CA2403017A1 - 5-amide substituted diarylamines as mex inhibitors - Google Patents
5-amide substituted diarylamines as mex inhibitors Download PDFInfo
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- CA2403017A1 CA2403017A1 CA002403017A CA2403017A CA2403017A1 CA 2403017 A1 CA2403017 A1 CA 2403017A1 CA 002403017 A CA002403017 A CA 002403017A CA 2403017 A CA2403017 A CA 2403017A CA 2403017 A1 CA2403017 A1 CA 2403017A1
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Abstract
Diarylamines, such as 5-amide substituted diarylamines of formula (I) or formula (II) wherein A is hydroxy, C1-6 alkoxy, or NR6OR7; X is OR12, NR13R12, or NR14; 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.
Description
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 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.
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 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.
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;
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;
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, 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.
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, 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.
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.
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.
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)-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, WO 01/68619 -$- PCT/USO1/07816 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 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, 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.
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-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.
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, 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.
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, 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-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 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).
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.
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
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)-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, WO 01/68619 -$- PCT/USO1/07816 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 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, 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.
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-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.
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, 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.
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, 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-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 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).
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.
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 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 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:
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 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 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.
Table I
O A
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 -NHMe -OH -CI -NMe2 -OH -CI
-OH -CI
~N~
-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 -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
N
F ~ / 1 O F
-R~ -X
-Me -NH2 -Me -NHMe -Me -NMe2 -Me -Me ~N~
-Me ~N~
WO 01/68619 -2$- PCT/USO1/07816 -Me -NCH3nBu -Me ~N~
LN
-Me -N[(CH2)20CH2CH3]2 _Me ~N~N I
~NJ
-Me ~ ~oH
~N
'F
-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.
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 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 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, 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;
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 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 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 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%).
7.8-Difluoro-1-L-iodo-2-methyl phenyl)-4-oxo-1.4-dihydro-2l~benzo[d][1.3loxazine-6-carboxylic acid methvlamide.
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 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
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, 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
N'-Cyclo~~ropylmethoxy-4.5-difluoro-6-i(4-iodo-2-methyl-phenylamino)~-N3-methyl-isophthalamide .
H
~O.N O H
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
(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
7.8-Difluoro-1-(4-iodo-2-methyl-phenyl)-4-oxo-1.4-dihydro-21-~benzo[dl[1.3]oxazine-6-carboxylic acid dimethylamide.
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
N'-Cyclopropylmethoxy-4.5-difluoro-6~4-iodo-2-methyl-~phenylamino)-N3.N3-dimet~rl-isophthalamide (27~
.N O
CH3 ~ N
H3C~N I ~ F I ~ I
O F
Prepared in the manner of Example 4 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
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).
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
C2o H~~ F2 I N2 04, MS (APCI)m+1 = 515 in vitro MEK assay: 34% inhibition @ 1 wM
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
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
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 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
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 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
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
C2s H2s F2 I N2 04, MS (APCI)m+1 = 557 in vitro MEK assay: 23% inhibition @ 1 wM
WO 01/68619 '41- PCT/USO1/07816 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 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
7.8-Difluoro-1-(4-iodo-2-methyl-phenyl)-4-oxo-1.4-dihyd ro-2H-3.1-benzoxazine-carboxylic acid methyl-(1-methyl-piperidin-4-yl)I-amide o ' ~~ cH3 CfH3 \ N \
N I ~ F I ~ I
H C~N~ O F
C23 HZa F2 I N3 03, MS (APCI)m+1 = 556 in vitro MEK assay: 0% inhibition @ 1 ~,M
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, 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
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
7.8-Difluoro-1-(4-iodo-2-methyl-phenylJl-4-oxo-1.4-dihydro-2H-3.1-benzoxazine-carboxylic acid [2-(1-methyl-~yrrolidin-2-y~-ethyl]-amide \ 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
7.8-Difluoro-1-(4-iodo-2-methyl-phenyl)I-4-oxo-1.4-dihydro-2H-3.1-benzoxazine-carboxylic acid (2-hydroxyl)-methyl-amide 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
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 \ N \
S N I / F I / I
N-N O F
C~8 H~~ F2 I N4 03 S, MS (APCI)m+1 = 529 in vitro MEK assay: 31 % inhibition @ 1 ~M
7.8-Difluoro-1-(4-iodo-2-methyl-phenyl)~-4-oxo-1.4-dihvrdro-2H-3.1-benzoxazine-carboxylic acid (2-methoxy-1-methyl-ethyl)-amide N
H3C N I ~ F I ~ I
OJ O F
C2o His F2 I N2 04, MS (APCI)m+1 = 517 in vitro MEK assay: 46% inhibition @ 1 ~M
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
C24 H~6 F2 I N3 03 S, MS (APCI)m+1 = 592 in vitro MEK assay: 26% inhibition @ 1 wM
WO 01/68619 '45- PCT/USO1/07816 .8-Difluoro-1-(4-iodo-2-methyl-phenyl)-4-oxo-1.4-d ihyd ro-2H-3.1-benzoxazine-carboxylic acid (4-methyl-thiazol-2-yl)-amide 0 01 cH3 N
H
SYN I ~ F I ~ I
~N O F
CZO H~4 F2 I N3 03 S, MS (APCI)m+1 = 542 in vitro MEK assay: 20% inhibition @ 1 p,M
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
C2o H~5 F2 I N4 03 S2, MS (APCI)m+1 = 589 in vitro MEK assay: 5% inhibition @ 1 wM
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 o °1 H
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
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
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
WO 01/68619 '47- PCT/USO1/07816 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
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
7.8-Difluoro-1-~4-iodo-2-methyl-phenyl)-4-oxo-1.4-dihydro-2H-3.1-benzoxazine-6-carboxylic acid thiazol-2-vlamide 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
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 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
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
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
WO 01/68619 -5~- PCT/USO1/07816 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
7.8-Difluoro-1-(4-iodo-2-methyl-phenyl)i-4-oxo-1,4-dihydro-2H-3.1-benzoxazine-carbox, li~c acid~1 H-pvrazol-3-yl)-amide 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
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 \ 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
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
7.8-Difluoro-6-(3-hyd roxymethyl-piperidine-1-carbony ~-1-(4-iodo-2-methyl-pheny~-1.2-dihydro-3.1-benzoxazin-4-one 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
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
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
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
WO 01/68619 -~- PCT/USO1/07816 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
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 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
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
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 C22 H~6 F2 I N3 04, MS (APCI)m+1 = 552 in vitro MEK assay: 43% inhibition @ 1 pM
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
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
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
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
WO 01/68619 -5$- PCT/USO1/07816 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
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 \ 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
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
C24 H2o F2 I N3 03, MS (APCI)m+1 = 564 in vitro MEK assay: 61 % inhibition @ 1 ~.M
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
7.8-Difluoro-1-(4-iodo-2-methyl-phenyl)-4-oxo-1.4-d ihYdro-2H-3.1-benzoxazine-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
7.8-Difluoro-1-(4-iodo-2-methyl-phenyl)I-6-(piperazine-1-carbonyl)-1.2-dihydro-3.1-benzoxazin-4-one ° °~ H
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
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
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
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
7.8-difluoro-1-(4-iodo-2-methyl-phenyl)i-4-oxo-1.4-dihydro-2H-3.1-benzoxazine-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 afford the corresponding isophthalamic acids. LC/MS was performed on a CPI
120SE (C18) column (4.6x50mm, 5 ~.m).
