U.S. patent application number 10/967427 was filed with the patent office on 2005-03-03 for 4-bromo or 4-iodo phenylamino benzhydroxamic acid derivatives and their use as mek inhibitors.
Invention is credited to Barrett, Stephen Douglas, Bridges, Alexander James, Doherty, Annette Marian, Dudley, David Thomas, Saltiel, Alan Robert, Tecle, Haile.
Application Number | 20050049429 10/967427 |
Document ID | / |
Family ID | 26729421 |
Filed Date | 2005-03-03 |
United States Patent
Application |
20050049429 |
Kind Code |
A1 |
Barrett, Stephen Douglas ;
et al. |
March 3, 2005 |
4-Bromo or 4-iodo phenylamino benzhydroxamic acid derivatives and
their use as MEK inhibitors
Abstract
Phenylamino benzhydroxamic acid derivatives of the formula 1
where R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, and R.sub.6 are
hydrogen or substituent groups such as alkyl, and where R.sub.7 is
hydrogen or an organic radical, are potent inhibitors of MEK and,
as such, are effective in treating cancer and other proliferative
diseases such as psoriasis and restenosis.
Inventors: |
Barrett, Stephen Douglas;
(Livonia, MI) ; Bridges, Alexander James; (Saline,
MI) ; Doherty, Annette Marian; (Paris, FR) ;
Dudley, David Thomas; (Ann Arbor, MI) ; Saltiel, Alan
Robert; (Ann Arbor, MI) ; Tecle, Haile; (Ann
Arbor, MI) |
Correspondence
Address: |
WARNER-LAMBERT COMPANY
2800 PLYMOUTH RD
ANN ARBOR
MI
48105
US
|
Family ID: |
26729421 |
Appl. No.: |
10/967427 |
Filed: |
October 18, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10967427 |
Oct 18, 2004 |
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10163890 |
Jun 4, 2002 |
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6821963 |
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10163890 |
Jun 4, 2002 |
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09462239 |
Jan 4, 2000 |
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09462239 |
Jan 4, 2000 |
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PCT/US98/13106 |
Jun 24, 1998 |
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60051440 |
Jul 1, 1997 |
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Current U.S.
Class: |
560/315 |
Current CPC
Class: |
C07D 211/22 20130101;
C07C 259/10 20130101; C07D 307/42 20130101; C07D 333/16 20130101;
C07D 295/088 20130101; C07D 309/06 20130101; C07D 213/30
20130101 |
Class at
Publication: |
560/315 |
International
Class: |
C07C 239/22 |
Claims
What is claimed is:
1. The compounds are defined by Formula I 12wherein: R.sub.1 is
hydrogen, hydroxy, C.sub.1-C.sub.8 alkyl, C.sub.1-C.sub.8 alkoxy,
halo, trifluoromethyl, or CN; R.sub.2 is hydrogen; R.sub.3,
R.sub.4, and R.sub.5 independently are hydrogen, hydroxy, halo,
trifluoromethyl, C.sub.1-C.sub.8 alkyl, C.sub.1-C.sub.8 alkoxy,
nitro, CN, or (O or NH).sub.m--(CH.sub.2).sub.n--R.sub.9, where
R.sub.9 is hydrogen, hydroxy, CO.sub.2H or NR.sub.10R.sub.11; n is
0 to 4; m is 0 or 1; R.sub.10 and R.sub.11 independently are
hydrogen or C.sub.1-C.sub.8 alkyl, or taken together with the
nitrogen to which they are attached can complete a 3- to 10-member
cyclic ring optionally containing one, two, or three additional
heteroatoms selected from O, S, NH, or N--C.sub.1-C.sub.8 alkyl;
R.sub.6 is hydrogen, C.sub.1-C.sub.8 alkyl, 13aryl, aralkyl, or
C.sub.3-C.sub.10 cycloalkyl; R.sub.7 is C.sub.3-C.sub.10
cycloalkyl; and wherein any of the foregoing alkyl groups can be
unsubstituted or substituted by cycloalkyl (or cycloalkyl
optionally containing a heteroatom selected from O, S, or
NR.sub.9), aryl, aryloxy, heteroaryl, or heteroaryloxy.
2. A compound according to claim 1 wherein R.sub.1 is
C.sub.1-C.sub.8 alkyl or halo.
3. A compound according to claim 2 wherein R.sub.6 is hydrogen.
4. A compound according to claim 3 wherein R.sub.1 is methyl.
5. A compound according to claim 4 having the formula 14
6. A compound of claim 5 wherein R.sub.4 is fluoro, and R.sub.3 and
R.sub.5 are hydrogen.
7. A compound of claim 6 which is:
4-Fluoro-2-(4-iodo-2-methyl-phenylamino-
)-N-(cyclopropylmethoxy)-benzamide; or
4-Fluoro-2-(4-iodo-2-methyl-phenyla-
mino)-N-(cyclopentoxy)-benzamide.
8. A compound of claim 5 wherein R.sub.3 and R.sub.4 are fluoro,
and R.sub.5 is hydrogen.
9. A compound of claim 8 which is:
3,4-Difluoro-2-(4-iodo-2-methyl-phenyla-
mino)-N-(cyclopropyl-methoxy)-benzamide;
3,4-Difluoro-2-(4-iodo-2-methyl-p-
henylamino)-N-(cyclobutyloxy)-benzamide;
3,4-Difluoro-2-(4-iodo-2-methyl-p-
henylamino)-N-(cyclopentyloxy)-benzamide;
3,4-Difluoro-2-(2-chloro-4-iodo--
phenylamino)-N-cyclobutylmethoxy-benzamide; or
3,4-Difluoro-2-(2-chloro-4--
iodo-phenylamino)-N-cyclopropylmethoxy-benzamide.
10. A compound of claim 5 wherein R.sub.3 and R.sub.4 are fluoro,
and R.sub.5 is bromo.
11. A compound according to claim 10 which is:
5-Bromo-3,4-difluoro-2-(4-i-
odo-2-methyl-phenylamino)-N-(cyclopropylmethoxy)-benzamide;
5-Bromo-N-cyclohexylmethoxy-3,4-difluoro-2-(4-iodo-2-methyl-phenylamino)--
benzamide;
5-Bromo-N-cyclopentylmethoxy-3,4-difluoro-2-(4-iodo-2-methyl-ph-
enylamino)-benzamide; or
5-Bromo-N-cyclobutylmethoxy-3,4-difluoro-2-(4-iod-
o-2-methyl-phenylamino)-benzamide.
12. A compound of claim 5 wherein R.sub.3 and R.sub.4 are hydrogen,
and R.sub.5 is halo.
13. A compound of claim 4 having the formula 15
14. A compound of claim 13 wherein R.sub.3 and R.sub.4 are fluoro,
and R.sub.5 is hydrogen.
15. A compound according to claim 14 which is:
3,4-Difluoro-2-(4-bromo-2-m-
ethyl-phenylamino)-N-(cyclobutoxy)-benzamide;
3,4-Difluoro-2-(4-bromo-2-me-
thyl-phenylamino)-N-(cyclopropyl-methoxy)-benzamide; or
3,4-Difluoro-2-(4-bromo-2-methyl-phenylamino)-N-(cyclopentoxy)-benzamide.
16. A compound according to claim 1 which is:
N-Cyclopropylmethoxy-3,4,5-t-
rifluoro-2-(4-iodo-2-methyl-phenylamino)-benzamide;
5-Chloro-N-cyclopropylmethoxy-3,4-difluoro-2-(4-iodo-2-methyl-phenylamino-
)-benzamide;
5-Bromo-N-cyclopropylmethoxy-3,4-difluoro-2-(2-fluoro-4-iodo--
phenylamino)-benzamide;
N-Cyclopropylmethoxy-2-(4-iodo-2-methyl-phenylamin-
o)-4-nitro-benzamide;
N-Cyclopropylmethoxy-3,4,5-trifluoro-2-(2-fluoro-4-i-
odo-phenylamino)-benzamide;
5-Chloro-N-cyclopropylmethoxy-3,4-difluoro-2-(-
2-fluoro-4-iodo-phenylamino)-benzamide;
5-Bromo-2-(2-chloro-4-iodo-phenyla-
mino)-N-cyclopropylmethoxy-3,4-difluoro-benzamide;
N-Cyclopropylmethoxy-2--
(2-fluoro-4-iodo-phenylamino)-4-nitro-benzamide;
2-(2-Chloro-4-iodo-phenyl-
amino)-N-cyclopropylmethoxy-3,4,5-trifluoro-benzamide;
5-Chloro-2-(2-chloro-4-iodo-phenylamino)-N-cyclopropylmethoxy-3,4-difluor-
o-benzamide;
2-(2-Bromo-4-iodo-phenylamino)-N-cyclopropylmethoxy-3,4,5-tri-
fluoro-benzamide;
2-(2-Bromo-4-iodo-phenylamino)-5-chloro-N-cyclopropylmet-
hoxy-3,4-difluoro-benzamide
2-(2-Bromo-4-iodo-phenylamino)-N-cyclopropylme-
thoxy-4-nitro-benzamide;
N-Cyclopropylmethoxy-4-fluoro-2-(2-fluoro-4-iodo--
phenylamino)-benzamide;
N-Cyclopropylmethoxy-3,4-difluoro-2-(2-fluoro-4-io-
do-phenylamino)-benzamide;
2-(2-Chloro-4-iodo-phenylamino)-N-cyclopropylme-
thoxy-4-fluoro-benzamide;
2-(2-Chloro-4-iodo-phenylamino)-N-cyclopropylmet-
hoxy-3,4-difluoro-benzamide;
2-(2-Bromo-4-iodo-phenylamino)-N-cyclopropylm-
ethoxy-4-fluoro-benzamide;
2-(2-Bromo-4-iodo-phenylamino)-N-cyclopropylmet-
hoxy-3,4-difluoro-benzamide;
N-Cyclopropylmethoxy-3,4,5-trifluoro-2-(4-iod-
o-2-methyl-phenylamino)-benzamide;
2-(2-Chloro-4-iodo-phenylamino)-N-cyclo-
butylmethoxy-3,4-difluoro-benzamide;
2-(2-Chloro-4-iodo-phenylamino)-N-cyc-
lopropylmethoxy-3,4-difluoro-benzamide; or
5-Bromo-2-(2-chloro-4-iodo-phen-
ylamino)-N-cyclopropylmethoxy-3,4-difluoro-benzamide.
17. A pharmaceutical formulation comprising a compound of claim 1
admixed with a pharmaceutically acceptable excipient, diluent, or
carrier.
18. A formulation of claim 17 comprising a compound of the formula
16
19. A formulation of claim 17 comprising a compound of the formula
17
20. A method for inhibiting MEK enzymes in a mammal comprising
administering an MEK inhibiting amount of a compound of claim
1.
21. A method of treating a mammal suffering from a proliferative
disease and in need of treatment comprising administering an
antiproliferative amount of a compound of claim 1.
22. A method according to claim 21 wherein the proliferative
disease is psoriasis, restenosis, autoimmune disease, or
atherosclerosis.
23. A method according to claim 21 wherein the proliferative
disease is cancer.
24. A method for treating a mammal suffering from stroke and in
need of treatment comprising administering an effective amount of a
Compound of claim 1.
