Note: Descriptions are shown in the official language in which they were submitted.
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5-AMIDE SUBSTITUTED DIARYLAMINES AS MEX INHIBITORS
The present invention relates to diarylamines, such as 5-amide
substituted diarylamines, and methods of use thereof.
BACKGROUND OF THE INVENTION
Mitogen ERK Kinase ("MEK") enzymes are dual specificity kinases
involved in, for example, immunomodulation, inflammation, and proliferative
diseases such as cancer and restenosis.
Proliferative diseases are caused by a defect in the intracellular
signaling system, or the signal transduction mechanism of certain proteins.
Defects include a change either in the intrinsic activity or in the cellular
concentration of one or more signaling proteins in the signaling cascade. 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, a G-protein that is activated
when bound to GTP, and inactivated when bound to GDP. 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.
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 (e.g., MEK1 and MEK2) which then activates the MAP kinase, ERK
(ERK1 and ERK2). Activation of MAP kinase by mitogens appears to be
essential for proliferation; constitutive activation of this kinase is
sufficient to
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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, S21$ and S222 in the case of MEK-1,
which are the prerequisite for activation of MEK as a kinase. MEK in tum
phosphorylates MAP kinase on both a tyrosine, Y185, and a threonine
residue, T183, separated by a single amino acid. This double phosphorylation
activates MAP kinase at least 100-fold. Activated MAP kinase can then
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, such as
a
kinase, a transcription factor, or another cellular protein. In addition to
Raf 1
and MEKK, other kinases activate MEK, and MEK itself appears to be a signal
integrating kinase. Current understanding is that MEK is highly specific for
the phosphorylation of MAP kinase. In fact, no substrate for MEK other than
the MAP kinase, ERK, 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.
It has been found that the compounds of the present invention are
inhibitors of MEK and are useful in the treatment of a variety of
proliferative
disease states, such as conditions related to the hyperactivity of MEK, as
well
as diseases modulated by the MEK cascade.
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SUMMARY
The present invention provides compounds of formula 1 and II:
A O H R' O O
1 R,
\ N \ \ N \
X ~ , ~ / x ~ ~ /
/ R I /~ I
O R; ~ R / Rs
O
wherein
R1 is hydrogen, C1-g alkyl, C1-g alkoxy, halo, C1-2 haloalkyl, or CN;
R3 and R4 are each independently hydrogen, halo, C1-2 haloalkyl,
C1-g alkyl, C1-g alkoxy, nitro, CN, or (O or NH)k-(CH2)j-Rg,
where Rg is hydrogen, hydroxy, C02H or NR1pR11
jisOto4;
kis0or1;
R1p and R11 are each independently hydrogen or C1-g alkyl, or
together with the nitrogen to which they are attached form a 3-
to 10-member cyclic ring optionally containing one, two, or three
additional heteroatoms selected from the group consisting of O,
S, NH, and N-C1-g alkyl;
A is hydroxy, C1-g alkoxy, or NRgOR~;
R6 is hydrogen, C1-g alkyl, (CO)-C1-g alkyl, phenyl, naphthyl,
phenyl(C1-g alkyl), or C3-10 cYcloalkyl;
R~ is hydrogen, C1-g alkyl, C2-g alkenyl, C2-g alkynyl,
03-10 cYcloalkyl or Cg-10 cYcloalkyl optionally containing a
heteroatom selected from the group consisting of O, S, and
N Rg;
X is OR12, NR13R12, or NR14;
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R12 and R13 are each independently hydrogen, C1-g alkyl, C2-6
alkenyl, C2-g alkynyl, C4-g cycloalkyl, [(CH2)nY(CH2)m]qCH3, phenyl,
naphthyl, (C1-g alkyl)phenyl, -[(CH2)nY(CH2)m]qphenyl, C2-g heteroaryl,
(C1-g alkyl)C2-6 heterocyclic radical, or [(CH2)nY(CH2)m]q C2-g heterocyclic
radical;
Y is N or O;
R14 taken with N is a 5- to 7-membered heterocyclic radical with
between 0 and 3 additional heteroatoms or heteroatom combinations in the
ring selected from the group consisting of O, S, SO, S02, NH, and NMe;
0<_ n,m<_ 6,n+m<_8,1<_ q<_5;and
wherein the above alkyl, alkenyl, alkynyl, heterocyclic radical, aryl, and
cycloalkyl groups can be optionally substituted with between 1 and 4
substituents independently selected from the group consisting of hydroxy,
C1-4 alkyl, fluoro, chloro, iodo, bromo, amino, and C1-4 alkoxy, and NRaRb;
wherein Ra and Rb are each independently selected from the group
consisting of hydrogen and C1-g alkyl; and
the pharmaceutically acceptable salts thereof.
The invention also provides a pharmaceutical composition comprising
a compound of formula I or II and a pharmaceutically acceptable carrier.
Additionally, the invention provides a method of treating a proliferative
disease in a patient in need thereof comprising administering a
therapeutically
effective amount of a compound of formula I or II.
The invention also provides the use of a compound of formula I or II for
the manufacture of a medicament for the treatment of a proliferative disease.
Furthermore, the invention provides methods of treating cancer,
restenosis, psoriasis, autoimmune disease, atherosclerosis, osteoarthritis,
rheumatoid arthritis, heart failure, chronic pain, and neuropathic pain in a
patient in need thereof comprising administering a therapeutically effective
amount of a compound of formula I or II.
The invention also provides the use of a compound of formula I or II for
the manufacture of a medicament for the treatment of cancer, restenosis,
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psoriasis, autoimmune disease, atherosclerosis, osteoarthritis, rheumatoid
arthritis, heart failure, chronic pain, and neuropathic pain.
In addition, the invention provides a method for treating cancer in a
patient in need thereof comprising administering a therapeutically effective
amount of a compound of formula I or II in combination with radiation therapy
or at least one chemotherapeutic agent.
The invention also features synthetic intermediates and methods
disclosed herein.
Other aspects of the invention are provided in the description,
examples, and claims below.
DETAILED DESCRIPTION
The invention features diarylamine compounds, pharmaceutical
compositions thereof, and methods of using such compounds and
compositions.
Certain terms are defined below and by their usage throughout this
disclosure.
Alkyl groups, such as C1-g alkyl, include aliphatic chains (i.e.,
hydrocarbyl or hydrocarbon radical structures containing hydrogen and
carbon atoms) with a free valence. Alkyl groups are understood to include
straight chain and branched structures. Examples include methyl, ethyl,
propyl, isopropyl, butyl, n-butyl, isobutyl, t-butyl, pentyl, isopentyl, 2,3-
dimethylpropyl, hexyl, 2,3-dimethylhexyl, 1,1-dimethylpentyl, heptyl, and
octyl.
The term "C1-g alkyl" includes within its definition the terms "C1-g alkyl"
and
"C1-4 alkyl".
Cycloalkyl groups, such as C3-10 cYcloalkyl, include cyclopropyl,
cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. The term
'~C3'10 cYcloalkyl" includes within its definition the terms °C4-g
cycloalkyl".
The term "halo" as used herein refers to fluoro, chloro, bromo, or iodo.
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The term "haloalkyl" as used herein refers to a straight or branched
alkyl chain with 1, 2 or 3 halo atoms attached to it. The term "C1-2
haloalkyl"
as used herein refers to a straight or branched alkyl chain having from one to
two carbon atoms with 1, 2 or 3 halo atoms attached to it. Typical C1-2
haloalkyl groups include chloromethyl, 2-bromoethyl, difluoromethyl,
trifluoromethyl and the like.
The term "alcoxy" as used herein refers to a straight or branched alkyl
chain attached to an oxygen atom. The term "C1-g alcoxy" as used herein
refers to a straight or branched alkyl chain having from one to eight carbon
atoms attached to an oxygen atom. Typical C1-g alcoxy groups include
methoxy, ethoxy, propoxy, isopropoxy, butoxy, t-butoxy, pentoxy and the like.
The term "C1-g alcoxy" includes within its definition the terms "C1-g alcoxy"
and "C1-4 alcoxy".
Alkyl and cycloalkyl groups can be substituted with 1, 2, 3 or more
substituents which are independently selected from hydroxy, alkyl, halo,
amino, alkoxy, alkylamino, dialkylamino, cycloalkyl, aryl, aryloxy,
arylalkyloxy,
heterocyclic radical, and (heterocyclic radical)oxy. Specific examples include
fluoromethyl, hydroxyethyl, 2,3-dihydroxyethyl, (2- or 3-furanyl)methyl,
cyclopropylmethyl, benzyloxyethyl, (3-pyridinyl)methyl, (2- or 3-
furanyl)methyl;
(2-thienyl)ethyl, hydroxypropyl, aminocyclohexyl, 2-dimethylaminobutyl,
methoxymethyl, ll~pyridinylethyl, diethylaminoethyl, and cyclobutylmethyl.
In some embodiments, each hydrocarbon radical above is optionally
substituted with between 1 and 3 or more substituents independently selected
from halo, hydroxyl or hydroxy, amino, (amino)sulfonyl, and N02. In another
embodiment, each heterocyclic radical above is optionally substituted with
between 1 and 3 or more substituents independently selected from halo, C1~
alkyl, C3_g cycloalkyl, C3~ alkenyl, Cg~ alkynyl, phenyl, hydroxyl or hydroxy,
C~-C4 alkoxy amino, (amino)sulfonyl, and N02, wherein each substituent alkyl,
cycloalkyl, alkenyl, alkynyl or phenyl is in turn optionally substituted with
between 1 and 2 substituents independently selected from halo, C1_2 alkyl,
hydroxyl or hydroxy, amino, and N02.
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More general forms of substituted hydrocarbon radicals include
hydroxyalkyl, hydroxyalkenyl, hydroxyalkynyl, hydroxycycloalkyl, hydroxyaryl,
and corresponding forms for the prefixes amino-, halo-, vitro-, alkyl-, phenyl-
,
cycloalkyl- and so on, or combinations of substituents. According to formula I
and II, therefore, substituted alkyls include hydroxyalkyl, aminoalkyl,
nitroalkyl,
haloalkyl, alkylalkyl (branched alkyls, such as methylpentyl),
(cycloalkyl)alkyl,
phenylalkyl, alkoxyalkyl, alkylaminoalkyl, dialkylaminoalkyl, arylalkyl,
aryloxyalkyl, arylalkyloxyalkyl, (heterocyclic radical)alkyl, and
(heterocyclic
rad ica l )oxya l kyl .
R~ thus includes hydroxyalkyl, hydroxyalkenyl, hydroxyalkynyl,
hydroxycycloalkyl, hydroxyaryl, aminoalkyl, aminoalkenyl, aminoalkynyl,
aminocycloalkyl, aminoaryl, alkylalkenyl, (alkylaryl)alkyl, (haloaryl)alkyl,
(hydroxyaryl)alkynyl, and so forth. Similarly, Ra includes hydroxyalkyl and
aminoaryl, and Rb includes hydroxyalkyl, aminoalkyl, and
hydroxyalkyl(heterocyclic radical)alkyl.
Alkenyl groups are analogous to alkyl groups, but have at least one
double bond (two adjacent sp2 carbon atoms). Depending on the placement
of a double bond and substituents, if any, the geometry of the double bond
may be entgegen (E), or zusammen (Z), cis, or traps. Similarly, alkynyl
groups have at least one triple bond (two adjacent sp carbon atoms).
Unsaturated alkenyl or alkynyl groups may have one or more double or triple
bonds, respectively, or a mixture thereof; like alkyl groups, unsaturated
groups may be straight chain or branched, and they may be substituted as
described both above for alkyl groups and throughout the disclosure by
example. Examples of alkenyls, alkynyls, and substituted forms include cis-2-
butenyl, traps-2-butenyl, 3-butynyl, 3-phenyl-2-propynyl, 3-(2'-fluorophenyl)-
2-
propynyl, 3-methyl(5-phenyl)-4-pentynyl, 2-hydroxy-2-propynyl, 2-methyl-2-
propynyl; 2-propenyl, 4-hydroxy-3-butynyl, 3-(3-fluorophenyl)-2-propynyl, and
2-methyl-2-propenyl. In formulas I and II, alkenyls and alkynyls can be C2~,
C2_g or C2_g, for example, and are preferably C3~ or C3_g.
Heterocyclic radicals, which include but are not limited to heteroaryls,
such as C3_g and C2_g heteroaryls, include: furyl, oxazolyl, isoxazolyl,
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thiophenyl, thiazolyl, pyrrolyl, imidazolyl, 1,3,4-triazolyl, tetrazolyl,
pyridinyl,
pyrimidinyl, pyridazinyl, indolyl, and their nonaromatic counterparts. Further
examples of heterocyclic radicals include piperidyl, quinolyl, isothiazolyl,
piperidinyl, morpholinyl, piperazinyl, tetrahydrofuryl, tetrahydropyrrolyl,
pyrrolidinyl, octahydroindolyl, octahydrobenzothiofuranyl, and
octahydrobenzofuranyl. Heterocyclic radicals may be substituted as described
both above for alkyl groups and throughout the disclosure by example.
Heterocyclic radicals include heteroaryls such as substituted or
unsubstituted radicals of pyran, pyrazole, triazole, indazole, pyrazine,
oxadiazole, oxathiadiazole; heterocycles also include heteroalkyls such as
substituted and unsubstituted radicals of tetrahydropyran, pyrrolidone,
imidazoline, and tetrahydrothiophene.
The present invention includes pharmaceutically acceptable salts,
amides, and esters of the disclosed compounds. The invention also features a
pharmaceutically acceptable salt or C1_g ester of a disclosed compound. For
example, the disclosed alcohol compounds may form esters having the
structure obtained by replacing the H of a hydroxyl group with a -C(=O)C1 _7
acyl group.
The invention provides the disclosed compounds and closely related,
pharmaceutically acceptable forms of the disclosed compounds, such as
salts, esters, amides, hydrates or solvated forms thereof; masked or protected
forms; and racemic mixtures, or enantiomerically or optically pure forms.
Pharmaceutically acceptable salts, esters, and amides include
carboxylate salts (e.g., C1_g alkyl, cycloalkyl, aryl, heteroaryl, or non-
aromatic
heterocyclic), amino acid addition salts, esters, and amides which are within
a
reasonable benefit/risk ratio, pharmacologically effective, and suitable for
contact with the tissues of patients without undue toxicity, irritation, or
allergic
response. Representative salts include hydrobromide, hydrochloride, sulfate,
bisulfate, nitrate, acetate, oxalate, valerate, oleate, palmitate, stearate,
laurate, borate, benzoate, lactate, phosphate, tosylate, citrate, maleate,
fumarate, succinate, tartrate, naphthylate, mesylate, glucoheptonate,
lactiobionate, and laurylsulfonate. These may include alkali metal and alkali
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earth rations such as sodium, potassium, calcium, and magnesium, as well
as non-toxic ammonium, quaternary ammonium, and amine rations such as
tetramethyl ammonium, methylamine, trimethylamine, and ethylamine. See,
for example, S.M. Berge, et al., "Pharmaceutical Salts," J. Pharm. Sri., 1977,
66:1-19 which is incorporated herein by reference. Representative
pharmaceutically acceptable amides of the invention include those derived
from ammonia, primary C1_g alkyl amines and secondary di (C1_g alkyl)
amines. Secondary amines include 5- or 6-membered heterocyclic or
heteroaromatic ring moieties containing at least one nitrogen atom and
optionally between 1 and 2 additional heteroatoms. Preferred amides are
derived from ammonia, C1_3 alkyl primary amines, and di(C1_2 alkyl)amines.
Representative pharmaceutically acceptable esters of the invention include
C1_7 alkyl, C5_7 cycloalkyl, phenyl, and phenyl(C1_g)alkyl esters. Preferred
esters include methyl esters.
The present invention includes compounds having one or more
functional groups (e.g., hydroxyl, amino, or carboxyl) masked by a protecting
group. Examples of protecting groups used to protect functional groups and
their preparation are disclosed by T.W. Green, "Protective Groups in Organic
Synthesis," John Wiley & Sons, 1981. Choice of the protecting group used
will depend upon the substituent to be protected and the conditions that will
be employed in subsequent reaction steps wherein protection is required, and
is well within the knowledge of one of ordinary skill in the art. Protecting
groups include, but are not limited to, the list provided below.
H dy roxyl protecting groups
Hydroxyl protecting groups include: ethers, esters, and protection for
1,2- and 1,3-diols. The ether protecting groups include: methyl, substituted
methyl ethers, substituted ethyl ethers, substituted benzyl ethers, silyl
ethers
and conversion of silyl ethers to other functional groups.
Substituted methyl ethers include: methoxymethyl, methylthiomethyl, t-
utylthiomethyl, (phenyldimethylsilyl) methoxymethyl, benzyloxymethyl, ~
ethoxybenzyloxymethyl, (4-methoxyphenoxy) methyl, guaiacolmethyl, t
butoxymethyl, 4-pentenyloxymethyl, siloxymethyl, 2-methoxyethoxymethyl,
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2,2,2-trichloroethoxymethyl, bis(2-chloro- ethoxy)methyl, 2-
(trimethylsilyl)ethoxymethyl, tetrahydropyranyl, 3-bromotetrahydro-pyranyl,
tetrahydrothiopyranyl, 1-methoxycyclohexyl, 4-methoxytetrahydropyranyl, 4-
methoxytetrahydrothio-pyranyl, 4-methoxytetrahydrothiopyranyl S,S-dioxido,
1-[(2-chloro-4-methyl)phenyl]-4-methoxypiperidin-4-yl, 1,4-dioxan-2-yl,
tetrahydrofuranyl, tetrahydrothiofuranyl, and 2,3,3a,4,5,6,7,7a-octahydro-
7,8,8-trimethyl-4,7-ethanobenzofuran-2-yl.
Substituted ethyl ethers include: 1-ethoxyethyl, 1-
(2,chloroethoxy)ethyl,
1-methyl-1-methoxyethyl, 1-methyl-1-benzyloxyethyl, 1-methyl-1-benzyloxy-2-
fluoroethyl, 2,2,2-trichloroethyl, 2-trimethylsilyethyl, 2-
(phenylselenyl)ethyl, t
butyl, allyl, p-chlorophenyl, p-methoxyphenyl, 2,4-dinitrophenyl, and benzyl.
