WO2005007616A1 - Diphenylamino ketone derivatives as mek inhibitors - Google Patents

Diphenylamino ketone derivatives as mek inhibitors Download PDF

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WO2005007616A1
WO2005007616A1 PCT/IB2004/002281 IB2004002281W WO2005007616A1 WO 2005007616 A1 WO2005007616 A1 WO 2005007616A1 IB 2004002281 W IB2004002281 W IB 2004002281W WO 2005007616 A1 WO2005007616 A1 WO 2005007616A1
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fluoro
difluoro
phenylamino
compound
alkyl
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PCT/IB2004/002281
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French (fr)
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Stephen Douglas Barrett
Michael David Kaufman
Mark Stephen Plummer
Jessica Elizabeth Reed
Bruce David Roth
Kevin Matthew Schlosser
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Warner-Lambert Company Llc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D277/00—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings
    • C07D277/02—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings
    • C07D277/20—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
    • C07D277/22—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to ring carbon atoms
    • C07D277/24—Radicals substituted by oxygen atoms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/06—Antipsoriatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C225/00—Compounds containing amino groups and doubly—bound oxygen atoms bound to the same carbon skeleton, at least one of the doubly—bound oxygen atoms not being part of a —CHO group, e.g. amino ketones
    • C07C225/22—Compounds containing amino groups and doubly—bound oxygen atoms bound to the same carbon skeleton, at least one of the doubly—bound oxygen atoms not being part of a —CHO group, e.g. amino ketones having amino groups bound to carbon atoms of six-membered aromatic rings of the carbon skeleton
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/24—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with substituted hydrocarbon radicals attached to ring carbon atoms
    • C07D213/44—Radicals substituted by doubly-bound oxygen, sulfur, or nitrogen atoms, or by two such atoms singly-bound to the same carbon atom
    • C07D213/46—Oxygen atoms
    • C07D213/50—Ketonic radicals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D233/00—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings
    • C07D233/54—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members
    • C07D233/64—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members with substituted hydrocarbon radicals attached to ring carbon atoms, e.g. histidine
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D263/00—Heterocyclic compounds containing 1,3-oxazole or hydrogenated 1,3-oxazole rings
    • C07D263/02—Heterocyclic compounds containing 1,3-oxazole or hydrogenated 1,3-oxazole rings not condensed with other rings
    • C07D263/30—Heterocyclic compounds containing 1,3-oxazole or hydrogenated 1,3-oxazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
    • C07D263/32—Heterocyclic compounds containing 1,3-oxazole or hydrogenated 1,3-oxazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to ring carbon atoms

Definitions

  • the present invention relates to diphenylamino ketone derivatives, pharmaceutical compositions and methods of use thereof.
  • MAPK/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.
  • 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., MEK ⁇ and MEK2) which then activates the MAP kinase, ERK (ERK-
  • MAP kinase 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.
  • Ras is not itself a protein kinase, it participates in the activation of Raf and other kinases, most likely through a phosphorylation mechanism.
  • Raf and other kinases phosphorylate MEK on two closely adjacent serine residues, S 218 and S 222 j n the case of MEK-1 , which are the prerequisite for activation of MEK as a kinase.
  • MEK in turn phosphorylates MAP kinase on both a tyrosine,
  • MAP kinase 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 kinaes.
  • Many of these MAP kinase phosphorylations are mitogenically activating for the target protein, such as a kinase, a transcription factor, or another cellular protein.
  • target protein such as a kinase, a transcription factor, or another cellular protein.
  • other kinases activate MEK, and MEK itself appears to be a signal integrating kinase.
  • MEK is highly specific for the phosphorylation of MAP kinase.
  • 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.
  • the present invention provides a compound of formula
  • Q is C ⁇ _ 6 alkyl, C 2 . 6 alkenyl, C 2 . 6 alkynyl, or C 2 -5 heteroaryl, wherein the C-
  • Ri and R 5 are each independently hydrogen or fluorine;
  • R 2 is hydrogen, chlorine, fluorine or methyl;
  • Ri is bromine, chlorine, fluorine, iodine, C ⁇ e alkyl, C 2 - 6 alkenyl, C 2 - 6 alkynyl, C 3 . 6 cycloalkyl, -(CH 2 )-C 3 . 6 cycloalkyl, cyano, -0-(C ⁇ alkyl), -S-(C 1 .
  • 6 alkynyl are optionally substituted with between 1 and 3 substituents independently selected from hydroxy and alkyl;
  • R 6 and R 7 are each independently hydrogen, methyl, or ethyl;
  • m is 1 to 4;
  • n is 1 to 2;
  • p is 0 to 2;
  • t is O to 1;
  • v is 1 to 5; and pharmaceutically acceptable salts, C ⁇ _ 6 amides and C ⁇ - 6 esters thereof.
  • An embodiment of the present invention provide a compound of formula I, as defined above, and pharmaceutically acceptable salts thereof.
  • compounds of Formula I wherein: Q is C 1 - 6 alkyl, C 2 . 6 alkenyl, or C 2 .
  • heteroaryl is optionally substituted with C M alkyl, and further wherein the C ⁇ alkyl is optionally substituted with between 1 and 3 substituents independently selected from hydroxy and amino; or Q is methyl, -CH 2 OH, -(CH 2 ) 4 OH, butenyl, methyl-substituted oxazolyl, thiazolyl, methyl-substituted imidazolyl, or pyridinyl.
  • the present invention also provides compounds of Formula I wherein Ri is fluorine; R 2 is fluorine; R 5 is fluorine; or R, and R 5 are fluorine. Additionally, the present invention provides compounds of Formula I, wherein R is: iodine, C ⁇ .
  • 3 alkynyl are optionally substituted with hydroxy; iodine; C- ⁇ - 4 alkyl, C 2 _ 3 alkenyl, or C M alkynyl, and further wherein the C-M alkyl, C M alkenyl, and C 2 . 3 alkynyl are optionally substituted with hydroxy; or iodine, ethyl, -CH 2 OH, ethenyl, or acetyl.
  • the invention also provides a pharmaceutical composition comprising a compound of
  • 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. Furthermore, the invention provides methods of treating cancer, restenosis, psoriasis, autoimmune disease, atherosclerosis, osteoarthritis, rheumatoid arthritis, heart failure, chronic p ⁇ in, and neuropathic pain in a patient in need thereof comprising administering a therapeutically effective amount of a compound of Formula I. 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 in combination with radiation therapy or at least one chemotherapeutic agent.
  • halogen or halo in the present invention refer to a fluorine, bromine, chlorine, and iodine atom or fluoro, bromo, chloro, and iodo.
  • fluorine and fluoro for example, are understood to be equivalent herein.
  • Alkyl groups such as "C ⁇ - ⁇ 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, isobutyl, sec-butyl, t-butyl, pentyl, 2-pentyl,
  • C ⁇ alkyl includes within its definition the terms "C M alkyl” and "C ⁇ - 2 alkyl”.
  • Alkenyl groups such as C 2 . 6 alkenyl, 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
  • E Electrode
  • Z Visual
  • alkynyl groups such as C 2 . 6 alkynyl, 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. Examples of alkenyls and alkynyls include vinyl, allyl, 2-methyl-2-propenyl, cis-2-butenyl, trans-2-butenyl, and acetyl. The term
  • C 2 . 6 alkenyl includes within its definition the term “C 2 . 3 alkenyl” and the term “C 2 . 6 alkynyl” includes within its definition the term “C 2 . 3 alkynyl”.
  • Cycloalkyl groups such as C 3 . 6 cycloalkyl, refer to a saturated hydrocarbon ring structure containing from 3 to 6 atoms. Typical C 3 . 6 cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like.
  • heteroaryl or “C 2 _ 5 heteroaryl” as used herein, unless otherwise indicated, includes monocyclic aromatic heterocycles containing five or six ring members, of which from
  • 1 to 4 can be heteroatoms selected, independently, from N, S and O, and bicyclic aromatic heterocycles containing from eight to twelve ring members, of which from 1 to 4 can be heteroatoms selected, independently, from N, S and O.
  • Heterocyclic radicals which include but are not limited to heteroaryls, include: furyl, (is)oxazolyl, isoxazolyl, thiophenyl, thiazolyl, pyrrolyl, imidazolyl, 1 ,3,4-triazolyl, tetrazolyl, pyridinyl, pyrimidinyl, pyridazinyl, indolyl, and their nonaromatic counterparts.
  • heterocyclic radicals include thienyl, piperidyl, quinolyl, isothiazolyl, piperidinyl, morpholinyl, piperazinyl, tetrahydrofuryl, tetrahydropyrrolyl, pyrrolidinyl, octahydroindolyl, octahydrobenzothiofuranyl, octahydrobenzofuranyl, (iso)quinolinyl, naphthyridinyl, benzimidazolyl, and benzoxazolyl.
  • the present invention includes the hydrates and the pharmaceutically acceptable salts and solvates of the compounds defined by Formula I.
  • the compounds of this invention can possess a sufficiently basic functional group, and accordingly react with any of a number of inorganic and organic acids, to form a pharmaceutically acceptable salt.
  • pharmaceutically acceptable salt refers to salts of the compounds of Formula I which are substantially non-toxic to living organisms.
  • Typical pharmaceutically acceptable salts include those salts prepared by reaction of the compounds of the present invention with a pharmaceutically acceptable mineral or organic acid. Such salts are also known as acid addition salts.
  • Such salts include the pharmaceutically acceptable salts listed in Journal of Pharmaceutical Science, 1977;66:2-19, which are known to the skilled artisan.
  • Acids commonly employed to form acid addition salts are inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, and the like, and organic acids such as p-toluenesulfonic, methanesulfonic acid, benzenesulfonic acid, oxalic acid, p-bromophenylsulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid, acetic acid, and the like.
  • inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, and the like
  • organic acids such as p-toluenesulfonic, methanesulfonic acid, benzenesulfonic acid, oxalic acid, p-bromophenylsulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid, acetic acid, and
  • Example of such pharmaceutically acceptable salts are the sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, bromide, hydrobromide, iodide, acetate, propionate, decanoate, caprate, caprylate, acrylate, ascorbate, formate, hydrochloride, dihydrochloride, isobutyrate, caproate, heptanoate, propiolate, glucuronate, glutamate, propionate, phenylpropionate, salicylate, oxalate, malonate, succinate, suberate, sebacate, fumarate, malate, maleate, hydroxymateate, mandelate, mesylate, nicotinate, isonicotinate, cinnamate, hippurate, nitrate, stearate, phthalate,
  • a preferred pharmaceutically acceptable salt is hydrochloride. It should be recognized that the particular counterion forming a part of any salt of this inventions is usually not of a critical nature, so long as the salt as a whole is pharmacologically acceptable and as long as the counterion does not contribute undesired qualities to the salt as a whole. It is further understood that such salts may exist as a hydrate.
  • the enantiomers of compounds of the present invention can be resolved by one of ordinary skill in the art using standard techniques well-known in the art, such as those described by J. Jacques, et al., “Enantiomers, Racemates, and Resolutions", John Wiley and Sons, Inc 1981. Examples of resolutions include recrystallization techniques or chiral chromatography.
  • the compounds of the present invention have one or more chiral centers and may exist in a variety of stereoisomeric configurations. As a consequence of these chiral centers, the compounds of the present invention occur as racemates, mixtures of enantiomers and as individual enantiomers, as well as diastereomers and mixtures of diastereomers. All such racemates, enantiomers, and diastereomers are within the scope of the present invention.
  • the compounds of Formula I 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 below. These schemes are not intended to limit the scope of the invention in any way.
  • a suitable benzoic acid (1) is coupled with a suitable aniline (2) to provide a 2-(arylamino)-benzoic acid or diphenylamine (3).
  • the aniline (2) and the benzoic acid (1 ) are dissolved in a suitable organic solvent with an acid catalyst and heated at reflux for several hours.
  • Preferred solvents are polar solvents such as ethanol, and preferred acid catalysts are mineral acids such as concentrated HCI.
  • the reaction is typically complete within about 12 to 36 hours.
  • the product (3) is typically isolated by filtration after cooling of the reaction mixture, and further purified, if desired, by standard methods such as chromatography or crystallization.
  • an acid chloride (4) is prepared from 2-(arylamino)-benzoic acid (3) by reacting the benzoic acid (3) with oxalyl chloride in a suitable solvent, such as DMF.
  • a suitable solvent such as DMF.
  • Step C nucleophilic addition to an acid chloride (4), followed by deprotection and decarboxylation provides a compound of formula I.
  • an acid chloride (4) is combined with tris(trimethylsiloxy)ethylene and stirred under nitrogen.
  • dioxane and hydrochloric acid are added to the mixture and stirred to produce a compound of formula I.
  • the product can be isolated by filtration and removing the solvent, for example by evaporation under reduced pressure, and further purified, if desired, by standard methods such as chromatography, crystallization, or distillation.
  • Step A the benzoic acid (3) is converted to the benzaldehyde (5).
  • the benzoic acid (3) is reduced with lithium aluminum hydride in a suitable solvent, such as tetrahydrofuran providing an intermediate alcohol which is then oxidized with, for example, manganese dioxide in chloroform providing benzaldehyde (5).
  • a suitable solvent such as tetrahydrofuran
  • the Wittig reaction is followed by dihydroxylation to provide compound (7).
  • the dihydroxylation is performed using a stoichiometric amount of 4-methyl-morpholine 4-oxide and a catalytic amount of Os0 .
  • Step D compound (7) is protected under procedures generally known in the art to provide the protected compound (8).
  • Step E the protected compound (8) is oxidized according to procedures known in the art followed by a standard deprotection in Step F to provide a compound of formula la.
  • R 4 is halogen
  • R 4 is not halogen
  • the compounds of formula I, wherein R 4 is not halogen are prepared from the compounds of formula I wherein R is halogen, by transition metal-promoted coupling with reagent M-R 4 wherein R 4 is non-halogen (12) in a suitable solvent or solvents such as triethylamine, tetrahydrofuran or dimethylformamide.
  • the transition metal-promoted coupling may be carried out with a palladium(O) or palladium (II) coupling agent, such as (Ph 3 P) Pd or (Ph 3 P) 2 PdCI 2 .
  • the entire mixture is stirred from about 2 to 24 hours at room temperature.
  • M is defined as a functional group known to transfer a carbon radical fragment in transition metal-promoted coupling processes.
  • a suitable M group include trialkylstannyl, trialkylsilyl, trimethylsilyl, zinc, tin, copper, boron, magnesium and lithium.
  • Preferred halogens, when R 4 is halogen are bromine and iodine.
  • the resulting compound of formula l, as well as the protected Formula I compound can be isolated by removing the solvent, for example by evaporation under reduced pressure, and further purified, if desired, by standard methods such as chromatography, crystallization, or distillation. It would be understood by one of skill in the art that the substituent R 4 , when R is non-halogen, may be further transformed, such as by oxidation, reduction, deprotection, or hydrogenation.
  • a compound wherein R is C 2 - 4 alkenyl may be transformed to a compound wherein R 4 is hydroxy-substituted alkyl by treating the double bond of the alkene with ozone and NaBH 4 .
  • a compound wherein R is C M alkenyl may be transformed to a compound wherein R is alkyl substituted with 2 hydroxy substituents by treating the double bond of the alkene with Os0 4 .
  • a compound wherein R 4 is an alkene or alkyne derivative may be reduced under conditions known in the art, such as through hydrogenation, such as with Pd/C under an atmosphere of hydrogen.
  • the alkyne derivative is dissolved in a suitable solvent, such as absolute ethanol, in the presence of a metal catalyst, such as palladium on carbon. This mixture is stirred under an atmosphere of hydrogen from about 1 to 24 hours at room temperature to provide the fully saturated derivative.
  • the alkyne derivative is partially reduced via hydrogenation to provide the alkene derivative.
  • the alkyne derivative is dissolved in a suitable solvent, such as tetrahydrofuran, in the presence of a catalyst, such as Lindlar catalyst or palladium on carbon and, if desired, a suitable compound which disrupts the actions of the catalyst, such as quinoline or pyridine.
  • a catalyst such as Lindlar catalyst or palladium on carbon
  • a suitable compound which disrupts the actions of the catalyst such as quinoline or pyridine.
  • This mixture is stirred under an atmosphere of hydrogen from about 1 to 24 hours at room temperature to provide the alkene derivative.
  • the substituent R 4 may also be transformed into a different R 4 through standard synthetic procedures known to one of skill in the art.
  • R 4 may be transformed before the coupling of the ester (1 ) and aniline (2) as shown in Scheme 1 , Step A. Further transformations of R 4 are shown in Scheme 5 below.
  • R-l 2 is NR 6 R 7 or OR 6
  • step A the compound of formula Ig is dissolved in a suitable solvent such as tetrahydrofuran and reacted with methanesulfonyl chloride to give the intermediate mesylate, then Nal in EtOAc to give the iodide compound (13).
  • step B the iodide compound (13) is reacted with a suitable amine, such as methylamine or dimethylamine, or a suitable alkoxide to give compounds of formula Ih.
  • a suitable amine such as methylamine or dimethylamine
  • an aniline (2) may be prepared to include the desired R 4 .
  • aniline (2) can be prepared by techniques and procedures readily available to one of ordinary skill in the art and by following the procedures as set forth in the following Schemes, or analogous variants thereof. Additionally, anilines (2) are taught in USSN 10/349,801 filed January 23, 2003 and USSN 10/349,826 filed January 23, 2003, the disclosure of which is hereby incorporated by reference. These Schemes are not intended to limit the scope of the invention in any way.
  • Step A a suitable amine or alkoxide (14) is reacted with a 4-tert- butoxycarbonylamino-3-substituted-benzyl bromide (13), such as 4-tert-butoxycarbonylamino- 3-fluorobenzyI bromide (J. Med. Chem., 2000/43:5017).
  • Step B the BOC protecting group of compound of structure (15) is hydrolized with, for example, TFA, to provide the desired aniline (2a).
  • the desired aniline (2e) wherein R 2 is methyl, fluorine or chlorine, using compound (21 ) as the starting material can be prepared.
  • R 2 is fluorine
  • the sulfonyl chloride derivative (21 ) is a compound known in the literature (German Patent DE 2630060, 1978).
  • the sulfonyl chloride derivative (21) is also known in the literature (German Patent. DE 2750170, 1978).
  • the sulfonyl chloride derivative (21 ) where R 2 is chlorine is commercially available.
  • one of ordinary skill in the art would appreciate that there are numerous ways of acetylating anilines.
  • Compounds of the present invention include, but are not limited to the following compounds: 1-[3,4-Difluoro-2-(2-fluoro-4-iodo-phenylamino)-phenyl]-2-hydroxy-ethanone; [2-(4-Ethynyl-2-fluoro-phenylamino)-3,4-difluoro-phenyl]-pyridin-2-yl-methanone; [2-(4-Ethynyl-2-fluoro-phenylamino)-3,4-difluoro-phenyl]-thiazol-2-yl-methanone; [2-(4-Ethynyl-2-fluoro-phenylamino)-3,4-difluoro-phenyl]-(1-methyl-1 H-imidazol-2-yl)- methanone; [
  • 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.
  • treating for purposes of the present invention refers to treatment, prophylaxis or prevention, amelioration or elimination of a named condition once the condition has been established.
  • Selective MEK 1 or MEK 2 inhibitors are those compounds which inhibit the MEK 1 or
  • a selective MEK 1 or MEK 2 inhibitor has an IC 50 for MEK 1 or MEK 2 that is at least one- fiftieth (1/50) that of its IC 50 for one of the above-named other enzymes.