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
4.5-Difluoro-6-(4-iodo-2-methyl phenvlamino)- 11~f3-(2-oxo-pyrrolidin-1-yl)-propyl]-isophthalamic acid HO O H
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
4.5-Difluoro-6-(4-iodo-2-methyl-phenylamino)- N-(1-methyl-piperidin-4-y~-isophthalamic acid 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
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
4.5-Difluoro-N f2-(2-hydroxy-ethoxY -ethyl]-6-(4-iodo-2-meth I-~ ahenylamino)-iso~hthalamic acid HO' 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
4.5-Difluoro-6-(4-iodo-2-meth~phenylaminoy- N-/2-i(1-meth~~yrrolidin-2-y~-ethyll-isophthalamic acid 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
4.5-Difluoro-6-~4-iodo-2-methyl-phenylamino)- N-(2-p rids in-2-yl-ethyl)-isophthalamic acid /
N ~ ' HO O H
N
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
N-Butyl-4.5-difluoro-6-(4-iodo-2-methyl-phenylamino)- N-(2-mercapto-ethyl)-isophthalamic acid 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
3.4-Difluoro-5-,[1-[2-(2-hydroxy-ethylLpi~~eridin-1-yl]-methanoy~-~4-iodo-2-methyl-phenylamino)-benzoic acid HO O H
\ 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
3.4-Difluoro-5-[1-(2-hydroxymethyl-piperidin-1-yl)-methanoyl]-2-(4-iodo-2-methyl-phen~rlamino)-benzoic acid \ 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
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
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
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 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
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
1-[3.4-Difluoro-5-[1-(3-h day-piperidin-1-~,)-methanoyl]-2-(4-iodo-2-methyl-hhen lay mino~phenyl]-ethanone 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
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
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
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
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
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
3.4-Difluoro-5-[1-~(4-hydroxy-piperidin-1-~)-methanol]-2-(4-iodo-2-methyl-phenylamino~ benzoic acid HO O H
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
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 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
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
~2-Ethyl-2H-pyrazol-3-yl~-4.5-difluoro-6-i(4-iodo-2-meth~phen~aminol isophthalamic acid HO O H
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
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
4,5-Difluoro-N-(1 H-indazol-6-yl)i-6-~(4-iodo-2-methyl-phenylamino)-isophthalamic acid N.
r NH HO O H
\ / ~ ~ 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
4 5-Difluoro-11~(2-hydroxy-1-meth~yly-6-(4-iodo-2-methyl-phenylamino~
isophthalamic acid ~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
4.5-Difluoro-11~[,2,~- 1 H-imidazol-4-yl~-ethyl]-6-y4-iodo-2-methyl-phenvlamino~
isophthalamic acid H
H
N
HN I ~ F I ~ I
O F
C2o H» FZ I N4 03, MS (APCI)m+1 = 527 in vitro MEK assay: 60% inhibition @ 1 p.M
in vitro MEK assay: IC5o = 1.4 wM
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
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
4.5-Difluoro-6-~4-iodo-2-methyl-phenylamino)-11~~(3-morpholin-4-yl-aropyl)-isophthalamic acid ~N HO O CH
\ 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
N (4-Dimethylamino-phenyl)I-4.5-difluoro-6-(iodo-methyl-phenylamino)-isophthalamic acid H3C.N.CH3 .