Substituted benzyl ethers include: p-methoxybenzyl, 3,4-
dimethoxybenzyl, o-nitrobenzyl, p-nitrobenzyl, p-halobenzyl, 2,6-
dichlorobenzyl, p-cyanobenzyl, p-phenylbenzyl, 2- and 4-picolyl, 3-methyl-2-
picolyl N~xido, diphenylmethyl, p, p'-dinitrobenzhydryl, 5-dibenzosuberyl,
triphenylmethyl, a-naphthyldiphenyl-methyl, p-methoxyphenyldiphenylmethyl,
di(p-methoxyphenyl)phenylmethyl, tri-(p-methoxyphenyl)methyl, 4-(4'-
bromophenacyloxy)phenyldiphenylmethyl, 4,4',4"-tris(4,5-
dichlorophthalimidophenyl)methyl, 4,4',4"-tris(levulinoyloxyphenyl) methyl,
4,4',4"tris(benzoyloxyphenyl)methyl, 3-(imidazol-1-ylmethyl)bis(4',4"-
dimethoxyphenyl)-methyl, 1,1-bis(4-methoxyphenylr1'-pyrenylmethyl, 9-
anthryl, 9-(9-phenyl) xanthenyl, 9-(9-phenyl-10-oxo) anthryl, 1,3-
benzodithiolan-2-yl, and benzisothiazolyl S,S-dioxido.
Silyl ethers include: trimethylsilyl, triethylsilyl, triisopropylsilyl,
dimethylisopropylsilyl, diethylisopropylsilyl, dimethylthexylsilyl, t-
butyldimethylsilyl, t butyldiphenylsilyl, tribenzylsilyl, tri-p-xylylsilyl,
triphenylsilyl, diphenylmethylsilyl, and t butylmethoxyphenylsilyl.
Ester protecting grounds
Ester protecting groups include: esters, carbonates, assisted
cleavage, miscellaneous esters, and sulfonates.
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Examples of protective esters include: formate, benzoylformate,
acetate, chloroacetate, dichloroacetate, trichloroacetate, trifluoroacetate,
methoxyacetate, triphenylmethoxyacetate, phenoxyacetate, p-
chlorophenoxyacetate, p-P-phenylacetate, 3-phenylpropionate, 4-
oxopentanoate (levulinate), 4,4-(ethylenedithio) pentanoate, pivaloate,
adamantoate,crotonate,4-methoxycrotonate, benzoate, p-phenylbenzoate,
and 2,4,6-trimethylbenzoate (mesitoate).
Carbonates include: methyl, 9-fluorenylmethyl, ethyl, 2,2,2-
trichloroethyl,
2-(trimethylsilyl) ethyl, 2-(phenylsulfonyl) ethyl, 2-(triphenylphosphonio)
ethyl,
isobutyl, vinyl, allyl, p-nitrophenyl, benzyl, p-methoxybenzyl, 3,4-
dimethoxybenzyl, o-nitrobenzyl, p-nitrobenzyl, S-benzyl thiocarbonate, 4-
ethoxy-1-naphthyl, and methyl dithiocarbonate.
Examples of assisted cleavage protecting groups include: 2-
iodobenzoate, 4-azido-butyrate, 4-vitro-4-methylpentanoate, o-
(dibromomethyl) benzoate, 2-formylbenzene-sulfonate, 2-(methylthiomethoxy)
ethyl carbonate, 4-(methylthiomethoxymethyl) benzoate, and 2-
(methylthiomethoxymethyl) benzoate.
In addition to the above classes, miscellaneous esters include: 2,6-
dichloro-4-methylphenoxyacetate, 2,6-dichloro-4-(1,1,3,3-tetramethylbutyl)
phenoxyacetate, 2,4-bis(1,1-dimethylpropyl) phenoxyacetate,
chlorodiphenylacetate, isobutyrate, monosuccinoate, (E~-2-methyl-2-
butenoate (tigloate), o-(methoxycarbonyl) benzoate, p-P-benzoate, a-
naphthoate, nitrate, alkyl N,N,N' N'-tetramethylphosphorodiamidate, N-
phenylcarbamate, borate, dimethylphosphinothioyl, and 2,4-
dinitrophenylsulfenate.
Protective sulfates include: sulfate, methanesulfonate(mesylate),
benzylsulfonate, and tosylate.
Protection for 1.2 and 1.3-diols
The protection for 1,2 and 1,3-diols group includes: cyclic acetals and
ketals, cyclic ortho esters, and silyl derivatives.
Cyclic acetals and ketals include: methylene, ethylidene, 1-t-
butylethylidene, 1-phenylethylidene, (4-methoxyphenyl) ethylidene, 2,2,2-
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trichloroethylidene, acetonide (isopropylidene), cyclopentylidene,
cyclohexylidene, cycloheptylidene, benzylidene, p-methoxybenzylidene, 2,4-
dimethoxybenzylidene, 3,4-dimethoxybenzylidene, and 2-nitrobenzylidene:
Cyclic ortho esters include: methoxymethylene, ethoxymethylene,
dimethoxy-methylene, 1-methoxyethylidene, 1-ethoxyethylidine, 1,2-
dimethoxyethylidene, a-methoxybenzylidene, 1-(N,N-
dimethylamino)ethylidene derivative, a-(N,N~iimethylamino) benzylidene
derivative, and 2-oxacyclopentylidene.
Protection for the Carboxyl Group
Ester protecting groups include: esters, substituted methyl esters, 2-
substituted ethyl esters, substituted benzyl esters, silyl esters, activated
esters, miscellaneous derivatives, and stannyl esters.
Substituted methyl esters include: 9-fluorenylmethyl, methoxymethyl,
methylthiomethyl, tetrahydropyranyl, tetrahydrofuranyl, methoxyethoxymethyl,
2-(trimethylsilyl)ethoxy-methyl, benzyloxymethyl, phenacyl, p-bromophenacyl,
a-methylphenacyl, p-methoxyphenacyl, carboxamidomethyl, and 11N
phthalimidomethyl.
2-Substituted ethyl esters include: 2,2,2-trichloroethyl, 2-haloethyl, ~-
chloroalkyl, 2-(trimethylsily)ethyl, 2-methylthioethyl, 1,3-dithianyl-2-
methyl,
2(p-nitrophenylsulfenyl)-ethyl, 2-(p toluenesulfonyl)ethyl, 2-(2'-
pyridyl)ethyl, 2-
(diphenylphosphino)ethyl, 1-methyl-1-phenylethyl, t butyl, cyclopentyl,
cyclohexyl, allyl, 3-buten-1-yl, 4-(trimethylsily)-2-buten-1-yl, cinnamyl, a-
methylcinnamyl, phenyl, p-(methylmercapto)-phenyl, and benzyl.
Substituted benzyl esters include: triphenylmethyl, diphenylmethyl,
bis(o-nitrophenyl)methyl, 9-anthrylmethyl, 2-(9,10-dioxo)anthrylmethyl, 5-
dibenzo-suberyl, 1-pyrenylmethyl,2-(trifluoromethyl)-6-chromylmethyl, 2,4,6-
trimethylbenzyl, p-bromobenzyl, o-nitrobenzyl, p-nitrobenzyl, p-
methoxybenzyl, 2,6-dimethoxybenzyl, 4-(methylsulfinyl)benzyl, 4-sulfobenzyl,
piperonyl, and 4-P-benzyl.
Silyl esters include: trimethylsilyl, triethylsilyl, t butyldimethylsilyl, i-
propyldimethylsilyl, phenyldimethylsilyl, and di- t-butylmethylsilyl.
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Miscellaneous derivatives includes: oxazoles, 2-alkyl-1,3-oxazolines,
4-alkyl-5-oxo-1,3-oxazolidines, 5-alkyl-4-oxo-1,3-dioxolanes, ortho esters,
phenyl group, and pentaaminocobalt(III) complex.
Examples of stannyl esters include: triethylstannyl and tri-n-
butylstannyl.
Amides and Hydrazides
Amides include: N,N-dimethyl, pyrrolidinyl, piperidinyl, 5,6-
dihydrophenanthridinyl, o-nitroanilides, N-7-nitroindolyl, N-8-nitro-1,2,3,4-
tetrahydroquinolyl, and ~P-benzenesulfonamides. Hydrazides include: 11~
phenyl, N,N'-diisopropyl and other dialkyl hydrazides.
Protection for the Amino Group
Carbamates include: carbamates, substituted ethyl, assisted cleavage,
photolytic cleavage, urea-type derivatives, and miscellaneous carbamates.
Carbamates include: methyl and ethyl, 9-fluorenylmethyl, 9-(2-
sulfo)fluorenylmethyl, 9-(2,7-dibromo)fluorenylmethyl, 2,7-di-t butyl-[9-
(10,10-
dioxo-10,10,10,10-tetrahydro- thioxanthyl)]methyl, and 4-methoxyphenacyl.
Substituted ethyl protective groups include: 2,2,2-trichloroethyl, 2-
trimethylsilylethyl, 2-phenylethyl, 1-(1-adamantyl~1-methylethyl, 1,1-dimethyl-
2-haloethyl, 1,1dimethyl-2,2-dibromoethyl, 1,1-dimethyl-2,2,2-trichloroethyl,
1-
methyl-1-(4-biphenylyl)ethyl, 1-(3,5-di-t butylphenyl)-1-methylethyl, 2-(2'-
and
4'-pyridyl)ethyl, 2-(N,ll~icyclohexylcarboxamido)- ethyl, t butyl, 1-
adamantyl,
vinyl, allyl, 1-isopropylallyl, connamyl, 4-nitrocinnamyl, quinolyl, 11~
hydroxypiperidinyl, alkyldithio, benzyl, ~o-methoxybenzyl, p-nitrobenzyl, ~
bromobenzyl, p-chlorobenzyl, 2,4dichlorobenzyl, 4-methylsulfinylbenzyl,
9-anthrylmethyl, and diphenylmethyl.
Protection via assisted cleavage includes: 2-methylthioethyl,
2-methylsulfonylethyl, 2-(~toluenesulfonyl)ethyl, [2-(1,3-dithianyl)]methyl,
4-methylthiophenyl, 2,4-dimethyl-thiophenyl, 2-phosphonioethyl,
2-triphenylphosphonioisopropyl, 1,1-dimethyl-2cyanoethyl, m-chloro-~r
acyloxybenzyl, p-(dihydroxyboryl)benzyl, 5-benzisoxazolyl-methyl, and
2-(trifluoromethyl)-6-chromonylmethyl.
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Photolytic cleavage methods use groups such as: m-nitrophenyl, 3,5-
dimethoxybenzyl, o-nitrobenzyl, 3,4-dimethoxy-6-nitrobenzyl, and phenyl(o-
nitrophenyl)methyl.
Examples of urea-type derivatives include: phenothiazinyl-(10r
carbonyl derivative, N'-p-toluenesulfonylaminocarbonyl, and N'-
phenylaminothiocarbonyl.
In addition to the above, miscellaneous carbamates include: t amyl, S-
benzyl thiocarbamate, p-cyanobenzyl, cyclobutyl, cyclohexyl, cyclopentyl,
cyclopropylmethyl, p-decyloxy-benzyl, diisopropylmethyl, 2,2-
dimethoxycarbonylvinyl, o-(N,ll~dimethyl-carboxamido)-benzyl, 1,1-dimethyl-
3(N,ll~dimethylcarboxamido)propyl, 1,1-dimethyl-propynyl, di(2-pyridyl)methyl,
2-furanylmethyl, 2-iodoethyl, isobornyl, isobutyl, isonicotinyl, p(p'-
methoxyphenyl- azo)benzyl, 1-methylcyclobutyl, 1-methylcyclohexyl, 1-
methyl-1-cyclopropyl- methyl, 1-methyl-(3,5-dimethoxyphenyl)ethyl, 1-methyl-
1(p-henylazophenyl)- ethyl, 1-methyl-1-phenylethyl, 1-methyl-1-(4-
pyridyl)ethyl, phenyl,
p-(phenylazo)benzyl, 2,4,6-tri-t butylphenyl, 4-(trimethylammonium) benzyl,
and 2,4,6-trimethylbenzyl.
Amides
Amides includes: N formyl, 11f=acetyl, Iwchloroacetyl, ll~trichloroacetyl,
IU~trifluoroacetyl, I~phenylacetyl, N-3-phenylpropionyl, Iwpicolinoyl, Iw3-
pyridyl-carboxamide, N-benzoylphenylalanyl derivative, N benzoyl, and I~p-
phenylbenzoyl.
Assisted cleavage groups include: ll~o-nitrophenylacetyl, Iwo-
nitrophenoxyacetyl, llwacetoacetyl, (N=dithiobenzyloxycarbonylamino)acetyl,
113-(p-hydroxphenyl) propionyl, 113-(o-nitrophenyl)propionyl, 112-methyl-2-
(o-nitrophenoxy)propionyl, 112-methyl-2-(o-phenylazophenoxy)propionyl, N-4-
chlorobutyryl, 113-methyl-3-nitrobutyryl, Iwo-nitrocinnamoyl, 11~
acetylmethionine derivative, N-o-nitrobenzoyl, 11~o-
(benzoyloxymethyl)benzoyl, and 4,5-Biphenyl-3-oxazolin-2-one.
Cyclic imide derivatives include: ll~phthalimide, ll~dithiasuccinoyl,
112,3-Biphenyl-maleoyl, IU~2,5-dimethylpyrrolyl,
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N-1,1,4,4-tetramethyldisilylazacyclopentane adduct, 5-substituted
1,3-dimethyl-1,3,5-triazacyclohexan-2-one, 5-substituted 1,3-dibenzyl-
1,3,5-triazacyclohexan-2-one, and 1-substituted 3,5-dinitro-4-pyridonyl.
Special -NH Protective Groups
Protective groups for - NH include: 111~alkyl and ll~aryl amines, imine
derivatives, enamine derivatives, and N-hetero atom derivatives (such as N-
metal, N-N, N-P, N-Si, and N-S), ll~sulfenyl, and N-sulfonyl.
llNalkyl and ll~aryl amines include: 11f=methyl, llNallyl,
I~[2-(trimethylsilyl)ethoxyl]-methyl, 113-acetoxypropyl,
N-(1-isopropyl-4-vitro-2-oxo-3-pyrrolin-3-yl), quaternary ammonium salts,
N-benzyl, ll~di(4-methoxyphenyl)methyl, 115-dibenzosuberyl,
N-triphenylmethyl, 11~(4-methoxyphenyl)diphenylmethyl, 119-phenylfluorenyl,
112,7-dichloro-9-fluorenylmethylene, N-ferrocenylmethyl, and
N-2-picolylamine N'-oxide.
Imine derivatives include: 111,1-dimethylthiomethylene,
ll~benzylidene,
ll~~methoxybenzylidene, I~diphenylmethylene,
11~[(2-pyridyl)mesityl]methylene,
I~(N',N'-dimethylaminomethylene), N,N'-isopropylidene,
ll~~nitrobenzylidene,
ll~salicylidene, N-5-chlorosalicylidene, 11~(5-chloro-2-hydroxyphenyl)phenyl-
methylene, and 11f=cyclohexylidene.
An example of an enamine derivative is I~(5,5-dimethyl -3-oxo-1-
cyclohexenyl).
ll~metal derivatives include: ll~borane derivatives, N-diphenylborinic
acid derivative, N-[phenyl(pentacarbonylchromium- or -tungsten)]carbenyl,
and ll~copper or N-zinc chelate. Examples of N-N derivatives include:
ll~nitro, ll~nitroso, and ll~oxide. Examples of 11~P derivatives include:
11f=diphenylphosphinyl, ll~dimethylthiophosphinyl, ll~diphenylthiophosphinyl,
N-dialkyl phosphoryl, N-dibenzyl phosphoryl, and I~diphenyl phosphoryl.
Examples of N-sulfenyl derivatives include: 11f=benzenesulfenyl,
11f=o-nitrobenzenesulfenyl, 11f=2,4-dinitrobenzenesulfenyl,
ll~pentachlorobenzenesulfenyl, N-2-vitro-4-methoxy-benzenesulfenyl, N-
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triphenylmethylsulfenyl, and N-3-nitropyridinesulfenyl. N-sulfonyl derivatives
include: N ~.toluenesulfonyl, N-benzenesulfonyl, N-2,3,6-trimethyl-
4-methoxybenzenesulfonyl, N-2,4,6-trimethoxybenzenesulfonyl, N-
2,6-dimethyl-4-methoxy-benzenesulfonyl, N-pentamethylbenzenesulfonyl, N-
2,3,5,6-tetramethyl-4-methoxybenzene- sulfonyl, N-
4-methoxybenzenesulfonyl, N-2,4,6-trimethylbenzenesulfonyl, N-
2,6-dimethoxy- 4-methylbenzenesulfonyl, N-
2,2,5,7,8-pentamethylchroman-6-sulfonyl, N-methanesulfonyl,
N /i-trimethylsilylethanesulfonyl, N-9-anthracenesulfonyl, N-
4-(4',8'-dimethoxynaphthylmethyl)-benzenesulfonyl, N-benzylsulfonyl, N-
trifluoromethylsulfonyl, and N-phenacylsulfonyl.
Disclosed compounds which are masked or protected may be prodrugs,
compounds metabolized or otherwise transformed in vivo to yield a disclosed
compound, e.g., transiently during metabolism. This transformation may be a
hydrolysis or oxidation which results from contact with a bodily fluid such as
blood, or
the action of acids, or liver, gastrointestinal, or other enzymes.
Some of these masked or protected compounds are pharmaceutically
acceptable; others will be useful as intermediates. Synthetic intermediates
and processes disclosed herein, and minor modifications thereof, are also
within the scope of the invention.
The compounds of formulas I and II can be prepared by techniques
and procedures readily available to one of ordinary skill in the art, for
example
by following the procedures as set forth in the following Schemes or
analogous variants thereof. These synthetic strategies are further exemplified
in Examples 1-6 below. These Schemes are not intended to limit the scope of
the invention in any way.
As used herein, the following terms have the meanings indicated:
"LiBH4" refers to lithium borohydride; "TMSCI" refers to trimethylsilyl
chloride;
"TBDPSCI" refers to tent butyldiphenylsilyl chloride; "sBuLi" refers to seo-
butyllithium; "TBAF" refers to tetrabutylammonium fluoride; "HOAc" refers to
acetic acid; "KMn04" refers to potassium permanganate; "LiHMDS" refers to
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lithium 1,1,1,3,3,3-hexamethyl-disilazane. All other terms and substituents,
unless otherwise indicated, are previously defined. The reagents and starting
materials are readily available to one of ordinary skill in the art. Scheme I
provides a synthesis of the compound of structure (2).
SCHEMEI
COZH
a) LiBH4, TMSCI
F b) TBDPSCI
c) sBuLi
'F d) COZ
F e) TBAF, HOAc F
f) KMN04 (2)
(1)
In Scheme I, step a, the compound of structure (1 ), which is 2,3,4-
trifluorobenzoic acid, is reduced with in situ prepared borane under
conditions
described in Angew. Chem. Int. Ed. (1989), 28, 218 to provide the
corresponding alcohol.
In Scheme I, step b, the alcohol is protected with a suitable hydroxyl
protecting group, such as tert-butyldiphenylsilyl chloride. A suitable
hydroxyl
protecting group will be stable to basic conditions.