  • a selective inhibitor has an IC 50 that is at least 1/100, more preferably 1/500, and even more preferably 1/1000, 1/5000, or less than that of its IC 5Q or one or more of the above-named enzymes.
  • 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.
  • diseases or conditions modulated by the MEK cascade 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.
  • 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.
  • 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),
  • 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).
  • B hepatitis virus
  • HPV human papilloma virus
  • CMV cytomegalovirus
  • EBV Epstein-Barr virus
  • 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.
  • 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, 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.
  • 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.
  • 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, 200, 300, or 400 mg can be administered according to the disclosed methods.
  • compositions 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 Formula I and a pharmaceutically acceptable carrier.
  • the active ingredient such as a compound of Formula I
  • the 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.
  • 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 Q) 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.
  • 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
  • absorption-enhancing agents are readily available to one of ordinary skill in the art.
  • Step B Preparation of 3,4-difluoro-2-[r2-fluoro-4(trimethylsilylethvnyl)phenvn aminolbenzoic acid
  • Step C Preparation of 3.4-difluoro-2-r(4-ethvnyl-2-fluorophenyl)amino1benzoic acid
  • 3,4-difluoro-2-[[2-fluoro-4-(trimethylsilylethynyl) phenyljaminojbenzoic acid (3.99 g, 11.0 mmol) was dissolved in MeOH (200 mL), to which was added K 2 C0 3 (3.03 g, 22.0 mmol). This mixture was stirred at room temperature for 15 hours, then the reaction solvent removed under reduced pressure.
  • the reaction mixture was stirred for ten minutes and was concentrated in vacuo to the yellow solid 3,4-difluoro-2-(2- fluoro-4-iodo-phenylamino)-benzoyl chloride.
  • the yellow solid was suspended in tris(trimethylsiloxy)ethylene (10 g, 33 mmol) and the stirring mixture was brought to 90 °C under a nitrogen atmosphere for six hours.
  • the mixture was cooled slightly, and to it was added a solution consisting of dioxane (25 mL) and 10% aqueous hydrochloric acid (10 mL). Vigorous liberation of gas ensued and the mixture was stirred for ten minutes at 85 °C.
  • the product of of preparation 1 (2.2 g, 7.6 mmol) was dissolved in dichloromethane (30 mL) and N-methylmorpholine (3.5 mL). To the resultant solution was added N,0- dimethylhydroxylamine hydrochloride (0.95 g, 9.7 mmol) and PyBOP (3.9 g, 7.5 mmol). The resultant reaction mixture was stirred for 2 h at ambient temperature. An additional portion of PyBOP (1.3 g, 2.5 mmol) was added and stirring was continued. After 2 h, the reaction was diluted with ethyl acetate (100 mL) and washed with water (100 mL x 3) and saturated brine (100 mL x 3).
  • Step B Preparation of .2-(4-ethvnyl-2-fluoro-phenylamino)-3.4-difluoro-phenv ⁇ - thiazol-2-yl-methanone A solution of thiazole (0.148 mL, 2.08 mmol) in tetrahydrofuran (10 mL) was cooled to
  • the resultant reaction mixture was stirred 15 min at -78 °C and 2 h at ambient temperature and was then transferred to a -78 °C solution of the product of preparation 1 , 2-(4-ethynyl-2- fluoro-phenylamino)-3,4-difluoro-benzoic acid (200 mg, 0.69 mmol) in tetrahydrofuran (5 mL).
  • the resultant reaction mixture was stirred 30 min at -78 °C and was allowed to warm to ambient temperature overnight.
  • the reaction was partitioned between water (20 mL) and ethyl acetate (30 mL).
  • Step A Synthesis of 3,4-Difluoro-2-(2-fluoro-4-iodo-phenylamino)-benzoyl chloride A solution of 3,4-Difluoro-2-(2-fluoro-4-iodo-phenylamino)-benzoic acid (5.0 g, 12.7 mmol) in thionyl chloride (75 mL) containing 10 drops of dimethylformamide was heated at reflux for 15 min.
  • Step B Synthesis of [3,4-Difluoro-2-(2-fluoro-4-iodo-phenylaminoVphenylH4-methyl- oxazol-2-yl)-methanone .
  • Step A Synthesis of f3.4-Difluoro-2-(2-fluoro-4-iodod-phenylamino)-phenyl]- methanol.
  • Step D Synthesis of 1-(3,4-difluoro-2-(2-fluoro-4-iodo-phenylamino)-phenyll-ethane- 1.2-diol.
  • Step F Synthesis of 1-[3.4-difluoro-2-(2-fluoro-4-iodo-phenylamino)-phenyll-2- triisopropylsilanyloxy-ethanone.
  • Step G Synthesis of 1-r3.4-difluoro-2-(2-fluoro-4-vinyl-phenylamino)-phenyll-2- hydroxy-ethanone.
  • Step A Synthesis of 1-r3.4-difluoro-2-(2-fluoro-4-vinyl-phenylamino)-phenv-l-2- triisopropylsilanyloxy-ethanol.
  • Step A Synthesis of 1-(3,4-difluoro-2-r2-fluoro-4-(2-hvdroxy-ethyl)-phenylamino1- phenyll-2-triisopropylsilanyloxy-ethanol.
  • Step B Synthesis of 1-(3.4-difluoro-2-[2-fluoro-4-(2-triisopropylsilanyloxy-ethyl)- phenylaminol-phenyll-2-triisopropylsilanyloxy-ethanol.
  • Step D Synthesis of 1-(3.4-difluoro-2-.2-fluoro-4-(2-hvdroxy-ethv0-phenylaminoy phenyl)-2-hvdroxyl-ethanone.
  • Step A synthesis of 1-f3.4-difluoro-2-(2-fluoro-4-iodo-phenylamino)- ⁇ henv ⁇ -ethanol.
  • Step A Preparation of 1-r3,4-Difluoro-2-(2-fluoro-4-trimethylsilanylethynyl- phenylaminoVphenvn-ethanone 3,4-Difluoro-2-(2-fluoro-4-trimethylsilanylethynyl-phenylamino)-N-methoxy-N-methyl- benzamide (1 g, 2.46 mmol) was dissolved in tetrahydrofuran (12 mL). Methylmagnesium bromide (4.01 mL, 1.4M in toluene/THF, 5.61 mmol) was added and the reaction stirred at room temperature for 1 hour.
  • Step B Preparation of 1-r2-(4-Ethvnyl-2-fluoro-phenylamino)-3.4-difluoro-phenyll- ethanone 1-[3,4-Difluoro-2-(2-fluoro-4-trimethylsilanylethynyl-phenylamino)-phenyl]-ethanone (0.89g, 2.46 mmol) was dissolved in tetrahydrofuran (10 mL).
  • Step A Preparation of 2-(4-Ethyl-2-fluoro-phenylamino)-3.4-difluoro-N-methoxy-N- methyl-benzamide 2-(4-Ethynyl-2-fluoro-phenylamino)-3,4-difluoro-N-methoxy-N-methyl-benzamide (4.11g, 12.2 mmol) and 10% Pd/C (1.01g) were taken up in tetrahydrofuran (100 mL) and stirred under and atmosphere of hydrogen for 16 hours.
  • Step B Preparation of 1-r2-(4-Ethyl-2-fluoro-phenylamino)-3.4-difluoro-phenvn-pent- 4-en-1-one 2-(4-Ethyl-2-fluoro-phenylamino)-3,4-difluoro-N-methoxy-N-methyl-benzamide (1.5g, 4.43 mmol) was dissolved in THF. 3-Butenylmagnesium chloride (23.3 mL, 11.7 mmol) was added and the reaction stirred at room temperature for 30 minutes. Water was added and the mixture was extracted with ethyl acetate, dried with MgS0 4 , and concentrated.
  • Step C Preparation of 1-r2-(4-Ethyl-2-fluoro-phenylamino)-3.4-difluoro-phenyll-5- hydroxy-pentan-1 -one
  • 1-[2-(4-Ethyl-2-fluoro-phenylamino)-3,4-difluoro-phenyl]-pent-4-en-1-one (0.83g, 2.49 mmol) was cooled to 0°C.
  • BH 3 -THF (2.12 mL, 1.0 M in THF) was added and the reaction warmed to room temperature over 2 hours.
  • MAP kinase in murine colon 26 (C26) carcinoma cells. Since ERK1 and ERK2 represent the only known substrates for MEKIand MEK2, the measurement of inhibition of ERK phosphorylation in cells provides direct read out of cellular MEK inhibition by the compounds of the invention. Detection of phosphorylation of ERK was carried out either by Western blot or ELISA format. Briefly, the assays involve treatment of exponentially growing C26 cells with varying concentrations of the test compound (or vehicle control) for one hour at 37° C.
  • cells were rinsed free of compound/vehicle and lysed in a solution containing 70 mM NaCI, 50 mM glycerol phosphate, 10 mM HEPES, pH 7.4, 1% Triton X-100, 1 mM Na 3 V0 4 , 100 ⁇ M PMSF, 10 ⁇ M leupeptin and 10 ⁇ M pepstatin. Supernatants were then subjected to gel electrophoresis and hybridized to a primary antibody recognizing dually phosphorylated ERK1 and ERK2.
  • blots were subsequently 'stripped' and re-probed with a 1 :1 mixture of polyclonal antibodies recognizing unphosphorylated ERK1 and ERK2.
  • pERK TiterZyme Enzyme immunometric Assay kits were acquired from Assay Designs, Inc (Ann Arbor, Ml). Briefly, cells were harvested in lysis solution containing 50mM ⁇ -glycerophosphate, 10mM HEPES, pH7.4, 70mM NaCI, 2mM EDTA and 1 %SDS and protein lysates were diluted 1 :15 with supplied Assay buffer prior to the execution of the assay.