25. A method for treating a mammal suffering from heart failure and
in need of treatment comprising administering an effective amount
of a Compound of claim 1.
26. A method for treating a mammal suffering from hepatomegaly and
in need of treatment comprising administering an effective amount
of a Compound of claim 1.
27. A method for treating a mammal suffering from cardiomegaly and
in need of treatment comprising administering an effective amount
of a Compound of claim 1.
28. A method for treating a mammal suffering from diabetes and in
need of treatment comprising administering an effective amount of a
Compound of claim 1.
29. A method for treating a mammal suffering from Alzheimer's
disease and in need of treatment comprising administering an
effective amount of a Compound of claim 1.
30. A method for treating a mammal suffering from cancer comprising
administering an effective amount of a compound of claim 1 in
conjunction with conventional radiation therapy.
31. A method for treating a mammal suffering from cystic fibrosis
and in need of treatment comprising administering an effective
amount of a compound of claim 1.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional application of U.S. Ser.
No. 10/163,890 filed Jun. 4, 2002, now allowed, which is a
continuation application of U.S. Ser. No. 09/462,239 filed Jan. 4,
2000, now abandoned, which is a 371 application of PCT/US98/13106
filed Jun. 24, 1998, now abandoned, which claims the benefit of
priority to U.S. Provisional Ser. No. 60/051,440 filed Jul. 1,
1997, now abandoned.
FIELD OF THE INVENTION
[0002] This invention provides certain hydroxamic acid derivatives
of anthranilic acids which inhibit certain dual specificity kinase
enzymes involved in proliferative diseases such as cancer and
restenosis.
BACKGROUND OF THE INVENTION
[0003] Proliferative diseases are caused by a defect in the
intracellular signaling system, or the signal transduction
mechanism of certain proteins. Cancer, for example, is commonly
caused by a series of defects in these signaling proteins,
resulting from a change either in their intrinsic activity or in
their cellular concentrations. The cell may produce a growth factor
that binds to its own receptors, resulting in an autocrine loop,
which continually stimulates proliferation. Mutations or
overexpression of intracellular signaling proteins can lead to
spurious mitogenic signals within the cell. Some of the most common
mutations occur in genes encoding the protein known as Ras, which
is a G-protein that is activated when bound to GTP, and inactivated
when bound to GDP.
[0004] The above mentioned growth factor receptors, and many other
mitogenic receptors, when activated, lead to Ras being converted
from the GDP-bound state to the GTP-bound state. This signal is an
absolute prerequisite for proliferation in most cell types. Defects
in this signaling system, especially in the deactivation of the
Ras.GTP complex, are common in cancers, and lead to the signaling
cascade below Ras being chronically activated.
[0005] Activated Ras leads in turn to the activation of a cascade
of serine/threonine kinases. One of the groups of kinases known to
require an active Ras.GTP for its own activation is the Raf family.
These in turn activate MEK, which then activates MAP kinase.
Activation of MAP kinase by mitogens appears to be essential for
proliferation, and constitutive activation of this kinase is
sufficient to induce cellular transformation. Blockade of
downstream Ras signaling, for example by use of a dominant negative
Raf-1 protein, can completely inhibit mitogenesis, whether induced
from cell surface receptors or from oncogenic Ras mutants. Although
Ras is not itself a protein kinase, it participates in the
activation of Raf and other kinases, most likely through a
phosphorylation mechanism. Once activated, Raf and other kinases
phosphorylate MEK on two closely adjacent serine residues,
S.sup.218 and S.sup.222 in the case of MEK-1, which are the
prerequisite for activation of MEK as a kinase. MEK in turn
phosphorylates MAP kinase on both a tyrosine, Y.sup.185, and a
threonine residue, T.sup.183, separated by a single amino acid.
This double phosphorylation activates MAP kinase at least 100-fold,
and it can now catalyze the phosphorylation of a large number of
proteins, including several transcription factors and other
kinases. Many of these MAP kinase phosphorylations are
mitogenically activating for the target protein, whether it be
another kinase, a transcription factor, or other cellular protein.
MEK is also activated by several kinases other than Raf-1,
including MEKK, and itself appears to be a signal integrating
kinase. As far as is currently known, MEK is highly specific for
the phosphorylation of MAP kinase. In fact, no substrate for MEK
other than MAP kinase has been demonstrated to date, and MEK does
not phosphorylate peptides based on the MAP kinase phosphorylation
sequence, or even phosphorylate denatured MAP kinase. MEK also
appears to associate strongly with MAP kinase prior to
phosphorylating it, suggesting that phosphorylation of MAP kinase
by MEK may require a prior strong interaction between the two
proteins. Both this requirement and the unusual specificity of MEK
are suggestive that it may have enough difference in its mechanism
of action to other protein kinases that selective inhibitors of
MEK, possibly operating through allosteric mechanisms rather than
through the usual blockade of the ATP binding site, may be
found.
[0006] This invention provides compounds which are highly specific
inhibitors of the kinase activity of MEK. Both in enzyme assays and
whole cells, the compounds inhibit the phosphorylation of MAP
kinase by MEK, thus preventing the activation of MAP kinase in
cells in which the Ras cascade has been activated. The results of
this enzyme inhibition include a reversal of transformed phenotype
of some cell types, as measured both by the ability of the
transformed cells to grow in an anchorage-independent manner and by
the ability of some transformed cell lines to proliferate
independently of external mitogens.
[0007] The compounds provided by this invention are phenylamino
benzhydroxamic acid derivatives in which the phenyl ring is
substituted at the 4-position with bromo or iodo. U.S. Pat. No.
5,155,110 discloses a wide variety of fenamic acid derivatives,
including certain phenylamino benzhydroxamic acid derivatives, as
anti-inflammatory agents. The reference fails to describe the
compound of this invention or their kinase inhibitory activity.
SUMMARY OF THE INVENTION
[0008] This invention provides 4-bromo and 4-iodo phenylamino
benzhydroxamic acid derivatives which are kinase inhibitors and as
such are useful for treating proliferative diseases such as cancer,
psoriasis, and restenosis. The compounds are defined by Formula I
2
[0009] wherein:
[0010] R.sub.1 is hydrogen, hydroxy, C.sub.1-C.sub.8 alkyl,
C.sub.1-C.sub.8 alkoxy, halo, trifluoromethyl, or CN;
[0011] R.sub.2 is hydrogen;
[0012] R.sub.3, R.sub.4, and R.sub.5 independently are hydrogen,
hydroxy, halo, trifluoromethyl, C.sub.1-C.sub.8 alkyl,
C.sub.1-C.sub.8 alkoxy, nitro, CN, or (O or
NH).sub.m--(CH.sub.2).sub.n--R.sub.9, where R.sub.9 is hydrogen,
hydroxy, CO.sub.2H or NR.sub.10R.sub.11;
[0013] n is 0 to 4;
[0014] m is 0 or 1;
[0015] R.sub.10 and R.sub.11 independently are hydrogen or
C.sub.1-C.sub.8 alkyl, or taken together with the nitrogen to which
they are attached can complete a 3- to 10-member cyclic ring
optionally containing one, two, or three additional heteroatoms
selected from O, S, NH, or N--C.sub.1-C.sub.8 alkyl;
[0016] R.sub.6 is hydrogen, C.sub.1-C.sub.8 alkyl, 3
[0017] aryl, aralkyl, or C.sub.3-C.sub.10cycloalkyl;
[0018] R.sub.7 is hydrogen, 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.10 (cycloalkyl or
cycloalkyl optionally containing a heteroatom selected from O, S,
or NR.sub.9); or R.sub.6 and R.sub.7 taken together with the N--O
to which they are attached can complete a 5- to 10-membered cyclic
ring, optionally containing one, two, or three additional
heteroatoms selected from O, S, or NR.sub.10R.sub.11;
[0019] and wherein any of the foregoing alkyl, alkenyl, and alkynyl
groups can be unsubstituted or substituted by cycloalkyl (or
cycloalkyl optionally containing a heteroatom selected from O, S,
or NR.sub.9), aryl, aryloxy, heteroaryl, or heteroaryloxy.
[0020] Preferred compounds have Formula II 4
[0021] where R.sub.1, R.sub.3, R.sub.4, R.sub.5, R.sub.6, and
R.sub.7 are as defined above. Especially preferred are compounds
wherein R.sub.1 is methyl or halo, and R.sub.3, R.sub.4, and
R.sub.5 are halo such as fluoro or bromo.
[0022] Another preferred group of compounds have Formula III 5
[0023] wherein R.sub.1, R.sub.3, R.sub.4, R.sub.5, and R.sub.7 are
as defined above.
