-carboxylate
(0.32 g; Example 5, Step 1) in 10 mL acetonitrile and 3 mL water
was added 0.315 g
(E)-1-(benzyloxyimino)-2,3-dihydro-1H-inden-5-ylboronic acid
(prepared as described in example 2, step 4) and 0.30 g potassium
carbonate. The reaction mixture was purged with nitrogen and 84 mg
tetrakis(triphenyphosphine)palladium(0) catalyst was added. The
reaction mixture heated to 80.degree. C. for 2 hours, filtered
through celite, and the filter cake was washed with acetonitrile.
The filtrate was evaporated under vacuum to yield crude product.
The residue was purified by chromatography, eluting with ethyl
acetate to afford 0.36 g (83% yield) product as a colorless oil.
.sup.1H NMR (CDCl.sub.3) was consistent with the desired structure.
LC/MS showed one peak with a M+1 molecular ion of m/z 598.3 by
positive ESI.
[0229] Step 3: Preparation of
5-(1-(piperidin-4-yl)-3-pyridin-4-yl-1H-pyrazol-4-yl)-2,3-dihydroinden-1--
oneoxime 105: To
(E)-benzyl-4-(4-(1-(benzyloxyimino)-2,3-dihydro-1H-inden-5-yl)-5-(pyridin-
-4-yl)-1H-pyrazol-1-yl)piperidine-1-carboxylate (0.35 g; Example 5,
Step 2) in 10 mL methanol was added 390 .mu.L 6M hydrochloric acid
and 40 mg palladium hydroxide (20% by weight on carbon). A
hydrogen-filled balloon was attached to the reaction vessel and the
mixture was stirred at ambient temperature for 1.5 hours. The
mixture was filtered through celite and the celite was washed with
methanol. The filtrate was concentrated under vacuum and the
residue was dissolved in 10 mL dichloromethane and 10 mL methanol,
treated with 1.64 g of MP-carbonate resin (2.86 mmol/g loading) for
0.5 hours, filtered and evaporated under vacuum. The crude product
was purified by trituration with dichloromethane to yield 0.1492 g
(68% yield) of a white solid. .sup.1H NMR (d4 MeOH) was consistent
with the desired structure 105. LC/MS showed one peak with a M+1
molecular ion of m/z 374.4 by positive ESI.
Example 6
5-(1-(5-(aminomethyl)pyridin-2-yl)-3-pyridin-4-yl-1H-pyrazol-4-yl)-2,3-dih-
ydroinden-1-one oxime 117
##STR00093##
[0231] Step 1: Preparation of
6-(4-bromo-3-(pyridin-4-yl)-1H-pyrazol-1-yl)nicotinonitrile: To
4-(4-bromo-1H-pyrazol-3-yl)-pyridine (200 mg; Example 4, Step 1) in
5 mL DMF was added slowly 0.043 g of a 60% suspension of sodium
hydride in mineral oil. After 20 minutes 0.124 g
6-chloronicotinonitrile was added. The mixture was heated at
80.degree. C. overnight. The cooled reaction mixture was diluted
with water and resulting solid was collected by vacuum filtration
to yield 0.26 g (89% yield) product as a tan solid. .sup.1H NMR
(CDCl.sub.3) was consistent with the desired structure. LC/MS
showed one peak with a M+1 molecular ion of m/z 328.5 by positive
ESI.
[0232] Step 2: Preparation of
(E)-6-(4-(1-(benzyloxyimino)-2,3-dihydro-1H-inden-5-yl)
3-(pyridin-4-yl)-1H-pyrazol-1-yl)nicotinonitrile: To
6-(4-bromo-3-(pyridin-4-yl)-1H-pyrazol-1-yl)nicotinonitrile (0.26
g; Example 6, step 1) in 16 mL acetonitrile and 4 mL water was
added (E)-1-(benzyloxyimino)-2,3-dihydro-1H-inden-5-ylboronic acid
(0.34 g; Example 2, Step 4) and 0.33 g potassium carbonate. The
reaction mixture was purged with nitrogen and 46 mg
tetrakis(triphenyphosphine)palladium(0) catalyst was added. The
reaction mixture was heated to 80.degree. C. for 1.5 hours,
filtered through celite, the filter cake was washed with
acetonitrile. The filtrate was evaporated to yield crude product,
which was purified by chromatography, eluting with a 10:1 mixture
of dichloromethane-methanol to afford 0.15 g (36% yield) product as
a pale yellow solid. .sup.1H NMR (CDCl.sub.3) was consistent with
the desired structure. LC/MS showed one peak with a M+1 molecular
ion of m/z 483.2 by positive ESI.