  • EXAMPLE 14 Carraqeenan-induced Footpad Edema (CFE) Rat Model Male outbred Wistar rats (135-150 g, Charles River Labs) are dosed orally with 10 mL/kg vehicle or test compound 1 hour prior to administration of a sonicated suspension of carrageenan (1 mg/0.1 mL saline). Carrageenan is injected into the subplantar region of the right hind paw. Paw volume is determined by mercury plethysmography immediately after injection and again five hours after carrageenan injection. Percent inhibition of edema is determined, and the ID40 calculated by linear regression. Differences in swelling compared to control animals are assessed by a 1-way ANOVA, followed by Dunnett's test.
  • Type II collagen-induced arthritis 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 ⁇ g 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).
  • stage 1 erythema and edema
  • stage 2 joint distortion
  • stage 3 joint ankylosis
  • 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.
  • mice and rats describe methods for transplanting split-heart neonatal cardiac grafts into the ear pinna of mice and rats (J. Pharm. and Toxic. Meth., 1998;39:9-17).
  • Compounds are dissolved in solutions containing combinations of absolute ethanol, 0.2% hydroxypropyl methylcellulose in water, propylene glycol, cremophor, and dextrose, or other solvent or suspending vehicle.
  • Mice are dosed orally or intraperitoneally once, twice or three times daily from the day of transplant (Day 0) through Day 13 or until grafts have been rejected.
  • Rats are dosed once, twice, or three times daily from Day 0 through Day 13.
  • Each animal is anesthetized and an incision is made at the base of the recipient ear, cutting only the dorsal epidermis and dermis.
  • the incision is spread open and down to the cartilage parallel to the head, and sufficiently wide to accommodate the appropriate tunneling for a rat or insertion tool for a mouse.
  • a neonatal mouse or rat pup less than 60 hours old is anesthetized and cervically dislocated.
  • the heart is removed from the chest, rinsed with saline, bisected longitudinally with a scalpel, and rinsed with sterile saline.
  • the donor heart fragment is placed into the preformed tunnel with the insertion tool and air or residual fluid is gently expressed from the tunnel with light pressure.
  • Implants are examined at 10- to 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 to 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.
  • a control compound such as cyclosporine, tacrolimus, or orally-administered lefluonomide.
  • EXAMPLE 18 The analgesic activity of the compounds of the present invention is assessed by a test with rats. Rats weighing from 175 to 200 g are injected with carrageenan (2% in 0.9% sodium chloride aqueous solution, 100 ⁇ L injection volume) into the footpad of one hind limb. The rats are placed on a glass plate with illumination from a halogen lamp placed directly under the injected paw. The time (in seconds) from beginning illumination until the hindlimb was withdrawn from the glass was measured and scored as Paw Withdrawal Latency (PWL). Drug substances were given by oral gavage injection 2% hours after carrageenan injection to the footpad.
  • PWL Paw Withdrawal Latency
  • PWL was measured prior to carrageenan injection, just prior to drug injection, and 1 , 2 (and sometimes 3) hours after drug injection.
  • Carrageenan a polysaccharide extracted from seaweed
  • hyperalgesia Pain-related behavioral responses of greater intensity than expected
  • This hyperalgesia is maximal 2 to 3 hours after injection.
  • Treatment of rats with various analgesic drugs reduces hyperalgesia measured in this way and is a conventional test for detection of analgesic activity in rats.

Abstract

The present invention relates to diphenylamino ketone derivatives, pharmaceutical compositions and methods of use thereof.

Description

DIPHENYLAMINO KETONE DERIVATIVES AS MEK INHIBITORS FIELD OF THE INVENTION The present invention relates to diphenylamino ketone derivatives, pharmaceutical compositions and methods of use thereof. BACKGROUND OF THE INVENTION MAPK/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., MEK^ and MEK2) which then activates the MAP kinase, ERK (ERK-| 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, S218 and S222 jn the case of MEK-1 , which are the prerequisite for activation of MEK as a kinase. MEK in turn phosphorylates MAP kinase on both a tyrosine,
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 kinaes. 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 OF THE INVENTION The present invention provides a compound of formula
Figure imgf000003_0001
wherein Q is Cι_6 alkyl, C2.6 alkenyl, C2.6 alkynyl, or C2-5 heteroaryl, wherein the C-|.6 alkyl, C2-6 alkenyl, and C2-6 alkynyl groups are optionally substituted with between 1 and 2 hydroxy substituents, and the C2.5 heteroaryl is optionally substituted with CM alkyl, and further wherein the Cι.4 alkyl is optionally substituted with between 1 and 3 substituents independently selected from hydroxy and amino; Ri and R5 are each independently hydrogen or fluorine; R2 is hydrogen, chlorine, fluorine or methyl; Ri is bromine, chlorine, fluorine, iodine, C^e alkyl, C2-6 alkenyl, C2-6 alkynyl, C3.6 cycloalkyl, -(CH2)-C3.6 cycloalkyl, cyano, -0-(C^ alkyl), -S-(C1.2 alkyl), -SOCH3, -S02CH3, - S02NR6R7, -C≡C-(CH2)nNH2, -C---C-(CH2)nNHCH3, -C≡C-(CH2)nN(CH3)2, -C≡C-CH2OCH3, -C=C(CH2)nOH, -C=C-(CH2)nNH2, (Z)-CHCHCH2OCH3, -(Z)-CHCH-(CH2)nNHCH3, (Z)-CHCH- (CH2)nN(CH3)2, -(CH2)pC02R6, 0(0)0,-3 alkyl, C(0)NHCH3, -(CH2)mNH2, -(CH2)mNHCH3, -(CH2)mN(CH3)2, -(CH2)mOR6, -CH2S(CH2)t(CH3), -(CH2)PCF3, -C≡CCF3 , -CH=CHCF3, - CH2CHCF2, -CH=CF2, -(CF2)VCF3, -CH2(CF2)nCF3, -(CH2)tCF(CF3)2, -CH(CF3)2, -CF2CF(CF3)2, or -C(CF3)3, wherein the C-i-s alkyl and C2.6 alkynyl are optionally substituted with between 1 and 3 substituents independently selected from hydroxy and alkyl; R6 and R7 are each independently hydrogen, methyl, or ethyl; m is 1 to 4; n is 1 to 2; p is 0 to 2; t is O to 1; v is 1 to 5; and pharmaceutically acceptable salts, Cι_6 amides and Cι-6 esters thereof. An embodiment of the present invention provide a compound of formula I, as defined above, and pharmaceutically acceptable salts thereof. Additionally provided by the present invention are compounds of Formula I, wherein: Q is C1-6 alkyl, C2.6 alkenyl, or C2.5 heteroaryl, wherein the C^ alkyl group is optionally substituted with between 1 and 2 hydroxy substituents and the C2.5 heteroaryl is optionally substituted with methyl; Q is C.|.4 alkyl optionally substituted with between 1 and 2 hydroxy substituents; Q is C2.5 heteroaryl containing from one to two heteroatoms independently selected from the group consisting of nitrogen, oxygen and sulfur, and further wherein the C2.5 heteroaryl is optionally substituted with CM alkyl, and further wherein the C^ alkyl is optionally substituted with between 1 and 3 substituents independently selected from hydroxy and amino; or Q is methyl, -CH2OH, -(CH2)4OH, butenyl, methyl-substituted oxazolyl, thiazolyl, methyl-substituted imidazolyl, or pyridinyl. The present invention also provides compounds of Formula I wherein Ri is fluorine; R2 is fluorine; R5 is fluorine; or R, and R5 are fluorine. Additionally, the present invention provides compounds of Formula I, wherein R is: iodine, Cι.6 alkyl, C2.6 alkenyl, or C2.6 alkynyl, and further wherein the C^ alkyl, C2.6 alkenyl, and C2.6 alkynyl are optionally substituted with hydroxy; iodine, C-ι. alkyl, C2^ alkenyl, C2.3 alkynyl, or S-CH3, and further wherein the C-M alkyl, C2.3 alkenyl, and C2.3 alkynyl are optionally substituted with hydroxy; iodine; C-ι-4 alkyl, C2_3 alkenyl, or CM alkynyl, and further wherein the C-M alkyl, CM alkenyl, and C2.3 alkynyl are optionally substituted with hydroxy; or iodine, ethyl, -CH2OH, ethenyl, or acetyl. The invention also provides a pharmaceutical composition comprising a compound of
Formula I 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. Furthermore, the invention provides methods of treating cancer, restenosis, psoriasis, autoimmune disease, atherosclerosis, osteoarthritis, rheumatoid arthritis, heart failure, chronic p^in, and neuropathic pain in a patient in need thereof comprising administering a therapeutically effective amount of a compound of Formula I. 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 in combination with radiation therapy or at least one chemotherapeutic agent. The invention also provides the use of a compound of Formula I for the manufacture of a medicament for the treatment of the disease states or diseases provided above. DETAILED DESCRIPTION OF THE INVENTION Certain terms are defined below and by their usage throughout this disclosure. The terms "halogen" or "halo" in the present invention refer to a fluorine, bromine, chlorine, and iodine atom or fluoro, bromo, chloro, and iodo. The terms fluorine and fluoro, for example, are understood to be equivalent herein. Alkyl groups, such as "Cι-β 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, isobutyl, sec-butyl, t-butyl, pentyl, 2-pentyl,
3-pentyl, isopentyl, neopentyl, (R)-2-methylbutyl, (S)-2-methylbutyl, 3-methylbutyl,