In Scheme I, step c, directed metallation of the protected alcohol
provides the anion. In step d, the resulting anion is quenched with carbon
dioxide to provide the monoacid.
In Scheme I, steps a and f, the protected monoacid is deprotected and
oxidized under conditions well known in the art to provide the symmetrical
diacid (2) which is 4,5,6-trifluoro-isophthalic acid.
These synthetic strategies are further exemplified in Example 1, steps
a-d.
Scheme II provides a synthesis of the compound of structure (4).
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SCHEME II
C 02H a) LiBH4, TMSCI
bj TBDPSCI, Imidazole
F c) sBuLi F
\
/ d) TMSCI
e) sBuLi
F ~ .C02
g) TBAF
(3) h) KMn04 (4)
In Scheme II, step a, the compound of structure (3), which is 2,4-
difluorobenzoic acid, is reduced with in situ prepared borane under conditions
described in Angew. Chem. Int. Ed. (1989), 28, 218 to provide the
corresponding alcohol.
In Scheme II, step b, the alcohol is protected with a suitable hydroxyl
protecting group, such as Pert butyldiphenylsilyl chloride. A suitable
hydroxyl
protecting group will be stable to basic conditions.
In Scheme II, step c, directed metallation of the protected alcohol
provides the anion. In step d, the resulting anion is quenched with a
silylating
agent, such as trimethylsilyl chloride to provide the monoacid.
In Scheme II, step e, directed metallation of the protected alcohol
provides the anion. In step f, the resulting anion is quenched with carbon
dioxide to provide the monoacid.
In Scheme II, steps g and h, the protected monoacid is deprotected
and oxidized under conditions well known in the art to provide the symmetrical
diacid (4) which is 4,6-difluoca-isophtha~ic acid.
Scheme III provides a synthesis of the compounds of formula I, which
includes formulas la and Ib, and formula II.
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SCHEME III
R1 Step A, Couplln~ R1
HzN ~ wlth a Diacid (6) Step B, Protectio
I~ ~ \
I :H
halo ~ I
(S) HO=C R~
Rs (7) Rs
(g)
(8)
O O
Step C, Activation ~ R1
of the Acld Step D, Amide
Formation I ~ N I \
\ I x ~ I
/ O R Rs
Fs s
(9) Formula II
Fs
Step F, Activation /
Step E, Deprotecti of the Acid
X
Formula la X
(10)
Step G, Amide
Formation R1
I\
X / I
..s
Formula Ib
In Scheme III, step A, a suitable aniline (5), such as 4-iodo-2
methylaniline, 4-iodo-2-chloroaniline, or 4-iodo-2-fluoroaniline is coupled
with
a symmetrical diacid (6) to provide the compound of structure (7). Examples
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of suitable diacids (6) include, but are not limited to, compound (2) as shown
in Scheme I, compound (4) as shown in Scheme II and 4-fluoro-isophthalic
acid which can be prepared by one of ordinary skill in the art following
generally the procedure disclosed by Chu~rina. G.N. et al.. Uzh. Vses. Khim:
O-va, 19(5), 598-9 (1974). It is within the skill of one of ordinary art to
identify
additional diacids (6) useful in the preparation of compounds of the present
invention. For example, compound (6) and compound (5), in separate flasks,
are each suspended in a suitable organic solvent, such as tetrahydrofuran, at
-78 °C under nitrogen. Each suspension is treated with an excess of a
suitable base, such as 2 equivalents of lithium 1,1,1,3,3,3-hexamethyl-
disilazane or lithium amide. After both solutions are stirred for about 30
minutes at -78 °C, the diacid solution was transferred by cannula into
the
aniline solution and allowed to warm to room temperature. After stirring from
about 4 to 12 hours, the mixture was precipitated with a suitable solution,
such as a saturated HCI diethyl ether solution or combined with 1 N HCI and
extracted with ethyl acetate. The resulting precipitate was filtered and
concentrated under vacuum to provide the anthranilic diacid (7).
In Scheme III, step B, the acidic groups of the diacid (7) are
differentiated by protection using suitable aldehyde, such as formalin or
paraformaldehyde when R1 is methyl; or by using methyl bromide and cesium
fluoride when R1 is a halogen, such as chloride or fluoride. For example, the
diacid (7), a suitable aldehyde, such as paraformaldehyde, and a suitable
acid, such as para-toluenesulfonic acid monohydrate were combined in a
suitable solvent, such as dichloromethane. In a roundbottom flask attached
with a Dean-Stark apparatus, the solution is allowed to reflux for about 3
hours. The resulting solution is concentrated and the residue is suspended in
a suitable solvent, such as methanol. The aldehyde is filtered off, the
filtrate
is collected and concentrated under vacuum to provide the free acid (8).
In Scheme III, step C, the free acid (8) is activated, such as by the
addition of trifluoroacetic acid pentafluorophenyl ester. For example, to a
suspension of the free acid (8) in a suitable solvent, such as in N,11~
dimethylformamide is added trifluoroacetic acid pentafluorophenyl ester and a
suitable base, such as pyridine. The reaction mixture is stirred for about 4
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hours, diluted with a suitable solvent, such as ethyl acetate, and washed with
a series of solutions, such as 3 times with 1.0 M HCI solution, 3 times with
5%
aqueous NaHC03 solution, 2 times with water and once with saturated brine
solution. The organic extracts are combined, dried over sodium sulfate,
filtered and concentrated under vacuum to provide the activated free ester
(9).
In Scheme III, step D, amines are added to the free ester (9) to provide
the amide or the ester, which is a compound of formula II. For example, the
free ester (9) is suspended in a suitable solvent, such as tetrahydrofuran. To
the resulting suspension, a suitable alcohol, such as methanol, or suitable
amines, such as methylamine hydrochloride and a suitable tertiary amine
base, such as triethylamine and N,N-diisopropylethylamine are added. After
stirring from about 12 to 17 hours, the reaction mixture was diluted with a
suitable solvent, such as ethyl acetate and washed using a series of
solutions,
such as 2 times with water and 2 times with saturated brine solution. The
organic extracts are combined, dried over sodium sulfate, filtered and
concentrated under vacuum to provide the amide of formula II.
Examples of X may be derived by one of ordinary skill in the art from
commercially available reagents that include, but are not limited to, the
following:
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trans-2-aminocyclohexanol hydrochloride ~ 2-amino-5-mercapto-1,3,4-thiadiazole
2-amino-1,3,4-thiadiazole 2-amino-5-methyl-1,3,4-thiadiazole
3-amino-1-phenyl-2-pyrazolin-5-one 2-amino-5-ethyl-1,3,4-thiadiazole
5-amino-3-methylisoxazole 2-amino-6-methoxybenzothiazole
3-amino-5-methylisoxazole 2-amino-6-ethoxybenzothiazole
5-amino-3-phenyl-1,2,4-thiadiazole 2-amino-6-methylbenzothiazole
2-(2-aminoethyl)-1-methylpyrrolidine 2-amino-4-methylbenzothiazole
2-(aminomethyl)-1-ethylpyrrolidine 4-aminobenzo-2,1,3-thiadiazole
1-(2-aminoethyl)pyrrolidine 4-amino-6-chloro-2-
methylmercaptopyrimidine
pseudothiohydantoin 2-aminopyrimidine
1-(3-aminopropyl)-2-pyrrolidinone 2-amino-4,6-dihydroxypyrimidine
furfurylamine 4-aminopyrimidine
1-aminomethyl-1-cyclohexanol aminopyrazine
hydrochloride
histamine 4-morpholinoaniline
3-amino-1,2,4-triazole 4-(2-aminoethyl)morpholine
3-amino-5-mercapto-1,2,4-triazole n-(3-aminopropyl)morpholine
3-amino-5-methylthio-1,2,4-triazole 5-amino-2-chloropyridine
3-aminopyrazole 5-amino-2-methoxypyridine
3-amino-4-carbethoxypyrazole 2-aminopyridine
2-amino-2-thiazoline 2-aminopyridine
2-aminothiazole 2-(aminomethyl)pyridine
2-amino-4-methylthiazole 2-(2-aminoethyl)pyridine
ethyl 2-amino-4-thiazoleacetate 3-aminopyridine
d-cycloserine 3-(aminomethyl)pyridine
tetrahydrofurfurylamine 4-aminopyridine
thiophene-2-methylamine 4-(aminomethyl)pyridine
2-aminopurine 3-amino-1,2,4-triazine
2-aminobenzimidazole 1-(2-aminoethyl)piperidine
5-methoxytryptamine 3,4-ethylenedioxyaniline
6-methoxytryptamine 2-aminophenethyl alcohol
6-aminoindazole N,N-dimethyl-p-phenylenediamine
8-azaadenine N,N-diethyl-1,4-phenylenediamine
2-aminobenzothiazole 2-aminobenzenesulfonamide
2-(2-aminoethoxy)ethanol sulfanilamide
2-(3,4-dimethoxyphenyl)ethylamine2-amino-1-methoxypropane
3-isopropoxypropylamine dl-2-amino-1-propanol
methyl3-aminothiophene-2-carboxylate4-hydroxypiperidine
n-(3-aminopropyl)imidazole 4-piperidineethanol
3-aminopyrazine-2-carboxylic 1-methyl-4-(methylamino)piperidine
acid methyl
ester
5-amino-1-ethylpyrazole N-methyl-p-anisidine
3-amino-5-hydroxypyrazole methylaminoacetaldehyde
dimethylacetal
2-amino-5-(ethylthio)-1,3,4-thiadiazole(S)-(+)-2-(methoxymethyl)pyrrolidine
dl-cycloserine 1-methylpiperazine dihydrochloride
3-amino-5-methylpyrazole 3-hydroxypiperidine hydrochloride
4-chloro-n-methylaniline dl-nornicotine
2-(methylamino)ethanol 4-hydroxypiperidine hydrochloride
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N,N'-bis(2-hydroxyethyl)ethylenediamine ~ 4-(1-pyrrolidinyl)piperidine
diethanolamine N-ethylpiperazine
2-(butylamino)ethanethiol d-prolinol
thiazolidine thialdine
I-prolinol (R)-3-hydroxypyrrolidine
3-pyrrolidinol (R)-(-)-3-pyrrolidinol hydrochloride
N-omega-methyltryptamine (R)-(+)-3-hydroxypiperidine
hydrochloride
piperazine (S)-3-hydroxypyrrolidine
1-formylpiperazine thialdine
1-methylpiperazine
1-benzylpiperazine
N-(2-hydroxyethyl)piperazine
morpholine
thiomorpholine
2-piperidinemethanol
2-piperidineethanol
3-piperidinemethanol
In Scheme III, step E, the acid of the compound of formula II is deprotected
under acidic conditions using a polymer bound glycol as a quench reagent to
provide the compound of formula la. For example, to a suspension of formula II
in a
suitable solvent, such as tetrahydrofuran, is added a suitable quench agent,
such as
polymer bound glycerol, and a suitable acidic solution, such as about 10 mL of
1.0
M hydrochloric acid solution. After stirring for about 48 hours at room
temperature,
the resin is filtered off and the filtrate is transferred to a separatory
funnel and
partitioned with a suitable solvent, such as ethyl acetate. The organics are
washed
using a series of solutions, such as twice with 1.0 M HCI and twice with
saturated
brine solution. The organic extracts are collected, dried over sodium sulfate,
filtered
and concentrated under vacuum to provide the compound of formula la.
In Scheme III, step F, the deprotected acid of formula la is activated, such
as
by the addition of trifluoroacetic acid pentafluorophenyl ester and reacted
with an
appropriately substituted hydroxyl amine, to allow the formation of the
hydroxamate,
which is the compound of formula 1 b. For example, to a suspension of formula
1 a
in a suitable solvent, such as in N,N dimethylformamide is added
trifluoroacetic acid
pentafluorophenyl ester and a suitable base, such as pyridine. The reaction
mixture
is stirred for about 17 hours, diluted with a suitable solvent, such as ethyl
acetate,
and washed using a series of solutions, such as 3 times with 1.0 M HCI
solution, 3
times with 5% aqueous NaHC03 solution, 2 times with water and once with
saturated brine solution. The organic extracts are combined, dried over sodium
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sulfate, filtered and concentrated under vacuum to provide the activated free
ester
(10).
In Scheme III, step G, amines are added to the free ester (10) to provide the
amide, which is a compound of forri~ula Ib. For example, the free ester (10)
is
suspended in a suitable solvent, such as tetrahydrofuran. To the resulting
suspension, a suitable alcohol, such as methanol, or suitable amines, such as
methylamine hydrochloride and cyclopropylmethylamine hydrochloride, and a
suitable tertiary amine base, such as triethylamine and
N,llf=diisopropylethylamine
are added. After stirring from about 12 to 17 hours, the reaction mixture was
partitioned between a suitable solvent, such as ethyl acetate and a suitable
acid,
such as 1.0 M HCI solution. The organic layer was washed using a series of
solutions, such as 2 times with water and 2 times with saturated brine
solution. The
organic extracts are combined, dried over magnesium sulfate, filtered and
concentrated under vacuum to provide the hydroxamate of formula Ib.
One aspect of the invention features the disclosed compounds shown in
formulas I and II. Preferred compounds of formulas I or II are those in which
R1 is
C1_g alkyl or halo, preferably C1_g alkyl, more preferably fluoro, chloro, or
methyl,
and most preferably methyl; R3 and R4 are each independently hydrogen or halo,
preferably fluoro; A is hydroxy or NRgOR7; X is NR1 gRl2 or NR14; and R12 and
R1g are each independently [(CH2)nY(.CH2)m]qCHg, (C1-6 alkyl)phenyl, -
[(CH2)nY(CH2)m]qphenyl, or (C1-g alkyl)C2-g heterocyclic radical.
Also preferred are compounds of formula I or formula II in which heterocyclic
radicals include heteroaryls such as substituted or unsubstituted radicals of
pyrrole,
furan, pyran, thiophene, pyrazole, imidazole, triazole, tetrazole, indole,
isoxazole,
indazole, pyridine, pyrazine, oxazole, thiazole, oxadiazole, oxathiadiazole;
heterocycles also include heteroalkyls such as substituted and unsubstituted
radicals of morpholine, piperidine, piperazine, tetrahydrofuran,
tetrahydropyran,
pyrrolidone, imidazoline, and tetrahydrothiophene.
Table I and Table II provide examples of preferred compounds of the present
invention.
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Table I
O A
R1
X
F ~ I
O F
-A -R~ -X
-OH -Me -NH2
-OH -Me -NHMe
-OH -Me -NMe2
-OH -Me
w
-OH -Me
~N~
-OH -Me
~N~
r
-OH -Me -N
CH3nBu
-OH -Me ~N~
LN
-OH -Me -N[(CH2)20CH2CH3]2
-OH -Me
~NJ
-OH -Me ~N~OH
~NJ
-OH -CI -NH2
-OH -CI -NHMe
-OH -CI -NMe2
-OH -CI
-OH -CI
~N~
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-OH -CI
~N~
c
-OH -CI -N
CH3nBu
-OH -CI ~N~
~N
-OH -CI -N[(CH2)20CH2CH3]2
-OH -CI N
~N~~
~NJ
-OH -CI ~N~OH
LN
'~
-NHOCH2cPr -Me -NH2
-NHOCH2cPr -Me -NHMe
-NHOCH2cPr -Me -NMe2
-NHOCHZcPr -Me
~N~
-NHOCH2cPr -Me
~N~
-NHOCH2cPr -Me
~N~
-NHOCH2cPr -Me -NCH3nBu
-NHOCH2cPr -Me ~N~
LN
-NHOCH2cPr -Me -N[(CH2)20CH2CH3]2
-NHOCH2cPr -Me ~N~N ~
~NJ
-NHOCH2cPr -Me ~N/~OH
~ INJ
-NHOCH2cPr -CI -NH2
-NHOCH2cPr -CI -NHMe
-NHOCH2cPr -CI -NMe2
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-NHOCH2cPr -CI
w
-NHOCH2cPr -CI
N~
~
-NHOCH2cPr -CI
N~
~
-NHOCH2cPr -CI -NCH3nBu
-NHOCH2cPr -CI ~N~
~N
-NHOCH2cPr -CI -N[(CH2)20CH2CH3]2
-NHOCH2cPr -CI N
r N- 'N-
~ INJ
-NHOCH2cPr -CI ~N/~OH
LN
Table II
O O
R1
N
F ~ / 1
O F
-R~ -X
-Me -NH2
-Me -NHMe
-Me -NMe2
-Me
-Me
~N~
-Me
~N~
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-Me -NCH3nBu
-Me ~N~
LN
-Me -N[(CH2)20CH2CH3]2
_Me ~N~N I
~NJ
-Me ~ ~oH
~N
'F
-CI -NH2
-CI -NHMe
-CI -NMe2
-CI
-CI
~N~
-CI
~N~
r
-CI -N
CH3nBu
-CI ~N/
~NJ
-CI -N[(CH2)20CH2CH3]2
-CI ~N.~N I
~NJ
~N/~OH
~NrJ
As used herein, the term "patient" refers to any warm-blooded animal such
as, but not limited to, a human, horse, dog, guinea pig, or mouse. Preferably,
the
patient is human.
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The term "treating" for purposes of the present invention refers to
prophylaxis
or prevention, amelioration or elimination of a named condition once the
condition
has been established.
According to one aspect of the invention, the compounds are MEK inhibitors.
MEK inhibition assays include the in vitro cascade assay for inhibitors of MAP
kinase pathway described at column 6, line 36 to column 7, line 4 of U.S.
Patent
Number 5,525,625 and the in vitro MEK assay at column 7, lines 4-27 of the
same
patent, the entire disclosure of which is incorporated by reference (see also
Examples 163-173 below).
Selective MEK 1 or MEK 2 inhibitors are those compounds which inhibit the
MEK 1 or MEK 2 enzymes, respectively, without substantially inhibiting other
enzymes such as MKK3, PKC, Cdk2A, phosphorylase kinase, EGF, and PDGF
receptor kinases, and C-src. In general, a selective MEK 1 or MEK 2 inhibitor
has
an IC5o for MEK 1 or MEK 2 that is at least one-fiftieth (1/50) that of its
IC5o for one
of the above-named other enzymes. Preferably, a selective inhibitor has an
ICso
that is at least 1/100, more preferably 1/500, and even more preferably
1/1000,
1/5000, or less than that of its IC5oor one or more of the above-named
enzymes.
The disclosed compositions are useful as both prophylactic and therapeutic
treatments for diseases or conditions related to the hyperactivity of MEK, as
well as
diseases or conditions modulated by the MEK cascade. Examples include, but are
not limited to, stroke, septic shock, heart failure, osteoarthritis,
rheumatoid arthritis,
organ transplant rejection, and a variety of tumors such as ovarian, lung,
pancreatic,
brain, prostatic, and colorectal.