[0233] Step 3: Preparation of
5-(1-(5-(aminomethyl)pyridin-2-yl)-3-pyridin-4-yl-1H-pyrazol-4-yl)-2,3-di-
hydroinden-1-one oxime 117: To
(E)-6-(4-(1-(benzyloxyimino)-2,3-dihydro-1H-inden-5-yl)
3-(pyridin-4-yl)-1H-pyrazol-1-yl)nicotinonitrile (0.15 g; Example
6, Step 2) in 10 mL methanol was added 205 .mu.L 6M hydrochloric
acid and 11 mg palladium hydroxide (20% by weight on carbon). A
hydrogen-filled balloon was attached to the reaction vessel and the
mixture was stirred at ambient temperature for 2 hours. The mixture
was filtered through celite and the celite was washed with
methanol. The filtrate was concentrated under vacuum and the
residue was dissolved in 10 mL methanol, treated with 1.00 g of
MP-carbonate resin (2.86 mmol/g loading) for 20 minutes, filtered
and evaporated under vacuum. The crude product was purified by
chromatography, eluting with a mixture of 50:10:1
dichloromethane-methanol-triethylamine to yield 0.088 g (71% yield)
of a white solid. .sup.1H NMR (d.sub.6 DMSO) was consistent with
the desired structure 117. LC/MS showed one peak with a M+1
molecular ion of m/z 397.3 by positive ESI.
Example 7
5-(2-(pDYidin-4-yl)pyrazolo[1.5-a]pfrimidin-3-yl)-2,3-dihydroinden-1-one
oxime 124
##STR00094##
[0235] Step 1: Preparation of 3-oxo-3-(pyridin-4-yl)propanenitrile:
To 20.00 g methyl isonicotinate in 290 mL dry toluene, was added a
solution of 50 mL 4.37M sodium methoxide in methanol, followed by
19.2 mL acetonitrile. The resulting mixture was refluxed for 16
hours and concentrated to dryness. The solids were then taken up in
a minimal amount of water and the pH adjusted to pH 5-6 with
concentrated hydrochloric acid. The mixture was diluted with water
and extracted with a mixture of 1:10 isopropyl
alcohol-dichloromethane. The organic extracts dried over sodium
sulfate, filtered, and evaporated to yield 3.9 g product as a dark
foam. .sup.1H NMR (d6 DMSO) was consistent with the desired
structure. MS showed a M+1 molecular ion of m/z 145.2 by positive
APCI (atmospheric pressure chemical ionization mass
spectrometry).
[0236] Step 2: Preparation of 5-(pyridin-4-yl)-1-H-pyrazol-3-amine:
To 3-oxo-3-(pyridin-4-yl)propanenitrile (3.40 g; Example 7, Step 1)
suspended in 230 mL ethanol was added 2.92 mL hydrazine. The
mixture refluxed for 3 hours and concentrated to dryness. The
residue was purified by chromatography, eluting with a mixture of
10% methanol in dichloromethane to yield 0.80 g (21% yield) product
as a light yellow solid. MS showed a M+1 molecular ion of m/z 161.3
by positive APCI.
[0237] Step 3: Preparation of
2-(pyridin-4-yl)pyrazolo[1.5-a]pyrimidine: To
5-(pyridin-4-yl)-1-H-pyrazol-3-amine (0.50 g; Example 7, Step 2) in
31 mL ethanol was added 0.182 g zinc chloride, 0.54 mL
malonaldehyde, and 7.8 mL concentrated hydrochloric acid. The
mixture was refluxed for 1 hour, cooled, and concentrated to
dryness. The residue taken up in concentrated ammonium hydroxide,
diluted with water, and extracted with a mixture of 10% methanol in
ethyl acetate. The extracts dried over sodium sulfate and
evaporated to afford 0.50 g (81% yield) product as a tan solid.