I \ HO O CH
\ 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
5-~1-(4-Ethvl-pi~erazin-1-vl)-methanoyl]-3.4-difluoro-2-(4-iodo-2-methyl-phen~rlamino)I-benzoic acid H
H3C~N~ \ N \
~N I / F I / I
O F
WO 01/68619 -7$- PCT/USO1/07816 C2~ H22 F2 I N3 03, MS (APCI)m+1 = 530 in vitro MEK assay: 70% inhibition @ 1 wM
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
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
O F
C2s H2z F2 I N3 03, MS (APCI)m+1 = 590 in vitro MEK assay: 77% inhibition @ 1 ~M
4.5-Difluoro-6-(4-iodo-2-methyl-phenylamino)-l1r' (4-sulfamoyl-phenyly-isophthalamic acid q O= -NH2 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
4.5-Difluoro-6-(4-iodo-2-methyl-phenylamino -11~(tetrahydro-furan-2-~rlmethyy-isophthalamic acid 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
3.4-Difluoro-2-~(4-iodo-2-meth~phenylaminoy-5-( 1-thiazolid in-3-yl-methano~-benzoic acid 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
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
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 WO 01/68619 -$1- PCT/USO1/07816 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
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
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 WO 01/68619 -$2- PCT/USO1/07816 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 3.4-Difluoro-2-(4-iodo-2-methYl~henylamino~ 5-[~4-pyridin-2 yl-piperazin-1-yl~
methanoyl]-benzoic acid 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 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 5-~1-[4-i(2-Amino-ether)-piperazin-1-yll methanoyl~-3,4-difluoro-2-(4-iodo-2-methyl-phenylaminol-benzoic acid WO 01/68619 -$3- PCT/USO1/07816 3.4-Difluoro-5-{1-[4-(2-hydroxy-2-methyl-propyl)i-piperazin-1-vl]-methanoyl]~-~4-iodo-2-methyl-phenylamino)-benzoic acid 5-{1-[4-L,4-Dimethoxy-phenyl)i-piperazin-1-~]-methanol}-3.4-difluoro-2-(4-iodo-methyl-phenylamino)-benzoic acid 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 3.4-Difluoro-2-(4-iodo-2-methyl-phenylamino)-5-(1-.(4-[propane-1-sulfonYl)-phenyl-piperazin-1-y~~-methanoyly-benzoic acid 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 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 WO 01/68619 -$4- PCT/USO1/07816 H
~N1 C2~ H28 F2 I N5 03 , MS (APCI)m+1 = 636 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
N~ \ N \
~N I / F I / I
O F
C24 H2s F2 I N4 03, MS (APCI)m+1 = 587 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 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 3.4-Difluoro-2-l4-iodo-2-methyl-phenylamino~-5-{1-f4-y3-phenox~,vridin-2-xl)-piperazin-1-yl]-methanoyl}-benzoic acid 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 3.4-Difluoro-2-l4-iodo-2-meth~phenylaminoL(1-[4-(3-phenoxy~pyridin-4-yl?-j1-4]diazepan-1-~l-methanoyl}-benzoic acid 5-.(1-[4-(3-Chloro-4-hydroxymethyl-phenyl)-piperazin-1-~]-methanoyl}-3.4-difluoro-2-~4-iodo-2-methyl-phenylamino)-benzoic acid 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
WO 01/68619 -$6- PCT/USO1/07816 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 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 5-[1-(1,1-Dioxo-16-thiomorpholin-4-yl)i-methanoyl]-3.4-difluoro-2-(4-iodo-2-methyl-phenylamino)-benzoic acid 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
N \ N \
~N I / F I / 1 i i O F
C25 H29 F2 I N4 03 , MS (APCI)m+1 = 599 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 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
N~'CH ~ N
~TN I / F I / I
C23 H2s F2 I N3 04 , MS (APCI)m+1 = 574 3.4-Difluoro-5-{1-[4 ~2-hydroxy-ethyl)-2.6-dimethyl-piperazin-1-yl]-methanoyl}-2-(4-iodo-2-meth~phenylamino~ benzoic acid 3,4-Difluoro-2-(4-iodo-2-methyl-phenylamino)-5-[1-(4-isopropyl-2-methyl~piperazin-1-yl -methanov~-benzoic acid 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 3.4-Difluoro-5-{1-(4-(5-hydroxy-pentyl)-piperazin-1-yl]-methanoylj~-~4-iodo-2-methyl-phenylamino)-benzoic acid 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 3.4-Difluoro-2-~(4-iodo-2-methyl-phenylamino)-5-.[1-~4-methyl-pyridin-2-vIL
piperazin-1-yl]-methanoyl}-benzoic acid 5-{1-[4-(2-sec-Butox~phen~Lpiperazin-1-yl]-methanoyl}-3.4-difluoro-2-i(4-iodo-methyl-phenylamino)-benzoic acid 3.4-Difluoro-2-(4-iodo-2-methyl-phenylamino -~5-,(1-j4-(2-isobutox~phenyl)i-piperazin-1-yll-methano~~-benzoic acid 5-[1-~4-Benzothiazol-2-~piperazin-1-yl~-methanoy]-3.4-difluoro-2-i(4-iodo-2-methyl-phenylamino)-benzoic acid 5-~(1-j4-i(6-Ethoxv-p rid~~lpiperazin-1-Yl-methanoyl}-3.4-difluoro-2-(4-iodo-2-methyl-phenylamino-benzoic acid WO 01/68619 -$9- PCT/USO1/07816 5-f 1-(4-Benzooxazol-2-yl-piperazin-1-yl)i-methanoyl]-3.4-difluoro-2-(4-iodo-2-methyl-phenylamino)-benzoic acid 3.4-Difluoro-2-(4-iodo-2-methyl-phen lay mino)-5~1-[4-(3-meth~c~uinoxalin-2-~)-piperazin-1-YI]-methano~l]~-benzoic acid 5-j1-i~3'.6'-Dimethyl-2.3.5.6-tetrahydro-[1.2')bipvrazinyl-4yl)-methanoyl]-3.4-difluoro-2-(4-iodo-2-methyl-phenylamino)-benzoic acid 3.4-Difluoro-2-(4-iodo-2-methyl-phen lay mino)I-5~1-[4-(6-methyl-pyridazin-3-vl)-piperazin-1-y,-methanoyl}-benzoic acid 3.4-Difluoro-5-(1-~(4-[3-~2-hydrox -ey thoxy)-phenyl]'-piperazin-1-~rl]~-methanoy~-2-~4-iodo-2-methyl-phenylamino)-benzoic acid 3.4-Difluoro-5-[1-(2-hydroxy-ethyl)-imidazolidin-2-ylidene-hvdrazinocarbonyl]-2-(4-iodo-2-methyl-phenylamino)-benzoic acid 3.4-Difluoro-2~4-iodo-2-meth-phenvlamino -L5-(1-(4 j3-(propane-1-sulfon~)-phenyl]-piperazin-1-~I}-methanoyl)i-benzoic acid O OH CH
HsC~S~ ~ N Ws N
O vN I ~ I ~ I
'F
O F
WO 01/68619 -9~- PCT/USO1/07816 C2$ H28 F2 I N3 05 S , MS (APCI)m+1 = 684 3.4-Difluoro-2~(4-iodo-2-methyl-phenvlamino)-5-.(1-[4 ~3-methanesulfonyl-~henyl)-piperazin-1-yl]-methanoyl~~-benzoic acid 3.4-Difluoro-2-(4-iodo-2-methyl-phenylamino)-5-(1-~[4-(2 (propane-1-sulfonylL
phenyl]-piperazin-1-XI -methanol)-benzoic acid 5-~(1-(4-(4.5-Dimethyl-thiazol-2-yl~~ piperazin-1-yl~ methanoy~-3.4-difluoro-2-(4-iodo-2-methyl-phenylamino)-benzoic acid 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 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
\ N~ \ N \
O ~N I / F I / I
O F
C24 H2o F2 I N3 05 , MS (APCI)m+1 = 596 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 3.4-Difluoro-2-(4-iodo-2-methyl-phenvlamino)i-5-{1-[4-(3-phosphono-propyl)-~ir~erazin-1-yll-methanoy~-benzoic acid 3.4-Difluoro-5-{1-f~2-hydroxv-ethyl)-piperazin-1-yl]-methanoyl)~-2-(4-iodo-2-meth phenylamino)-benzoic acid 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 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 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 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
4.5-Difluoro-N -~( 1-hydroxvmeth~rl-cyclopentylZ 6-(4-iodo-2-methyl-phenylamino)-isophthalamic acid 4.5-Difluoro-N- -(4-hydroxy-cyclohexyl)-6-L4-iodo-2-methyl-phenylaminol isophthalamic acid 4.5-Difluoro-N -(SR)-2-hvdroxy-cyclohex~)i-6-(4-iodo-2-methyl-phenylamino)-isophthalamic acid 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 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 N (1.1-Dioxo-tetrahydro-1 16-thiophen-3-yIJ~-4,5-difluoro-6-(4-iodo-2-methvl-phenylamino)-isophthalamic acid Biological Examples 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.