The invention further relates to a method for treating proliferative diseases,
such as cancer, restenosis, psoriasis, autoimmune disease, and
atherosclerosis.
Other aspects of the invention include methods for treating MEK-related
(including
ras-related) cancers, whether solid or hematopoietic. Examples of cancers
include
brain, breast, lung, such as non-small cell lung, ovarian, pancreatic,
prostate, renal,
colorectal, cervical, acute leukemia, and gastric cancer. Further aspects of
the
invention include methods for treating or reducing the symptoms of xenograft
(cell(s), skin, limb, organ or bone marrow transplant) rejection,
osteoarthritis,
rheumatoid arthritis, cystic fibrosis, complications of diabetes (including
diabetic
retinopathy and diabetic nephropathy), hepatomegaly, cardiomegaly, stroke
(such
as acute focal ischemic stroke and global cerebral ischemia), heart failure,
septic
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shock, asthma, Alzheimer's disease, and chronic or neuropathic pain. Compounds
of the invention are also useful as antiviral agents for treating viral
infections such
as HIV, hepatitis (B) virus (HBV), human papilloma virus (HPV),
cytomegalovirus
(CMV), and Epstein-Barr virus (EBV). These methods include the step of
administering to a patient in need of such treatment, or suffering from such a
disease or condition, a pharmaceutically or therapeutically effective amount
of a
disclosed compound or pharmaceutical composition thereof.
The term "chronic pain" for purposes of the present invention includes, but is
not limited to, neuropathic pain, idiopathic pain, and pain associated with
chronic
alcoholism, vitamin deficiency, uremia, or hypothyroidism. Chronic pain is
associated with numerous conditions including, but not limited to,
inflammation,
arthritis, and post-operative pain.
As used herein, the term "neuropathic pain" is associated with numerous
conditions which include, but are not limited to, inflammation, postoperative
pain,
phantom limb pain, burn pain, gout, trigeminal neuralgia, acute herpetic and
postherpetic pain, causalgia, diabetic neuropathy, plexus avulsion, neuroma,
vasculitis, viral infection, crush injury, constriction injury, tissue injury,
limb
amputation, post-operative pain, arthritis pain, and nerve injury between the
peripheral nervous system and the central nervous system.
The invention also features methods of combination therapy, such as a
method for treating cancer, wherein the method further includes providing
radiation
therapy or chemotherapy, for example, with mitotic inhibitors such as a taxane
or a
vinca alkaloid. Examples of mitotic inhibitors include paclitaxel, docetaxel,
vincristine, vinblastine, vinorelbine, and vinflunine. Other therapeutic
combinations
include a MEK inhibitor of the invention and an anticancer agent such as
cisplatin,
5-fluorouracil or 5-fluoro-2-4(1 H,3H)-pyrimidinedione (5FU), flutamide, and
gemcitabine.
The chemotherapy or radiation therapy may be administered before,
concurrently, or after the administration of a disclosed compound according to
the
needs of the patient.
Those skilled in the art will be able to determine, according to known
methods, the appropriate therapeutically-effective amount or dosage of a
compound
of the present invention to administer to a patient, taking into account
factors such
as age, weight, general health, the compound administered, the route of
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administration, the type of pain or condition requiring treatment, and the
presence of
other medications. In general, an effective amount or a therapeutically-
effective
amount will be between about 0.1 and about 1000 mg/kg per day, preferably
between about 1 and about 300 mg/kg body weight, and daily dosages will be
between about 10 and about 5000 mg for an adult subject of normal weight.
Commercially available capsules or other formulations (such as liquids and
film-
coated tablets) of 100 mg, 200 mg, 300 mg, or 400 mg can be administered
according to the disclosed methods.
The compounds of the present invention are preferably formulated prior to
administration. Therefore, another aspect of the present invention is a
pharmaceutical composition comprising a compound of formulas I or II and a
pharmaceutically acceptable carrier. In making the compositions of the present
invention, the active ingredient, such as a compound of formula I or formula
II, will
usually be mixed with a carrier, or diluted by a carrier or enclosed within a
carrier.
Dosage unit forms or pharmaceutical compositions include tablets, capsules,
pills,
powders, granules, aqueous and nonaqueous oral solutions and suspensions, and
parenteral solutions packaged in containers adapted for subdivision into
individual
doses.
Dosage unit forms can be adapted for various methods of administration,
including controlled release formulations, such as subcutaneous implants.
Administration methods include oral, rectal, parenteral (intravenous,
intramuscular,
subcutaneous), intracisternal, intravaginal, intraperitoneal, intravesical,
local (drops,
powders, ointments, gels, or cream), and by inhalation (a buccal or nasal
spray).
Parenteral formulations include pharmaceutically acceptable aqueous or
nonaqueous solutions, dispersion, suspensions, emulsions, and sterile powders
for
the preparation thereof. Examples of carriers include water, ethanol, polyols
(propylene glycol, polyethylene glycol), vegetable oils, and injectable
organic esters
such as ethyl oleate. Fluidity can be maintained by the use of a coating such
as
lecithin, a surfactant, or maintaining appropriate particle size. Carriers for
solid
dosage forms include (a) fillers or extenders, (b) binders, (c) humectants,
(d)
disintegrating agents, (e) solution retarders, (f) absorption acccelerators,
(g)
adsorbants, (h) lubricants, (i) buffering agents, and (j) propellants.
Compositions may also contain adjuvants such as preserving, wetting,
emulsifying, and dispensing agents; antimicrobial agents such as parabens,
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chlorobutanol, phenol, and sorbic acid; isotonic agents such as a sugar or
sodium
chloride; absorption-prolonging agents such as aluminum monostearate and
gelatin;
and absorption-enhancing agents.
The following examples represent typical syntheses of the compounds of
formula I and II as described generally above. These examples are illustrative
only
and are not intended to limit the invention in any way. The reagants and
starting
materials are readily available to one of ordinary skill in the art. As used
herein, the
following terms have the meanings indicated: "g" refers to grams; "mg" refers
to
milligrams; "mL" refers to milliliters; "mmol" refer to millimoles;
"°C" refers to degrees
Celsius; "APCI" refers to atmospheric pressure chemical ionization; and "THF"
refers to tetrahydrofuran;
EXAMPLE 1
7,8-Difluoro-1-(4-iodo-2-methyl-phen~)-4-oxo-1.4-dihydro-21-
~benzoLdlf1,3]oxazine-
6-carboxylic acid
Step a: To a suspension of (2,3,4-trifluoro-phenyl)-methanol (prepared as in
Angew.
Chem. Int. Ed. (1989), 28, 218) (8.7g, 54 mmol) in 50 mL of dichloromethane
was
added tert-butyldiphenylsilyl chloride (15.4 mL, 59 mmol) and imidazole (4.02
g, 59
mmol). After 17 hours, the reaction was poured into 100 mL of 1 M HCI solution
and extracted into dichloromethane. The organic layer was washed 2 times with
1
M HCI solution and 2 times with brine~solution. The organic phase was
collected
and dried over Na2S04, filtered, and concentrated in vacuo. The white, oily
solid
(18.8 g) was purified by column silica chromatography eluting with 9:1
hexane:ethyl
acetate to afford 14.37 g (68.5%) tert-butyl-Biphenyl-(2,3,4-trifluoro-
benzyloxy)-
silane.
Step b: To a suspension of tert-butyl-Biphenyl-(2,3,4-trifluoro-benzyloxy)-
silane
(7.42g, 18.5 mmol) in freshly distilled tetrahydrofuran (50 mL) at -78
°C under
nitrogen is added 1.3 M seo-butyllithium in cyclohexane (18.5 mL, 24.0 mmol).
The
reaction was allowed to stir at -78 °C for 1 hour and quenched with C02
gas
(lecture bottle) directly into the solution for 30 minutes and the reaction
mixture was
slowly brought to room temperature. After 3 hours, the reaction was
partitioned
between ethyl acetate and 1 M HCI solution and washed with brine solution. The
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organic phase was collected and dried over Na2S04, filtered and concentrated
in
vacuo to afford 8.17 g (99.6%) of 5-(tert-butyldiphenyl-silanyloxymethyl~
2,3,4-
trifluoro-benzoic acid as a white, waxy solid.
Step c: To a suspension of 5-(Pert-butyldiphenyl-silanyloxymethyl)-2,3,4-
trifluoro-
benzoic acid (8.17g, 18.4 mmol) in freshly distilled THF (20 mL) was added a
solution of tetrabutylammonium fluoride (1.0 M in THF, 40.0 mL, 40.0 mmol).
After
stirring at room temperature for 2 hours the reaction mixture was concentrated
in
vacuo and redissolved in ethyl acetate, transferred to a separatory funnel and
washed 3 times with 1 M HCI solution, 2 times with saturated brine solution.
The
organic layers were collected, dried over Na2S04, filtered and concentrated in
vacuo. To the resulting residue was added hexanes affording a white solid,
which
was washed several times with hexanes, collected and dried in vacuo affording
2,3,4-tifluoro-5-hydroxymethyl-benzoic acid (1.98g, 52.2%).
Stets d: To a refluxing suspension of trifluoro-5-hydroxymethyl-benzoic acid
(1.908,
9.22 mmol) in acetone is added a solution of potassium permanganate (4.3g,
27.7
mmol) in water (5 mL). After refluxing for 6 hours the reaction is allowed to
cool and
an aqueous solution of NaHS03 (SmL, 1.0 M) and an aqueous solution of H2S04
(SmL, 1.0 M) is added which clears the reaction solution. This mixture is
transferred
to a separatory funnel and extracted several times with ethyl acetate. The
organic
layers are collected, dried over Na2S04, filtered and concentrated in vacuo
affording
4,5,6-trifluoro-isophthalic acid as a light yellow solid (1.03 g, 50.7%).
Step e: A suspension of 4,5,6-trifluoro-isophthalic acid (1.03 g, 4.68 mmol)
in freshly
distilled THF (20 mL) at -78 °C under nitrogen is treated with 2.0
equivalents of
freshly prepared 1 M LiHMDS solution (HMDS, 2.07 mL, 9.83 mmol; n-
butyllithium,
3.5 mL, 9.36 mmol) in THF. In a second flask is suspended 4-iodo-2-
methylaniline
(1.09 g, 4.68 mmol) in 20 mL of freshly distilled THF, cooled to -78 °C
under
nitrogen and treated with 2.0 equiv. of freshly prepared 1 M LiHMDS solution
(HMDS, 2.07 mL, 9.83 mmol; n-butyllithium, 3.5 mL, 9.36 mmol) in THF. After
both
solutions stirred for 30 minutes at -78 °C, the benzoic acid solution
was cannula
transferred into the aniline solution and allowed to slowly warm to room
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temperature. After stirring for 4 hours, the reaction mixture was poured into
200 mL
of a saturated HCI diethyl ether solution affording a white precipitate. The
solid is
filtered off and the remaining filtrate is collected and concentrated in vacuo
affording
4,5-difluoro-6-(4-iodo-2-methyl-phenylamino)-isophthalic acid (1.55 g, 77%).
Step f: A suspension of 4,5-difluoro-6-(4-iodo-2-methyl-phenylamino)-
isophthalic
acid (1.1 g, 2.54 mmol), paraformaldehyde (5.0g), and para-toluenesulfonic
acid
monohydrate (15.0 mg) in dichloromethane (250 mL) in a roundbottom flask
attached with a Dean-Stark apparatus is allowed to reflux for 3 hours. The
cooled
solution is then concentrated and the residue is redissolved in methanol and
paraformaldehyde is filtered off and filtrate is collected and concentrated in
vacuo
affording 7,8-difluoro-1-(4-iodo-2-methyl-phenyl)-4-oxo-1,4-dihydro-2H-
benzo[d][1,3]oxazine-6-carboxylic acid as a red solid (0.80 g, 70.0%).
EXAMPLE 2
7.8-Difluoro-1-L-iodo-2-methyl phenyl)-4-oxo-1.4-dihydro-
2l~benzo[d][1.3loxazine-
6-carboxylic acid methvlamide.
O 01 CH3
H3 \ 1N \
HN I / F I / I
O F
To a suspension of 7,8-difluoro-1-(4-iodo-2-methyl-phenyl)-4-oxo-1,4-
dihydro-2h~-benzo[d][1,3]oxazine-6-carboxylic acid (0.3 g, 0.67 mmol) in N,N-
dimethylformamide (3 mL) is added trifluoroacetic acid pentafluorophenyl ester
(0.127 mL, 0.74 mmol) and pyridine (0.60 mL, 0.74 mmol). After stirring for 4
hours
the reaction mixture is diluted with ethyl acetate and washed 3 times with 1.0
M HCI
solution, 3 times with 5% aqueous NaHC03 solution, 2 times with water and once
with saturated brine solution. The organic layers were collected and dried
over
Na2S04, filtered and concentrated in vacuo affording 7,8-difluoro-1-(4-iodo-2-
methyl-phenyl)-4-oxo-1,4-dihydro-21+benzo[d][1,3]oxazine-6-carboxylic acid
pentafluorophenylester as a dark orange oil (0.34g, 83.1 %). To a suspension
of
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7,8-difluoro-1-(4-iodo-2-methyl-phenyl)-4-oxo-1,4-dihydro-
2h~benzo[d][1,3]oxazine-
6-carboxylic acid pentafluorophenylester (0.33 g, 0.54 mmol) in freshly
distilled THF
(10 mL) is added methylamine hydrochloride (0.037 g, 0.54 mmol) and N,N-
diisopropylethylamine (0.019 mL, 1.08 mmol). After stirring for 17 hours, the
reaction mixture was diluted with ethyl acetate and washed 2 times with water
and 2
times with saturated brine solution. The organic layers were collected and
dried
over NaZS04, filtered and concentrated in vacuo affording 7,8-difluoro-1-(4-
iodo-2-
methyl-phenyl)-4-oxo-1,4-dihydro-2h~benzo[d][1,3]oxazine-6-carboxylic acid
methylamide as a yellow solid (0.19 g, 76.9%); mp 219-223 °C;'NMR
(400MHz;
DMSO-d6) 8.40 (s,1 H), 8.06 (d, 1 H, J=6.8), 7.74 (s, 1 H), 7.50 (d, 1 H,
J=8.4), 6.87
(d, 1 H, J=8.0), 5.61 (s, 2H), 2.76 (d, 3H, J=4.4), 2.25 (s, 3H);
MS(APCI)m+1=459;
Anal.calcd/found for C»H~3F21N203 C 45.00/45.39, H 3.01/3.16, N 6.05/5.88.
in vitro MEK assay: ICS = 6.6~.M
EXAMPLE 3
4 5-Difluoro-6-(-4-iodo-2-methyl-phenylamino)-I~meth I-~r isophthalamic acid.
HO O CHs
H
CH3 ~ N
HN I ~ F I ~ I
O F
To a suspension of 7,8-difluoro-1-(4-iodo-2-methyl-phenyl)-4-oxo-1,4-
dihydro-21+benzo[d][1,3]oxazine-6-carboxylic acid methylamide in THF (10 mL)
is
added polymer bound glycerol, (0.33g, 200-400 mesh) and 10 mL of 1.0 M HCI
solution. After stirring for 48 hours at room temperature, the resin is
filtered off and
the filtrate is transferred to a separatory funnel and partitioned with ethyl
acetate.
The organics are washed twice with 1.0 M HCI, twice with saturated brine
solution,
collected, dried over Na2S04, filtered and concentrated in vacuo affording 4,5-
difluoro-6-(-4-iodo-2-methyl-phenylamino)-ll~methyl-isophthalamic acid (0.11g,
76.2%); mp 254-259 °C;'H NMR (400MHz; DMSO-d6) 9.31 (s, 1H), 8.19 (s,
1H),
8.07 (d, 1 H, J=7.2), 7.56 (s, 1 H), 6.69 (t, 1 H, J=5.6), 2.74 (d, 3H,
J=4.4), 2.20 (s,
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3H); MS (APCI)m+1=447; Anal.calcd/found for C~6H~3FZIN203, C 43.07/43.26, H
2.94/3.07. N 6.28/6.10.
in vitro MEK assay: IC5o = 2.4p.M
EXAMPLE 4
N'-Cyclo~~ropylmethoxy-4.5-difluoro-6-i(4-iodo-2-methyl-phenylamino)~-N3-
methyl-
isophthalamide .
H
~O.N O H
H 3
H3 \ N \
HN
O F
To a suspension of 4,5-difluoro-6-(-4-iodo-2-methyl-phenylamino)-N-methyl-
isophthalamic acid (0.138, 0.29 mmol) in N,N-dimethylformamide (5mL) is added
trifluoroacetic acid pentafluorophenyl ester (0.055 mL, 0.32 mmol) and
pyridine
(0.03 mL, 0.32 mmol). After stirring for 17 hours the reaction mixture is
diluted with
ethyl acetate and transferred to a separatory funnel, washed twice with 1.0 M
HCI,
twice with 5% aqueous NaHC03 solution, 2 times with water and once with
saturated brine solution. The organic layers were collected and dried over
Na2S04,
filtered and concentrated in vacuo affording 4,5-difluoro-6-(-4-iodo-2-methyl-
phenylamino)-ll~methyl-isophthalamic acid pentafluorophenyl ester (0.088,
45.2%).
To a suspension of 4,5-difluoro-6-(-4-iodo-2-methyl-phenylamino)-ll~methyl-
isophthalamic acid pentafluorophenyl ester (0.088, 0.13 mmol) in freshly
distilled
THF (3 mL) is added cyclopropylmethylamine hydrochloride (0.0168, 0.13mmol),
and N,ll~diisopropylethylamine (0.07 mL, 0.39 mmol). After stirring at room
temperature for 17 hours the reaction mixture was partitioned between ethyl
acetate
and 1.0 M HCI solution. The organic layer was washed twice with water, twice
with
saturated brine solution, dried over MgS04, filtered and concentrated in
vacuo.
Purification was performed by silica column chromatography in 2:1 ethyl
acetate:hexanes affording N'-cyclopropylmethoxy-4,5-difluoro-6-(4-iodo-2-
methyl-
phenylamino)-N3-methyl-isophthalamide (0.033, 42.8%); mp 198-202 °C;'H
NMR
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(400MHz; DMSO-d6) 8.59 (s, 1 H), 8.01 (s, 1 H), 7.44 (d, 1 H, J=5.2), 7.32 (s,
1 H),
7.18 (d, 1 H, J=7.6), 6.36 (m, 1 H), 3.34 (d, 2H, J=6.8), 2.56 (d, 3H, J=4.0),
1.99 (s,
3H), 0.82 (m, 1H), 0.27 (d, 2H, J=8.4), 0.00 (m, 2H); MS(APCI)m+1=516;
Anal.calcd/found for C2oH2oF21N303 C 47.08/46.85, H 4.22/4.02 N 7.68/7.29.
in vitro MEK assay: ICSO = 1.1 wM
EXAMPLE 5
7.8-Difluoro-1-(4-iodo-2-methyl-phenyl)-4-oxo-1.4-dihydro-21-
~benzo[dl[1.3]oxazine-
6-carboxylic acid dimethylamide.