[0024] The most preferred compounds are those wherein R.sub.1 is
methyl or halo such as F, Br, Cl, and I, R.sub.3 is hydrogen or
halo such as fluoro, R.sub.4 is halo such as fluoro, and R.sub.5 is
hydrogen or halo such as fluoro or bromo. Such compounds have the
formulas 6
[0025] Specific compounds provided by the invention include the
following:
[0026]
3,4,5-Trifluoro-N-hydroxy-2-(4-iodo-2-methyl-phenylamino)-benzamide-
;
[0027]
5-Chloro-3,4-difluoro-N-hydroxy-2-(4-iodo-2-methyl-phenylamino)-ben-
zamide;
[0028]
5-Bromo-3,4-difluoro-2-(2-fluoro-4-iodo-phenylamino)-N-hydroxy-benz-
amide;
[0029]
N-Hydroxy-2-(4-iodo-2-methyl-phenylamino)-4-nitro-benzamide;
[0030]
3,4,5-Trifluoro-2-(2-fluoro-4-iodo-phenylamino)-N-hydroxy-benzamide-
;
[0031]
5-Chloro-3,4-difluoro-2-(2-fluoro-4-iodo-phenylamino)-N-hydroxy-ben-
zamide;
[0032]
5-Bromo-2-(2-chloro-4-iodo-phenylamino)-3,4-difluoro-N-hydroxy-benz-
amide;
[0033]
2-(2-Fluoro-4-iodo-phenylamino)-N-hydroxy-4-nitro-benzamide;
[0034]
2-(2-Chloro-4-iodo-phenylamino)-3,4,5-trifluoro-N-hydroxy-benzamide-
;
[0035]
5-Chloro-2-(2-chloro-4-iodo-phenylamino)-3,4-difluoro-N-hydroxy-ben-
zamide;
[0036]
5-Bromo-2-(2-bromo-4-iodo-phenylamino)-3,4-difluoro-N-hydroxy-benza-
mide;
[0037]
2-(2-Chloro-4-iodo-phenylamino)-N-hydroxy-4-methyl-benzamide;
[0038]
2-(2-Bromo-4-iodo-phenylamino)-3,4,5-trifluoro-N-hydroxy-benzamide;
[0039]
2-(2-Bromo-4-iodo-phenylamino)-5-chloro-3,4-difluoro-N-hydroxy-benz-
amide;
[0040]
2-(2-Bromo-4-iodo-phenylamino)-N-hydroxy-4-nitro-benzamide;
[0041]
4-Fluoro-2-(2-fluoro-4-iodo-phenylamino)-N-hydroxy-benzamide;
[0042]
3,4-Difluoro-2-(2-fluoro-4-iodo-phenylamino)-N-hydroxy-benzamide;
[0043]
2-(2-Chloro-4-iodo-phenylamino)-4-fluoro-N-hydroxy-benzamide;
[0044]
2-(2-Chloro-4-iodo-phenylamino)-3,4-difluoro-N-hydroxy-benzamide;
[0045]
2-(2-Bromo-4-iodo-phenylamino)-4-fluoro-N-hydroxy-benzamide;
[0046]
2-(2-Bromo-4-iodo-phenylamino)-3,4-difluoro-N-hydroxy-benzamide;
[0047]
N-Cyclopropylmethoxy-3,4,5-trifluoro-2-(4-iodo-2-methyl-phenylamino-
)-benzamide;
[0048]
5-Chloro-N-cyclopropylmethoxy-3,4-difluoro-2-(4-iodo-2-methyl-pheny-
lamino)-benzamide;
[0049]
5-Bromo-N-cyclopropylmethoxy-3,4-difluoro-2-(2-fluoro-4-iodo-phenyl-
amino)-benzamide;
[0050]
N-Cyclopropylmethoxy-2-(4-iodo-2-methyl-phenylamino)-4-nitro-benzam-
ide;
[0051]
N-Cyclopropylmethoxy-3,4,5-trifluoro-2-(2-fluoro-4-iodo-phenylamino-
)-benzamide;
[0052]
5-Chloro-N-cyclopropylmethoxy-3,4-difluoro-2-(2-fluoro-4-iodo-pheny-
lamino)-benzamide;
[0053]
5-Bromo-2-(2-chloro-4-iodo-phenylamino)-N-cyclopropylmethoxy-3,4-di-
fluoro-benzamide;
[0054]
N-Cyclopropylmethoxy-2-(2-fluoro-4-iodo-phenylamino)-4-nitro-benzam-
ide;
[0055]
2-(2-Chloro-4-iodo-phenylamino)-N-cyclopropylmethoxy-3,4,5-trifluor-
o-benzamide;
[0056]
5-Chloro-2-(2-chloro-4-iodo-phenylamino)-N-cyclopropylmethoxy-3,4-d-
ifluoro-benzamide;
[0057]
5-Bromo-2-(2-bromo-4-iodo-phenylamino)-N-ethoxy-3,4-difluoro-benzam-
ide;
[0058]
2-(2-Chloro-4-iodo-phenylamino)-N-ethoxy-4-nitro-benzamide;
[0059]
2-(2-Bromo-4-iodo-phenylamino)-N-cyclopropylmethoxy-3,4,5-trifluoro-
-benzamide;
[0060]
2-(2-Bromo-4-iodo-phenylamino)-5-chloro-N-cyclopropylmethoxy-3,4-di-
fluoro-benzamide
[0061]
2-(2-Bromo-4-iodo-phenylamino)-N-cyclopropylmethoxy-4-nitro-benzami-
de;
[0062]
N-Cyclopropylmethoxy-4-fluoro-2-(2-fluoro-4-iodo-phenylamino)-benza-
mide;
[0063]
N-Cyclopropylmethoxy-3,4-difluoro-2-(2-fluoro-4-iodo-phenylamino)-b-
enzamide;
[0064]
2-(2-Chloro-4-iodo-phenylamino)-N-cyclopropylmethoxy-4-fluoro-benza-
mide;
[0065]
2-(2-Chloro-4-iodo-phenylamino)-N-cyclopropylmethoxy-3,4-difluoro-b-
enzamide;
[0066]
2-(2-Bromo-4-iodo-phenylamino)-N-cyclopropylmethoxy-4-fluoro-benzam-
ide;
[0067]
2-(2-Bromo-4-iodo-phenylamino)-N-cyclopropylmethoxy-3,4-difluoro-be-
nzamide;
[0068]
4-Fluoro-N-hydroxy-2-(4-iodo-2-methyl-phenylamino)-N-isopropyl-benz-
amide;
[0069]
N-Cyclopropylmethoxy-3,4,5-trifluoro-2-(4-iodo-2-methyl-phenylamino-
)-benzamide;
[0070]
4-Fluoro-N-hydroxy-2-(4-iodo-2-methyl-phenylamino)-N-methyl-benzami-
de;
[0071]
4-Fluoro-N-hydroxy-2-(4-iodo-2-methyl-phenylamino)-5-nitro-benzamid-
e;
[0072]
2-(2-Chloro-4-iodo-phenylamino)-N-hydroxy-4-nitro-benzamide;
[0073]
3,4-Difluoro-N-hydroxy-2-(4-iodo-2-methyl-phenylamino)-benzamide;
[0074] 2-(2-Chloro-4-iodo-phenylamino)-4-fluoro-N-hydroxy-benzamide
(HCl salt);
[0075]
2-(2-Chloro-4-iodo-phenylamino)-4-fluoro-N-(tetrahydro-pyran-2-ylox-
y)-benzamide;
[0076]
3,4-Difluoro-2-(2-chloro-4-iodo-phenylamino)-N-cyclobutylmethoxy-be-
nzamide;
[0077]
5-Bromo-2-(2-chloro-4-iodo-phenylamino)-N-(2-dimethylamino-ethoxy)--
3,4-difluoro-benzamide monohydrochloride salt;
[0078]
5-Bromo-2-(2-chloro-4-iodo-phenylamino)-3,4-difluoro-N-hydroxy-benz-
amide;
[0079]
3,4-Difluoro-2-(2-chloro-4-iodo-phenylamino)-N-cyclopropylmethoxy-b-
enzamide;
[0080]
5-Bromo-2-(2-chloro-4-iodo-phenylamino)-N-cyclopropylmethoxy-3,4-di-
fluoro-benzamide;
[0081]
5-Bromo-N-cyclohexylmethoxy-3,4-difluoro-2-(4-iodo-2-methyl-phenyla-
mino)-benzamide;
[0082]
5-Bromo-N-cyclopentylmethoxy-3,4-difluoro-2-(4-iodo-2-methyl-phenyl-
amino)-benzamide; and
[0083]
5-Bromo-N-cyclobutylmethoxy-3,4-difluoro-2-(4-iodo-2-methyl-phenyla-
mino)-benzamide.
[0084] This invention also provides pharmaceutical formulations
comprising a compound of Formula I together with a pharmaceutically
acceptable excipient, diluent, or carrier. Preferred formulations
include any of the foregoing preferred compounds together with an
excipient, diluent, or carrier.
[0085] The compounds of Formula I are potent and selective
inhibitors of kinase enzymes, particularly MEK.sub.1 and MEK.sub.2.
They are, therefore, useful to treat subjects suffering from cancer
and other proliferative diseases such as psoriasis, restenosis,
autoimmune disease, and atherosclerosis. The compounds are
especially well-suited to treat cancers such as breast cancer,
colon cancer, prostate cancer, skin cancer, and pancreatic cancer.
The compounds can also be used to treat stroke, diabetes,
hepatomegaly, cardiomegaly, Alzheimer's disease, cystic fibrosis,
and viral disease. The invention provides a method of inhibiting
MEK enzymes and the foregoing diseases by administering to a
subject an effective amount of a compound of Formula I.
DETAILED DESCRIPTION OF THE INVENTION
[0086] As used herein, the term "aryl" means a cyclic, bicyclic, or
tricyclic aromatic ring moiety having from five to twelve carbon
atoms. Examples of typical aryl groups include phenyl, naphthyl,
and fluorenyl. The aryl may be substituted by one, two, or three
groups selected from fluoro, chloro, bromo, iodo, alkyl, hydroxy,
alkoxy, nitro, or amino. Typical substituted aryl groups include
3-fluorophenyl, 3,5-dimethoxyphenyl, 4-nitronaphthyl,
2-methyl-4-chloro-7-aminofluorenyl, and the like.
[0087] The term "aryloxy" means an aryl group bonded through an
oxygen atom, for example phenoxy, 3-bromophenoxy, naphthyloxy, and
4-methyl-1-fluorenyloxy.
[0088] "Heteroaryl" means a cyclic, bicyclic, or tricyclic aromatic
ring moiety having from four to eleven carbon atoms and one, two,
or three heteroatoms selected from O, S, or N. Examples include
furyl, thienyl, pyrrolyl, pyrazolyl, triazolyl, thiazolyl,
xanthenyl, pyronyl, indolyl, pyrimidyl, naphthyridyl, pyridyl, and
triazinyl. The heteroaryl groups can be unsubstituted or
substituted by one, two, or three groups selected from fluoro,
chloro, bromo, iodo, alkyl, hydroxy, alkoxy, nitro, or amino.
Examples of substituted heteroaryl groups include chloropyranyl,
methylthienyl, fluoropyridyl, amino-1,4-benzisoxazinyl,
nitroisoquinolinyl, and hydroxyindolyl.
[0089] The heteroaryl groups can be bonded through oxygen to make
heteroaryloxy groups, for example thienyloxy, isothiazolyloxy,
benzofuranyloxy, pyridyloxy, and 4-methylisoquinolinyloxy.
[0090] The term "C.sub.1-C.sub.8 alkyl" means straight and branched
chain aliphatic groups having from one to eight carbon atoms.
Typical C.sub.1-C.sub.8 alkyl groups include methyl, ethyl,
isopropyl, tert.-butyl, 2,3-dimethylhexyl, and 1,1-dimethylpentyl.
The alkyl groups can be unsubstituted or substituted by cycloalkyl,
cycloalkyl containing a heteroatom selected from O, S, or NR.sub.9,
aryl, aryloxy, heteroaryl, or heteroaryloxy, as those terms are
defined above. Examples of aryl and aryloxy substituted alkyl
groups include phenylmethyl, 2-phenylethyl, 3-chlorophenylmethyl,
1,1-dimethyl-3-(2-nitrophenoxy)butyl, and
3,4,5-trifluoronaphthylmethyl. Examples of alkyl groups substituted
by a heteroaryl or heteroaryloxy group include thienylmethyl,
2-furylethyl, 6-furyloxyoctyl, 4-methylquinolyloxymethyl, and
6-isothiazolylhexyl. Cycloalkyl substituted alkyl groups include
cyclopropylmethyl, 2-cyclopentylethyl, 2-piperidin-1-ylethyl,
3-(tetrahydropyran-2-yl)propyl- , and cyclobutylmethyl.
[0091] "C.sub.2-C.sub.8 Alkenyl" means a straight or branched
carbon chain having one or more double bonds. Examples include
but-2-enyl, 2-methyl-prop-2-enyl, 1,1-dimethyl-hex-4-enyl,
3-ethyl-4-methyl-pent-2-en- yl, and 3-isopropyl-pent-4-enyl. The
alkenyl groups can be substituted by aryl, aryloxy, heteroaryl, or
heteroyloxy, for example 3-phenylprop-2-enyl, 6-thienyl-hex-2-enyl,
2-furyloxy-but-2-enyl, and 4-naphthyloxy-hex-2-enyl.
[0092] "C.sub.2-C.sub.8 Alkynyl" means a straight or branched
carbon chain having from two to eight carbon atoms and at least one
triple bond. Typical alkynyl groups include prop-2-ynyl,
2-methyl-hex-5-ynyl, 3,4-dimethyl-hex-5-ynyl, and
2-ethyl-but-3-ynyl. The alkynyl groups can be substituted by aryl,
aryloxy, heteroaryl, or heteroaryloxy, for example
4-(2-fluorophenyl)-but-3-ynyl, 3-methyl-5-thienylpent-4-ynyl,
3-phenoxy-hex-4-ynyl, and 2-furyloxy-3-methyl-hex-4-ynyl.