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.
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.
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 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.
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.
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.
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.
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 WO 01/68619 -9$- PCT/USO1/07816 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.
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 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.
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.
WO 01/68619 -1 ~~- PCT/USO1/07816 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.
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 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 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:
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 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 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.
Table I
O A
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 -NHMe -OH -CI -NMe2 -OH -CI
-OH -CI
~N~
-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 -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
N
F ~ / 1 O F
-R~ -X
-Me -NH2 -Me -NHMe -Me -NMe2 -Me -Me ~N~
-Me ~N~
WO 01/68619 -2$- PCT/USO1/07816 -Me -NCH3nBu -Me ~N~
LN
-Me -N[(CH2)20CH2CH3]2 _Me ~N~N I
~NJ
-Me ~ ~oH
~N
'F
-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.
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 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 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, 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;
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 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 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 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%).
7.8-Difluoro-1-L-iodo-2-methyl phenyl)-4-oxo-1.4-dihydro-2l~benzo[d][1.3loxazine-6-carboxylic acid methvlamide.
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 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
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, 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
N'-Cyclo~~ropylmethoxy-4.5-difluoro-6-i(4-iodo-2-methyl-phenylamino)~-N3-methyl-isophthalamide .
H
~O.N O H
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
(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
7.8-Difluoro-1-(4-iodo-2-methyl-phenyl)-4-oxo-1.4-dihydro-21-~benzo[dl[1.3]oxazine-6-carboxylic acid dimethylamide.
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
N'-Cyclopropylmethoxy-4.5-difluoro-6~4-iodo-2-methyl-~phenylamino)-N3.N3-dimet~rl-isophthalamide (27~
.N O
CH3 ~ N
H3C~N I ~ F I ~ I
O F
Prepared in the manner of Example 4 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
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).
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
C2o H~~ F2 I N2 04, MS (APCI)m+1 = 515 in vitro MEK assay: 34% inhibition @ 1 wM
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
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
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 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
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 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
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
C2s H2s F2 I N2 04, MS (APCI)m+1 = 557 in vitro MEK assay: 23% inhibition @ 1 wM
WO 01/68619 '41- PCT/USO1/07816 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 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
7.8-Difluoro-1-(4-iodo-2-methyl-phenyl)-4-oxo-1.4-dihyd ro-2H-3.1-benzoxazine-carboxylic acid methyl-(1-methyl-piperidin-4-yl)I-amide o ' ~~ cH3 CfH3 \ N \
N I ~ F I ~ I
H C~N~ O F
C23 HZa F2 I N3 03, MS (APCI)m+1 = 556 in vitro MEK assay: 0% inhibition @ 1 ~,M
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, 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
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
7.8-Difluoro-1-(4-iodo-2-methyl-phenylJl-4-oxo-1.4-dihydro-2H-3.1-benzoxazine-carboxylic acid [2-(1-methyl-~yrrolidin-2-y~-ethyl]-amide \ 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
7.8-Difluoro-1-(4-iodo-2-methyl-phenyl)I-4-oxo-1.4-dihydro-2H-3.1-benzoxazine-carboxylic acid (2-hydroxyl)-methyl-amide 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
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 \ N \
S N I / F I / I
N-N O F
C~8 H~~ F2 I N4 03 S, MS (APCI)m+1 = 529 in vitro MEK assay: 31 % inhibition @ 1 ~M
7.8-Difluoro-1-(4-iodo-2-methyl-phenyl)~-4-oxo-1.4-dihvrdro-2H-3.1-benzoxazine-carboxylic acid (2-methoxy-1-methyl-ethyl)-amide N
H3C N I ~ F I ~ I
OJ O F
C2o His F2 I N2 04, MS (APCI)m+1 = 517 in vitro MEK assay: 46% inhibition @ 1 ~M
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
C24 H~6 F2 I N3 03 S, MS (APCI)m+1 = 592 in vitro MEK assay: 26% inhibition @ 1 wM
WO 01/68619 '45- PCT/USO1/07816 .8-Difluoro-1-(4-iodo-2-methyl-phenyl)-4-oxo-1.4-d ihyd ro-2H-3.1-benzoxazine-carboxylic acid (4-methyl-thiazol-2-yl)-amide 0 01 cH3 N
H
SYN I ~ F I ~ I
~N O F
CZO H~4 F2 I N3 03 S, MS (APCI)m+1 = 542 in vitro MEK assay: 20% inhibition @ 1 p,M
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
C2o H~5 F2 I N4 03 S2, MS (APCI)m+1 = 589 in vitro MEK assay: 5% inhibition @ 1 wM
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 o °1 H
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
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
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
WO 01/68619 '47- PCT/USO1/07816 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
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
7.8-Difluoro-1-~4-iodo-2-methyl-phenyl)-4-oxo-1.4-dihydro-2H-3.1-benzoxazine-6-carboxylic acid thiazol-2-vlamide 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
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 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
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
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
WO 01/68619 -5~- PCT/USO1/07816 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
7.8-Difluoro-1-(4-iodo-2-methyl-phenyl)i-4-oxo-1,4-dihydro-2H-3.1-benzoxazine-carbox, li~c acid~1 H-pvrazol-3-yl)-amide 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
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 \ 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
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
7.8-Difluoro-6-(3-hyd roxymethyl-piperidine-1-carbony ~-1-(4-iodo-2-methyl-pheny~-1.2-dihydro-3.1-benzoxazin-4-one 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
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
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
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
WO 01/68619 -~- PCT/USO1/07816 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
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 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
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
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 C22 H~6 F2 I N3 04, MS (APCI)m+1 = 552 in vitro MEK assay: 43% inhibition @ 1 pM
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
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
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
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
WO 01/68619 -5$- PCT/USO1/07816 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
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 \ 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
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
C24 H2o F2 I N3 03, MS (APCI)m+1 = 564 in vitro MEK assay: 61 % inhibition @ 1 ~.M
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
7.8-Difluoro-1-(4-iodo-2-methyl-phenyl)-4-oxo-1.4-d ihYdro-2H-3.1-benzoxazine-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
7.8-Difluoro-1-(4-iodo-2-methyl-phenyl)I-6-(piperazine-1-carbonyl)-1.2-dihydro-3.1-benzoxazin-4-one ° °~ H
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
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
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
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
7.8-difluoro-1-(4-iodo-2-methyl-phenyl)i-4-oxo-1.4-dihydro-2H-3.1-benzoxazine-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 afford the corresponding isophthalamic acids. LC/MS was performed on a CPI
120SE (C18) column (4.6x50mm, 5 ~.m).