0 01 H
3
CH3 ~ N
H3C~N I ~ F I ~ 1
O F
Prepared in the manner of Example 4
'H NMR (400MHz; CDCI3) 7.96 (dd, 1 H, J=2.2, 6.6), 7.65 (t , 1 H, J=1.5), 7.48
(dd,
1 H, J=1.7, 8.3), 6.70 (d, 1 H, J=8.3), 5.37 (s, 2H), 3.11 (s, 3H), 2.96 (s,
3H), 2.3 (s,
3H); MS(APCI)m+1=473; Anal.calcd/found for C~gF~5F2IN2O3 C 46.66/47.05, H
3.80/3.65, N 5.34/5.58.
in vitro MEK assay: ICSO = 5.8wM
EXAMPLE 6
N'-Cyclopropylmethoxy-4.5-difluoro-6~4-iodo-2-methyl-~phenylamino)-N3.N3-
dimet~rl-isophthalamide (27~
.N O
O H H3
CH3 ~ N
H3C~N I ~ F I ~ I
O F
Prepared in the manner of Example 4
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mp 78-80 °C; 'H NMR (400MHz; DMSO-ds) 8.52 (s, 1 H), 7.32 (s, 1 H),
7.15 (m, 1 H),
6.39 (m, 1 H), 3.35 (d, 2H, J=6.8), 2.79 (s, 3H), 2.72 (s, 3H), 2.00 (s, 3H),
0.93 (m,
1H), 0.27 (d, 2H, J=8.0), 0.00 (m, 2H); MS(APCI)m+1=530.
in vitro MEK assay: IC5o = 4.3~M
EXAMPLE 7
The following were prepared using parallel synthetic techniques in the
following
manner:
Step A:
A solution of 7,8-difluoro-1-(4-iodo-2-methyl-phenyl)-4-oxo-1,4-dihydro-21-I-
benzo[d][1,3]oxazine-6-carboxylic acid pentafluorophenylester in a 2:1 mixture
of
THF to N,Iwdimethylformamide (0.32M, 19.36g) was prepared. In preweighed 2-
dram glass vials was added the corresponding amine (0.35mmol) and then the
prepared 7,8-difluoro-1-(4-iodo-2-methyl-phenyl)-4-oxo-1,4-dihydro-2l~
benzo[d][1,3]oxazine-6-carboxylic acid pentafluorophenylester solution
(0.3mmol).
To each vial was added a morpholine polystyrene resin (0.2g), capped with
Teflon
coated caps and placed on an orbital shaker for 24 hours. The individual
reactions
were then charged with polyamine polystyrene resin (0.2 g) and isocyanate
polystyrene resin (0.1 g) and dichloromethane (2 mL) and allowed to shake for
another 17 hours. The reactions were filtered and concentrated in vacuo to
afford
the corresponding 7,8-difluoro-1-(4-iodo-2-methyl-phenyl)-4-oxo-1,4-dihydro-
21+
benzo[d][1,3]oxazine-6-carboxylic acid amides. LC/MS was performed on a CPI
120SE (C18) column (4.6x50mm,5 pm).
EXAMPLE 8
7.8-Difluoro-6-y(R -~ydrox~pyrrolidine-1-carbonyl~1 ~4-iodo-2-methyl-phenyl~~-
1.2-dihydro-3.1-benzoxazin-4-one
0 01 cH3
HO I ' N
~N
'F I
O F
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C2o H~~ F2 I N2 04, MS (APCI)m+1 = 515
in vitro MEK assay: 34% inhibition @ 1 wM
EXAMPLE 9
7,8-Difluoro-1-(4-iodo-2-methyl-~henyl~-~,(S)-2-methoxymethyl-pYrrolidine-1-
carbon-1,2-dihydro-3.1-benzoxazin-4-one
HaC-O O Ol CH3
N
N I ~ F ' ~ I
O F
C~ H2~ F2 I NZ 04, MS (APCI)m+1 = 543
in vifro MEK assay: 93% inhibition @ 1 ~M
EXAMPLE 10
7.8-Difluoro-1-(4-iodo-2-methyl-phenyl)-4-oxo-1.4-dihydro-2H-3.1-benzoxazine-6-
carboxylic acid (2-hiperidin-1-yl-ethyl)-amide
0 0l cH3
N
NON I ~ F I ~ I
O F
C2s H24 F2 I N3 03, MS (APCI)m+1 = 556
in vitro MEK assay: 44% inhibition @ 1 wM
EXAMPLE 11
7.8-Difluoro-1-(4-iodo-2-methyl-phen~)-4-oxo-1.4-dihydro-2H-3.1-benzoxazine-6-
carboxylic acid i(2-pyrrolidin-1-yl-ethyl)-amide
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WO 01/68619 -4~- PCT/USO1/07816
0 01 cH3
N
NON I ~ F I / I
G of
C22 H22 F2 I N3 03, MS (APCI)m+1 = 542
in vitro MEK assay: 33% inhibition @ 1 wM
EXAMPLE 12
7 8-Difluoro-1-(4-iodo-2-meth~phenyl)-4-oxo-1.4-dih~rdro-2H-3.1-benzoxazine-6-
carboxylic acid (3-(2-oxo-pyrrolidin-1-yl)-pro~,yll-amide
0 01 H
3
N
~N~N I / F I / I
O O F
C2s Hz2 F2 I N3 04, MS (APCI)m+1 = 570
in vitro MEK assay: 21 % inhibition @ 1 p,M
EXAMPLE 13
7.8-Difluoro-1-(4-iodo-2-methyl-pheny,-4-oxo-1.4-dihydro-2H-3.1-benzoxazine-6-
carboxylic acid (1-hydrox~ clue ohexylmeth~)-amide
0 01 cH3
N
V'vN I / F I / I
OH 0 F
C2s H2s F2 I N2 04, MS (APCI)m+1 = 557
in vitro MEK assay: 23% inhibition @ 1 wM
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EXAMPLE 14
7.8-Difluoro-1-(4-iodo-2-methyl-phenyl)-4-oxo-1.4-dihydro-2H-3.1-benzoxazine-6-
carboxylic acid pyridin-2-ylmethvl)-amide
C2~ H24 F2 I N3 03, MS (APCI)m+1 = 604
EXAMPLE 15
4-[7.8-Difluoro-1-~~4-iodo-2-methyl-phenyl)-4-oxo-1,4-dihydro-2H-3.1-
benzoxazine-6-
carbonyll-aiaerazine-1-carbaldeh~
0 01 cH3
O~N~ \ N \
~N I / F I / 1
O F
C2~ H~8 F2 I N3 04, MS (APCI)m+1 = 542
in vitro MEK assay: 13% inhibition @ 1 wM
EXAMPLE 16
7.8-Difluoro-1-(4-iodo-2-methyl-phenyl)-4-oxo-1.4-dihyd ro-2H-3.1-benzoxazine-
6-
carboxylic acid methyl-(1-methyl-piperidin-4-yl)I-amide
o ' ~~ cH3
CfH3 \ N \
N I ~ F I ~ I
H C~N~ O F
3
C23 HZa F2 I N3 03, MS (APCI)m+1 = 556
in vitro MEK assay: 0% inhibition @ 1 ~,M
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EXAMPLE 17
7~8-Difluoro-1-(4-iodo-2-methyl-~henvl)-6-(4-meth~piperazine-1-carbonyl)-1.2-
dihydro-3.1-benzoxazin-4-one
0 0,
3
H3C'N~ \ N \
~N I / F I / I
O F
C2~ H2o F2 I N3 03, MS (APCI)m+1 = 528
in vitro MEK assay: 78% inhibition @ 1 ~,M
EXAMPLE 18
7.8-Difluoro-1-(4-iodo-2-methyl-phenyl]-4-oxo-1.4-dihydro-2H-3.1-benzoxazine-6-
carboxylic acid j2-(2-hydroxy-ethoxy)-ethyl]-amide
0 01 cH3
\ N \
N I / F I / I
O F
OH
C2o H~9 F2 I N2 05, MS (APCI)m+1 = 533
in vitro MEK assay: 43% inhibition @ 1 ~,M
EXAMPLE 19
7.8-Difluoro-1-(4-iodo-2-methyl-phenylJl-4-oxo-1.4-dihydro-2H-3.1-benzoxazine-
6-
carboxylic acid [2-(1-methyl-~yrrolidin-2-y~-ethyl]-amide
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0 01
3
\ N \
N I / F I / I
O F
N
~CH3
CZS HZa F2 I N3 03, MS (APCI)m+1 = 556
in vitro MEK assay: 28% inhibition @ 1 p.M
EXAMPLE 20
7.8-Difluoro-1-(4-iodo-2-methyl-phenyl)I-4-oxo-1.4-dihydro-2H-3.1-benzoxazine-
6-
carboxylic acid (2-hydroxyl)-methyl-amide
0 01
3
CH3 \ N \
N I / F I / I
HO~ O F
C~9 H» F2 I N2 04, MS (APCI)m+1 = 503
in vitro MEK assay: 41 % inhibition @ 1 p,M
EXAMPLE 21
7.8-Difluoro-1-(4-iodo-2-methyl-phenyl)-4-oxo-1.4-dihydro-2H-3.1-benzoxazine-6-
carboxylic acid 1.3.4-thiadiazol-2-, lay mide
0 01
3
\ N \
S N I / F I / I
N-N O F
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C~8 H~~ F2 I N4 03 S, MS (APCI)m+1 = 529
in vitro MEK assay: 31 % inhibition @ 1 ~M
EXAMPLE 22
7.8-Difluoro-1-(4-iodo-2-methyl-phenyl)~-4-oxo-1.4-dihvrdro-2H-3.1-benzoxazine-
6-
carboxylic acid (2-methoxy-1-methyl-ethyl)-amide
0 01 H
3
N
H3C N I ~ F I ~ I
OJ O F
CH3
C2o His F2 I N2 04, MS (APCI)m+1 = 517
in vitro MEK assay: 46% inhibition @ 1 ~M
EXAMPLE 23
7.8-Difluoro-1-(4-iodo-2-methyl-phenyl)-4-oxo-1.4-dihydro-2H-3.1-benzoxazine-6-
carboxylic acid j4-methyl-benzothiazol-2-yl)-amide
0 0l cH3
N
H
SYN I ~ F I ~ I
/ ~ N O F
CH3
C24 H~6 F2 I N3 03 S, MS (APCI)m+1 = 592
in vitro MEK assay: 26% inhibition @ 1 wM
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EXAMPLE 24
.8-Difluoro-1-(4-iodo-2-methyl-phenyl)-4-oxo-1.4-d ihyd ro-2H-3.1-benzoxazine-
6-
carboxylic acid (4-methyl-thiazol-2-yl)-amide
0 01 cH3
N
H
SYN I ~ F I ~ I
~N O F
H3C
CZO H~4 F2 I N3 03 S, MS (APCI)m+1 = 542
in vitro MEK assay: 20% inhibition @ 1 p,M
EXAMPLE 25
7.8-Difluoro-1-(4-iodo-2-methyl-phenyl-4-oxo-1.4-dihvd ro-2H-3.1-benzoxazine-6-
carboxylic acid (5-ethylsulfanyl-1.3.4-thiadiazol-2-yy-amide
0 0l cH3
N
H
NYN I ~ F I ~ I
N
~S O F
~S
H3C
C2o H~5 F2 I N4 03 S2, MS (APCI)m+1 = 589
in vitro MEK assay: 5% inhibition @ 1 wM
EXAMPLE 26
7.8-Difluoro-1-(4-iodo-2-methyl-phenyll-4-oxo-1.4-dihydro-2H-3.1-benzoxazine-6-
carboxylic acid (5-ethyl-1.3.4-thiadiazol-2-~)-amide
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o °1 H
3
N
H
H3C~\ S N I ~ F I ~ I
'N-N O F
C2o H~5 F2 I N4 03 S, MS (APCI)m+1 = 557
in vitro MEK assay: 20% inhibition @ 1 wM
EXAMPLE 27
7.8-Difluoro-1-(4-iodo-2-methyl-phen~)-4-oxo-1.4-dihydro-2H-3.1-benzoxazine-6-
carboxylic acid (5-mercapto-1.3.4-thiadiazol-2-yl)-amide
o~ °1 cH3
N
N I ~ F I ~ I
O F
C~8 H» F2 I N4 03 S2, MS (APCI)m+1 = 561
in vitro MEK assay: 27% inhibition @ 1 wM
EXAMPLE 28
7.8-Difluoro-1-(4-iodo-2-methyl-phenyl)-4-oxo-1,4-dihydro-2H-3.1-benzoxazine-6-
carboxylic acid (6-ethoxy-benzothiazol-2-yl)-amide
o °1 cH3
N
S~N I ~ F I ~ I
° / ~ N O F
H3C.J
C25 H~8 F2 I N3 04 S, MS (APCI)m+1 = 622
in vitro MEK assay: 9% inhibition @ 1 wM
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EXAMPLE 29
7.8-Difluoro-1-(4-iodo-2-methyl-phenyly-4-oxo-1.4-dihydro-2H-3.1-benzoxazine-6-
carboxylic acid benzothiazol-2-ylamide
0 01 cH3
N
SYN I ~ F I ~ I
I ~ N O F
C23 H~4 F2 I N3 03 S, MS (APCI)m+1 = 578
in vitro MEK assay: 21 % inhibition @ 1 wM
EXAMPLE 30
7.8-Difluoro-1-(4-iodo-2-methyl-phenyl)-4-oxo-1.4-dihydro-2H-3.1-benzoxazine-6-
carboxylic acid [2-(2-by d~ rox,y-ethyl)i-phenyl]-amide
OH
C24 H~9 F2 I N2 04, MS (APCI)m+1 = 565
in vitro MEK assay: 55% inhibition @ 1 p,M
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EXAMPLE 31
7.8-Difluoro-1-~4-iodo-2-methyl-phenyl)-4-oxo-1.4-dihydro-2H-3.1-benzoxazine-6-
carboxylic acid thiazol-2-vlamide
0 01 H
3
N
H
S~N I ~ F I ~ I
~N O F
C,9 H,2 F2 I N3 03 S, MS (APCI)m+1 = 528
in vitro MEK assay: 32% inhibition @ 1 wM
EXAMPLE 32
7.8-Difluoro-6-[2-(2-hydrox -~ethy~-piperidine-1-carbonyl]-~4-iodo-2-methyl-
phenyl -1.~--2-dihydro-3.1-benzoxazin-4-one
Ho 0 01
H3
N
N I ~ F I ~ I
O F
C23 HZS F2 I N2 04, MS (APCI)m+1 = 557
in vitro MEK assay: 76% inhibition @ 1 p.M
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EXAMPLE 33
7.8-Difluoro-6-(2-h~ roxymethvl-piperidine-1-carbonyl)-1-(4-iodo-2-methyl-
phenyll-
1.2-dihydro-3.1-benzoxazin-4-one
\ N \
N I ~ F I ~ I
O F
C22 H2~ F2 I N2 04, MS (APCI)m+1 = 543
in vitro MEK assay: 79% inhibition @ 1 wM
EXAMPLE 34
3~r7.8-Difluoro-1-(4-iodo-2-methyl-phen~)-4-oxo-1.4-dihydro-2H-3.1-benzoxazine-
6-carbonyl-amino}-1 H-pyrazole-4-carboxylic acid ethyl ester
H3c1 0 0l
O O ~ \ N ~ \a
/ N / F ~1
H-N O F
C22 H" F2 I N4 05, MS (APCI)m+1 = 583
in vitro MEK assay: 72% inhibition @ 1 p,M
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EXAMPLE 35
7.8-Difluoro-1-(4-iodo-2-methyl-phenyl)-4-oxo-1.4-dihydro-2H-3.1-benzoxazine-6-
carboxylic acid (5-methylsulfan~-1 H-1.2.4-triazol-3-~)-amide
0 01 cH3
N
H3Cg~N~
I / F I / I
N.N O F
H
C~9 H~4 F2 I N5 03 S, MS (APCI)m+1 = 558
in vitro MEK assay: 76% inhibition @ 1 wM
EXAMPLE 36
7.8-Difluoro-1-(4-iodo-2-methyl-phenyl)i-4-oxo-1,4-dihydro-2H-3.1-benzoxazine-
6-
carbox, li~c acid~1 H-pvrazol-3-yl)-amide
0 01 H
3
N
~N ( / F I / I
N-N O F
H
C~9 H~3 F2 I N4 03, MS (APCI)m+1 = 511
in vitro MEK assay: 60% inhibition @ 1 wM
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EXAMPLE 37
7.8-Difluoro-1-(4-iodo-2-meth~phen~L-4-oxo-1.4-di~dro-2H-3.1-benzoxazine-6-
carboxylic acid pyridin-3-vlamide
0 01 H
3
\ N \
\ N I / F I / I
IN O F
C2~ H~4 F2 I N3 03, MS (APCI)m+1 = 522
in vitro MEK assay: 20% inhibition @ 1 wM
EXAMPLE 38
7.8-Difluoro-6-~(3-hvdroxy-piperidine-1-carbonyl)I-1-(4-iodo-2-methyl-phenyl)-
1.2-
dihydro-3.1-benzoxazin-4-one
0 01 cH3
\ N \
~N I / F I / I
HO O F
C2~ H~9 F2 I N2 04, MS (APCI)m+1 = 529
in vitro MEK assay: 48% inhibition @ 1 ~M
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EXAMPLE 39
7.8-Difluoro-6-(3-hyd roxymethyl-piperidine-1-carbony ~-1-(4-iodo-2-methyl-
pheny~-
1.2-dihydro-3.1-benzoxazin-4-one
HO O O1 H
I 3
N
N I ~ F I ~ I
O F
C22 H2~ FZ I N2 04, MS (APCI)m+1 = 543
in vitro MEK assay: 71 % inhibition @ 1 ~M
EXAMPLE 40
7.8-Difluoro-6-(3-hydroxy-pyrrolidine-1-carbonyl)-~4-iodo-2-methyl-phenyl)-1.2-
dihvdro-3,1-benzoxazin-4-one
0 01 cH3
HO
N
~N I ~ F I / I