[0093] The alkenyl and alkynyl groups can have one or more double
bonds or triple bonds, respectively, or a combination of double and
triple bonds. For example, typical groups having both double and
triple bonds include hex-2-en-4-ynyl,
3-methyl-5-phenylpent-2-en-4-ynyl, and
3-thienyloxy-hex-3-en-5-ynyl.
[0094] The term "C.sub.3-C.sub.10 cycloalkyl" means a non-aromatic
ring or fused rings containing from three to ten carbon atoms.
Examples include cyclopropyl, cyclobutyl, cyclopenyl, cyclooctyl,
bicycloheptyl, adamantyl, and cyclohexyl. The ring can optionally
contain a heteroatom selected from O, S, or NR.sub.9. Such groups
include tetrahydrofuryl, tetrahydropyrrolyl, octahydrobenzofuranyl,
octahydroindolyl, and octahydrobenzothiofuranyl.
[0095] R.sub.3, R.sub.4, and R.sub.5 can include groups defined by
the term (O or NH).sub.m--(CH.sub.2).sub.n--R.sub.9. Examples of
such groups are aminomethyl, 2-aminoethyl, 2-aminoethylamino,
3-aminopropoxy,N,N-diet- hylamino,
3-(N-methyl-N-isopropylamino)-propylamino,
2-(N-acetylamino)-ethoxy, 4-(N-dimethylaminocarbonylamino)-butoxy,
and 3-(N-cyclopropylamino)-propoxy.
[0096] The 4-bromo and 4-iodo phenylamino benzhydroxamic acid
derivatives of Formula I can be prepared from commercially
available starting materials utilizing synthetic methodologies
well-known to those skilled in organic chemistry. A typical
synthesis is carried out by reacting a 4-bromo or 4-iodo aniline
with a benzoic acid having a leaving group at the 2-position to
give a phenylamino benzoic acid, and then reacting the benzoic acid
phenylamino derivative with a hydroxylamine derivative. This
process is depicted in Scheme 1. 7
[0097] where L is a leaving group, for example halo such as fluoro,
chloro, bromo or iodo, or an activated hydroxy group such as a
diethylphosphate, trimethylsilyloxy, p-nitrophenoxy, or
phenylsulfonoxy.
[0098] The reaction of the aniline derivative and the benzoic acid
derivative generally is accomplished by mixing the benzoic acid
with an equimolar quantity or excess of the aniline in an
unreactive organic solvent such as tetrahydrofuran, or toluene, in
the presence of a base such as lithium diisopropylamide, n-butyl
lithium, sodium hydride, and sodium amide. The reaction generally
is carried out at a temperature of about -78.degree. C. to about
25.degree. C., and normally is complete within about 2 hours to
about 4 days. The product can be isolated by removing the solvent,
for example by evaporation under reduced pressure, and further
purified, if desired, by standard methods such as chromatography,
crystallization, or distillation.
[0099] The phenylamino benzoic acid next is reacted with a
hydroxylamine derivative HNR.sub.6OR.sub.7 in the presence of a
peptide coupling reagent. Hydroxylamine derivatives that can be
employed include methoxylamine, N-ethyl-isopropoxy amine, and
tetrahydro-oxazine. Typical coupling reagents include
2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ),
1,3-dicyclohexylcarbodiimide (DCC), bromo-tris(pyrrolidino)-phosp-
honium hexafluorophosphate (PyBrOP) and
(benzotriazolyloxy)tripyrrolidino phosphonium hexafluorophosphate
(PyBOP). The phenylamino benzoic acid and hydroxylamino derivative
normally are mixed in approximately equimolar quantities in an
unreactive organic solvent such as dichloromethane,
tetrahydrofuran, chloroform, or xylene, and an equimolar quantity
of the coupling reagent is added. A base such as triethylamine or
diisopropylethylamine can be added to act as an acid scavenger if
desired. The coupling reaction generally is complete after about 10
minutes to 2 hours, and the product is readily isolated by removing
the reaction solvent, for instance by evaporation under reduced
pressure, and purifying the product by standard methods such as
chromatography or crystallizations from solvents such as acetone,
diethyl ether, or ethanol.
[0100] An alternative method for making the invention compounds
involves first converting a benzoic acid to a hydroxamic acid
derivative, and then reacting the hydroxamic acid derivative with
an aniline. This synthetic sequence is depicted in Scheme 2. 8
[0101] where L is a leaving group. The general reaction conditions
for both of the steps in Scheme 2 are the same as those described
above for Scheme 1.
[0102] Yet another method for making invention compounds comprises
reacting a phenylamino benzhydroxamic acid with an ester forming
group as depicted in Scheme 3. 9
[0103] where L is a leaving group such as halo, and a base is
triethylamine or diisopropylamine.
[0104] The synthesis of invention compounds of Formula I is further
illustrated by the following detailed examples.
EXAMPLE 1
4-Fluoro-N-hydroxy-2-(4-iodo-2-methyl-phenylamino)-benzamide
(a) Preparation of 4-Fluoro-2-(4-iodo-2-methyl-phenylamino)-benzoic
acid
[0105] To a stirred solution containing 3.16 g (0.0133 mol) of
2-amino-5-iodotoluene in 5 mL of tetrahydrofuran at -78.degree. C.
was added 10 mL (0.020 mol) of a 2.0 M lithium diisopropylamide in
tetrahydrofuran/heptane/ethylbenzene (Aldrich) solution. The
resulting green suspension was stirred vigorously for 15 minutes,
after which time a solution of 1.00 g (0.00632 mol) of
2,4-difluorobenzoic acid in 10 mL of tetrahydrofuran was added. The
reaction temperature was allowed to increase slowly to room
temperature, at which temperature the mixture was stirred for 2
days. The reaction mixture was concentrated by evaporation of the
solvent under reduced pressure. Aqueous HCl (10%) was added to the
concentrate, and the solution was extracted with dichloromethane.
The organic phase was dried (MgSO.sub.4) and then concentrated over
a steambath to low volume (10 mL) and cooled to room temperature.
The off-white fibers which formed were collected by vacuum
filtration, rinsed with hexane, and dried in a vacuum-oven
(76.degree. C.; ca. 10 mm of Hg) to afford 1.10 g (47%) of the
desired material; mp 224-229.5.degree. C.;
[0106] .sup.1H NMR (400 MHz, DMSO): .delta. 9.72 (s, 1H), 7.97 (dd,
1H, J=7.0, 8.7 Hz), 7.70 (d, 1H, J=1.5 Hz), 7.57 (dd, 1H, J=8.4,
1.9 Hz), 7.17 (d, 1H, J=8.2 Hz), 6.61-6.53 (m, 2H), 2.18 (s,
3H);
[0107] .sup.13C NMR (100 MHz, DMSO): .delta. 169.87, 166.36 (d,
J.sub.C--F=249.4 Hz), 150.11 (d, J.sub.C--F=11.4 Hz), 139.83,
138.49, 136.07, 135.26 (d, J.sub.C--F=11.5 Hz), 135.07, 125.60,
109.32, 104.98 (d, J.sub.C--F=21.1 Hz), 99.54 (d, J.sub.C--F=26.0
Hz), 89.43, 17.52;
[0108] .sup.19F NMR (376 MHz, DMSO): .delta. -104.00 to -104.07
(m);
[0109] IR (KBr) 1670 (C.dbd.O stretch)cm.sup.-1;
[0110] MS (CI) M+1=372.
[0111] Analysis calculated for C.sub.14H.sub.11FINO.sub.2:
[0112] C, 45.31; H, 2.99; N, 3.77.
[0113] Found: C, 45.21; H, 2.77; N, 3.64.
(b) Preparation of
4-Fluoro-N-hydroxy-2-(4-iodo-2-methyl-phenylamino)-benz- amide
[0114] To a stirred solution of
4-fluoro-2-(4-iodo-2-methyl-phenylamino)-b- enzoic acid (0.6495 g,
0.001750 mol), O-(tetrahydro-2H-pyran-2-yl)-hydroxy- lamine (0.2590
g, 0.002211 mol), and diisopropylethylamine (0.40 mL, 0.0023 mol)
in 31 mL of an equivolume tetrahydrofuran-dichloromethane solution
was added 1.18 g (0.00227 mol) of solid PyBOP
([benzotriazolyloxy]tripyrrolidino phosphonium hexafluorophosphate,
Advanced ChemTech) directly. The reaction mixture was stirred for
30 minutes after which time it was concentrated in vacuo. The brown
oil was treated with 10% aqueous hydrochloric acid. The suspension
was extracted with ether. The organic extraction was washed with
10% sodium hydroxide followed by another 10% hydrochloric acid
wash, was dried (MgSO.sub.4) and concentrated in vacuo to afford
1.0 g of a light-brown foam. This intermediate was dissolved in 25
mL of ethanolic hydrogen chloride, and the solution was allowed to
stand at room temperature for 15 minutes. The reaction mixture was
concentrated in vacuo to a brown oil that was purified by flash
silica chromatography. Elution with
dichloromethane.fwdarw.dichloromethane-methanol (166:1) afforded
0.2284 g of a light-brown viscous oil. Scratching with
pentane-hexanes and drying under high vacuum afforded 0.1541 g
(23%) of an off-white foam; mp 61-75.degree. C.;
[0115] .sup.1H NMR (400 MHz, DMSO): .delta. 11.34 (s, 1H), 9.68 (s,
1H), 9.18 (s, 1H), 7.65 (d, 1H, J=1.5 Hz), 7.58 (dd, 1H, J=8.7, 6.8
Hz), 7.52 (dd, 1H, J=8.4, 1.9 Hz), 7.15 (d, 1H, J=8.4 Hz), 6.74
(dd, 1H, J=11.8, 2.4 Hz), 6.62 (ddd, 1H, J=8.4, 8.4, 2.7 Hz), 2.18
(s, 3H);
[0116] .sup.13C NMR (100 MHz, DMSO): .delta. 165.91, 164.36 (d,
J.sub.C--F=247.1 Hz), 146.78, 139.18, 138.77, 135.43, 132.64,
130.60 (d, J.sub.C--F=11.5 Hz), 122.23, 112.52, 104.72 (d, J=22.1
Hz), 100.45 (d, J.sub.C--F=25.2 Hz), 86.77, 17.03;
[0117] .sup.19F NMR (376 MHz, DMSO): .delta. -107.20 to -107.27
(m);
[0118] IR (KBr) 3307 (broad, O--H stretch), 1636 (C.dbd.O stretch)
cm.sup.-1;
[0119] MS (CI) M+1=387.
[0120] Analysis calculated for
C.sub.14H.sub.12FIN.sub.2O.sub.2:
[0121] C, 43.54; H, 3.13; N, 7.25.
[0122] Found: C, 43.62; H, 3.24; N, 6.98.