2,3-dimethylpropyl, hexyl, and the like. The term "C^ alkyl" includes within its definition the terms "CM alkyl" and "Cι-2 alkyl". Alkenyl groups, such as C2.6 alkenyl, 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 trans. Similarly, alkynyl groups, such as C2.6 alkynyl, 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. Examples of alkenyls and alkynyls include vinyl, allyl, 2-methyl-2-propenyl, cis-2-butenyl, trans-2-butenyl, and acetyl. The term
"C2.6 alkenyl" includes within its definition the term "C2.3 alkenyl" and the term "C2.6 alkynyl" includes within its definition the term "C2.3 alkynyl". Cycloalkyl groups, such as C3.6 cycloalkyl, refer to a saturated hydrocarbon ring structure containing from 3 to 6 atoms. Typical C3.6 cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like. The term "heteroaryl" or "C2_5 heteroaryl" as used herein, unless otherwise indicated, includes monocyclic aromatic heterocycles containing five or six ring members, of which from
1 to 4 can be heteroatoms selected, independently, from N, S and O, and bicyclic aromatic heterocycles containing from eight to twelve ring members, of which from 1 to 4 can be heteroatoms selected, independently, from N, S and O. Heterocyclic radicals, which include but are not limited to heteroaryls, include: furyl, (is)oxazolyl, isoxazolyl, thiophenyl, thiazolyl, pyrrolyl, imidazolyl, 1 ,3,4-triazolyl, tetrazolyl, pyridinyl, pyrimidinyl, pyridazinyl, indolyl, and their nonaromatic counterparts. Further examples of heterocyclic radicals include thienyl, piperidyl, quinolyl, isothiazolyl, piperidinyl, morpholinyl, piperazinyl, tetrahydrofuryl, tetrahydropyrrolyl, pyrrolidinyl, octahydroindolyl, octahydrobenzothiofuranyl, octahydrobenzofuranyl, (iso)quinolinyl, naphthyridinyl, benzimidazolyl, and benzoxazolyl. The present invention includes the hydrates and the pharmaceutically acceptable salts and solvates of the compounds defined by Formula I. The compounds of this invention can possess a sufficiently basic functional group, and accordingly react with any of a number of inorganic and organic acids, to form a pharmaceutically acceptable salt. The term "pharmaceutically acceptable salt" as used herein, refers to salts of the compounds of Formula I which are substantially non-toxic to living organisms. Typical pharmaceutically acceptable salts include those salts prepared by reaction of the compounds of the present invention with a pharmaceutically acceptable mineral or organic acid. Such salts are also known as acid addition salts. Such salts include the pharmaceutically acceptable salts listed in Journal of Pharmaceutical Science, 1977;66:2-19, which are known to the skilled artisan. Acids commonly employed to form acid addition salts are inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, and the like, and organic acids such as p-toluenesulfonic, methanesulfonic acid, benzenesulfonic acid, oxalic acid, p-bromophenylsulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid, acetic acid, and the like. Example of such pharmaceutically acceptable salts are the sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, bromide, hydrobromide, iodide, acetate, propionate, decanoate, caprate, caprylate, acrylate, ascorbate, formate, hydrochloride, dihydrochloride, isobutyrate, caproate, heptanoate, propiolate, glucuronate, glutamate, propionate, phenylpropionate, salicylate, oxalate, malonate, succinate, suberate, sebacate, fumarate, malate, maleate, hydroxymateate, mandelate, mesylate, nicotinate, isonicotinate, cinnamate, hippurate, nitrate, stearate, phthalate, teraphthalate, butyne- 1 ,4-dioate, butyne-1 ,4-dicarboxylate, hexyne-1 ,4-dicarboxylate, hexyne-1 ,6-dioate, benzoate, chlorobenzoate, methylbenzoate, hydrozybenzoate, methoxybenzoate, dinitrobenzoate, o- acetoxybenzoate, naphthalene-2-benzoate, phthalate, p-toluenesulfonate, p- bromobenzenesulfonate, p-chlorobenzenesulfonate, xylenesulfonate, phenylacetate, trifluoroacetate, phenylpropionate, phenylbutyrate, citrate, lactate, α-hydroxybutyrate, glycolate, tartrate, hemi-tartrate, benzenesulfonate, methanesulfonate, ethanesulfonate, propanesulfonate, hydroxyethanesulfonate, 1-naphthalenesulfonate, 2-naphthalenesulfonate, 1 ,5-naphthalenedisulfonate, mandelate, tartarate, and the like. A preferred pharmaceutically acceptable salt is hydrochloride. It should be recognized that the particular counterion forming a part of any salt of this inventions is usually not of a critical nature, so long as the salt as a whole is pharmacologically acceptable and as long as the counterion does not contribute undesired qualities to the salt as a whole. It is further understood that such salts may exist as a hydrate. The enantiomers of compounds of the present invention can be resolved by one of ordinary skill in the art using standard techniques well-known in the art, such as those described by J. Jacques, et al., "Enantiomers, Racemates, and Resolutions", John Wiley and Sons, Inc 1981. Examples of resolutions include recrystallization techniques or chiral chromatography. Some of the compounds of the present invention have one or more chiral centers and may exist in a variety of stereoisomeric configurations. As a consequence of these chiral centers, the compounds of the present invention occur as racemates, mixtures of enantiomers and as individual enantiomers, as well as diastereomers and mixtures of diastereomers. All such racemates, enantiomers, and diastereomers are within the scope of the present invention. The compounds of Formula I 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 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: "AcOH" refers to acetic acid; "CDI" refers to 1 ,1'-carbonyldiimidazole; Celite® refers to a filter agent which is acid washed and approximately 95% Si02; "CHCI3" refers to chloroform; "CH2CI2" and "DCM" refer to dichloromethane; "cone." refers to concentrated; "DABCO" refers to 1 ,4- diazabicyclo[2.2.2]octane; "DIEA" refers to N,N-diisopropylethylamine; "DMA" refers to N,N- dimethylacetamide; "DMF" refers to N,N-dimethylformamide; "DMSO" refers to methyl sulfoxide; "DMT-MM" refers to 4-(4,6-dimethoxy-1 ,3,5-triazin-2-yl)-4-methylmorpholinium chloride; "EtOAc" refers to ethyl acetate; ΕtOH" refers to ethanol; "Et20" refers to diethyl ether; "FMOC" refers to 9W-fluoren-9-ylmethyl ester; "h" refers to hours; "HCI" refers to hydrochloric acid; "Me" refers to methyl; "MeOH" refers to methanol; "Me2S04" refers to dimethyl sulfate; "min" refers to minutes; "NaOH" refers to sodium hydroxide' "Na2S04 refers to sodium sulfate; "N-MM" refers to N-methylmorpholine; "Pd/C refers to palladium on carbon; "PE" refers to petroleum ether which can be substituted with hexanes; "(Ph3P)2PdCI2" refers to dichlorobis (triphenylphosphine)palladium(ll); "(Ph3P)4Pd" refers to tetrakis (triphenylphosphine) palladium (0); "PS" refers to polymer - supported; "R.T." refers to room temperature; "sat" refers to saturated; "TEA" refers to triethylamine; "TFA" refers to trifluoroacetic acid; "THF" refers to "tetrahydrofuran; "TIPSCI" refers to 1 ,3-dichloro-1 , 1 ,3,3,- tetraisopropyl-disiloxane; "TLC" refers to thin layer chromatography and "TMS" refers to trimethylsilyl. All other terms and substituents, unless otherwise indicated, are previously defined. 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. Schemes 1 and 2 provide syntheses of the compounds of Formula I. Scheme 1
Figure imgf000008_0001
(4) formula I In Scheme 1 , Step A, a suitable benzoic acid (1) is coupled with a suitable aniline (2) to provide a 2-(arylamino)-benzoic acid or diphenylamine (3). For example, the aniline (2) and the benzoic acid (1 ) are dissolved in a suitable organic solvent with an acid catalyst and heated at reflux for several hours. Preferred solvents are polar solvents such as ethanol, and preferred acid catalysts are mineral acids such as concentrated HCI. The reaction is typically complete within about 12 to 36 hours. The product (3) is typically isolated by filtration after cooling of the reaction mixture, and further purified, if desired, by standard methods such as chromatography or crystallization. In Scheme 1 , Step B, an acid chloride (4) is prepared from 2-(arylamino)-benzoic acid (3) by reacting the benzoic acid (3) with oxalyl chloride in a suitable solvent, such as DMF. In Scheme 1 , Step C, nucleophilic addition to an acid chloride (4), followed by deprotection and decarboxylation provides a compound of formula I. For example, an acid chloride (4) is combined with tris(trimethylsiloxy)ethylene and stirred under nitrogen. After the reaction is complete, dioxane and hydrochloric acid are added to the mixture and stirred to produce a compound of formula I. The product can be isolated by filtration and removing the solvent, for example by evaporation under reduced pressure, and further purified, if desired, by standard methods such as chromatography, crystallization, or distillation. Scheme 2
Figure imgf000009_0001
(3) (5) (6)
Figure imgf000009_0002
Figure imgf000009_0003
formula la
Figure imgf000009_0004