i i
O F
C2o H» F2 I N2 04, MS (APCI)m+1 = 515
in vitro MEK assay: 42% inhibition @ 1 ~M
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EXAMPLE 41
7.8-Difluoro-1-(4-iodo-2-methyl-phenyl)-6-(4-pvrrolidin-1-~~'Iperidine-1-
carbonyl)-
1.2-dihydro-3.1-benzoxazin-4-one
0 01 cH3
~N \ N \
~N I / F I / 1
i i
O F
CZS HZS F2 I N3 03, MS (APCI)m+1 = 582
in vitro MEK assay: 7% inhibition @ 1 p,M
EXAMPLE 42
7.8-Difluoro-1-(4-iodo-2-meth-phenyl)-4-oxo-1.4-dih)rdro-2H-3.1-benzoxazine-6-
carboxylic acid (2-morpholin-4-vl-ethyl)i-amide
0 0l cH3
\ N \
~N~N I / F I / 1
OJ O F
C22 H22 F2 I N3 04, MS (APCI)m+1 = 558
in vitro MEK assay: 15% inhibition @ 1 wM
CA 02403017 2002-09-10
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EXAMPLE 43
7.8-Difluoro-6-i(4-hydroxy-piperidine-1-carbonylL(4-iodo-2-meth~phenyl -1.2-
dih~rdro-3.1-benzoxazin-4-one
0 01 cH3
HO ~ N
~N I / F I / I
i
O F
C2~ H~9 F2 I N2 04, MS (APCI)m+1 = 529
in vitro MEK assay: 52% inhibition @ 1 ~M
EXAMPLE 44
7.8-Difluoro-6-[4-(2-hydroxy-ethyl)-piperidine-1-carbon)-1-(4-iodo-2-methyl-
phenyl)-1.2-dihydro-3.1-benzoxazin-4-one
HO O 01 H
I 3
N
N I / F I / I
O F
CZS H23 F2 I N2 04, MS (APCI)m+1 = 557
in vitro MEK assay: 69% inhibition @ 1 ~,M
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EXAMPLE 45
7.8-Difluoro-1-~4-iodo-2-methyl-phenyy-4-oxo-1.4-dihydro-2H-3.1-benzoxazine-6-
carboxylic acid ~6-chloro-pyridin-3-yl -amide
C2~ H~3 CI F2 I N3 03, MS (APCI)m+1 = 556
in vitro MEK assay: 62% inhibition @ 1 p,M
EXAMPLE 46
7.8-Difluoro-1-(4-iodo-2-meth~phenyl)-4-oxo-1.4-dihydro-2H-3.1-benzoxazine-6-
carboxylic acid 6-methoxy-pyridin-3-~ -amide
CH3
C22 H~6 F2 I N3 04, MS (APCI)m+1 = 552
in vitro MEK assay: 43% inhibition @ 1 pM
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EXAMPLE 47
7.8-Difluoro-1-(4-iodo-2-methyl-phenyl)-4-oxo-1.4-dihydro-2H-3.1-benzoxazine-6-
carboxylic acid j2-(5-methoxy-1 H-indol-3-~)-ethyl]-amide
0 01 cH3
H3C-O H \ N \
N I ~ F I ~ I
O F
N
H
C2~ H22 F2 I N3 04, MS (APCI)m+1 = 618
in vitro MEK assay: 0% inhibition @ 1 wM
EXAMPLE 48
7.8-Difluoro-1-(4-iodo-2-methyl-phen~)-4-oxo-1.4-dihydro-2H-3.1-benzoxazine-6-
carboxylic acid [2-(6-methoxy-1 H-indol-3-vl)-ethyl]-amide
0 01 cH3
_ \ N \
HC/ \ ~ N I ~ F I ~ I
NJ O F
H
C2~ H22 F2 I N3 04, MS (APCI)m+1 = 618
in vitro MEK assay: 12% inhibition @ 1 ~.M
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EXAMPLE 49
7.8-Difluoro-1-(4-iodo-2-methyl-~henyly-4-oxo-1.4-dihydro-2H-3.1-benzoxazine-6-
carboxylic acid bis-~2-h~roxy-ethyl)-amide
old °~ cH3
I\ N I\
~N / F / I
HO ° F
C2o H~9 F2 I N2 05, MS (APCI)m+1 = 533
in vitro MEK assay: 50% inhibition @ 1 wM
EXAMPLE 50
7.8-Difluoro-1-(4-iodo-2-methyl-phenyl)-4-oxo-1.4-dihydro-2H-3.1-benzoxazine-6-
carboxylic acid [2-(1 H-imidazol-4-yl)-ethvll-amide
o °1 cH3
\ N \
N I ~ F I ~ I
O F
HNV N
C2~ H» F2 I N4 03, MS (APCI)m+1 = 539
in vitro MEK assay: 44% inhibition @ 1 ~.M
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EXAMPLE 51
7.8-Difluoro-1-(4-iodo-2-methxl-phenyl)-6-(morpholine-4-carbonyl)-1.2-dihydro-
3,1-
benzoxazin-4-one
0 01 cH3
\
~ F I ~ .I
O F
C2o H» F2 I N2 04, MS (APCI)m+1 = 515
in vitro MEK assay: 72% inhibition @ 1 wM
EXAMPLE 52
7.8-Difluoro-1-(4-iodo-2-methyl-phenyl)-4-oxo-1.4-dihydro-2H-3,1-benzoxazine-6-
carboxylic acid (3-imidazol-1-yl-propyl)-amide
0 01
3
\ N \
~ F I ~ I
O F
N
C~ H~9 F2 I N4 03, MS (APCI)m+1 = 553
in vitro MEK assay: 35% inhibition @ 1 wM
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EXAMPLE 53
7.8-Difluoro-1-(4-iodo-2-meth~~henvl)~-4-oxo-1.4-d ihydro-2H-3.1-benzoxazine-6-
carboxylic acid,~4-dimethylamino-phenyl)-amide
0 01 cH3
\ N \
I\ N I/ F I/ I
H3C.N~ O F
CH3
C24 H2o F2 I N3 03, MS (APCI)m+1 = 564
in vitro MEK assay: 61 % inhibition @ 1 ~.M
EXAMPLE 54
6-(4-Ethyl-eiperazine-1-carbon)-7.8-difluoro-1-(4-iodo-2-methyl-phenyl-1.2-
dihydro-3.1-benzoxazin-4-one
0 01 cH3
H3C~N~ \ N \
~N I / F I / I
O F
C22 H22 Fz I N3 03, MS (APCI)m+1 = 542
in vitro MEK assay: 56% inhibition @ 1 wM
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EXAMPLE 55
7.8-Difluoro-1-(4-iodo-2-methyl-phenyl)-4-oxo-1.4-d ihYdro-2H-3.1-benzoxazine-
6-
carboxylic acid (2-(1 H-indol-3-y~-eth~]-methyl-amide
o °1 cH3
CH3 I \ N
N / F / I
N I O F
H
C2~ H22 F2 I N3 03, MS (APCI)m+1 = 602
in vitro MEK assay: 41 % inhibition @ 1 ~M
EXAMPLE 56
7.8-Difluoro-1-(4-iodo-2-methyl-phenyl)I-6-(piperazine-1-carbonyl)-1.2-dihydro-
3.1-
benzoxazin-4-one
° °~ H
3
HN~ \ N \
~N ~ ~ F ~ ~ I
O F
C2o H~8 F2 I N3 03, MS (APCI)m+1 = 514
in vitro MEK assay: 1 % inhibition @ 1 ~M
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EXAMPLE 57
7.8-Difluoro-1=(4-iodo-2-meth~phenyl)-4-oxo-1,4-dihydro-2H-3.1-benzoxazine-6-
carboxylic acid (tetrahydro-furan-2-ylmethyl)-amide
0 01 cH3
N
~N I / F I / I
O F
C2~ H~9 F2 I N2 04, MS (APCI)m+1 = 529
in vitro MEK assay: 35% inhibition @ 1 wM
EXAMPLE 58
7.8-Difluoro-1-(4-iodo-2-methyl-phenyl)i-6-ythiazolidine-3-carbonyl-1.2-
dihydro-3.1-
benzoxazin-4-one
0 01 cH3
s~ w N w
~N I / F I / I
O F
C~9 H15 F2 I Nz 03 S, MS (APCI)m+1 = 517
in vitro MEK assay: 76% inhibition @ 1 ~,M
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EXAMPLE 59
7.8-Difluoro-1-(4-iodo-2-methyl-phenyl)~-6-(thiomoraholine-4-carbonyl)-1.2-
dihydro-
3.1-benzoxazin-4-one
0 01 cH3
\ N \
~N I / F I / I
O F
C2o H» F2 I N2 03 S, MS (APCI)m+1 = 531
in vitro MEK assay: 75% inhibition @ 1 ~M
EXAMPLE 60
7.8-difluoro-1-(4-iodo-2-methyl-phenyl)i-4-oxo-1.4-dihydro-2H-3.1-benzoxazine-
6-
carboxylic acid y(S)-2-hydroxy-cyclohexyl -amide
0 01 cH3
\ N \
N I / F I / I
~O~ F
C22 H2~ F2 I N2 04, MS (APCI)m+1 = 543
in vitro MEK assay: 30% inhibition @ 1 p.M
Step B:
To each of the 2 dram vials containing the 7,8-Difluoro-1-(4-iodo-2-methyl-
phenyl)-4-oxo-1,4-dihydro-2H benzo[d][1,3]oxazine-6-carboxylic acid amides was
added THF (1 mL) and aqueous HCI (1.0 M, 1 mL) and glycerol polystyrene resin
(0.2g), capped with Teflon coated caps and allowed to shake on an orbital
shaker at
50 °C for 5 days. The reactions were filtered and washed with ethyl
acetate (1.5
mL) and concentrated in vacuo. HPLC purification was performed in
acetonitrile/water (0.05%TFA) on a YM C30 (C18) column (100mm ODS-A) to
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afford the corresponding isophthalamic acids. LC/MS was performed on a CPI
120SE (C18) column (4.6x50mm, 5 ~.m).
EXAMPLE 61
3,4-Difluoro-5-[1-UR)-3-hydrox~pvrrolidin-1-yl -methanoyl]-~4-iodo-2-methyl-
phenylaminoy-benzoic acid
O OH CHa
HO . H
N
~N ( ~ I ~ I
'F
O F
C~9 H~~ FZ I N2 Oa, MS (APCI)m+1 = 503
in vitro MEK assay: 75% inhibition @ 1 wM
EXAMPLE 62
4.5-Difluoro-6-(4-iodo-2-methyl phenvlamino)- 11~f3-(2-oxo-pyrrolidin-1-yl)-
propyl]-
isophthalamic acid
HO O H
H 3
N
N I ~ F I ~ I
O F
OyN1
C22 H22 F2 I N3 04, MS (APCI)m+1 = 558
in vitro MEK assay: 78% inhibition @ 1 ~M
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EXAMPLE 63
4.5-Difluoro-6-(4-iodo-2-methyl-phenylamino)- N-(1-methyl-piperidin-4-y~-
isophthalamic acid
HO O CH3
H
\ N \
I
N I / F ( / I
O F
OH
C2z HZa F2 I N3 03, MS (APCI)m+1 = 544
in vitro MEK assay: 0% inhibition @ 1 p,M
EXAMPLE 64
3~4-Difluoro-2-~(4-iodo-2-methyl-phenylaminoLS-[1-(4-methyl-~'Iperazin-1-~)-
methanoyl]-benzoic acid
HO O H
H a
H3C'N~ \ N \
~N I / F I / I
O F
C2o H2o F2 I N3 03, MS (APCI)m+1 = 516
in vitro MEK assay: 57% inhibition @ 1 wM
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EXAMPLE 65
4.5-Difluoro-N f2-(2-hydroxy-ethoxY -ethyl]-6-(4-iodo-2-meth I-~ ahenylamino)-
iso~hthalamic acid
HO'
L HO O CH3
H
N
HN I ~ F I ~ I
O . F
C~9 H~9 F2 I N2 05, MS (APCI)m+1 = 521
in vitro MEK assay: 63% inhibition @ 1 ~.M
in vitro MEK assay: ICSO = 1.38~.M
EXAMPLE 66
4.5-Difluoro-6-(4-iodo-2-meth~phenylaminoy- N-/2-i(1-meth~~yrrolidin-2-y~-
ethyll-
isophthalamic acid
H3C-N HO O CH
H 3
N
HN I ~ F I ~ I
O F
C~ H24 F2 I N2 03, MS (APCI)m+1 = 544
in vitro MEK assay: 14% inhibition @ 1 ~M
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EXAMPLE 67
4.5-Difluoro-6-~4-iodo-2-methyl-phenylamino)- N-(2-p rids in-2-yl-ethyl)-
isophthalamic
acid
/
N ~ ' HO O H
H 3
N
HN I / F I / 1
O F
C22 H~8 FZ I N3 03, MS (APCI)m+1 = 538
in vitro MEK assay: 81 % inhibition @ 1 wM
in vitro MEK assay: IC5o = 3.3wM
EXAMPLE 68
N-Butyl-4.5-difluoro-6-(4-iodo-2-methyl-phenylamino)- N-(2-mercapto-ethyl)-
isophthalamic acid
H3C HO O
H Hs
N
N ~ / F I / 1
HSI O F
C2~ H23 F2 I N2 03 S, MS (APCI)m+1 = 549
in vitro MEK assay: 24% inhibition @ 1 wM
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EXAMPLE 69
3.4-Difluoro-5-,[1-[2-(2-hydroxy-ethylLpi~~eridin-1-yl]-methanoy~-~4-iodo-2-
methyl-
phenylamino)-benzoic acid
HO O H
H 3
\ N \
I
N I / F I / I
O F
OH
C22 HZS F2 I N2 04, MS (APCI)m+1 = 545
in vitro MEK assay: 78% inhibition @ 1 ~M
in vitro MEK assay: IC5o = 2.2wM
EXAMPLE 70
3.4-Difluoro-5-[1-(2-hydroxymethyl-piperidin-1-yl)-methanoyl]-2-(4-iodo-2-
methyl-
phen~rlamino)-benzoic acid
O H CH3
\ N \
N I / F I / I
O F
C2~ H2~ F2 I N2 04, MS (APCI)m+1 = 531
in vitro MEK assay: 49% inhibition @ 1 p.M
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EXAMPLE 71
4.5-Difluoro-6~4-iodo-2-methyl-ahenylamino)- N pvridin-3-ylmethyl-
isophthalamic
acid
N\ HO O CHs
H
~ N
HN I / F I / I
O F
C2~ H~6 F2 I N3 03, MS (APCI)m+1 = 524
in vitro MEK assay: 86% inhibition @ 1 ~,M
in vitro MEK assay: IC5o = 1.45~.M
EXAMPLE 72
X2.3-Dihydro-benzof 1.4ldioxin-6-y~-4.5-difluoro-6-(4-iodo-2-methyl-
phenylaminol
isophthalamic acid
~'o
O / HO O CH
I H s.
N
HN I ~ F I ~ I
O F
C23 H~~ F2 I N2 O5, MS (APCI)m+1 = 567
in vitro MEK assay: 65% inhibition @ 1 pM
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EXAMPLE 73
3-(~(1-[5-Carboxy-2.3-difluoro-4-(4-iodo-2-methyl-phen lay mino~hen~l-
methanoyl~-
amino)-1 H-pnrazole-4-carboxylic acid ethyl ester
H HO O CH
N s
H3CV0
O HN I ~ F I ~ I
O F
C2~ H~~ FZ I N4 05, MS (APCI)m+1 = 571
in vitro MEK assay: 86% inhibition @ 1 wM
in vitro MEK assay: IC5o = 1.1 wM
EXAMPLE 74
4,5-Difluoro-6-(4-iodo-2-meth~~hern lar mino)- N-pyridin-3-yl-iso~hthalamic
acid
N ~ HO O H CH3
~ w N ~ w
HN ~ F ~ I
O F
C2o H~4 FZ I N3 03, MS (APCI)m+1 = 510
in vitro MEK assay: 50% inhibition @ 1 ~,M
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EXAMPLE 75
1-[3.4-Difluoro-5-[1-(3-h day-piperidin-1-~,)-methanoyl]-2-(4-iodo-2-methyl-
hhen lay mino~phenyl]-ethanone
O OH CH3
H
N
vN ~ I I ~ I
HO ~ 'F
O F
C2o H~9 F2 I N2 04, MS (APCI)m+1 = 517
in vitro MEK assay: 92% inhibition @ 1 ~M
in vitro MEK assay: IC5o = 0.465wM
EXAMPLE 76
3.4-Difluoro-5-[1-(3-hydroxymethyl-piperidin-1-~ -methanoyl]-~4-iodo-2-meth
~henylamino)-benzoic acid
HO HO O CHs
H
N
N I ~ F I ~ I
O F
C2~ H2~ F2 I N2 04, MS (APCI)m+1 = 531
in vitro MEK assay: 88% inhibition @ 1 wM
in vitro MEK assay: IC5o = 0.300wM
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EXAMPLE 77
3.4-Difluoro-5-[1-(3-hydroxy-pyrrolidin-1-yy-methanoyll-2-(4-iodo-2-methyl-
phenylamino)-benzoic acid
HO O H
HO H
\ N \
~N
'F I
O F
C~9 H» F2 I Nz 04, MS (APCI)m+1 = 503
in vitro MEK assay: 83% inhibition @ 1 wM
in vitro MEK assay: ICS = 0.880wM
EXAMPLE 78
3.4-Difluoro-2-(4-iodo-2-meth~phenylamino)-5-[1-~(4-pyrrolidin-1-yl-piperidin-
1-~)-
methanol)-benzoic acid
HO O CH3.