EXAMPLE 2
5-Bromo-3,4-difluoro-N-hydroxy-2-(4-iodo-2-methyl-phenylamino)-benzamide
(a) Preparation of 5-Bromo-2,3,4-trifluorobenzoic acid
[0123] To a stirred solution comprised of
1-bromo-2,3,4-trifluorobenzene (Aldrich, 99%; 5.30 g, 0.0249 mol)
in 95 mL of anhydrous tetrahydrofuran cooled to -78.degree. C. was
slowly added 12.5 mL of 2.0 M lithium diisopropylamide in
heptane/tetrahydrofuran/ethylbenzene solution (Aldrich). The
mixture was stirred for 1 hour and transferred by canula into 700
mL of a stirred saturated ethereal carbon dioxide solution cooled
to -78.degree. C. The cold bath was removed, and the reaction
mixture was stirred for 18 hours at ambient temperature. Dilute
(10%) aqueous hydrochloric acid (ca. 500 mL) was poured into the
reaction mixture, and the mixture was subsequently concentrated on
a rotary evaporator to a crude solid. The solid product was
partitioned between diethyl ether (150 mL) and aq. HCl (330 mL, pH
0). The aqueous phase was extracted with a second portion (100 mL)
of diethyl ether, and the combined ethereal extracts were washed
with 5% aqueous sodium hydroxide (200 mL) and water (100 mL, pH
12). These combined alkaline aqueous extractions were acidified to
pH 0 with concentrated aqueous hydrochloric acid. The resulting
suspension was extracted with ether (2.times.200 mL). The combined
organic extracts were dried (MgSO.sub.4), concentrated in vacuo,
and subjected to high vacuum until constant mass was achieved to
afford 5.60 g (88% yield) of an off-white powder; mp
139-142.5.degree. C.;
[0124] .sup.1H NMR (400 MHz, DMSO): .delta. 13.97 (broad s, 1H,
8.00-7.96 (m, 1H);
[0125] .sup.13C NMR (100 MHz, DMSO): .delta. 162.96, 129.34,
118.47, 104.54 (d, J.sub.C--F=22.9 Hz);
[0126] .sup.19F NMR (376 MHz, DMSO): .delta. -120.20 to -120.31
(m), -131.75 to -131.86 (m), -154.95 to -155.07 (m);
[0127] IR (KBr) 1696 (C.dbd.O stretch)cm.sup.-1;
[0128] MS (CI) M+1=255.
[0129] Analysis calculated for
C.sub.74H.sub.21BrF.sub.3O.sub.2:
[0130] C, 32.97; H, 0.79; N, 0.00; Br, 31.34; F, 22.35.
[0131] Found: C, 33.18; H, 0.64; N, 0.01; Br, 30.14; F, 22.75.
(b) Preparation of
5-Bromo-3,4-difluoro-2-(4-iodo-2-methyl-phenylamino)-be- nzoic
acid
[0132] To a stirred solution comprised of 1.88 g (0.00791 mol) of
2-amino-5-iodotoluene in 10 mL of tetrahydrofuran at -78.degree. C.
was added 6 mL (0.012 mol) of a 2.0 M lithium diisopropylamide in
tetrahydrofuran/heptane/ethylbenzene (Aldrich) solution. The
resulting green suspension was stirred vigorously for 10 minutes,
after which time a solution of 1.00 g (0.00392 mol) of
5-bromo-2,3,4-trifluorobenzoic acid in 15 mL of tetrahydrofuran was
added. The cold bath was subsequently removed, and the reaction
mixture stirred for 18 hours. The mixture was concentrated, and the
concentrate was treated with 100 mL of dilute (10%) aqueous
hydrochloric acid. The resulting suspension was extracted with
ether (2.times.150 mL), and the combined organic extractions were
dried (MgSO.sub.4) and concentrated in vacuo to give an orange
solid. The solid was triturated with boiling dichloromethane,
cooled to ambient temperature, and collected by filtration. The
solid was rinsed with dichloromethane, and dried in the vacuum-oven
(80.degree. C.) to afford 1.39 g (76%) of a yellow-green powder; mp
259.5-262.degree. C.;
[0133] .sup.1H NMR (400 MHz, DMSO): .delta. 9.03 (s, 1H), 7.99 (dd,
1H, J=7.5, 1.9 Hz), 7.57 (dd, 1H, J=1.5 Hz), 7.42 (dd, 1H, J=8.4,
1.9 Hz), 6.70 (dd, 1H, J=8.4, 6.0 Hz), 2.24 (s, 3H);
[0134] .sup.19F NMR (376 MHz, DMSO): .delta. -123.40 to -123.47
(m); -139.00 to -139.14 (m);
[0135] IR (KBr) 1667 (C.dbd.O stretch)cm.sup.-1;
[0136] MS (CI) M+1=469.
[0137] Analysis calculated for
C.sub.14H.sub.9BrF.sub.2INO.sub.2:
[0138] C, 35.93; H, 1.94; N, 2.99; Br, 17.07; F, 8.12; I,
27.11.
[0139] Found: C, 36.15; H, 1.91; N, 2.70; Br, 16.40; F, 8.46; I,
26.05.
(c) Preparation of
5-Bromo-3,4-difluoro-N-hydroxy-2-(4-iodo-2-methyl-pheny-
lamino)-benzamide
[0140] To a stirred solution comprised of
5-bromo-3,4-difluoro-2-(4-iodo-2- -methyl-phenylamino)-benzoic acid
(0.51 g, 0.0011 mol), O-(tetrahydro-2H-pyran-2-yl)-hydroxylamine
(0.15 g, 0.0013 mol), and diisopropylethylamine (0.25 mL, 0.0014
mol) in 20 mL of an equivolume tetrahydrofuran-dichloromethane
solution was added 0.6794 g (0.001306 mol) of solid PyBOP (Advanced
ChemTech) directly. The reaction mixture was stirred at 24.degree.
C. for 10 minutes, and then was concentrated to dryness in vacuo.
The concentrate was suspended in 100 mL of 10% aqueous hydrochloric
acid. The suspension was extracted with 125 mL of diethyl ether.
The ether layer was separated, washed with 75 mL of 10% aqueous
sodium hydroxide, and then with 100 mL of dilute acid. The ether
solution was dried (MgSO.sub.4) and concentrated in vacuo to afford
0.62 g (100%) of an off-white foam. The foam was dissolved in ca.
15 mL of methanolic hydrogen chloride. After 5 minutes, the
solution was concentrated in vacuo to an oil, and the oil was
purified by flash silica chromatography. Elution with
dichloromethane.fwdarw.dichloromethane-methanol (99:1) afforded
0.2233 g (42%) of a yellow powder. The powder was dissolved in
diethyl ether and washed with dilute hydrochloric acid. The organic
phase was dried (MgSO.sub.4) and concentrated in vacuo to afford
0.200 g of a foam. This product was triturated with pentane to
afford 0.1525 g of a powder that was repurified by flash silica
chromatography. Elution with dichloromethane afforded 0.0783 g
(15%) of an analytically pure title compound, mp 80-90.degree.
C.;
[0141] .sup.1H NMR (400 MHz, DMSO): .delta. 11.53 (s, 1H), 9.38 (s,
1H), 8.82 (s, 1H), 7.70 (dd, 1H, J=7.0, 1.9 Hz), 7.53 (s, 1H), 7.37
(dd, 1H, J=8.4, 1.9 Hz), 6.55 (dd, 1H, J=8.2, 6.5 Hz), 2.22 (s,
3H);
[0142] .sup.19F NMR (376 MHz, DMSO): .delta. -126.24 to -126.29
(m), -137.71 to -137.77 (m);
[0143] IR (KBr) 3346 (broad, O--H stretch), 1651 (C.dbd.O
stretch)cm.sup.-1;
[0144] MS (CI) M+1=484.
[0145] Analysis calculated for C.sub.14H.sub.10
BrF.sub.2IN.sub.2O.sub.2:
[0146] C, 34.81; H, 2.09; N, 5.80.
[0147] Found: C, 34.53; H, 1.73; N, 5.52,
[0148] Examples 3 to 12 and 78 to 102 in the table below were
prepared by the general procedures of Examples 1 and 2.
EXAMPLES 13-77
[0149] Examples 13 to 77 were prepared utilizing combinatorial
synthetic methodology by reacting appropriately substituted
phenylamino benzoic acids (e.g., as shown in Scheme 1) and
hydroxylamines (e.g., 10
[0150] A general method is given below:
[0151] To a 0.8 mL autosampler vial in a metal block was added 40
.mu.L of a 0.5 M solution of the acid in DMF and 40 .mu.L of the
hydroxylamine (2 M solution in Hunig's base and 1 M in amine in
DMF). A 0.5 M solution of PyBrop was freshly prepared, and 50 .mu.L
were added to the autosampler vial. The reaction was allowed to
stand for 24 hours.
[0152] The reaction mixture was transferred to a 2 dram vial and
diluted with 2 mL of ethyl acetate. The organic layer was washed
with 3 mL of distilled water and the water layer washed again with
2 mL of ethyl acetate. The combined organic layers were allowed to
evaporate to dryness in an open fume hood.
[0153] The residue was taken up in 2 mL of 50% acetonitrile in
water and injected on a semi-prep reversed phase column (10
mm.times.25 cm, 5 .mu.M spherical silica, pore Size 115 A
derivatised with C-18, the sample was eluted at 4.7 mL/min with a
linear ramp to 100% acetonitrile over 8.5 minutes. Elution with
100% acetonitrile continued for 8 minutes.) Fractions were
collected by monitoring at 214 nM. The desired fractions were
evaporated using a Zymark Turbovap. The product was dissolved in
chloroform and transferred to a preweighed vial, evaporated, and
weighed again to determine the yield. The structure was confirmed
by mass spectroscopy.
1EXAMPLES 3-102 Example Melting MS No. Compound Point (.degree. C.)