In Scheme 2, Step A, the benzoic acid (3) is converted to the benzaldehyde (5). For example, the benzoic acid (3) is reduced with lithium aluminum hydride in a suitable solvent, such as tetrahydrofuran providing an intermediate alcohol which is then oxidized with, for example, manganese dioxide in chloroform providing benzaldehyde (5). In Scheme 1 , Steps B and C, the Wittig reaction, as known to one of skill in the art, is followed by dihydroxylation to provide compound (7). For example, the dihydroxylation is performed using a stoichiometric amount of 4-methyl-morpholine 4-oxide and a catalytic amount of Os0 . In Step D, compound (7) is protected under procedures generally known in the art to provide the protected compound (8). In Step E, the protected compound (8) is oxidized according to procedures known in the art followed by a standard deprotection in Step F to provide a compound of formula la. Scheme 4
Figure imgf000010_0001
R4 is halogen R4 is not halogen In Scheme 4, the compounds of formula I, wherein R4 is not halogen are prepared from the compounds of formula I wherein R is halogen, by transition metal-promoted coupling with reagent M-R4 wherein R4 is non-halogen (12) in a suitable solvent or solvents such as triethylamine, tetrahydrofuran or dimethylformamide. The transition metal-promoted coupling may be carried out with a palladium(O) or palladium (II) coupling agent, such as (Ph3P) Pd or (Ph3P)2PdCI2. The entire mixture is stirred from about 2 to 24 hours at room temperature. M is defined as a functional group known to transfer a carbon radical fragment in transition metal-promoted coupling processes. Examples of a suitable M group include trialkylstannyl, trialkylsilyl, trimethylsilyl, zinc, tin, copper, boron, magnesium and lithium. Examples of a suitable M-R4 reagent (12) when, R-t is C2^ alkenyl is allyltributyltin or tetravinyltin, and when R is hydroxy-substituted C2.6 alkynyl is propargyl alcohol. Preferred halogens, when R4 is halogen, are bromine and iodine. The resulting compound of formula l, as well as the protected Formula I compound, can be isolated by removing the solvent, for example by evaporation under reduced pressure, and further purified, if desired, by standard methods such as chromatography, crystallization, or distillation. It would be understood by one of skill in the art that the substituent R4, when R is non-halogen, may be further transformed, such as by oxidation, reduction, deprotection, or hydrogenation. A compound wherein R is C2-4 alkenyl may be transformed to a compound wherein R4 is hydroxy-substituted alkyl by treating the double bond of the alkene with ozone and NaBH4. Furthermore, a compound wherein R is CM alkenyl may be transformed to a compound wherein R is alkyl substituted with 2 hydroxy substituents by treating the double bond of the alkene with Os04. A compound wherein R4 is an alkene or alkyne derivative may be reduced under conditions known in the art, such as through hydrogenation, such as with Pd/C under an atmosphere of hydrogen. For example, the alkyne derivative is dissolved in a suitable solvent, such as absolute ethanol, in the presence of a metal catalyst, such as palladium on carbon. This mixture is stirred under an atmosphere of hydrogen from about 1 to 24 hours at room temperature to provide the fully saturated derivative. Alternately, the alkyne derivative is partially reduced via hydrogenation to provide the alkene derivative. For example, the alkyne derivative is dissolved in a suitable solvent, such as tetrahydrofuran, in the presence of a catalyst, such as Lindlar catalyst or palladium on carbon and, if desired, a suitable compound which disrupts the actions of the catalyst, such as quinoline or pyridine. This mixture is stirred under an atmosphere of hydrogen from about 1 to 24 hours at room temperature to provide the alkene derivative. The substituent R4 may also be transformed into a different R4 through standard synthetic procedures known to one of skill in the art. It would be understood by one of skill in the art that the transformation of R4 as shown in Scheme 4 may be performed at various steps throughout the synthesis of compounds of the present invention, as desired. For example, R4 may be transformed before the coupling of the ester (1 ) and aniline (2) as shown in Scheme 1 , Step A. Further transformations of R4 are shown in Scheme 5 below. Scheme 5
Figure imgf000011_0001
formula Ih
R-l2 is NR6R7 or OR6 In Scheme 5, step A, the compound of formula Ig is dissolved in a suitable solvent such as tetrahydrofuran and reacted with methanesulfonyl chloride to give the intermediate mesylate, then Nal in EtOAc to give the iodide compound (13). In Scheme 5, step B, the iodide compound (13) is reacted with a suitable amine, such as methylamine or dimethylamine, or a suitable alkoxide to give compounds of formula Ih. It would also be understood by one of skill in the art that an aniline (2) may be prepared to include the desired R4. The aniline (2) can be prepared by techniques and procedures readily available to one of ordinary skill in the art and by following the procedures as set forth in the following Schemes, or analogous variants thereof. Additionally, anilines (2) are taught in USSN 10/349,801 filed January 23, 2003 and USSN 10/349,826 filed January 23, 2003, the disclosure of which is hereby incorporated by reference. These Schemes are not intended to limit the scope of the invention in any way. Scheme 6
Figure imgf000012_0001
Bull. Soc. Chim. Belg., 95(2), 135-8 1986 In Scheme 6, a suitably substituted para-n itrostyrene is reacted with dimethyloxosulfonium methylide to form the substituted para-nitrocyclopropylbenzene. Reduction of para-nitrocyclopropylbenzene with iron in the presence of weak acid gives the desired aniline. Scheme 7
AlClq
Figure imgf000012_0002
Figure imgf000012_0003
n=1-4 In Scheme 7, the suitable orfήo-substituted acetamide is reacted with bromocyclobutane, bromocyclopropane, or bromocyclohexane under typical Friedel-Craft conditions, as known to one of skill in the art, to give the desired para-cycloalkylanilines. The acetamide is deprotected under conditions known to one of skill in the art to provide the desired para-cycloalkylmethylanilines. Scheme 8
Figure imgf000013_0001
(13) (15) (2a) In Scheme 8, Step A, a suitable amine or alkoxide (14) is reacted with a 4-tert- butoxycarbonylamino-3-substituted-benzyl bromide (13), such as 4-tert-butoxycarbonylamino- 3-fluorobenzyI bromide (J. Med. Chem., 2000/43:5017). In Step B, the BOC protecting group of compound of structure (15) is hydrolized with, for example, TFA, to provide the desired aniline (2a). Scheme 9
Figure imgf000013_0002
(16) (18) (2 ) In Scheme 9, Step A, a suitable 3-substituted-4-nitrophenol (16), such as 3-fluoro-
4-nitrophenol, is alkylated with a compound of structure (17) in the presence of a suitable base to provide a compound of structure (18). In Step B, compound (18) is reduced via hydrogenation in the presence of a metal catalyst, such as palladium on carbon, in an atmosphere of hydrogen to provide the desired aniline (2b). Scheme 10
(alk l)-X (17')
Figure imgf000013_0003
Figure imgf000013_0004
(19) (2c) In Scheme 10, a suitable 4-(aminophenyl)thiocyanate (19), is alkylated with a compound of structure (17') in the presence of a suitable nucleophilic base to provide an alkylthio compound of structure (2c). After reaction under standard conditions to form the diphenylamine (3), wherein R is -S-(alkyl), as in Scheme 1 above, this compound is then oxidized to the corresponding sulfonyl compound, also generally, the diphenylamine (3), wherein R4 is -S02-(alkyl). Scheme 11
Figure imgf000014_0001
Synlett, (11), 1743-1744; 1999
Figure imgf000014_0002
In Scheme 11 , the proper orfΛo-substituted or unsubstituted aniline (2d) is acetylated with acetic anhydride in the presence of trifluoromethanesulfonic acid indium salt to give the protected aniline (20). Chlorosulfonation in the typical manner, as known in the art, gives the sulfonyl chloride derivative (21 ) which is reacted with an excess of a suitable amine in a solvent such as dichloromethane or dichloroethane to give the protected para- aminobenzenesulfonamide (22). Acid-mediated deprotection in the appropriate solvent gives the desired aniline (2e). Alternatively, the desired aniline (2e) wherein R2 is methyl, fluorine or chlorine, using compound (21 ) as the starting material can be prepared. Where R2 is fluorine, the sulfonyl chloride derivative (21 ) is a compound known in the literature (German Patent DE 2630060, 1978). Similarly, where R2 is methyl, the sulfonyl chloride derivative (21) is also known in the literature (German Patent. DE 2750170, 1978). Finally, the sulfonyl chloride derivative (21 ) where R2 is chlorine is commercially available. In addition to the procedure described in Scheme 11 , one of ordinary skill in the art would appreciate that there are numerous ways of acetylating anilines. For example, heating the aniline and acetic anhydride together in a suitable solvent, such as acetic acid, would achieve the same result. Compounds of the present invention include, but are not limited to the following compounds: 1-[3,4-Difluoro-2-(2-fluoro-4-iodo-phenylamino)-phenyl]-2-hydroxy-ethanone; [2-(4-Ethynyl-2-fluoro-phenylamino)-3,4-difluoro-phenyl]-pyridin-2-yl-methanone; [2-(4-Ethynyl-2-fluoro-phenylamino)-3,4-difluoro-phenyl]-thiazol-2-yl-methanone; [2-(4-Ethynyl-2-fluoro-phenylamino)-3,4-difluoro-phenyl]-(1-methyl-1 H-imidazol-2-yl)- methanone; [3,4-Difluoro-2-(2-fluoro-4-iodo-phenylamino)-phenyl]-(4-methyl-oxazoI-2-yl)- methanone; 1-[3,4-Difluoro-2-(2-fluoro-4-iodo-phenylamino)-phenyl]-ethanone; 1-{3,4-Difluoro-2-[2-fluoro-4-(2-hydroxy-ethyl)-phenylamino]-phenyl}-2-hydroxy- ethanone; 1 -[2-(4-Ethyl-2-fluoro-phenylamino)-3,4-difluoro-phenyl]-2-hydroxy-ethanone; 1-[2-(4-Ethynyl-2-fluoro-phenylamino)-3,4-difluoro-phenyl]-ethanone; 1-[2-(4-Ethyl-2-fluoro-phenylamino)-3,4-difluoro-phenyl]-5-hydroxy-pentan-1-one; 1-[3,4-Difluoro-2-(2-fluoro-4-vinyl-phenylamino)-phenyl]-2-hydroxy-ethanone; and 1-[2-(4-Ethynyl-2-fluoro-phenylamino)-3,4-difluoro-phenyl]-pent-4-en-1-one. 