H
~N \ N \
~N I / F I / 1
~ i
O F
C2a HZS F2 I N3 03, MS (APCI)m+1 = 570
in vitro MEK assay: 33% inhibition @ 1 pM
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EXAMPLE 79
4.5-Difluoro-6~4-iodo-2-methyl-phenylamino)- N (2-morpholin-4-yl-ethy~-
isophthalamic acid
C°~ H° O
N H CHa
\ N ~ \
HN ~ F ~ I
O F
C2~ H22 F2 I N3 04, MS (APCI)m+1 = 546
in vitro MEK assay: 54% inhibition @ 1 ~.M
in vitro MEK assay: ICSO = 1.5 pM
EXAMPLE 80
4.5-Difluoro-6-(4-iodo-2-methyl-phenylamino~pyridin-4-vlmethyl-isophthalamic
acid
N, HO O CH
H a
\ \ N \
HN I ~ F I ~ I
O F
C2~ H~6 F2 1 N3 03, MS (APCI)m+1 = 524
in vitro MEK assay: 71 % inhibition @ 1 ~M
in vitro MEK assay: ICSO = 1.7 wM
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EXAMPLE 81
3.4-Difluoro-5-[1-~(4-hydroxy-piperidin-1-~)-methanol]-2-(4-iodo-2-methyl-
phenylamino~ benzoic acid
HO O H
H 3
HO ~ N
~N I / F I / I
O F
C2o H~9 F2 I N2 04, MS (APCI)m+1 = 517
in vitro MEK assay: 86% inhibition @ 1 wM
in vitro MEK assay: IC5o = 1.8 p,M
EXAMPLE 82
4.5-Difluoro-6-(4-iodo-2-methyl-ahenylamino)- N I(4-morpholin-4-yl-phenyl)-
isophthalamic acid
C25 H22 F2 I N3 04, MS (APCI)m+1 = 594
EXAMPLE 83
3.4-Difluoro-5-~[1-[4-(2-hydroxy-ethyl)-piperidin-1-yll-methanoyl}-2-(4-iodo-2-
methyl-
phen~lamino)-benzoic acid
HO HO O H
H 3
N
N I / F I / I
O F
C22 HZS F2 I N2 04, MS (APCI)m+1 = 545
in vitro MEK assay: 90% inhibition @ 1 ~M
in vitro MEK assay: IC5o = 0.150 wM
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EXAMPLE 84
~2-Ethyl-2H-pyrazol-3-yl~-4.5-difluoro-6-i(4-iodo-2-meth~phen~aminol
isophthalamic acid
HO O H
H 3
N
H3C~ N I /
F I
O F
CZO H~~ F2 I N4 03, MS (APCI)m+1 = 527
in vitro MEK assay: 79% inhibition @ 1 wM
in vitro MEK assay: IC5o = 0.960 pM
EXAMPLE 85
N (6-Chloro-pyridin-3-yl)-4,5-difluoro-6-(4-iodo-2-methyl-phen I~amino~
isophthalamic acid
c1
~ ~-10 O H CH3
/ \ N ~ \
HN I / F / I
O F
C2o H~3 F2 I N3 03, MS (APCI)m+1 = 544
in vitro MEK assay: 90% inhibition @ 1 ~.M
in vitro MEK assay: IC5o = 0.970 wM
EXAMPLE 86
4,5-Difluoro-N-(1 H-indazol-6-yl)i-6-~(4-iodo-2-methyl-phenylamino)-
isophthalamic
acid
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N.
r NH HO O H
H 3
\ / ~ ~ N ~
F ~ I
O F
C22 H~5 F2 I N4 03, MS (APCI)m+1 = 549
in vitro MEK assay: 77% inhibition @ 1 wM
in vitro MEK assay: IC5o = 1.1 ~,M
EXAMPLE 87
4 5-Difluoro-11~(2-hydroxy-1-meth~yly-6-(4-iodo-2-methyl-phenylamino~
isophthalamic acid
OH HO O H CH3
~CH ( ~ N
H TN ~ F ~ I
O F
C~8 H» F2 I N2 04, MS (APCI)m+1 = 491
in vitro MEK assay: 66% inhibition @ 1 ~M
in vitro MEK assay: IC5o = 2.2 ~M
EXAMPLE 88
4.5-Difluoro-11~[,2,~- 1 H-imidazol-4-yl~-ethyl]-6-y4-iodo-2-methyl-
phenvlamino~
isophthalamic acid
H
N / HO O CH3
H
N
HN I ~ F I ~ I
O F
C2o H» FZ I N4 03, MS (APCI)m+1 = 527
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in vitro MEK assay: 60% inhibition @ 1 p.M
in vitro MEK assay: IC5o = 1.4 wM
EXAMPLE 89
3.4-Difluoro-2-(4-iodo-2-methyl-phenylamino~(1-morpholin-4-yl-methanovl)-
benzoic acid
HO O CHs
H
O~ \ N \
~N I / F I / I
O F
C~9 H» F2 I N2 04, MS (APCI)m+1 = 503
in vitro MEK assay: 89% inhibition @ 1 p.M
EXAMPLE 90
4,5-Difluoro-Iwy3-imidazol-1-yl ~ropyl)-6-(4-iodo-2-methyl-phenylamino)-
isophthalamic acid
N
'N HO O CHs
H
\ N \
HN I / F I / I
O F
C2~ H~9 F2 I N4 03, MS (APCI)m+1 = 541
in vitro MEK assay: 59% inhibition @ 1 pM
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EXAMPLE 91
4.5-Difluoro-6-~4-iodo-2-methyl-phenylamino)-11~~(3-morpholin-4-yl-aropyl)-
isophthalamic acid
0
~N HO O CH
H 3
\ N \
HN I / F I / I
O F
C22 H2a F2 I N3 04, MS (APCI)m+1 = 560
in vitro MEK assay: 58% inhibition @ 1 wM
EXAMPLE 92
N (4-Dimethylamino-phenyl)I-4.5-difluoro-6-(iodo-methyl-phenylamino)-
isophthalamic acid
H3C.N.CH3 .
I \ HO O CH
H 3
\ N \
HN I / F I / I
O F
C23 H2o F2 I N3 03, MS (APCI)m+1 = 552
in vitro MEK assay: 80% inhibition @ 1 wM
EXAMPLE 93
5-~1-(4-Ethvl-pi~erazin-1-vl)-methanoyl]-3.4-difluoro-2-(4-iodo-2-methyl-
phen~rlamino)I-benzoic acid
HO O CH3
H
H3C~N~ \ N \
~N I / F I / I
O F
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C2~ H22 F2 I N3 03, MS (APCI)m+1 = 530
in vitro MEK assay: 70% inhibition @ 1 wM
EXAMPLE 94
4,5-Difluoro-6~4-iodo-2-methyl-phen lad mino~4-methoxy-phenyl)- N-methyl-
isophthalamic acid
H3C.
/ HO O H
I H a
\ \ N \
I I
H3C.N / F / I
O F
C23 H~9 F2 I N2 04, MS (APCI)m+1 = 553
in vitro MEK assay: 92% inhibition @ 1 wM
EXAMPLE 95
4.5-Difluoro-N-[2-y1 H-indol-3-yl~-ethyl]-6-(4-iodo-2-methyl-phenylamino)-N
methyl-
isophthalamic acid
/ I
HN ~ HO O H CH3
\ N \
.N I / F I / I
H3C
O F
C2s H2z F2 I N3 03, MS (APCI)m+1 = 590
in vitro MEK assay: 77% inhibition @ 1 ~M
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EXAMPLE 96
4.5-Difluoro-6-(4-iodo-2-methyl-phenylamino)-l1r' (4-sulfamoyl-phenyly-
isophthalamic
acid
q
O= -NH2
HO O CH3
H
\ N \
HN I ~ F I ~ I
O F
C2~ H~6 F2 I N3 04 S, MS (APCI)m+1 = 588
in vitro MEK assay: 85% inhibition @ 1 ~M
EXAMPLE 97
4.5-Difluoro-6-(4-iodo-2-methyl-phenylamino -11~(tetrahydro-furan-2-~rlmethyy-
isophthalamic acid
O CH3
H
\ N \
HN I ~ F I ~ I
O F
C2o H~9 F2 I N2 04, MS (APCI)m+1 = 517
in vitro MEK assay: 81 % inhibition @ 1 wM
in vitro MEK assay: ICS = 0.150 wM
EXAMPLE 98
3.4-Difluoro-2-~(4-iodo-2-meth~phenylaminoy-5-( 1-thiazolid in-3-yl-methano~-
benzoic acid
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HO O CHa
H
S~ \ N \
~N I / F I / I
O F
C~8 H~5 F2 I N2 03 S, MS (APCI)m+1 = 505
in vitro MEK assay: 86% inhibition @ 1 ~.M
in vitro MEK assay: ICSO = 0.087 ~,M
EXAMPLE 99
3.4-Difluoro-2-i(4-iodo-2-methyl~henylamino)I-5-(1-thiomorpholin-4-yl-methano~-
benzoic acid
HO O CHs
H
g~ \ N \
~N I / F I / I
O F
C~9 H» F2 I N2 03 S, MS (APCI)m+1 = 519
in vitro MEK assay: 82% inhibition @ 1 ~,M
in vitro MEK assay: ICSO = 0.150 ~,M
EXAMPLE 100
4.5-Difluoro-N-y(S)-2-hydroxy-c cl~yly-6-(4-iodo-2-methyl-phern lay mino)-
isophthalamic acid
HO O CHs
H
HO \ N \
HN I / F I / I
O F
C2~ H2~ F2 I N2 04, MS (APCI)m+1 = 531
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EXAMPLE 101
3.4-Difluoro-5-f 1-(3-hydro ~-pioeridin-1-yl)-methanol-2-~(4-iodo-2-meth~-
phenylamino)-benzoic acid
0 01 cH3
g~ \ N \
~N I / F I / I
O F
C2oH~9F21N204, MS (APCI)m+1 = 517
in vitro MEK assay: 75% inhibition @ 1 p,M
EXAMPLE 102
4.5-Difluoro-6-(4-iodo-2-meth I-~enylamino)-N (2-piperidin-1-yl-ethyll
isophthalamic acid
HO O H
H
\ N \
HN I / F ( / I
O F
C~H24F2IN3O3, MS (APCI)m+1 = 544
in vitro MEK assay: 25% inhibition @ 1 wM
EXAMPLE 103
3.4-Difluoro-2-(4-iodo-2-methvl phenylamino,-5-(1~2-methyl-4-i(3-phenoxy-p
ri~din-
4-~)-piperazin-1 yl]-methanol}-benzoic acid
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EXAMPLE 104
10
2-Chloro-4-(4-{1 j2.3-difluoro-4-(4-iodo-2-methyl-phen~rlaminoLS-carboxy-
phenyll-
methanoyl]~-3-methyl-piperazin-1-yl)-benzoic acid
EXAMPLE 105
3.4-Difluoro-2-(4-iodo-2-methYl~henylamino~ 5-[~4-pyridin-2 yl-piperazin-1-yl~
methanoyl]-benzoic acid
EXAMPLE 106
5-[1-(4-Ethanesulfonyl-piperazin-1-yl)-methanoyl]-3,4-difluoro-2-(4-iodo-2-
meth
phen, la~i mino)-benzoic acid
HsC O OH CHs
O%S N H
\ N \
O vN I ~ F I ~ I
O F
C2~ H22F2 I N3 035 S, MS (APCI)m+1 = 594
EXAMPLE 107
5-~(1-[~2-Amino-ethyl )-2-oxo-imidazol id in-1-Yl-metha noy~-3.4-d ifluoro-2-
(4-iodo-2-
methyl-phenylamino)-benzoic acid
EXAMPLE 108
5-~1-[4-i(2-Amino-ether)-piperazin-1-yll methanoyl~-3,4-difluoro-2-(4-iodo-2-
methyl-
phenylaminol-benzoic acid
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EXAMPLE 109
3.4-Difluoro-5-{1-[4-(2-hydroxy-2-methyl-propyl)i-piperazin-1-vl]-methanoyl]~-
~4-
iodo-2-methyl-phenylamino)-benzoic acid
EXAMPLE 110
5-{1-[4-L,4-Dimethoxy-phenyl)i-piperazin-1-~]-methanol}-3.4-difluoro-2-(4-iodo-
2-
methyl-phenylamino)-benzoic acid
EXAMPLE 111
5-{1-[4-(2-Carboxy-2-methyl-propyl)i-piperazin-1-~1]-methanoyl}-3.4-difluoro-2-
~4-
iodo-2-methyl-phen lay minoy-benzoic acid
EXAMPLE 112
3.4-Difluoro-2-(4-iodo-2-methyl-phenylamino)-5-(1-.(4-[propane-1-sulfonYl)-
phenyl-piperazin-1-y~~-methanoyly-benzoic acid
EXAMPLE 113
3.4-Difluoro-2-(4-iodo-2-methyl~henylamino)-5-[1-(3'-methyl-2.3.5.6-tetrahydro-
L .2~bie, ry azinyl-4-yly-methanoyl~-benzoic acid
EXAMPLE 114
3.4-Difluoro-2-(4-iodo-2-methyl-phenylamino,)i-5-[1-(4-{2-[(pyridin-2-
ylmethvl~
amino]-ethyl}-p~erazin-1-y~-methanoyll-benzoic acid
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H
~N1
C2~ H28 F2 I N5 03 , MS (APCI)m+1 = 636
EXAMPLE 115
5-{1-[4-(3-Dimethylamino-propyl)-piperazin-1-yl]-methanoylj~-3.4-difluoro-2-y4-
iodo-
2-methyl-phenylamino)-benzoic acid
H3C.N.CH3
O OH H
H 3
N~ \ N \
~N I / F I / I
O F
C24 H2s F2 I N4 03, MS (APCI)m+1 = 587
EXAMPLE 116
3.4-Difluoro-5-a(1-[4-i(6-hydroxy-pyridin-2-yl)-piperazin-1-yl]-methanoylJ~-2-
(4-iodo-2-
methyl-phenylamino~ benzoic acid
EXAMPLE 117
5-(1-(4-[2~2.5-Dimethvl-p~rrrol-1-y,-ethY]-piperazin-1~r1~-methanoyl)-3.4-
difluoro-2-
(4-iodo-2-meth~phen lair mino)-benzoic acid
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EXAMPLE 118
3.4-Difluoro-2-l4-iodo-2-methyl-phenylamino~-5-{1-f4-y3-phenox~,vridin-2-xl)-
piperazin-1-yl]-methanoyl}-benzoic acid
EXAMPLE 119
3.4-Difluoro-2-(4-iodo-2-methyl phen lal~ minoy-5-{1-[4-j5-phenoxy-pyridin-2-
ylr
piperazin-1-~1-methanoy~-benzoic acid
I \ O I \ O OH CH3
H
/ ~N \ N \
~N I / F I / I
O F
C3o H25 F2 I N4 04 , MS (APCI)m+1 = 671
EXAMPLE 120
3.4-Difluoro-2-l4-iodo-2-meth~phenylaminoL(1-[4-(3-phenoxy~pyridin-4-yl?-
j1-4]diazepan-1-~l-methanoyl}-benzoic acid
EXAMPLE 121
5-.(1-[4-(3-Chloro-4-hydroxymethyl-phenyl)-piperazin-1-~]-methanoyl}-3.4-
difluoro-2-
~4-iodo-2-methyl-phenylamino)-benzoic acid
EXAMPLE 122
3,4-Difluoro-2-(4-iodo-2-methyl-phenvlamino)-5-j1-y8-trifluoromethyl-3.4-
dihydro-1 H-
benzoL4.5]imidazof1.2-alpyrazin-2-yl~-methanoyll-benzoic acid
F F
F \ N O OH H
H
N~ I \ N I \
~N / F / I
O F
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EXAMPLE 123
5-~1-f4-(5-Chloro-1.3-dimethyl-1 I-~-pyrazole-4-sulfonyl)-piperazin-1-yll-
methano~)~-
3.4-difluoro-2-(4-iodo-2-methyl-phenylamino~benzoic acid
O OH
CI O~~ ~O H CH3
SAN \ N \
HsC_N ~ ~N I / I /
N CH3 ~ ~F I
O F
C2a HZaCI F2 I N5 05 S , MS (APCI)m+1 = 694
EXAMPLE 124
3.4-Difluoro-5-(1-~(4-[2-y2-h d~r rox -~ethylsulfanyl)i-phen~)-piperazin-1-~}-
methanovl)-
2-(4-iodo-2-metal-phen~amino)-benzoic acid
C~9 H~~ F2 I N2 05 S , MS (APCI)m+1 = 551
EXAMPLE 125
5-[1-(1,1-Dioxo-16-thiomorpholin-4-yl)i-methanoyl]-3.4-difluoro-2-(4-iodo-2-
methyl-
phenylamino)-benzoic acid
EXAMPLE 126
3.4-Difluoro-2-(4-iodo-2-methyl-phen lal~ minoy-5-{1-(~1-methyl-piaeridin-4-
piperazin-1-yl]-methanoyl~benzoic acid
H3C.N~ O OH CH
H 3
N \ N \
~N I / F I / 1
i i
O F
C25 H29 F2 I N4 03 , MS (APCI)m+1 = 599
EXAMPLE 127
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3.4-Difluoro-2-(4-iodo-2-methyl-phenylamino)-5-[1-(4-oxo-1 phenyl-1.3.8-triaza-
spiro~4.5]dec-8-y~-methanol-benzoic acid
EXAMPLE 128
3.4-Difluoro-5-~~1,-[4-(2-hvdroxy-ethyl)-2.5-dimethyl-piperazin-1-yl]-
methanoyl}-2-~4-
iodo-2-methyl-phenylamino)-benzoic acid
HO' O OH
L H3
N~'CH ~ N
~TN I / F I / I
CH3 O F
C23 H2s F2 I N3 04 , MS (APCI)m+1 = 574
EXAMPLE 129
3.4-Difluoro-5-{1-[4 ~2-hydroxy-ethyl)-2.6-dimethyl-piperazin-1-yl]-methanoyl}-
2-(4-
iodo-2-meth~phenylamino~ benzoic acid
EXAMPLE 130
3,4-Difluoro-2-(4-iodo-2-methyl-phenylamino)-5-[1-(4-isopropyl-2-
methyl~piperazin-
1-yl -methanov~-benzoic acid
EXAMPLE 131
5-~(1-[4-(3-Chloro-4-h dy roxymethyl-phenyl)-2.6-dimethyl-piperazin-1-yl]-
methanoyl~
3.4-difluoro-2-(4-iodo-2-methyl-phenylamino)-benzoic acid
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EXAMPLE 132
3.4-Difluoro-5-{1-(4-(5-hydroxy-pentyl)-piperazin-1-yl]-methanoylj~-~4-iodo-2-
methyl-phenylamino)-benzoic acid
EXAMPLE 133
3.4-Difluoro-5-(1-{4-[2-(2-hydroxy-ethoxy)-phenyl]-piperazin-1-yl}-methanol)-2-
(4-
iodo-2-meth~phen~rlamino)i-benzoic acid
EXAMPLE 134
3.4-Difluoro-2-~(4-iodo-2-methyl-phenylamino)-5-.[1-~4-methyl-pyridin-2-vIL
piperazin-1-yl]-methanoyl}-benzoic acid
EXAMPLE 135
5-{1-[4-(2-sec-Butox~phen~Lpiperazin-1-yl]-methanoyl}-3.4-difluoro-2-i(4-iodo-
2-
methyl-phenylamino)-benzoic acid
EXAMPLE 136
3.4-Difluoro-2-(4-iodo-2-methyl-phenylamino -~5-,(1-j4-(2-isobutox~phenyl)i-
piperazin-1-yll-methano~~-benzoic acid
EXAMPLE 137
5-[1-~4-Benzothiazol-2-~piperazin-1-yl~-methanoy]-3.4-difluoro-2-i(4-iodo-2-
methyl-
phenylamino)-benzoic acid
EXAMPLE 138
5-~(1-j4-i(6-Ethoxv-p rid~~lpiperazin-1-Yl-methanoyl}-3.4-difluoro-2-(4-iodo-2-
methyl-phenylamino-benzoic acid
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EXAMPLE 139
5-f 1-(4-Benzooxazol-2-yl-piperazin-1-yl)i-methanoyl]-3.4-difluoro-2-(4-iodo-2-
methyl-
phenylamino)-benzoic acid
EXAMPLE 140
3.4-Difluoro-2-(4-iodo-2-methyl-phen lay mino)-5~1-[4-(3-meth~c~uinoxalin-2-~)-
piperazin-1-YI]-methano~l]~-benzoic acid
EXAM PLE 141
5-j1-i~3'.6'-Dimethyl-2.3.5.6-tetrahydro-[1.2')bipvrazinyl-4yl)-methanoyl]-3.4-
difluoro-
2-(4-iodo-2-methyl-phenylamino)-benzoic acid
EXAMPLE 142
3.4-Difluoro-2-(4-iodo-2-methyl-phen lay mino)I-5~1-[4-(6-methyl-pyridazin-3-
vl)-
piperazin-1-y,-methanoyl}-benzoic acid
EXAMPLE 143
3.4-Difluoro-5-(1-~(4-[3-~2-hydrox -ey thoxy)-phenyl]'-piperazin-1-~rl]~-
methanoy~-2-~4-
iodo-2-methyl-phenylamino)-benzoic acid
EXAMPLE 144
35
3.4-Difluoro-5-[1-(2-hydroxy-ethyl)-imidazolidin-2-ylidene-hvdrazinocarbonyl]-
2-(4-
iodo-2-methyl-phenylamino)-benzoic acid
EXAMPLE 145
3.4-Difluoro-2~4-iodo-2-meth-phenvlamino -L5-(1-(4 j3-(propane-1-sulfon~)-
phenyl]-piperazin-1-~I}-methanoyl)i-benzoic acid
O OH CH
HsC~S~ ~ N Ws
N
O vN I ~ I ~ I
'F
O F
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C2$ H28 F2 I N3 05 S , MS (APCI)m+1 = 684
EXAMPLE 146
3.4-Difluoro-2~(4-iodo-2-methyl-phenvlamino)-5-.(1-[4 ~3-methanesulfonyl-
~henyl)-
piperazin-1-yl]-methanoyl~~-benzoic acid
EXAMPLE 147
3.4-Difluoro-2-(4-iodo-2-methyl-phenylamino)-5-(1-~[4-(2 (propane-1-sulfonylL
phenyl]-piperazin-1-XI -methanol)-benzoic acid
EXAMPLE 148
5-~(1-(4-(4.5-Dimethyl-thiazol-2-yl~~ piperazin-1-yl~ methanoy~-3.4-difluoro-2-
(4-iodo-
2-methyl-phenylamino)-benzoic acid
EXAMPLE 149
5-{1-(4-(5-Ethyl-(1.3.4]thiadiazol-2-yl)-piperazin-1-yl]-methanoyl}-3.4-
difluoro-2-(4-
iodo-2-methyl-phenylamino)-benzoic acid
N~N O OH CH3
~/S~N~_ \ N I \
vN I ~ F ~ I
O F
C23 H~ F2 I N5 03 S , MS (APCI)m+1 = 614
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EXAMPLE 150
3,4-Difluoro-5-.[1-[4-(1-furan-2-yl-methanoyl)~-piperazin-1-vl]-methanoyl}-2-
(4-iodo-2-
meth-ahenylamino)-benzoic acid
O O OH CH
H 3
\ N~ \ N \
O ~N I / F I / I
O F
C24 H2o F2 I N3 05 , MS (APCI)m+1 = 596
EXAMPLE 151