.sup.1H NMR (d.sub.6 DMSO) was consistent with the desired
structure. MS showed a M+1 molecular ion of m/z 197.4 by positive
APCI.
[0238] Step 4: Preparation of
3-bromo-2-(pyridin-4-yl)pyrazolo[1.5-a]pyrimidine: To
2-(pyridin-4-yl)pyrazolo[1.5-a]pyrimidine (0.30 g) in 15 mL
chloroform was added dropwise a solution of 86 .mu.L bromine in 1
mL chloroform. After 1.5 hours. at ambient temperature, the mixture
was diluted with dichloromethane, washed with 10% aqueous potassium
carbonate, dried over sodium sulfate, filtered, and evaporated to
afford 386 mg (92% yield) product as a tan solid. MS showed a M+1
molecular ion of m/z 277.4 by positive APCI.
[0239] Step 5: Preparation of
5-(2-(pyridin-4-yl)pyrazolo[1.5-a]pyrimidin-3-yl)-2,3-dihydroinden-1-one
O-methyl oxime: To
3-bromo-2-(pyridin-4-yl)pyrazolo[1.5-a]pyrimidine (0.150 g; Example
7, step 4) and
(E)-1-(methoxyimino)-2,3-dihydro-1H-inden-5-ylboronic acid (0.224
g; Example 1, Step 5) in 5.5 mL 1,2-dimethoxyethane, was added
0.167 mL triethylamine and 27 mg
1,1'-bis(diphenylphosphino)ferrocene palladium (II) chloride
catalyst. The mixture was heated at 60.degree. C. for 16 hours,
diluted with ethyl acetate, and washed with 10% aqueous potassium
carbonate, dried over sodium sulfate, filtered, and evaporated to
yield 94 mg (49% yield) product as a yellow solid. .sup.1H NMR
(CDCl.sub.3) was consistent with the desired structure. MS showed a
M+1 molecular ion of m/z 356.2 by positive APCI.
[0240] Step 6: Preparation of
5-(2-(pyridin-4-yl)pyrazolo[1.5-a]pyrimidin-3-yl)-2,3-dihydroinden-1-one:
To
5-(2-(pyridin-4-yl)pyrazolo[1.5-a]pyrimidin-3-yl)-2,3-dihydroinden-1-o-
ne O-methyl oxime (0.090 g; Example 7, Step 5) in 1.3 mL dioxane
was added 1.3 mL 4M hydrochloric acid and the mixture was heated to
100.degree. C. for 5 hours. The cooled reaction mixture was diluted
with ethyl acetate, washed with 10% aqueous potassium carbonate,
dried over sodium sulfate, filtered, and evaporated to yield 75 mg
product as a yellow-brown solid. This material was carried on
without further purification.
[0241] Step 7: Preparation of
5-(2-(pyridin-4-yl)pyrazolo[1.5-a]pyrimidin-3-yl)-2,3-dihydroinden-1-one
oxime 124: To
5-(2-(pyridin-4-yl)pyrazolo[1.5-a]pyrimidin-3-yl)-2,3-dihydroinden-1-one
(0.050 g; Example 7, step 6) in 1.5 mL ethanol was added 16 mg
hydroxylamine hydrochloride and 19 .mu.L pyridine. The resulting
mixture was heated at 80.degree. C. for 3 hours. The cooled
reaction mixture was diluted with ethyl acetate, washed with 10%
aqueous potassium carbonate, dried over sodium sulfate, filtered,
and evaporated to yield crude product. The crude material was
purified by preparative thin-layer chromatography, eluting with a
mixture of 10% methanol in dichloromethane to afford 10 mg product
as a tan solid. .sup.1H NMR (d.sub.6 DMSO) was consistent with the
desired compound 124. MS showed a M+1 molecular ion of m/z 342.2 by
positive APCI.
Example 8
B-Raf IC.sub.50 Assay Protocol
[0242] Activity of human recombinant B-Raf protein may be assessed
in vitro by assay of the incorporation of radiolabelled phosphate
to recombinant MAP kinase (MEK), a known physiologic substrate of
B-Raf, according to US 2004/127496 and WO 03/022840. Catalytically
active human recombinant B-Raf protein is obtained by purification
from sf9 insect cells infected with a human B-Raf recombinant
baculovirus expression vector. To ensure that all substrate
phosphorylation resulted from B-Raf activity, a catalytically
inactive form of MEK was utilized. This protein is purified from
bacterial cells expression mutant inactive MEK as a fusion protein
with glutathione-S-transferase (GST-kdMEK).