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
4.5-Difluoro-6-(4-iodo-2-methyl phenvlamino)- 11~f3-(2-oxo-pyrrolidin-1-yl)-propyl]-isophthalamic acid HO O H
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
4.5-Difluoro-6-(4-iodo-2-methyl-phenylamino)- N-(1-methyl-piperidin-4-y~-isophthalamic acid 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
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
4.5-Difluoro-N f2-(2-hydroxy-ethoxY -ethyl]-6-(4-iodo-2-meth I-~ ahenylamino)-iso~hthalamic acid HO' 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
4.5-Difluoro-6-(4-iodo-2-meth~phenylaminoy- N-/2-i(1-meth~~yrrolidin-2-y~-ethyll-isophthalamic acid 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
4.5-Difluoro-6-~4-iodo-2-methyl-phenylamino)- N-(2-p rids in-2-yl-ethyl)-isophthalamic acid /
N ~ ' HO O H
N
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
N-Butyl-4.5-difluoro-6-(4-iodo-2-methyl-phenylamino)- N-(2-mercapto-ethyl)-isophthalamic acid 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
3.4-Difluoro-5-,[1-[2-(2-hydroxy-ethylLpi~~eridin-1-yl]-methanoy~-~4-iodo-2-methyl-phenylamino)-benzoic acid HO O H
\ 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
3.4-Difluoro-5-[1-(2-hydroxymethyl-piperidin-1-yl)-methanoyl]-2-(4-iodo-2-methyl-phen~rlamino)-benzoic acid \ 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
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
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
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 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
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
1-[3.4-Difluoro-5-[1-(3-h day-piperidin-1-~,)-methanoyl]-2-(4-iodo-2-methyl-hhen lay mino~phenyl]-ethanone 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
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
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
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
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
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
3.4-Difluoro-5-[1-~(4-hydroxy-piperidin-1-~)-methanol]-2-(4-iodo-2-methyl-phenylamino~ benzoic acid HO O H
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
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 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
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
~2-Ethyl-2H-pyrazol-3-yl~-4.5-difluoro-6-i(4-iodo-2-meth~phen~aminol isophthalamic acid HO O H
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
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
4,5-Difluoro-N-(1 H-indazol-6-yl)i-6-~(4-iodo-2-methyl-phenylamino)-isophthalamic acid N.
r NH HO O H
\ / ~ ~ 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
4 5-Difluoro-11~(2-hydroxy-1-meth~yly-6-(4-iodo-2-methyl-phenylamino~
isophthalamic acid ~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
4.5-Difluoro-11~[,2,~- 1 H-imidazol-4-yl~-ethyl]-6-y4-iodo-2-methyl-phenvlamino~
isophthalamic acid H
H
N
HN I ~ F I ~ I
O F
C2o H» FZ I N4 03, MS (APCI)m+1 = 527 in vitro MEK assay: 60% inhibition @ 1 p.M
in vitro MEK assay: IC5o = 1.4 wM
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
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
4.5-Difluoro-6-~4-iodo-2-methyl-phenylamino)-11~~(3-morpholin-4-yl-aropyl)-isophthalamic acid ~N HO O CH
\ 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
N (4-Dimethylamino-phenyl)I-4.5-difluoro-6-(iodo-methyl-phenylamino)-isophthalamic acid H3C.N.CH3 .