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 treatment, prophylaxis or prevention, amelioration or elimination of a named condition once the condition has been established. 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 IC50 for MEK 1 or MEK 2 that is at least one- fiftieth (1/50) that of its IC50 for one of the above-named other enzymes. Preferably, a selective inhibitor has an IC50 that is at least 1/100, more preferably 1/500, and even more preferably 1/1000, 1/5000, or less than that of its IC5Q or 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 therapeutically effective amount of a disclosed compound of formula I 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, 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, 200, 300, 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 Formula I and a pharmaceutically acceptable carrier. In making the compositions of the present invention, the active ingredient, such as a compound of Formula I, 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 Q) 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 the present invention as described generally above. These examples are illustrative only and are not intended to limit the invention in any way. The reagents and starting materials are readily available to one of ordinary skill in the art. PREPARATION 1
Figure imgf000018_0001
3.4-difluoro-2-r(4-ethvnyl-2-fluorophenyl)aminolbenzoic acid Step A: Preparation of 2-fluoro-4-f(trimethylsilyl)ethvnyl1aniline
Figure imgf000018_0002
2-Fluoro-4-iodoaniline (5.00 g, 21.1 mmol), Cul (90 mg, 0.42 mmol), and (Ph3P)2PdCI2 (300 mg, 0.42 mmol) were weighed into a flask which was sealed and flushed with N2. A solution of TMS-acetylene (2.28 g, 23.2 mmol) in TEA (20 mL) was added, then the entire mixture stirred 15 hours at room temperature. The reaction mixture was diluted with diethyl ether (200 mL), filtered through Celite®, then all solvents removed under reduced pressure. The resulting dark brown oil was purified by filtration through a plug of flash silica (5% EtOAc/hexanes as eluant) to afford the desired product as a pale brown oil which rapidly solidified to give a crystalline solid (3.85g, 88%); m.p. (EtOAc/hexanes) 45-47°C. 1H NMR (400 MHz, CDCI3) δ 7.10 (dd, J = 11.7, 1.8 Hz, 1 H), 7.06 (ddd, J = 8.3, 1.8, 1.0 Hz, 1 H), 6.66 (dd, J = 9.4, 8.3 Hz, 1 H), 3.86 (br s, 2 H), 0.22 (s, 9 H). Anal. Calcd for CnH^FNSi: C, 63.7; H, 6.8; N, 6.8. Found: C, 63.7; H, 6.9; N, 6.7. Step B: Preparation of 3,4-difluoro-2-[r2-fluoro-4(trimethylsilylethvnyl)phenvn aminolbenzoic acid
Figure imgf000018_0003
A mixture of the product of Step A, 2-fluoro-4-[(trimethylsilyl)ethynyl]aniline (3.85 g, 18.6 mmol) and 2,3,4-trifluorobenzoic acid (3.27 g, 18.6 mmol) was dissolved in dry THF (25 mL). The flask was fitted with a pressure-equalising dropping funnel and the entire apparatus evacuated and flushed with N2. The solution was then cooled to -78°C (acetone/dry ice) and a solution of 1.06 M LiHMDS (52.64 mL, 55.8 mmol) was added dropwise from the dropping funnel. Following this addition, the reaction mixture was allowed to warm to room temperature and stirred for a further 15 hours. The reaction solvent was removed under reduced pressure and the resulting residue partitioned between 1 M HCI (100 mL) and EtOAc (2 x 100 mL). The combined EtOAc fractions were then washed with water (100 mL) and saturated NaCI (100 mL), dried (Na2S04), and the EtOAc removed under reduced pressure to afford a crude product which was purified by chromatography on flash silica (10% EtOAc/hexanes as eluant), giving the desired product as a pale yellow solid (3.99 g, 59%); m.p. (EtOAc/hexanes) 164-167°C. 1H NMR [400 MHz, (CD3)2SO] δ 13.70 (br s, 1 H), 9.31 (br s, 1 H), 7.82 (ddd, J = 9.1 , 6.1 , 2.0 Hz, 1 H), 7.34 (dd, J = 12.0, 1.9 Hz, 1 H), 7.18 (ddd, J = 8.3, 1.9, 0.8 Hz, 1 H), 7.16 (td, J = 9.5, 7.3 Hz, 1 H), 6.93 (ddd, J = 8.9, 8.3, 5.4 Hz, 1 H), 0.22 (s, 9 H). Anal. Calcd for C18H16F3N02Si: C, 59.5; H, 4.4; N, 3.9. Found: C, 59.7; H, 4.7; N, 3.9. Step C: Preparation of 3.4-difluoro-2-r(4-ethvnyl-2-fluorophenyl)amino1benzoic acid The product of Step B, 3,4-difluoro-2-[[2-fluoro-4-(trimethylsilylethynyl) phenyljaminojbenzoic acid (3.99 g, 11.0 mmol), was dissolved in MeOH (200 mL), to which was added K2C03 (3.03 g, 22.0 mmol). This mixture was stirred at room temperature for 15 hours, then the reaction solvent removed under reduced pressure. The resulting residue was dissolved in water (50 mL), to which was added 1 M HCI until the pH = 4. The resulting pale brown precipitate was collected by filtration and dried to afford the desired product (3.17 g, 99%); m.p. (EtOAc/hexanes) 160-162°C. 1H NMR [400 MHz, (CD3)2SO] δ 13.70 (br s, 1 H), 9.24 (br s, 1 H), 7.82 (ddd, J = 9.2, 6.1 , 2.1 Hz, 1 H), 7.38 (dd, J = 12.0, 1.9, 1 H), 7.21 (ddd, J = 8.4, 1.9, 0.8 Hz, 1 H), 7.16 (td, J = 9.5, 7.3 Hz, 1 H), 6.96 (ddd, J = 8.9, 8.4, 5.4 Hz, 1 H), 4.15 (s, 1 H). Anal. Calcd for C15H8F3N02: C, 62.4; H, 3.1; N, 4.7. Found: C, 62.4; H, 3.2; N, 4.6. EXAMPLE 1
Figure imgf000019_0001
1-r3,4-Difluoro-2-(2-fluoro-4-iodo-phenylamino)-phenvn-2-hvdroxy-ethanone To a stirring suspension comprised of 3,4-difluoro-2-(2-fluoro-4-iodo-phenylamino)- benzoic acid (1.33 g, 3.4 mmol, which can be prepared according to the procedure of WO 02/06213) and oxalyl chloride (1.4 mL, 16 mmoles) in dichloromethane (10 mL) at ambient temperature was added 0.025 mL of Λ/,Λ/-dimethylformamide. The reaction mixture was stirred for ten minutes and was concentrated in vacuo to the yellow solid 3,4-difluoro-2-(2- fluoro-4-iodo-phenylamino)-benzoyl chloride. The yellow solid was suspended in tris(trimethylsiloxy)ethylene (10 g, 33 mmol) and the stirring mixture was brought to 90 °C under a nitrogen atmosphere for six hours. The mixture was cooled slightly, and to it was added a solution consisting of dioxane (25 mL) and 10% aqueous hydrochloric acid (10 mL). Vigorous liberation of gas ensued and the mixture was stirred for ten minutes at 85 °C. Brine (50 mL) was added and the mixture was extracted twice with ether (2 x 150 mL). The combined ether phases were washed twice with saturated aqueous sodium bicarbonate (2 x 150 mL). The ether phase was dried (MgS04) and was concentrated in vacua to 1.4 g of an orange oil that was purified by flash chromatography. Elution with a gradient (100% hexanes to 1 :3 hexanes/ethyl acetate over 72 minutes) afforded a solid with minor impurities. The solid was dissolved in dichloromethane and vacuum filtered through silica to afford 0.135 g (9.8 % yield over two steps) of a yellow solid; mp 128-128.5 °C; 1H-NMR (400 MHz; CDCI3) δ 9.80 (s, 1H), 7.40 (m, 3H), 6J7 (m, 2H), 4.83 (d, 2H, J=3.9 Hz), 3.44 (t, 1 H, J=4.1 Hz); 19F- NMR (376 MHz; CDCI3) δ -124.52 (t, 1 F, J=10.1 Hz), -125.27 (dd, 1 F, J=10.1 , 5.1 Hz), - 144.11 (d, 1 F, J=7J Hz); MS (APCI+) 407.8 (M+1 , 28), 389.8 (100); (APCI-) 405.8 (M-1 , 100), 402.8 (53); IR 1662, 1529, 1503, 1260, 1067 cm"1; %C(calculated for C14H9F3IN02/found) 41.30/41.52, %H 2.23/2.08, %N 3.44/3.45. EXAMPLE 2
Figure imgf000020_0001
r2-(4-Ethvnyl-2-fluoro-phenylamino)-3,4-difluoro-phenyll-pyridin-2-yl-methanone n-Butyllithium (1.6 M in hexanes, 3.0 mL, 4.8 mmol) was added rapidly to a -78 °C solution of 2-bromopyridine (0.46 mL, 4.82 mmol) in tetrahydrofuran (5 mL). The resultant brown-colored reaction mixture was stirred 30 min at -78 °C. A solution of the product of preparation 1 , 2-(4-ethynyl-2-fluoro-phenylamino)-3,4-difluoro-benzoic acid (284 mg, 0.975 mmol), in tetrahydrofuran (5 mL) was added via cannula and the resultant brown-colored slurry was warmed to ambient temperature. After 1 h, the reaction was partitioned between water and ethyl acetate. The organics were washed with 1 M aqueous hydrochloric acid (2 x 2 mL), water, and brine, were dried over magnesium sulfate and concentrated in vacuo. Chromatography on silica gel (dichloromethane) afforded [2-(4-ethynyl-2-fluoro-phenylamino)- 3,4-difluoro-phenyl]-pyridin-2-yl-methanone (158 mg, 46 % yield) as a yellow film. Recrystallization from ether-hexanes afforded yellow needles: m.p. 109-111 °C; 1H NMR (400 MHz, acetone-d6) δ 8J7 (s, 1 H), 8.60 (ddd, J = 4.9, 1.7, 1.0 Hz, 1 H), 8.04-7.96 (m, 2 H), 7.71 (ddd, j = 8.9, 5.9, 2.1 Hz, 1 H), 7.59 (ddd, J = 7.3, 4.6, 1.5 Hz, 1 H), 7.21-7.14 (m, 3 H), 6.95 (td, J = 8.8, 4.9 Hz, 1 H), 3.59 (s, 1 H); 19F NMR (376 MHz, acetone-d6) δ -132.2, - 132J, -145.1. Anal. Calcd/Found for Ca-HnFaN- - C, 68.18/68.09; H, 3.15/3.13; 7.95/7.87.