5-(1-f4-[4-(4.5-Dihydro-1 I-~imidazol-2-yl~-butyl]-piperazin-1-yl}-methanoyl)-
3.4-
difluoro-2-(4-iodo-2-methyl-phen IaIL m!no)-benzoic acid
EXAMPLE 152
3.4-Difluoro-2-(4-iodo-2-methyl-phenvlamino)i-5-{1-[4-(3-phosphono-propyl)-
~ir~erazin-1-yll-methanoy~-benzoic acid
EXAMPLE 153
3.4-Difluoro-5-{1-f~2-hydroxv-ethyl)-piperazin-1-yl]-methanoyl)~-2-(4-iodo-2-
meth
phenylamino)-benzoic acid
EXAMPLE 154
3.4-Difluoro-2-(4-iodo-2-methyl-phenylamino)i-5-[1-(5-methyl-hexahydro-
pyrrolo[3.4-
clp rrvr ol-2-yl~-methanoyl]-benzoic acid
EXAMPLE 155
N (1.1-Dioxo-tetrahydro-1 16-thio~~hen-3-yl)I-4.5-difluoro-6-(4-iodo-2-methyl-
phenylamino)- N methyl-isophthalamic acid
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O OH CHs
H
~Hs \ N \
/~ N I ~ F I ~ I
S'
p ~ O F
C2o H~9 F2 I N2 05 S , MS (APCI)m+1 = 565
EXAMPLE 156
3-y{115-Carboxy-2.3-difluoro-4-i(4-iodo-2-meth~rl-phe ~lamino)-phenyl-
methano~~-
amino -tetrahydro-thiophene-3-carboxylic acid
O OH CHs
H
\ N \
N I ~ F I ~ I
S
OH F
O
EXAMPLE 157
4.5-Difluoro-N -~( 1-hydroxvmeth~rl-cyclopentylZ 6-(4-iodo-2-methyl-
phenylamino)-
isophthalamic acid
EXAMPLE 158
4.5-Difluoro-N- -(4-hydroxy-cyclohexyl)-6-L4-iodo-2-methyl-phenylaminol
isophthalamic acid
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EXAMPLE 159
4.5-Difluoro-N -(SR)-2-hvdroxy-cyclohex~)i-6-(4-iodo-2-methyl-phenylamino)-
isophthalamic acid
EXAMPLE 160
11~i(3-Cyclohexylamino-propel)-4,5-difluoro-6-(4-iodo-2-methyl-phenylamino)-
isophthalamic acid
O OH CHs
H
N
NON I ~ F I ~ I
O F
C2a His FZ I Ns Os , MS (APCI)m+1 = 572
EXAMPLE 161
4.5-Difluoro-6-(4-iodo-2-methyl-phenylamino)-N-(2-methylene-tetrahydro-
thiophen-
3-yl)-isophthalamic acid
O OH CHa
H
N
N I ~ F I ~ I
O F
O
C~9 H~5 F2 I NZ 04 S , MS (APCI)m+1 = 533
EXAMPLE 162
N (1.1-Dioxo-tetrahydro-1 16-thiophen-3-yIJ~-4,5-difluoro-6-(4-iodo-2-methvl-
phenylamino)-isophthalamic acid
Biological Examples
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EXAMPLE 163
Cascade assay for inhibitors of the MAP kinase pathway
Incorporation of 32P into myelin basic protein (MBP) is 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 contains 20 mM HEPES, pH 7.4, 10 mM MgCl2, 1
mM MnCl2, 1 mM EGTA, 50p,M ['y-32P]ATP, 10 wg GST-MEK, 0.5 ~.g GST-MAPK
and 40 ~.g MBP in a final volume of 100 p,L. Reactions are stopped after 20
minutes by addition of trichloroacetic acid and filtered through a GF/C filter
mat. 32P
retained on the filter mat is determined using a 120S Betaplate. Compounds are
assessed at 10 wM for ability to inhibit incorporation of 32P.
To ascertain whether compounds are inhibiting GST-MEK or GST MAPK, two
additional protocols are employed. In the first protocol, compounds are added
to
tubes containing GST-MEK, followed by addition of GST-MAPK, MBP and
['y-s2P]ATP. In the second protocol, compounds are added to tubes containing
both
GST-MEK and GST-MAPK, followed by MBP and [~ 32P]ATP.
Compounds that show activity in both protocols are scored as MAPK
inhibitors, while compounds showing activity in only the first protocol are
scored as
MEK inhibitors.
EXAMPLE 164
In vitro MAP kinase assay
Inhibitory activity can be confirmed in direct assays. For MAP kinase, 1 ~.g
GST-MAPK is incubated with 40 wg MBP in the presence or absence of test
compound for 15 minutes at 30 °C in a final volume of 50 ~,L containing
50 mM Tris
(pH 7.5), 10 wM MgC12, 2 ~,M EGTA, and 10 p.M ['y-3zP]ATP. The.reaction is
stopped by addition of Laemmli SDS sample buffer and phosphorylated MBP
resolved by electrophoresis on a 10% polyacrylamide gel. Radioactivity
incorporated into MBP is determined by both autoradiography, and scintillation
counting of excised bands.
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EXAMPLE 165
In vitro MEK assay
For evaluation of direct MEK activity, 10 p,g GST-MEK~ is incubated in the
presence of absence of test compound with 5 wg 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 are 15
minutes at 30 °C in a final volume of 50 p,L containing 50 mM Tris (pH
7.5), 10 ~M
MgCl2, 2 , wM EGTA, and 10 ~M [y-32P]ATP. The reaction is stopped by addition
of
Laemrnli SDS sample buffer. Phosphorylated GST-MAPK-KA is resolved by
electrophoresis on a 10% polyacrylamide gel. Radioactivity incorporated into
GST-MAPK-KA is determined by autoradiography, and subsequent scintillation
counting of excised bands.
Alternatively, an artificially activated MEK containing serine to glutamate
mutations at positions 218 and 222 (GST-MEK-2E) is used. When these two 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
wg
GST-MEK-2E is incubated with 5 wg GST-MAPK-KA for 15 minutes at 30 °C
in the
same reaction buffer as described above. Reactions are terminated and analyzed
as above.
EXAMPLE 166
Whole cell MAP kinase assay
To determine if compounds block activation of MAP kinase in whole cells, the
following protocol is used. Cells are plated in multi-well plates and grown to
confluence. Cells are serum-deprived overnight. Cells are exposed to the
desired
concentrations of compound or vehicle (DMSO) for 30 minutes, followed by
addition
of a growth factor, for example, PDGF (100 ng/mL). After a 5-minute treatment
with
the growth factor, cells are washed with PBS, and lysed in a buffer consisting
of 70
mM NaCI, 10 mM HEPES (pH 7.4), 50 mM glycerol phosphate, and 1% Triton
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X-100. Lysates are clarified by centrifugation at 13,000 x g for 10 minutes.
Five
micrograms of the resulting supernatants are incubated with 10 pg microtubule
associated protein-2 (Map2) for 15 minutes at 30 °C in a final volume
of 25 ~L
containing 50 mM Tris (pH 7.4), 10 mM MgCl2, 2 mM EGTA and 30 pM [~ 32P]ATP.
Reactions are terminated by addition of Laermmli sample buffer. Phosphorylated
Map2 is resolved on 7.5% acrylamide gels and incorporated radioactivity is
determined by scintillation counting of excised bands.
EXAMPLE 167
Monolayer growth
Cells are plated into multi-well plates at 10 to 20,000 cells/mL. Forty-eight
hours after seeding, test compounds are added to the cell growth medium and
incubation is continued for 2 additional days. Cells are then removed from the
wells
by incubation with trypsin and enumerated with a Coulter counter.
EXAMPLE 168
Growth in soft-agar
Cells are 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 are
transferred to a 37°C incubator. After 7 to 10 days' growth. visible
colonies are
manually enumerated with the aid of a dissecting microscope.
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EXAMPLE 169
Collagen-Induced Arthritis in Mice
Type II collagen-induced arthritis (CIA) in mice is an experimental model of
arthritis that has a number of pathologic, immunologic, and genetic features
in
common with rheumatoid arthritis. The disease is induced by immunization of
DBA/1 mice with 100 Ng type II collagen, which is a major component of joint
cartilage, delivered intradermally in Freund's complete adjuvant. The disease
susceptibility is regulated by the class II MHC gene locus, which is analogous
to the
association of rheumatoid arthritis with HLA-DR4.
A progressive and inflammatory arthritis develops in the majority of mice
immunized, characterized by paw width increases of up to 100%. A test compound
is administered to mice in a range of amounts, such as 20, 60, 100, and 200
mg/kg
body weight/day. The duration of the test can be several weeks to a few
months,
such as 40, 60, or 80 days. A clinical scoring index is used to assess disease
progression from erythema and edema (stage 1 ), joint distortion (stage 2), to
joint
ankylosis (stage 3). The disease is variable in that it can affect one or all
paws in
an animal, resulting in a total possible score of 12 for each mouse.
Histopathology
of an arthritic joint reveals synovitis, pannus formation, and cartilage and
bone
erosions. All mouse strains that are susceptible to CIA are high antibody
responders to type II collagen, and there is a marked cellular response to
CII.
EXAMPLE 170
SCW-induced monoarticular arthritis
Arthritis is induced as described by Schwab, et al., Infection and Immunity,
59:4436-4442 (1991 ) with minor modifications. Rats receive 6,ug sonicated SCW
[in 10 NI Dulbecco's PBS (DPBS)] by an intraarticular injection into the right
tibiotalar
joint on day 0. On day 21, the DTH is initiated with 100 Ng of SCW (250,u1)
administered i.v. For oral compound studies, compounds are suspended in
vehicle
(0.5% hydroxypropyl-methylcellulose/0.2% Tween 80), sonicated, and
administered
twice daily (10 ml/kg volume) beginning 1 hr prior to reactivation with SCW.
Compounds are administered in amounts between 10 and 500 mg/kg body
weight/day, such as 20, 30, 60, 100, 200, and 300 mg/kg/day. Edema
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measurements are obtained by determining the baseline volumes of the
sensitized
hindpaw before reactivation on day 21, and comparing them with volumes at
subsequent time points such as day 22, 23, 24, and 25. Paw volume is
determined
by mercury plethysmography.
EXAMPLE 171
Mouse ear-heart transplant model
Fey, T.A. et al. describe methods for transplanting split-heart neonatal
cardiac grafts into the ear pinna of mice and rats (J. Pharm. and Toxic. Meth.
39:9-
17 (1998)). Compounds are dissolved in solutions containing combinations of
absolute ethanol, 0.2% hydroxypropyl methylcellulose in water, propylene
glycol,
cremophor, and dextrose, or other solvent or suspending vehicle. Mice are
dosed
orally or intraperitoneally once, twice or three times daily from the day of
transplant
(day 0) through day 13 or until grafts have been rejected. Rats are dosed
once,
twice, or three times daily from day 0 through day 13. Each animal is
anesthetized
and an incision is made at the base of the recipient ear, cutting only the
dorsal
epidermis and dermis. The incision is spread open and down to the cartilage
parallel to the head, and sufficiently wide to accommodate the appropriate
tunneling
for a rat or insertion tool for a mouse. A neonatal mouse or rat pup less than
60
hours old is anesthetized and cervically dislocated. The heart is removed from
the
chest, rinsed with saline, bisected longitudinally with a scalpel, and rinsed
with
sterile saline. The donor heart fragment is placed into the preformed tunnel
with the
insertion tool and air or residual fluid is gently expressed from the tunnel
with light
pressure. No suturing, adhesive bonding, bandaging, or treatment with
antibiotics is
required.
Implants are examined at 10-20-fold magnification with a stereoscopic
dissecting microscope without anesthesia. Recipients whose grafts are not
visibly
beating may be anesthetized and evaluated for the presence of electrical
activity
using Grass E-2 platinum subdermal pin microelectodes placed either in the
pinna
or directly into the graft and a tachograph. Implants can be examined 1-4
times a
day for 10, 20, 30 or more days. The ability of a test compound to ameliorate
symptoms of transplant rejection can be compared with a control compound such
as cyclosporine, tacrolimus, or orally-administered lefluonomide.
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EXAMPLE 172
Murine ovalbumin-induced eosinophilia
Female C57BL/6 mice are obtained from the Jackson Laboratory (Bar
Harbor, ME). All animals are given food and water ad libitum. Mice are
sensitized
with a single i.p. injection of OVA (grade V, Sigma Chemical Company, St.
Louis,
MO) adsorbed to alum, (10 wg OVA + 9 mg alum in 200,u1 saline) or vehicle
control,
(9 mg alum in 200 NI saline) on day 0. On day 14, the mice are challenged with
a
12-minute inhalation of an aerosol consisting of 1.5% OVA (weight/volume) in
saline
produced by a nebulizer (small particle generator, model SPAG-2; ICN
Pharmaceuticals, Costa Mesa, CA). Groups of eight mice are dosed with oral
vehicle (0.5% hydroxypropylmethylcellulose / 0.25% TWEEN-80), or a test
compound at 10, 30, or 100 mg/kg in oral vehicle, 200,u1 per mouse p.o. Dosing
is
performed once per day starting on day 7 or day 13, and extending through day
16.
For determination of pulmonary eosinophilia, three days after the first OVA
aerosol challenge (day 17), the mice are anesthetized with an i.p. injection
of
anesthetic (Ketamine/Acepromazine/Xylazine), and the tracheae is exposed and
cannulated. The lungs and upper airways are lavaged twice with 0.5 ml of cold
PBS. A portion (200,u1) of the bronchoalveolar lavage (BAL) fluid is
enumerated
using a Coulter counter Model ZB1 (Coulter Electronics, Hialeah, FL). The
remaining BAL fluid is then centrifuged at 300 x g for five minutes, and the
cells are
resuspended in 1 ml of HBSS (Gibco BRL) containing 0.5% fetal calf serum
(HyClone) and 10 mM HEPES (Gibco BRL). The cell suspension is centrifuged in a
cytospin (Shandon Southern Instruments, Sewickley, PA) and stained by Diff
Quick
(American Scientific Products, McGraw Park, IL) to differentiate BAL
leukocytes into
neutrophil, eosinophil, monocyte or lymphocyte subsets. The number of
eosinophils
in the BAL fluid is determined by multiplying the percentage of eosinophils by
the
total cell count.
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EXAMPLE 173
Experimental compounds are added to 96 well format plates with filter
bottomed wells. Kinase-inactive ERK1 (K71 R mutant) in HEPES buffer is then
added to each well. After subsequent addition of MEK1 (2D mutant) diluted in a
Tris buffer before being added to the plate, and the reaction is initiated by
the
addition of radioactive ATP, diluted in 0.05% Tween 20. After 1 hour
incubation at
room temperature, Ice-cold 20% TCA is added to each well to stop the reaction
and
to precipitate the protein in solution. Filtration is done the following day
followed by
scintillation counting of the incorporated radioactivity using a Perkin Elmer
Wallac
microBeta 1450 counter. Inhibition is expressed as a percentage of the vehicle
control.
From the above disclosure and examples, and from the claims below, the
essential features of the invention are readily apparent. The scope of the
invention
also encompasses various modifications and adaptations within the knowledge of
a
person of ordinary skill. Examples include a disclosed compound modified by
addition or removal of a protecting group, or an ester, pharmaceutical salt,
hydrate,
acid, or amide of a disclosed compound. Publications cited herein are hereby
incorporated by reference in their entirety.