I \ HO O CH
\ 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
5-~1-(4-Ethvl-pi~erazin-1-vl)-methanoyl]-3.4-difluoro-2-(4-iodo-2-methyl-phen~rlamino)I-benzoic acid H
H3C~N~ \ N \
~N I / F I / I
O F
WO 01/68619 -7$- PCT/USO1/07816 C2~ H22 F2 I N3 03, MS (APCI)m+1 = 530 in vitro MEK assay: 70% inhibition @ 1 wM
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
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
O F
C2s H2z F2 I N3 03, MS (APCI)m+1 = 590 in vitro MEK assay: 77% inhibition @ 1 ~M
4.5-Difluoro-6-(4-iodo-2-methyl-phenylamino)-l1r' (4-sulfamoyl-phenyly-isophthalamic acid q O= -NH2 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
4.5-Difluoro-6-(4-iodo-2-methyl-phenylamino -11~(tetrahydro-furan-2-~rlmethyy-isophthalamic acid 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
3.4-Difluoro-2-~(4-iodo-2-meth~phenylaminoy-5-( 1-thiazolid in-3-yl-methano~-benzoic acid 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
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
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 WO 01/68619 -$1- PCT/USO1/07816 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
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
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 WO 01/68619 -$2- PCT/USO1/07816 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 3.4-Difluoro-2-(4-iodo-2-methYl~henylamino~ 5-[~4-pyridin-2 yl-piperazin-1-yl~
methanoyl]-benzoic acid 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 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 5-~1-[4-i(2-Amino-ether)-piperazin-1-yll methanoyl~-3,4-difluoro-2-(4-iodo-2-methyl-phenylaminol-benzoic acid WO 01/68619 -$3- PCT/USO1/07816 3.4-Difluoro-5-{1-[4-(2-hydroxy-2-methyl-propyl)i-piperazin-1-vl]-methanoyl]~-~4-iodo-2-methyl-phenylamino)-benzoic acid 5-{1-[4-L,4-Dimethoxy-phenyl)i-piperazin-1-~]-methanol}-3.4-difluoro-2-(4-iodo-methyl-phenylamino)-benzoic acid 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 3.4-Difluoro-2-(4-iodo-2-methyl-phenylamino)-5-(1-.(4-[propane-1-sulfonYl)-phenyl-piperazin-1-y~~-methanoyly-benzoic acid 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 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 WO 01/68619 -$4- PCT/USO1/07816 H
~N1 C2~ H28 F2 I N5 03 , MS (APCI)m+1 = 636 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
N~ \ N \
~N I / F I / I
O F
C24 H2s F2 I N4 03, MS (APCI)m+1 = 587 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 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 3.4-Difluoro-2-l4-iodo-2-methyl-phenylamino~-5-{1-f4-y3-phenox~,vridin-2-xl)-piperazin-1-yl]-methanoyl}-benzoic acid 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 3.4-Difluoro-2-l4-iodo-2-meth~phenylaminoL(1-[4-(3-phenoxy~pyridin-4-yl?-j1-4]diazepan-1-~l-methanoyl}-benzoic acid 5-.(1-[4-(3-Chloro-4-hydroxymethyl-phenyl)-piperazin-1-~]-methanoyl}-3.4-difluoro-2-~4-iodo-2-methyl-phenylamino)-benzoic acid 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
WO 01/68619 -$6- PCT/USO1/07816 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 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 5-[1-(1,1-Dioxo-16-thiomorpholin-4-yl)i-methanoyl]-3.4-difluoro-2-(4-iodo-2-methyl-phenylamino)-benzoic acid 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
N \ N \
~N I / F I / 1 i i O F
C25 H29 F2 I N4 03 , MS (APCI)m+1 = 599 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 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
N~'CH ~ N
~TN I / F I / I
C23 H2s F2 I N3 04 , MS (APCI)m+1 = 574 3.4-Difluoro-5-{1-[4 ~2-hydroxy-ethyl)-2.6-dimethyl-piperazin-1-yl]-methanoyl}-2-(4-iodo-2-meth~phenylamino~ benzoic acid 3,4-Difluoro-2-(4-iodo-2-methyl-phenylamino)-5-[1-(4-isopropyl-2-methyl~piperazin-1-yl -methanov~-benzoic acid 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 3.4-Difluoro-5-{1-(4-(5-hydroxy-pentyl)-piperazin-1-yl]-methanoylj~-~4-iodo-2-methyl-phenylamino)-benzoic acid 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 3.4-Difluoro-2-~(4-iodo-2-methyl-phenylamino)-5-.[1-~4-methyl-pyridin-2-vIL
piperazin-1-yl]-methanoyl}-benzoic acid 5-{1-[4-(2-sec-Butox~phen~Lpiperazin-1-yl]-methanoyl}-3.4-difluoro-2-i(4-iodo-methyl-phenylamino)-benzoic acid 3.4-Difluoro-2-(4-iodo-2-methyl-phenylamino -~5-,(1-j4-(2-isobutox~phenyl)i-piperazin-1-yll-methano~~-benzoic acid 5-[1-~4-Benzothiazol-2-~piperazin-1-yl~-methanoy]-3.4-difluoro-2-i(4-iodo-2-methyl-phenylamino)-benzoic acid 5-~(1-j4-i(6-Ethoxv-p rid~~lpiperazin-1-Yl-methanoyl}-3.4-difluoro-2-(4-iodo-2-methyl-phenylamino-benzoic acid WO 01/68619 -$9- PCT/USO1/07816 5-f 1-(4-Benzooxazol-2-yl-piperazin-1-yl)i-methanoyl]-3.4-difluoro-2-(4-iodo-2-methyl-phenylamino)-benzoic acid 3.4-Difluoro-2-(4-iodo-2-methyl-phen lay mino)-5~1-[4-(3-meth~c~uinoxalin-2-~)-piperazin-1-YI]-methano~l]~-benzoic acid 5-j1-i~3'.6'-Dimethyl-2.3.5.6-tetrahydro-[1.2')bipvrazinyl-4yl)-methanoyl]-3.4-difluoro-2-(4-iodo-2-methyl-phenylamino)-benzoic acid 3.4-Difluoro-2-(4-iodo-2-methyl-phen lay mino)I-5~1-[4-(6-methyl-pyridazin-3-vl)-piperazin-1-y,-methanoyl}-benzoic acid 3.4-Difluoro-5-(1-~(4-[3-~2-hydrox -ey thoxy)-phenyl]'-piperazin-1-~rl]~-methanoy~-2-~4-iodo-2-methyl-phenylamino)-benzoic acid 3.4-Difluoro-5-[1-(2-hydroxy-ethyl)-imidazolidin-2-ylidene-hvdrazinocarbonyl]-2-(4-iodo-2-methyl-phenylamino)-benzoic acid 3.4-Difluoro-2~4-iodo-2-meth-phenvlamino -L5-(1-(4 j3-(propane-1-sulfon~)-phenyl]-piperazin-1-~I}-methanoyl)i-benzoic acid O OH CH
HsC~S~ ~ N Ws N
O vN I ~ I ~ I
'F
O F
WO 01/68619 -9~- PCT/USO1/07816 C2$ H28 F2 I N3 05 S , MS (APCI)m+1 = 684 3.4-Difluoro-2~(4-iodo-2-methyl-phenvlamino)-5-.(1-[4 ~3-methanesulfonyl-~henyl)-piperazin-1-yl]-methanoyl~~-benzoic acid 3.4-Difluoro-2-(4-iodo-2-methyl-phenylamino)-5-(1-~[4-(2 (propane-1-sulfonylL