[0243] The activity/inhibition of V600E full-length B-Raf was
estimated by measuring the incorporation of radiolabeled phosphate
from [.gamma.-.sup.33P]ATP into FSBA-modified wild-type MEK. The
30-.mu.L assay mixtures contained 25 mM Na Pipes, pH 7.2, 100 mM
KCl, 10 mM MgCl.sub.2, 5 mM .beta.-glycerophosphate, 100 .mu.M Na
Vanadate, 4 .mu.M ATP, 500 nCi [.gamma.-.sup.33P]ATP, 1 .mu.M
FSBA-MEK and 20 nM V600E full-length B-Raf. Incubations were
carried out at 22.degree. C. in a Costar 3365 plate (Corning).
Prior to the assay, the B-Raf and FSBA-MEK were preincubated
together in assay buffer at 1.5.times. (20 .mu.L of 30 nM and 1.5
.mu.M, respectively) for 15 minutes, and the assay was initiated by
the addition of 10 .mu.L of 12 .mu.M ATP. Following the 60-minute
incubation, the assay mixtures were quenched by the addition of 200
.mu.L of 25% TCA, the plate was mixed on a rotary shaker for 10
minutes, and the product was captured on a Perkin-Elmer GF/B filter
plate using a Tomtec Mach III Harvester. After sealing the bottom
of the plate, 32 .mu.L of Bio-Safe II (Research Products
International) scintillation cocktail were added to each well and
the plate was top-sealed and counted in a Topcount NXT
(Packard).
Example 9
In Vitro B-Raf Assay
[0244] The activity of test compounds (Formulas Ia and Ib) as B-Raf
inhibitors may be determined by the following in vitro,
fluorescence anisotropy kinase binding assay, according to U.S.
2004/127496 and WO 03/022840 as follows.
[0245] The kinase enzyme, fluorescent ligand and a variable
concentration of test compound are incubated together to reach
thermodynamic equilibrium under conditions such that in the absence
of test compound the fluorescent ligand is significantly (>50%)
enzyme bound and in the presence of a sufficient concentration
(>10.times.Ki) of a potent inhibitor the anisotropy of the
unbound fluorescent ligand is measurably different from the bound
value.
[0246] The concentration of kinase enzyme is preferably greater
than or equal to 1.times.K.sub.f. The concentration of fluorescent
ligand required will depend on the instrumentation used, and the
fluorescent and physicochemical properties. The concentration used
must be lower than the concentration of kinase enzyme, and
preferably less than half the kinase enzyme concentration. A
typical protocol is:
[0247] 1. All compounds dissolved in Buffer of comparison 50 mM
HEPES, pharmaceutical 7.5, 1 mM CHAPS, 10 mM MgCl.sub.2.
[0248] 2. B-Raf Enzyme concentration: 1 nM
[0249] 3. Fluorescent ligand concentration: 0.5 nM
[0250] 4. Test compound concentration: 0.5 nM-100 .mu.M
[0251] 5. Components incubated in 10 .mu.L final volume in LJL HE
384 type B black microtitre plate until equilibrium reached (about
3 to 30 hours)
[0252] 6. Fluorescence anisotropy read by LJL Acquest.
[0253] K.sub.i=dissociation constant for inhibitor binding;
K.sub.f=dissociation constant for fluorescent ligand binding. The
fluorescent ligand may be a rhodamine- or fluorescein-type dye.
[0254] Alternative assay conditions of B-Raf catalytic activity
utilize 3 .mu.g of GST-kdMEK, 10 .mu.M ATP and 2 .mu.Ci
.sup.33P-ATP, 50 mM MOPS, 0.1 mM EDTA, 0.1M sucrose, 10 mM
MgCl.sub.2 plus 0.1% dimethylsulphoxide (containing compound where
appropriate) in a total reaction volume of 30 .mu.L. Reactions are
incubated at 25.degree. C. for 90 minutes and reactions terminated
by addition of EDTA to a final concentration of 50 .mu.M. 10 .mu.L
of reaction is spotted to P30 phosphocellulose paper and air dried.