(M - H.sup.+) 3 2-(4-bromo-2-methyl-phenylamino)-4-fluoro-N- 56-75
dec 523 hydroxy-benzamide 4 5-Chloro-N-hydroxy-2-(4-iodo-2-methyl-
65 dec phenylamino)-benzamide 5 5-Chloro-N-hydroxy-2-(4-iodo-2-me-
thyl- 62-67 phenylamino)-N-methyl-benzamide 6
5-Chloro-2-(4-iodo-2-methyl-phenylamino)-N- 105-108
(terahydropyran-2-yloxy)benzamide 7 5-Chloro-2-(4-iodo-2-methyl-ph-
enylamino)-N- 64-68 methoxybenzamide 8
4-Fluoro-N-hydroxy-2-(4-fluoro-2-methyl- 119-135
phenylamino)-benzamide 9 4-Fluoro-N-hydroxy-2-(2-methyl
phenylamino)- 101-103 benzamide 10 4-Fluoro-2-(4-fluor-2-m-
ethyl-phenylamino)-N- 142-146 (terahydropyran-2-yloxy)benzamide 11
4-Fluoro-N-hydroxy-2-(4-cluoro-2-methyl- 133.5-135
phenylamino)-benzamide 12 4-Fluoro-2-(4-iodo-2-methyl-phenylamino)-
-N- 107-109.5 phenylmethoxy-benzamide 13
4-Fluoro-2-(4-iodo-2-methyl-phenylamino)-N- 399 methoxy-benzamide
14 3,4-Difluoro-2-(4-iodo-2-methyl-phenylamino)- 417
N-methoxy-benzamide 15 2-(4-Bromo-2-methyl-phenylamin- o)- 369
3,4-difluoro-N-methoxy-benzamide 16
2-(4-Bromo-2-methyl-phenylamino)-N-ethoxy- 342*
3,4-difluoro-benzamide (M-EtO) 17 5-Bromo-N-ethoxy-3,4-difluoro-2-
-(4-iodo- 509 2-methyl-phenylamino)-benzamide 18
3,4-Difluoro-2-(4-iodo-2-methyl-phenylamino)- 445
N-isopropoxy-benzamide 19 2-(4-Bromo-2-methyl-phenylamino)- 397
3,4-difluoro-N-isopropoxy-benzamide 20
4-Fluoro-N-(furan-3-ylmethoxy)-2-(4-iodo- 465
2-methyl-phenylamino)-benzamide 21 3,4-Difluoro-N-(furan-3-ylmetho-
xy)-2-(4-iodo- 483 2-methyl-phenylamino)-benzamide 22
2-(4-Bromo-2-methyl-phenylamino)- 435 3,4-difluoro-N-(furan-3-yl-
methoxy)-benzamide 23 5-Bromo-3,4-difluoro-N-(furan-3-ylmethoxy)-
561 2-(4-iodo-2-methyl-phenylamino)-benzamide 24
5-Bromo-N-(but-2-enyloxy)-3,4-difluoro- 536
2-(4-iodo-2-methyl-phenylamino)-benzamide 25
4-Fluoro-2-(4-iodo-2-methyl-phenylamino)-N- 423
(prop-2-ynyloxy)-benzamide 26 3,4-Difluoro-2-(4-iodo-2-methyl-phen-
ylamino)- 441 N-(prop-2-ynyloxy)-benzamide 27
3,4-Difluoro-2-(4-iodo-2-methyl-phenylamino)- 455
N-(1-methyl-prop-2-ynyloxy)-benzamide 28 2-(4-Bromo-2-methyl-pheny-
lamino)- 407 3,4-difluoro-N-(1-methyl-prop-2-ynyloxy)- benzamide 29
N-(But-3-ynyloxy)-3,4-difluoro-2-(4-iodo- 455
2-methyl-phenylamino)-benzamide 30 2-(4-Bromo-2-methyl-phenylamin-
o)-N-(but- 407 3-ynyloxy)-3,4-difluoro-benzamide 31
5-Bromo-N-(but-3-ynyloxy)-3,4-difluoro- 533
2-(4-iodo-2-methyl-phenylamino)-benzamide 32
3,4-Difluoro-2-(4-iodo-2-methyl-phenylamino)- 517
N-(3-phenyl-prop-2-ynyloxy)-benzamide 33 3,4-Difluoro-2-(4-bromo-2-
-methyl- 469 phenylamino)-N-(3-phenyl-prop-2-ynyloxy)- benzamide 34
3,4-Difluoro-N-[3-(3-fluoro-phenyl)-prop- 535
2-ynyloxy]-2-(4-iodo-2-methyl-phenylamino)- benzamide 35
2-(4-Bromo-2-methyl-phenylamino)- 487 3,4-difluoro-N-[3-(3-fluor-
o-phenyl)-prop- 2-ynyloxy]-benzamide 36
3,4-Difluoro-N-[3-(2-fluoro-phenyl)-prop- 535
2-ynyloxy]-2-(4-iodo-2-methyl-phenylamino)- benzamide 37
5-Bromo-3,4-difluoro-N-[3-(2-fluoro-phenyl)- 613
prop-2-ynyloxy]-2-(4-iodo-2-methyl- phenylamino)-benzamide 39
2-(4-Bromo-2-methyl-phenylamino)- 510 3,4-difluoro-N-(3-methy-
l-5-phenyl-pent-2-en- 4-ynyloxy)-benzamide 40
N-Ethoxy-3,4-difluoro-2-(4-iodo-2-methyl- 431
phenylamino)-benzamide 41 2-(4-Bromo-2-methyl-phenylamino)-N-ethox-
y- 383 3,4-difluoro-benzamide 42 4-Fluoro-2-(4-iodo-2-meth-
yl-phenylamino)-N- 427 propoxy-benzamide 43
3,4-Difluoro-2-(4-iodo-2-methyl-phenylamino)- 445
N-propoxy-benzamide 44 2-(4-Bromo-2-methyl-phenylamino)- 397
3,4-difluoro-N-propoxy-benzamide 45 5-Bromo-3,4-difluoro-2-(4-i-
odo-2-methyl- 523 phenylamino)-N-propoxy-benzamide 46
4-Fluoro-2-(4-iodo-2-methyl-phenylamino)-N- 427
isopropoxy-benzamide 47 3,4-Difluoro-2-(4-iodo-2-methyl-phenylamin-
o)- 445 N-isopropoxy-benzamide 48 2-(4-Bromo-2-methyl-phen-
ylamino)- 397 3,4-difluoro-N-isopropoxy-benzamide 49
5-Bromo-3,4-difluoro-2-(4-iodo-2-methyl- 523
phenylamino)-N-isopropoxy-benzamide 50 N-Cyclobutyloxy-3,4-difluor-
o-2-(4-iodo- 457 2-methyl-phenylamino)-benzamide 51
2-(4-Bromo-2-methyl-phenylamino)-N- 409 cyclobutyloxy-3,4-difluo-
ro-benzamide 52 N-Cyclopentyloxy-4-fluoro-2-(4-iodo-2-methyl- 453
phenylamino)-benzamide 53 N-Cyclopentyloxy-3,4-difluoro-2--
(4-iodo- 471 2-methyl-phenylamino)-benzamide 54
2-(4-Bromo-2-methyl-phenylamino)-N- 423 cyclopentyloxy-3,4-diflu-
oro-benzamide 55 N-Cyclopropylmethoxy-4-fluoro-2-(4-iodo- 439
2-methyl-phenylamino)-benzamide 56 N-Cyclopropylmethoxy-3,4-dif-
luoro-2-(4-iodo- 457 2-methyl-phenylamino)-benzamide 57
2-(4-Bromo-2-methyl-phenylamino)-N- 409 cyclopropylmethoxy-3,4-d-
ifluoro-benzamide 58 5-Bromo-N-cyclopropylmethoxy-3,4-difluoro- 435
2-(4-iodo-2-methyl-phenylamino) 59
4-Fluoro-2-(4-iodo-2-methyl-phenylamino)-N- 505
(2-phenoxy-ethoxy)-benzamide 60 3,4-Difluoro-2-(4-iodo-2-methyl-ph-
enylamino)- 523 N-(2-phenoxy-ethoxy)-benzamide 61
2-(4-Bromo-2-methyl-phenylamino)- 475 3,4-difluoro-N-(2-phenoxy--
ethoxy)-benzamide 62 4-Fluoro-2-(4-iodo-2-methyl-phenylamino)-N-
481 (thiophen-2-ylmethoxy)-benzamide 63
3,4-Difluoro-2-(4-iodo-2-methyl-phenylamino)- 499
N-(thiophen-2-ylmethoxy)-benzamide 64 2-(4-Bromo-2-methyl-phenylam-
ino)- 451 3,4-difluoro-N-(thiophen-2-ylmethoxy)- benzamide 65
4-Fluoro-2-(4-iodo-2-methyl-phenylamino)-N- 439
(2-methyl-allyloxy)-benzamide 66 3,4-Difluoro-2-(4-iodo-2-methy-
l-phenylamino)- 457 N-(2-methyl-allyloxy)-benzamide 67
2-(4-Bromo-2-methyl-phenylamino)- 410 3,4-difluoro-N-(2-methyl-a-
llyloxy)-benzamide 68
N-(But-2-enyloxy)-4-fluoro-2-(4-iodo-2-methyl- - 439
phenylamino)-benzamide 69 N-(But-2-enyloxy)-3,4-difl-
uoro-2-(4-iodo- 457 2-methyl-phenylamino)-benzamide 70
2-(4-Bromo-2-methyl-phenylamino)-N-(but- 410
2-enyloxy)-3,4-difluoro-benzamide 71 3,4-Difluoro-2-(4-iodo-2-meth-
yl-phenylamino)- 441 N-(prop-2-ynyloxy)-benzamide 72
N-(But-3-ynyloxy)-3,4-difluoro-2-(4-iodo- 455
2-methyl-phenylamino)-benzamide 73 2-(4-Bromo-2-methyl-phenylamino-
)-N- 449 (4,4-dimethyl-pent-2-ynyloxy)-3,4-difluoro- benzamide 74
N-(But-2-enyloxy)-3,4-difluoro-2-(4-iodo- 457
2-methyl-phenylamino)-benzamide 75 2-(4-Bromo-2-methyl-phenylamin-
o)-N-(but- 410 2-enyloxy)-3,4-difluoro-benzamide 76
N-(3-tert.-butyl-propyn-2-yl)oxy-4-fluoro- 479
2-(4-iodo-2-methyl-phenylamino)-benzamide 77
4-Fluoro-2-(4-iodo-2-methyl-phenylamino)-N- 577*
phenylmethoxy-benzamide *CI 78 4-Fluoro-N-hydroxy-2-(4-iodo-2-met-
hyl- oil phenylamino)-N-isopropyl-benzamide 79
N-Cyclopropylmethoxy-3,4,5-trifluoro-2-(4- 125-127
iodo-2-methyl-phenylamino)-benzamide 80 4-Fluoro-N-hydroxy-2-(4-io-
do-2-methyl- 45-55 phenylamino)-N-methyl-benzamide 81
4-Fluoro-N-hydroxy-2-(4-iodo-2-methyl- 208-209
phenylamino)-5-nitro-benzamide (GLASS) 82 2-(2-Chloro-4-iodo-pheny-
lamino)-N-hydroxy-4- 199-200 nitro-benzamide 83
3,4-Difluoro-2-(4-iodo-2-methyl-phenylamino)- 163-165
N-(tetrahydro-pyran-2-yloxy)-benzamide 84 3,4-Difluoro-N-hydroxy-2-
-(4-iodo-2-methyl- 65-75 phenylamino)-benzamide 85
3,4-Difluoro-5-bromo-2-(4-iodo-2-methyl- 95
phenylamino)-N-(2-piperidin-1-yl-ethoxy)- benzamide 86
5-Bromo-3,4-difluoro-2-(4-iodo-2-methyl- 167-169
phenylamino)-N-(tetrahydro-pyran-2-yloxy)- benzamide 87
2-(2-Chloro-4-iodo-phenylamino)-4-fluoro-N- 165-169
hydroxy-benzamide (HCl salt) 88 2-(2-Chloro-4-iodo-phenylamino)-4--
fluoro-N- 166-167.5 (tetrahydro-pyran-2-yloxy)-benzamide 89
3,4-Difluoro-2-(2-chloro-4-iodo-phenylamino)- 173-174
N-cyclobutylmethoxy-benzamide 90 3,4-Difluoro-2-(2-chloro-4-iodo-p-
henylamino)- 121-122 N-(tetrahydro-pyran-2-yloxy)-benzamide 91
5-Bromo-2-(2-chloro-4-iodo-phenylamino)-N- 206-211.5
(2-dimethylamino-ethoxy)-3,4-difluoro- DEC benzamide
monohydrochloride salt 92 5-Bromo-N-(2-dimethylamino-propoxy)-3,4-
95-105 difluoro--2-(4-iodo-2-methyl-phenylamino)- benzamide 93
5-Bromo-2-(2-chloro-4-iodo-phenylamino)-3,4- 266-280
difluoro-N-hydroxy-benzamide DEC 94
5-Bromo-2-(2-chloro-4-iodo-phenylamino)-3,4- 167.5-169.5
difluoro-N-(tetrahydro-pyran-2-yloxy)- benzamide 95
3,4-Difluoro-2-(2-chloro-4-iodo-phenylamino)- 172.5-173.5
N-cyclopropylmethoxy-benzamide 96 5-Bromo-2-(2-chloro-4-iodo-pheny-
lamino)-N- 171-172.5 cyclopropylmethoxy-3,4-difluoro-benzamide 97
5-Bromo-3,4-difluoro-2-(4-iodo-2-methyl- 173.5-175
phenylamino)-N-(2-morpholin-4-yl-ethoxy)- benzamide 98
5-Bromo-N-(2-diethylamino-ethoxy)-3,4- 81 DEC
difluoro-(4-iodo-2-methyl-phenylamino)- benzamide 99
5-Bromo-3,4-difluoro-2-(4-iodo-2-methyl- 126-128
phenylamino)-N-isobutoxy-benzamide 100 5-Bromo-N-cyclohexylmethoxy-
-3,4-difluoro-2- 139-142 (4-iodo-2-methyl-phenylamino)-benzamide
101 5-Bromo-N-cyclopentylmethoxy-3,4-difluoro-2- 113-115
(4-iodo-2-methyl-phenylamino)-benzamide 102
5-Bromo-N-cyclobutylmethoxy-3,4-difluoro-2- 138-139
(4-iodo-2-methyl-phenylamino)-benzamide
[0154] The invention compounds are useful in treating cancer and
other proliferative diseases by virtue of their selective
inhibition of the dual specificity protein kinases MEK.sub.1 and
MEK.sub.2. The invention compound has been evaluated in a number of
biological assays which are normally utilized to establish
inhibition of proteins and kinases, and to measure mitogenic and
metabolic responses to such inhibition.