phenyl]-piperazin-1-XI -methanol)-benzoic acid 5-~(1-(4-(4.5-Dimethyl-thiazol-2-yl~~ piperazin-1-yl~ methanoy~-3.4-difluoro-2-(4-iodo-2-methyl-phenylamino)-benzoic acid 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 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
\ N~ \ N \
O ~N I / F I / I
O F
C24 H2o F2 I N3 05 , MS (APCI)m+1 = 596 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 3.4-Difluoro-2-(4-iodo-2-methyl-phenvlamino)i-5-{1-[4-(3-phosphono-propyl)-~ir~erazin-1-yll-methanoy~-benzoic acid 3.4-Difluoro-5-{1-f~2-hydroxv-ethyl)-piperazin-1-yl]-methanoyl)~-2-(4-iodo-2-meth phenylamino)-benzoic acid 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 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 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 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
4.5-Difluoro-N -~( 1-hydroxvmeth~rl-cyclopentylZ 6-(4-iodo-2-methyl-phenylamino)-isophthalamic acid 4.5-Difluoro-N- -(4-hydroxy-cyclohexyl)-6-L4-iodo-2-methyl-phenylaminol isophthalamic acid 4.5-Difluoro-N -(SR)-2-hvdroxy-cyclohex~)i-6-(4-iodo-2-methyl-phenylamino)-isophthalamic acid 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 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 N (1.1-Dioxo-tetrahydro-1 16-thiophen-3-yIJ~-4,5-difluoro-6-(4-iodo-2-methvl-phenylamino)-isophthalamic acid Biological Examples 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.
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.
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.
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 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.
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.
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.
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.
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 WO 01/68619 -9$- PCT/USO1/07816 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.
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 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.
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.
WO 01/68619 -1 ~~- PCT/USO1/07816 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.
Claims (32)
1. A compound of formula I or formula II:
wherein R1 is hydrogen, C1-8 alkyl, C1-8 alkoxy, halo, C1-2 haloalkyl, or CN;
R3 and R4 are each independently hydrogen, halo, C1-2 haloalkyl, C1-8 alkyl, C1-8 alkoxy, nitro, CN, or (O or NH)k-(CH2)j-R9, where R9 is hydrogen, hydroxy, CO2H or NR10R11;
j is 0 to 4;
k is 0 or 1;
R10 and R11 are each independently hydrogen or C1-8 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-C1-8 alkyl;
A is hydroxy, C1-6 alkoxy, or NR6OR7;
R6 is hydrogen, C1-8 alkyl, (CO)-C1-8 alkyl, phenyl, naphthyl, phenyl(C1-8 alkyl), or C3-10 cycloalkyl;
R7 is hydrogen, C1-8 alkyl, C2-8 alkenyl, C2-8 alkynyl, C3-10 cycloalkyl or 03-10 cycloalkyl optionally containing a heteroatom selected from the group consisting of O, S, and NR9;
X is OR12, NR13R12, or NR14;
R12 and R13 are each independently hydrogen, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C4-6 cycloalkyl, [(CH2)n Y(CH2)m]q CH3, phenyl, naphthyl, (C1-6 alkyl)phenyl, -[(CH2)n Y(CH2)m]q phenyl, C2-6 heteroaryl, (C1-6 alkyl)C2-6 heterocyclic radical, or [(CH2)n Y(CH2)m]q C2-6 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, SO2, 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 NR a R b;
wherein R a and R b are each independently selected from the group consisting of hydrogen and C1-6 alkyl; and the pharmaceutically acceptable salts thereof.
wherein R1 is hydrogen, C1-8 alkyl, C1-8 alkoxy, halo, C1-2 haloalkyl, or CN;
R3 and R4 are each independently hydrogen, halo, C1-2 haloalkyl, C1-8 alkyl, C1-8 alkoxy, nitro, CN, or (O or NH)k-(CH2)j-R9, where R9 is hydrogen, hydroxy, CO2H or NR10R11;
j is 0 to 4;
k is 0 or 1;
R10 and R11 are each independently hydrogen or C1-8 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-C1-8 alkyl;
A is hydroxy, C1-6 alkoxy, or NR6OR7;
R6 is hydrogen, C1-8 alkyl, (CO)-C1-8 alkyl, phenyl, naphthyl, phenyl(C1-8 alkyl), or C3-10 cycloalkyl;
R7 is hydrogen, C1-8 alkyl, C2-8 alkenyl, C2-8 alkynyl, C3-10 cycloalkyl or 03-10 cycloalkyl optionally containing a heteroatom selected from the group consisting of O, S, and NR9;
X is OR12, NR13R12, or NR14;
R12 and R13 are each independently hydrogen, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C4-6 cycloalkyl, [(CH2)n Y(CH2)m]q CH3, phenyl, naphthyl, (C1-6 alkyl)phenyl, -[(CH2)n Y(CH2)m]q phenyl, C2-6 heteroaryl, (C1-6 alkyl)C2-6 heterocyclic radical, or [(CH2)n Y(CH2)m]q C2-6 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, SO2, 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 NR a R b;
wherein R a and R b are each independently selected from the group consisting of hydrogen and C1-6 alkyl; and the pharmaceutically acceptable salts thereof.
2. A compound of claim 1 wherein R1 is C1-8 alkyl or halo.
3. A compound of claim 2 wherein R1 is methyl.
4. A compound of claim 1 wherein R3 and R4 are each independently selected from the group consisting of hydrogen and halo.
5. A compound of claim 4 wherein halo is fluoro.
6. A compound of claim 1 wherein A is NR6OR7.
7. A compound of claim 1 wherein X is NR13R12.
8. A compound of claim 1 wherein X is NR14,
9. A compound of claim 1 wherein R12 and R13 are each independently selected from the group consisting of [(CH2)n Y(CH2)m]q CH3, (C1-6 alkyl)phenyl, -[(CH2)n Y(CH2)m]q phenyl, and (C1-6 alkyl)C2-6 heterocyclic radical.