Following four washes in ice cold 10% trichloroacetic acid, 0.5%
phosphoric acid, papers are air dried prior to addition of liquid
scintillant and radioactivity is measured in a scintillation
counter.
[0255] The activity of compounds as Raf inhibitors may also be
determined by the assays described in WO 99/10325; McDonald, O. B.,
et al. (1999) Anal. Biochem. 268:318-329, and AACR meeting New
Orleans 1998 Poster 3793.
Example 10
Neuroprotection In Vitro Assay
[0256] The neuroprotective properties of B-Raf inhibitors may be
determined by the following in vitro assay in Rat Hippocampal Slice
Cultures, according to US 2004/127496; US 2004/082014; and WO
03/022840 as follows.
[0257] Organotypic cultures provide an intermediate between
dissociated neuronal cell cultures and in-vivo models of oxygen and
glucose deprivation (OGD). The majority of glial-neuronal
interactions and neuronal circuitry are maintained in cultured
hippocampal slices, so facilitating investigation of the patterns
of death among differing cell types in a model that resembles the
in vivo situation. These cultures allow the study of delayed
cellular damage and death 24 hours, or more, post-insult and permit
assessment of the consequences of long-term alterations in culture
conditions. A number of laboratories have reported delayed neuronal
damage in response to OGD in organotypic cultures of the
hippocampus (Vornov et al., Stroke, (1994) 25:57465; Newell et al.
(1995) Brain Res. 676:38-44). Several classes of compounds have
been shown to protect in this model, including EAA antagonists
(Strasser et al., Brain Res., (1995) 687:167-174), Na channel
blockers (Tasker et al., J. Neurosci., (1992) 12:98-4308) and Ca
channel blockers (Pringle et al. (1996) Stroke 7:2124-2130).
[0258] Method: Organotypic hippocampal slice cultures were prepared
using the method of Stoppini et al (1995) J. Neurosci. Methods
37:173-182. Briefly, 400 micron sections prepared from hippocampi
of 7-8 day postnatal Sprague Dawley rats are cultured on semiporous
membranes for 9-12 days. OGD is then induced by incubation in serum
and glucose-free medium in an anaerobic chamber for 45 minutes.
Cultures are then returned to the air/CO.sub.2 incubator for 23
hours before analysis. Propidium iodide (PI) is used as an
indicator of cell death. PI is nontoxic to neurones and has been
used in many studies to ascertain cell viability. In damaged
neurons PI enters and binds to nucleic acids. Bound PI shows
increased emission at 635 nm when excited at 540 nm. One PI
fluorescence image and one white light image are taken and the
proportion of cell death analyzed. The area of region CAl is
defined from the white light image and superimposed over the PI
image. The PI signal is thresholded and area of PI damage expressed
as a percentage of the CAI area. Correlation between PI
fluorescence and histologically confirmed cell death has been
validated previously by Nissl-staining using cresyl fast violet
(Newell et al. (1995) J. Neurosci. 15:7702-7711).
Example 11
Spectrophotometric ERK Inhibition Assay
[0259] The ERK inhibition properties of the compounds of the
invention may also be determined by the following
spectrophotometric coupled-enzyme assay (Fox et al. (1998) Protein
Sci 7:2249). In this assay, a fixed concentration of activated ERK2
(10 nM) is incubated with various concentrations of the compound in
DMSO (2.5%) for 10 minutes at 30.degree. C. in 0.1 M HEPES buffer,
pH 7.5, containing 10 mM MgCl.sub.2, 2.5 mM phosphoenolpyruvate,
200 .mu.M NADH, 150 .mu.g/mL pyruvate kinase, 50 .mu.g/mL lactate
dehydrogenase, and 200 .mu.M erktide peptide. The reaction is
initiated by the addition of 65 .mu.M ATP. The rate of decrease of
absorbance at 340 nm is monitored, which indicates the extent of
uninhibited enzyme present in the assay. The IC.sub.50 is evaluated
from the rate data as a function of inhibitor concentration.
Example 12
Cellular ERK 1/2 Phosphorylation Assay
[0260] The ERK inhibition properties of the compounds of the
invention may be determined by the following in vitro cellular
proliferation assay.