[0155] Enzyme Assays
[0156] Cascade Assay for Inhibitors of the MAP Kinase Pathway
[0157] Incorporation of .sup.32P into myelin basic protein (MBP)
was assayed in the presence of a glutathione S-transferase fusion
protein containing p44MAP kinase (GST-MAPK) and a glutathione
S-transferase fusion protein containing p45MEK (GST-MEK). The assay
solution contained 20 mM HEPES, pH 7.4, 10 mM MgCl.sub.2, 1 mM
MnCl.sub.2, 1 mM EGTA, 50 .mu.M [.gamma.-.sup.32P]ATP, 10 .mu.g
GST-MEK, 0.5 .mu.g GST-MAPK and 40 .mu.g MBP in a final volume of
100 .mu.L. Reactions were stopped after 20 minutes by addition of
trichloroacetic acid and filtered through a GF/C filter mat.
.sup.32P retained on the filter mat was determined using a 1205
Betaplate. Compounds were assessed at 10 .mu.M for ability to
inhibit incorporation of .sup.32P.
[0158] To ascertain whether compounds were inhibiting GST-MEK or
GST MAPK, two additional protocols were employed. In the first
protocol, compounds were added to tubes containing GST-MEK,
followed by addition of GST-MAPK, MBP and [.gamma.-.sup.32P]ATP. In
the second protocol, compounds were added to tubes containing both
GST-MEK and GST-MAPK, followed by MBP and [.gamma.-.sup.32P]ATP.
Compounds that showed activity in both protocols were scored as
MAPK inhibitors, while compounds showing activity in only the first
protocol were scored as MEK inhibitors.
[0159] In Vitro MAP Kinase Assay
[0160] Inhibitory activity was also confirmed in direct assays. For
MAP kinase, 1 .mu.g GST-MAPK was incubated with 40 .mu.g MBP for 15
minutes at 30.degree. C. in a final volume of 50 .mu.L containing
50 mM Tris (pH 7.5), 10 .mu.M MgCl.sub.2, 2 .mu.M EGTA, and 10
.mu.M [.gamma.-.sup.32P]ATP. The reaction was stopped by addition
of Laemmli SDS sample buffer and phosphorylated MBP resolved by
electrophoresis on a 10% polyacrylamide gel. Radioactivity
incorporated into MBP was determined by autoradiography, and
subsequently by excision of the bands followed by scintillation
counting.
[0161] In Vitro MEK Assay
[0162] For evaluation of direct MEK activity, 10 .mu.g GST-MEK1 was
incubated with 5 .mu.g of a glutathione S-transferase fusion
protein containing p44MAP kinase with a lysine to alanine mutation
at position 71 (GST-MAPK-KA). This mutation eliminates kinase
activity of MAPK, so only kinase activity attributed to the added
MEK remains. Incubations were 15 minutes at 30.degree. C. in a
final volume of 50 .mu.L containing 50 mM Tris (pH 7.5), 10 .mu.M
MgCl.sub.2, 2 .mu.M EGTA, and 10 .mu.M [.gamma.-.sup.32P]ATP. The
reaction was stopped by addition of Laemmli SDS sample buffer and
phosphorylated GST-MAPK-KA was resolved by electrophoresis on a 10%
polyacrylamide gel. Radioactivity incorporated into GST-MAPK-KA was
determined by autoradiography, and subsequently by excision of the
bands followed by scintillation counting. Additionally, an
artificially activated MEK was utilized that contained serine to
glutamate mutations at positions 218 and 222 (GST-MEK-2E). When
these sites are phosphorylated, MEK activity is increased.
Phosphorylation of these sites can be mimicked by mutation of the
serine residues to glutamate. For this assay, 5 .mu.g GST-MEK-2E
was incubated with 5 .mu.g GST-MAPK-KA for 15 minutes at 30.degree.
C. in the same reaction buffer as described above. Reactions were
terminated and analyzed as above.
[0163] Whole Cell MAP Kinase Assay
[0164] To determine if compounds were able to block activation of
MAP kinase in whole cells, the following protocol was used: Cells
were plated in multi-well plates and grown to confluence. Cells
were then serum-deprived overnight. Cells were exposed to the
desired concentrations of compound or vehicle (DMSO) for 30
minutes, followed by addition of a growth factor, eg, PDGF (100
ng/mL). After a 5-minute treatment with the growth factor, cells
were washed with PBS, then lysed in a buffer consisting of 70 mM
NaCl, 10 mM HEPES (pH 7.4), 50 mM glycerol phosphate, and 1% Triton
X-100. Lysates were clarified by centrifugation at 13,000.times.g
for 10 minutes. Five micrograms of the resulting supernatants were
incubated with 10 .mu.g microtubule associated protein-2 (Map2) for
15 minutes at 30.degree. C. in a final volume of 25 .mu.L
containing 50 mM Tris (pH 7.4), 10 mM MgCl.sub.2, 2 mM EGTA and 30
.mu.M [.gamma.-.sup.32P]ATP. Reactions were terminated by addition
of Laemmli sample buffer. Phosphorylated Map2 was resolved on 7.5%
acrylamide gels and incorporated radioactivity determined by
autoradiography and subsequent excision of the bands followed by
scintillation counting.
[0165] Immunoprecipitation and Antiphosphotyrosine Immunoblots
[0166] To determine the state of tyrosine phosphorylation of
cellular MAP kinase, cells were lysed, endogenous MAP kinase was
immunoprecipitated with a specific antibody, and the resulting
immunoprecipitate analyzed for the presence of phosphotyrosine as
follows: confluent cells were serum-deprived overnight and treated
with compounds and growth factors as described above. Cells were
then scraped and pelleted at 13,000.times.g for 2 minutes. The
resulting cell pellet was resuspended and dissolved in 100 .mu.L of
1% SDS containing 1 mM NaVO.sub.4. Following alternate boiling and
vortexing to denature cellular protein, 900 .mu.L RIPA buffer (50
mM Tris (pH 7.4), 150 mM NaCl, 1% Triton X-100, 0.1% deoxycholate,
and 10 mM EDTA) was added. To this mixture was added 60 .mu.L
agarose beads coupled with rabbit immunoglobulin G and 60 .mu.L
Pansorbin cells in order to clear the lysate of nonspecific binding
proteins. This mixture was incubated at 4.degree. C. for 15 minutes
then centrifuged at 13,000.times.g for 10 minutes. The resulting
supernatant was transferred to fresh tubes and incubated with 10
.mu.L of a polyclonal antisera raised against a fragment of MAP
kinase for a minimum of 1 hour at 4.degree. C. Seventy microliters
of a slurry of agarose beads coupled with protein G and protein A
was added and the incubation continued for an additional 30 minutes
at 4.degree. C. The beads were pelleted by centrifugation at
13,000.times.g for 5 minutes and washed three times with 1 mL RIPA
buffer. Laemmli sample buffer was added to the final bead pellet.
This mixture was boiled for 5 minutes then resolved on a 10%
acrylamide gel. Proteins on the gel were transferred to a
nitrocellulose membrane and nonspecific binding sites on the
membrane blocked by incubation with 1% ovalbumin and 1% bovine
serum albumin in TBST (150 mM NaCl, 10 mM Tris (pH 7.4), and 0.05%
Tween 20). The membrane was then incubated with a commercially
available antibody directed against phosphotyrosine. Antibody bound
on the membrane was detected by incubation with .sup.125I-protein
A, followed by autoradiography.
[0167] Cell Growth Assays
[0168] .sup.3H-Thyridine Incorporation
[0169] Cells were plated in multi-well plates and grown to near
confluence. The media was then removed and replaced with growth
media containing 1% bovine serum albumin. After 24-hour serum
starvation, compounds and specific growth factors were added and
incubations continued for an additional 24 hours. During the final
2 hours, .sup.3H-thymidine was added to the medium. To terminate
the incubations, the medium was removed and cell layers washed
twice with ice-cold phosphate-buffered saline. After the final
wash, ice-cold 5% trichloroacetic acid was added and the cells
incubated for 15 minutes at room temperature. The trichloroacetic
acid solution was then removed and the cell layer washed three
times with distilled water. After the final wash, the cell layer
was solubilized by addition of 2% sodium dodecylsulfate.
Radioactivity in this solution was determined by scintillation
counting.
[0170] In 3T3-L1 adipocyte cells, in which the inhibition blocks
MAPK activation by insulin with an IC.sub.50 of 3 .mu.M, the
compound had no effect on the insulin stimulated uptake of
radiolabeled 2-deoxyglucose, or on the insulin-stimulated synthesis
of either lipid or glycogen at 10 .mu.M concentration. This
demonstrates that the inhibitor shows selectivity between the
mitogenic and metabolic effects of insulin, and demonstrates that
the inhibitor will show less toxicity than an inhibitor which does
not show this surprising selectivity.
[0171] Monolayer Growth
[0172] Cells were plated into multi-well plates at 10 to 20,000
cells/mL. Forty-eight hours after seeding, compounds were added to
the cell growth medium and incubation was continued for 2
additional days. Cells were then removed from the wells by
incubation with trypsin and enumerated with a Coulter counter.
[0173] Growth in Soft-Agar
[0174] Cells were seeded into 35-mm dishes at 5 to 10,000
cells/dish using growth medium containing 0.3% agar. After chilling
to solidify the agar, cells were transferred to a 37.degree. C.
incubator. After 7 to 10 days growths visible colonies were
manually enumerated with the aid of a dissecting microscope.