10. A compound of claim 1 wherein the heterocyclic radical is a heteroaryl selected from the group consisting of a substituted or unsubstituted radical of pyrrole, furan, pyran, thiophene, pyrazole, imidazole, triazole, tetrazole, indole, isoxazole, indazole, pyridine, pyrazine, oxazole, thiazole, oxadiazole, and oxathiadiazole.
11. A compound of claim 1 wherein the heterocyclic radical is a heteroalkyl selected from the group consisting of a substituted or unsubstituted radical of morpholine, piperidine, piperazine, tetrahydrofuran, tetrahydropyran, pyrrolidone, imidazoline, and tetrahydrothiophene.
12. A pharmaceutical composition comprising a compound of claim 1 and a pharmaceutically acceptable carrier.
13. A method of treating a proliferative disease in a patient in need thereof comprising administering a therapeutically effective amount of a compound of claim 1.
14. A method of claim 13 wherein the proliferative disease is selected from the group consisting of cancer, restenosis, psoriasis, autoimmune disease, and atherosclerosis.
15. A method of treating psoriasis in a patient in need thereof comprising administering a therapeutically effective amount of a compound of claim 1.
16. A method of treating cancer in a patient in need thereof comprising administering a therapeutically effective amount of a compound of claim 1.
17. A method of claim 16 wherein the cancer is MEK-related.
18. A method of claim 16 wherein the cancer is brain, breast, lung, ovarian, pancreatic, prostate, renal, or colorectal cancer.
19. A method of treating osteoarthritis in a patient in need thereof comprising administering a therapeutically effective amount of a compound of claim 1.
20. A method of treating rheumatoid arthritis in a patient in need thereof comprising administering a therapeutically effective amount of a compound of claim 1.
21. A method of treating heart failure in a patient in need thereof comprising administering a therapeutically effective amount of a compound of claim 1.
22. A method of treating chronic pain in a patient in need thereof comprising administering a therapeutically effective amount of a compound of claim 1.
23. The method of claim 22, wherein the chronic pain is selected from the group consisting of neuropathic pain, idiopathic pain, and pain associated with chronic alcoholism, vitamin deficiency, uremia, and hypothyroidism.
24. The method of claim 22, wherein the chronic pain is associated with inflammation.
25. The method of claim 22, wherein the chronic pain is associated with arthritis.
26. The method of claim 22, wherein the chronic pain is associated with post-operative pain.
27. A method of treating neuropathic pain in a patient in need thereof comprising administering a therapeutically effective amount of a compound of claim 1.
28. The method of claim 27, wherein the neuropathic pain is associated with a condition selected from the group consisting of 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.
29. A method for treating cancer in a patient in need thereof comprising administering a therapeutically effective amount of a compound of claim 1 in combination with radiation therapy.
30. A method for treating cancer in a patient in need thereof comprising administering a therapeutically effective amount of a compound of claim 1 in combination with at least one chemotherapeutic agent.
31. A method of claim 30 wherein the chemotherapeutic agent is a mitotic inhibitor.
32. A method of claim 30 wherein the mitotic inhibitor is selected from the group consisting of paclitaxel, docetaxel, vincristine, vinblastine, vinorelbine, and vinflunine.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US18971400P | 2000-03-15 | 2000-03-15 | |
| US60/189,714 | 2000-03-15 | ||
| PCT/US2001/007816 WO2001068619A1 (en) | 2000-03-15 | 2001-03-12 | 5-amide substituted diarylamines as mex inhibitors |
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| Publication Number | Publication Date |
|---|---|
| CA2403017A1 true CA2403017A1 (en) | 2001-09-20 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002403017A Abandoned CA2403017A1 (en) | 2000-03-15 | 2001-03-12 | 5-amide substituted diarylamines as mex inhibitors |
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| Country | Link |
|---|---|
| JP (1) | JP2003527379A (en) |
| AU (1) | AU2001247372A1 (en) |
| BR (1) | BR0109188A (en) |
| CA (1) | CA2403017A1 (en) |
| MX (1) | MXPA02008103A (en) |
| WO (1) | WO2001068619A1 (en) |
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| WO1998037881A1 (en) * | 1997-02-28 | 1998-09-03 | Warner Lambert Company | Method of treating or preventing septic shock by administering a mek inhibitor |
| IL132840A (en) * | 1997-07-01 | 2004-12-15 | Warner Lambert Co | 4-bromo or 4-iodo phenylamino benzhydroxamic acid derivatives and pharmaceutical compositions comprising them for use as mek inhibitors |
| KR20010014360A (en) * | 1997-07-01 | 2001-02-26 | 로즈 암스트롱, 크리스틴 에이. 트러트웨인 | 2-(4-bromo or 4-iodo phenylamino)benzoic acid derivatives and their use as mek inhibitors |
| CA2349467A1 (en) * | 1999-01-13 | 2000-07-20 | Warner-Lambert Company | Sulphohydroxamic acids and sulphohydroxamates and their use as mek inhibitors |
| DE69926914T2 (en) * | 1999-01-13 | 2006-06-29 | Warner-Lambert Co. Llc | 1-HETEROCYCLE SUBSTITUTED DIARYLAMINES |
-
2001
- 2001-03-12 JP JP2001567711A patent/JP2003527379A/en active Pending
- 2001-03-12 MX MXPA02008103A patent/MXPA02008103A/en not_active Application Discontinuation
- 2001-03-12 BR BR0109188-3A patent/BR0109188A/en not_active IP Right Cessation
- 2001-03-12 AU AU2001247372A patent/AU2001247372A1/en not_active Abandoned
- 2001-03-12 WO PCT/US2001/007816 patent/WO2001068619A1/en not_active Application Discontinuation
- 2001-03-12 CA CA002403017A patent/CA2403017A1/en not_active Abandoned
Also Published As
| Publication number | Publication date |
|---|---|
| JP2003527379A (en) | 2003-09-16 |
| BR0109188A (en) | 2003-03-18 |
| WO2001068619A1 (en) | 2001-09-20 |
| AU2001247372A1 (en) | 2001-09-24 |
| MXPA02008103A (en) | 2002-11-29 |
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