[0261] Inhibition of basal ERK1/2 phosphorylation was determined by
incubating cells with compound for 1 hour and quantifying the
fluorescent pERK signal on fixed cells and normalizing to total ERK
signal.
[0262] Materials and Methods: Malme-3M cells were obtained from
ATCC and grown in RPMI-1640 supplemented with 10% fetal bovine
serum. Cells were plated in 96-well plates at 15,000 cells/well and
allowed to attach for 1-2 hours. Diluted compounds were then added
at a final concentration of 1% DMSO. After 1 hour, cells were
washed with PBS and fixed in 3.7% formaldehyde in PBS for 15
minutes. This was followed by washing in PBS/0.2% Triton X-100 and
permeabilizing in 100% MeOH for 15 minutes. Cells were blocked in
Odyssey blocking buffer (LI-COR Biosciences) for at least 1 hour.
Antibodies to phosphorylated ERK (Cell Signaling #9106, monoclonal)
and total ERK (Santa Cruz Biotechnology #sc-94, polyclonal) were
added to the cells and incubated for at least 1 hour. After washing
with PBS/0.2% TritonX-100, the cells were incubated with
fluorescently-labeled secondary antibodies (goat anti-rabbit
IgG-IRDye800, Rockland and goat anti-mouse IgG-Alexa Fluor 680,
Molecular Probes) for an additional hour. Cells were then washed
and analyzed for fluorescence at both wavelengths using the Odyssey
Infrared Imaging System (LI-COR Biosciences). Phosphorylated ERK
signal was normalized to total ERK signal.
[0263] Alternatively, the assay may comprise a complete media
prepared by adding 10% fetal bovine serum and
penicillin/streptomycin solution to RPMI 1640 medium (JRH
Biosciences), according to US 2003/0139452. Colon cancer cells,
e.g. HT-29 cell line, are added to each of 84 wells of a 96 well
plate at a seeding density of 10,000 cells/well/150 .mu.L. The
cells are allowed to attach to the plate by incubating at
37.degree. C. for 2 hours. A solution of test compound is prepared
in complete media by serial dilution to obtain the following
concentrations: 20 .mu.M, 6.7 .mu.M, 2.2 .mu.M, 0.74 .mu.M, 0.25
.mu.M, and 0.08 .mu.M. The test compound solution (50 .mu.L) is
added to each of 72 cell-containing wells. To the 12 remaining
cell-containing wells, only complete media (200 .mu.L) is added to
form a control group in order to measure maximal proliferation. To
the remaining 12 empty wells, complete media is added to form a
vehicle control group in order to measure background. The plates
are incubated at 37.degree. C. for 3 days. A stock solution of
.sup.3H-thymidine (1 mCi/mL, New England Nuclear, Boston, Mass.) is
diluted to 20 .mu.Ci/mL in RPMI medium then 20 .mu.L of this
solution is added to each well. The plates are further incubated at
37.degree. C. for 8 hours then harvested and analyzed for
.sup.3H-thymidine uptake using a liquid scintillation counter.
Example 13
Cell Viability Assay
[0264] Viable cells after a 3-day incubation with compound were
quantified using the MTS/PMS colorimetric assay from Promega.
[0265] Materials and Methods: Malme-3M cells were plated in 96 well
plates at a density of 20,000 cells/well. The cells were allowed to
attach for 1-2 hours. Diluted compounds were then added to the
cells at a final concentration of 0.5% DMSO. After 3 days, the
number of viable cells was determined using the MTS assay (Promega,
CellTiter 96 Aqueous Non-radioactive Cell Proliferation Assay).
Briefly, MTS reagents were added to the cells and incubated for 1
hour. Absorbance at 490 nm was then read using a microplate reader.
Background from medium only wells was subtracted.
[0266] The foregoing description is considered as illustrative only
of the principles of the invention. Further, since numerous
modifications and changes will be readily apparent to those skilled
in the art, it is not desired to limit the invention to the exact
construction and process shown as described above. Accordingly, all
suitable modifications and equivalents may be considered to fall
within the scope of the invention as defined by the claims that
follow.
[0267] The words "comprise," "comprising," "include," "including,"
and "includes" when used in this specification and in the following
claims are intended to specify the presence of stated features,
integers, components, or steps, but they do not preclude the
presence or addition of one or more other features, integers,
components, steps, or groups thereof.
* * * * *