[0175] Order of addition experiments established that the invention
compounds are inhibiting MEK and not MAP kinase. Experiments
looking at the phosphorylation of a kinase defective mutant of MAP
kinase as substrate (so that there can be no autophosphorylation of
the MAP kinase to complicate interpration) confirms that the
inhibitor inhibits MEK with an IC.sub.50 essentially identical to
that produced in the cascade assay.
[0176] Kinetic analysis demonstrates that the invention compounds
are not competitive with ATP. Thus, they do not bind at the ATP
binding site of the enzyme, which is probably the explanation as to
why these compounds do not show the nonspecific kinase inhibitory
activity typical of most kinase inhibitors, which do bind at the
ATP binding site and which are ATP competitive.
[0177] The in vitro and in vivo biological activity of several
representative compounds of Formula I in the foregoing assays is
presented in Table 1. Data for several known compounds is also
presented.
2TABLE 1 In vivo Compound of In vitro (cell culture) Example No.
IC.sub.50 (.mu.M) IC.sub.50 (.mu.M) 1 0.007 0.05 2 0.003 0.03 3
0.072 3 4 0.023 1 5 0.566 .about.30 6 0.345 .about.30 7 0.221
<30 8 7.13 3 9 0.409 1 11 0.334 0.5 12 0.826 13 0.243 14 0.061
>2 17 0.014 20 0.042 0.17 21 0.014 22 0.137 23 0.016 24 0.021
0.12 25 0.102 27 0.026 28 0.728 29 0.076 0.73 30 0.971 31 0.045 32
0.017 33 0.374 34 0.113 1.5 36 0.056 0.07 37 0.002 38 0.077 0.065
39 0.147 40 0.028 0.125 41 0.236 42 0.087 43 0.040 0.100 44 0.475
45 0.126 47 0.087 0.13 49 0.085 50 0.043 0.22 53 0.140 55 0.047 56
0.014 57 0.181 58 0.018 0.014 59 0.259 62 0.086 63 0.019 64 0.279
65 0.057 66 0.016 0.13 68 0.119 69 0.016 70 0.224 71 0.015 0.39 74
0.035 77 0.28 78 0.080 79 0.008 80 0.080 81 0.017 82 0.003 0.04 83
0.031 84 0.001 0.005 85 0.024 86 0.047 87 <0.001 88 0.069 89
0.005 0.30 90 0.055 91 0.020 92 0.033 93 0.010 0.05 94 0.038 95
0.001 96 <0.010 97 0.015 98 0.025 99 0.018 0.50 100 0.026 >1
101 0.008 >1 102 0.004 0.20
[0178] The following compounds, which are disclosed in U.S. Pat.
No. 5,155,110 were also evaluated in the foregoing assays, and each
such compound demonstrated little or no inhibitory activity.
3 11 % Inhibition R.sub.6 R.sub.7 In Vitro H H 9 at 1 .mu.M -3 at
10 .mu.M H CH.sub.3 -8 at 1 .mu.M 8 at 10 .mu.M CH.sub.3 H -5 at 1
.mu.M 19 at 10 .mu.M iPr H 17 at 1 .mu.M 9 at 10 .mu.M CH.sub.2--Ph
H -4 at 1 .mu.M 18 at 10 .mu.M H H 6 at 1 .mu.M -4 at 10 .mu.M H
CH.sub.3 -6 at 1 .mu.M 12 at 10 .mu.M CH.sub.3 H 13 at 1 .mu.M 19
at 10 .mu.M iPr H -11 at 1 .mu.M 7 at 10 .mu.M
EXAMPLE 103
[0179] The compound from Example 95,
2-(2-chloro-4-iodo-phenylamino)-N-cyc-
lopropylmethoxy-3,4-difluorobenzamide, was evaluated in animals
implanted with a murine colon tumor, C26/clone 10. Male CD2F1 mice
(NCI: Charles River, Kingston) were implanted subcutaneously with
tumor fragments (approximately 30 mg) in the region of the right
axilla on Day 0. The compound of Example 95 was administered
intraperitoneally (IP) or orally (PO) on Days 1 through 14,
post-implant, for a total of 14 days (6 mice per group). The
vehicle for the test compound, and for control animals, was 10%
EtOH/10% Cremophor-EL (Sigma)/80% H.sub.2O, pH 5.0. Tumor volumes
were recorded three times per week by measuring the length and
width of the individual tumors and calculating mass in milligrams
according to the formula (a.times.b.sup.2)/2, where a and b are the
length and width of the tumor. Percent treated/control (T/C) was
calculated based on the ratio of the median tumor volume of the
treated tumors compared with the median tumor volume of control
animals on specified measurement days.
[0180] In the trial in which the compound of Example 95 was
administered IP, the doses were 200, 124, 77, and 48 mg/kg/day. The
invention compound inhibited tumor growth by 59% to 100% as
assessed on Day 15. The median size of the control tumors on Day 15
was 1594 mg. Table 2 shows the number of animal deaths in each
treatment group, the change in body weight, the percent of the
median tumor volume of the treated group compared to the control
group, and the percent inhibition.
4TABLE 2 Change in Non-Specific Body Weight % T/C Dose Deaths
(grams) (Day 15) % Inhibition 200 1/6 +2 0 100 124 1/6 +3 4 96 77
2/5 +2 2 98 48 0/6 +3 41 59
[0181] In the test in which the compound of Example 95 was orally
administered, the doses were 300, 186, 115, and 71 mg/kg/day. The
invention compound inhibited tumor growth 64% to 83% as assessed on
Day 17. The median size of the control tumors on Day 17 was 1664
mg. Table 3 shows the number of animal deaths in each treatment
group, the change in body weight, the percent of the median tumor
volume of the treated group compared to the control group, and the
percent inhibition.
5TABLE 3 Change in Non-Specific Body Weight % T/C Dose Deaths
(grams) (Day 17) % Inhibition 300 0/6 +2 17 83 186 0/6 +2 25 75 115
1/6 +2 21 79 71 0/6 +2 36 64
[0182] The foregoing assay established that the invention compounds
of Formula I are particularly useful for treating cancers such as
colon cancer. The compounds are especially well-suited for use in
combination with radiation to treat and control cancers.
[0183] The invention compounds will be utilized to treat subjects
suffering from cancer and other proliferative diseases and in need
of treatment. The compounds are ideally suited to treating
psoriasis, restenosis, autoimmune disease, and atherosclerosis. The
compounds will generally be utilized as a pharmaceutical
formulation, in which the compound of Formula I is present in a
concentration of about 5% to about 95% by weight. The compounds can
be formulated for convenient oral, parenteral, topical, rectal, or
like routes of administration. The compound will be formulated with
common diluents, excipients, and carriers routinely utilized in
medicine, for instance, with polyols such as glycerin, ethylene
glycol, sorbitol 70; mono- and difatty acid esters of ethylene
glycol. Starches and sugars such as corn starch, sucrose, lactose,
and the like, can be utilized for solid preparations. Such solid
formulations can be in the form of tablets, troches, pills,
capsules, and the like. Flavoring agents such as peppermint, oil of
wintergreen, and the like can be incorporated.
[0184] Typical doses of active compound are those that are
effective to treat the cancer or other proliferative disorder
afflicting the mammal. Doses will generally be from about 0.1 mg
per kilogram body weight to about 500 mg per kilogram body weight.
Such doses will be administered from one to about four times a day,
or as needed to effectively treat the cancer, psoriasis,
restenosis, or other proliferative disorder.
[0185] A preferred method for delivering the invention compound is
orally via a tablet, capsule, solution, or syrup. Another method is
parenterally, especially via intravenous infusion of a solution of
the benzopyran in isotonic saline or 5% aqueous glucose.
[0186] Following are typical formulations provided by the
invention.
EXAMPLE 104
[0187]
6 Preparation of 50-mg Tablets Per 10,000 Per Tablet Tablets 0.050
g 4-fluoro-N-hydroxy-2-(4-iodo-2- -methyl- 500 g
phenylamino)-benzamide 0.080 g lactose 800 g 0.010 g corn starch
(for mix) 100 g 0.008 g corn starch (for paste) 80 g 0.002 g
magnesium stearate (1%) 20 g 0.150 g 1500 g
[0188] The benzhydroxamic acid, lactose, and corn starch (for mix)
are blended to uniformity. The corn starch (for paste) is suspended
in 600 mL of water and heated with stirring to form a paste. The
paste is used to granulate the mixed powders. The granules are
passed through a #8 screen and dried at 120.degree. F. The dry
granules are passed through a #16 screen. The mixture is lubricated
with 1% magnesium stearate and compressed into tablets. The tablets
are administered to a mammal for inhibiting MEK enzymes and
treating restenosis, atherosclerosis, and psoriasis.
EXAMPLE 105
[0189]
7 Preparation of Oral Suspension Ingredient Amount
5-Chloro-2-(4-iodo-2-methyl-phenylamino)-N- 500 mg
(methoxy)-benzamide Sorbitol solution (70% NF) 40 mL Sodium
benzoate 150 mg Saccharin 10 mg Red dye 10 mg Cherry flavor 50 mg
Distilled water qs ad 100 mL
[0190] The sorbitol solution is added to 40 mL of distilled water
and the benzhydroxamic acid derivative is suspended therein. The
saccharin, sodium benzoate, flavor, and dye are added and
dissolved. The volume is adjusted to 100 mL with distilled water.
Each milliliter of syrup contains 5 mg of the invention compound.
The syrup is administered to a mammal for treating proliferative
disease, especially breast cancer and skin cancer.
EXAMPLE 106
[0191] Preparation of Parenteral Solution
[0192] In a solution of 700 mL of propylene glycol and 200 mL of
water for injection is added 20.0 g of
4-fluoro-2-(4-bromo-2-methyl-phenylamino)-N-- (hydroxy)-benzamide.
The volume of the solution is adjusted to 1000 mL by addition of
water for injection. The formulation is heat sterilized, filled
into 50-mL ampoules each containing 2.0 mL (40 mg of
4-fluoro-2-(4-bromo-2-methyl-phenylamino)-N-(hydroxy)-benzamide),
and sealed under nitrogen.
[0193] The invention compounds thus formulated will be administered
to a mammal in need of treatment for a proliferative disorder such
as cancer, psoriasis, restenosis, atherosclerosis, and autoimmune
disease at a rate and dose effective to treat the condition. An
"antiproliferative amount" of an invention compound is that
quantity of compound that inhibits or reduces the rate of
proliferation of target cells. Typical cancers to be treated
according to this invention include breast cancer, colon cancer,
prostate cancer, skin cancer, and the like. The compound is
well-suited to the treatment of psoriasis, restenosis, and
atherosclerosis, and to inhibiting the activity of MEK enzymes,
especially MEK.sub.1 and MEK.sub.2. All that is required is to
administer to a mammal an MEK inhibiting amount of a compound of
the invention. An "MEK inhibiting amount" of an invention compound
is an amount that when administered to a mammal causes a measurable
inhibition of the MEK enzyme. Typical MEK inhibiting amounts will
be from about 0.1 .mu.g to about 500 mg of active compound per
kilogram body weight. For treating the proliferative diseases
mentioned above, typical doses will be from about 0.1 to about 50
mg/kg, normally given from one to about four times per day.
* * * * *