Figure imgf000021_0001
r2-(4-Ethvnyl-2-fluoro-phenylamino)-3,4-difluoro-phenyll-thiazol-2-yl-methanone Step A: Preparation of 2-(4-Ethvnyl-2-fluoro-phenylamino)- 3.4-difluoro-N-methoxy-N- methyl-benzamide
Figure imgf000021_0002
The product of of preparation 1 , (2.2 g, 7.6 mmol) was dissolved in dichloromethane (30 mL) and N-methylmorpholine (3.5 mL). To the resultant solution was added N,0- dimethylhydroxylamine hydrochloride (0.95 g, 9.7 mmol) and PyBOP (3.9 g, 7.5 mmol). The resultant reaction mixture was stirred for 2 h at ambient temperature. An additional portion of PyBOP (1.3 g, 2.5 mmol) was added and stirring was continued. After 2 h, the reaction was diluted with ethyl acetate (100 mL) and washed with water (100 mL x 3) and saturated brine (100 mL x 3). The organics were dried over anhydrous sodium sulfate, concentrated in vacuo, and purified by silica gel chromatography to afford the product (1.33 g, 53 % yield): 1H NMR [400 MHz, (CD3)2SO] δ 8.03 (br s, 1 H), 7.29J.22 (m, 3 H) 7.08 (dd, J = 8.3, 1.5 Hz, 1 H), 6.67 (td, J = 8.8, 2.0 Hz, 1 H), 4.05 (s, 1 H), 3.39 (s, 3 H), 3.07 (s, 3 H); MS (APCI+) for C17H13F3N202= 335.1 [M+1]. Step B: Preparation of .2-(4-ethvnyl-2-fluoro-phenylamino)-3.4-difluoro-phenvπ- thiazol-2-yl-methanone A solution of thiazole (0.148 mL, 2.08 mmol) in tetrahydrofuran (10 mL) was cooled to
-48 °C and treated with n-butyllithium (1.3 mL, 1.6 M in hexane, 2.08 mmol). The resultant purple-colored solution was stirred 15 min at -48 °C. To this reaction mixture was added a solution of 2-(4-ethynyl-2-fluoro-phenylamino)-3,4-difluoro-N-methoxy-N-methyl-benzamide (200 mg, 0.6 mmol) in tetrahydrofuran (10 mL). The resultant deep yellow-colored reaction mixture was stirred an additional 30 min and was quenched with saturated aqueous ammonium chloride (30 mL). The reaction was partitioned with ethyl acetate and the organic layer was separated and was washed with saturated aqueous bicarbonate (50 mL x 3). The organics were dried over anhydrous sodium sulfate, concentrated and chromatographed on silica gel. Recrystallization of the yellow solid from dichloromethane-hexanes afforded [2-(4- ethynyl-2-fluoro-phenylamino)-3,4-difluoro-phenyl]-thiazol-2-yl-methanone (88 mg, 41 % yield): m.p. 161 °C; 1H NMR (400 MHz, CDCI3) ϋ 9.50 (s, 1 H), 8.60 (ddd, J = 9.0, 5.9, 2.2 Hz, 1 H), 8.08 (d, J = 3.2 Hz, 1 H), 7.74 (d, J = 2.9 Hz, 1 H), 7.18 (m, 1 H), 6.91 (dt, J = 7.1 , 9.2 Hz, 1 H), 6.83 (dt, J = 5.9, 8.3 Hz, 1 H), 3.03 (s, 1 H); 19F NMR (376 MHz, CDCl3) δ - 126.6, -129.2 , -141.9. Anal. Calcd/Found for C18H9F3N2OS-0.12(C4H8O2): C, 60.17/60.04; H, 2.72/3.11 ; N, 7.59/7.55. EXAMPLE 4
Figure imgf000022_0001
2-(4-Ethvnyl-2-fluoro-phenylamino)-3.4-difluoro-phenvn-(1-methyl-1 H-imidazol-2-yl)- methanone A solution of N-methyl imidazole (0.27 mL, 3.4 mmol) in tetrahydrofuran (10mL) was cooled to -78 °C and treated with n-butyllithium (3.17 mL, 1.6 M in hexanes, 5.07 mmol). The resultant reaction mixture was stirred 15 min at -78 °C and 2 h at ambient temperature and was then transferred to a -78 °C solution of the product of preparation 1 , 2-(4-ethynyl-2- fluoro-phenylamino)-3,4-difluoro-benzoic acid (200 mg, 0.69 mmol) in tetrahydrofuran (5 mL). The resultant reaction mixture was stirred 30 min at -78 °C and was allowed to warm to ambient temperature overnight. The reaction was partitioned between water (20 mL) and ethyl acetate (30 mL). The organic layer was washed with water (40 mL x 3) and saturated brine (35 mL x 2), dried over anhydrous sodium sulfate and concentrated in vacuo. Silica gel chromatography afforded a yellow solid. Recrystallization from dichloromethane-hexanes afforded [2-(4-Ethynyl-2-fluoro-phenylamino)-3,4-difluoro-phenyl]-(1-methyl-1 H-imidazol-2-yl)- methanone (105 mg, 43% yield): m.p. 166 °C; 1H NMR (400 MHz, CDCI3) δ 9.08 (s, 1 H), 8.05 (ddd, J = 9.0, 5.8, 2.2 Hz, 1 H), 7.22 (d, J = 1.0 Hz, 1 H), 7.18-7.11 (m, 2 H), 7.11 (s, 1 H), 6.93 (dt, J = 6.9, 9.0 Hz, 1 H), 6.73 (td, J = 8.5, 6.3 Hz, 1 H), 4.03 (s, 3 H), 3.00 (s, 1 H); 19F NMR (376 MHz, CDCI3) δ -128.8, -131.0, -141.4; MS (APCI+) = 356.0. Anal. Calcd/Found for C19H12F3N3O 0.03(CH2CI2): C, 63.87/63.76; H, 3.40/3.26; N, 11.74/11.69 EXAMPLE 5
Figure imgf000023_0001
r3.4-Difluoro-2-(2-fluoro-4-iodo-phenylamino)-phenyll-(4-methyl-oxazol-2-yl)- methanone Step A: Synthesis of 3,4-Difluoro-2-(2-fluoro-4-iodo-phenylamino)-benzoyl chloride A solution of 3,4-Difluoro-2-(2-fluoro-4-iodo-phenylamino)-benzoic acid (5.0 g, 12.7 mmol) in thionyl chloride (75 mL) containing 10 drops of dimethylformamide was heated at reflux for 15 min. The solvent was removed in vacuo affording 3,4-Difluoro-2-(2-fluoro-4-iodo- phenylamino)-benzoyl chloride (5.3 g, which can be prepared according to the procedure of WO 02/06213) as a yellow solid. Step B: Synthesis of [3,4-Difluoro-2-(2-fluoro-4-iodo-phenylaminoVphenylH4-methyl- oxazol-2-yl)-methanone . A solution of 4-methyl-oxazole (0.080 mL, 1.0 mmol) in tetrahydrofuran (10 mL) was cooled to -78 °C and treated with n-butyllithium (1.6 M in hexane, 0.51 mL, 0.81 mmol). The resultant solution was stirred 25 min at -78 °C. Zinc chloride (1.45 mL, 1.0 M in ether) was added and the reaction mixture was allowed to warm to 0 °C over 45 min. Copper (I) iodide (185 mg, 0.97 mmol) was added and the reaction was stirred for 10 min. 3,4-Difluoro-2-(2- fluoro-4-iodo-phenylamino)-benzoyl chloride (200 mg, 0.49 mmol) was added and the reaction mixture was allowed to warm to ambient temperature overnight. The reaction was diluted with ethyl acetate and was washed with concentrated ammonium hydroxide-water (1 :1 , 40 mL), water (40 mL) and brine (40 mL). The organics were concentrated in vacuo and chromatographed on silica gel. Elution with hexanes ethyl acetate (gradient to 20% ethyl acetate) afforded [3,4-Difluoro-2-(2-fluoro-4-iodo-phenylamino)-phenyl]-(4-methyl-oxazol-2- yl)-methanone (21 mg) after crystallization from dichlormethane-hexanes: m.p. 127 °C; 1H NMR (400 MHz, CDCI3) δ 9.59 (s, 1 H), 8.56 (ddd, J = 9.3, 5.8, 2.1 Hz, 1 H), 7.62 (d, J = 1.2 Hz, 1 H), 7.40 (dd, J = 10.3, 2.0 Hz, 1 H), 7.34 (br d, J = 8.6 Hz, 1 H), 6.86 (dt, J = 7.1 , 9.2 Hz, 1 H), 6.69 (dt, J = 4.9, 8.7 Hz, 1 H), 2.30 (d, J = 1.2 Hz, 3 H); 9F NMR (376 MHz, CDCI3) δ -125.7, -125.9, -143.2; MS (APCI+)= 459.0. Anal. Calcd/Found for
C17H10F3IN2O2-0.1(C6H14): C, 45.29/45.38; H, 2.46/2.32; N, 6.00/5.73. EXAMPLE 6
Figure imgf000024_0001
1-[3.4-difluoro-2-(2-fluoro-4-vinyl-phenylamino)-phenvn-2-hydroxy-ethanone Step A: Synthesis of f3.4-Difluoro-2-(2-fluoro-4-iodod-phenylamino)-phenyl]- methanol.
Figure imgf000024_0002
To 3,4-difluoro-2-(2-fluoro-4-iodo-phenylamino)-benzoic acid ( 10.0g, 25.4mmol) in tetrahydrofuran was added borane in tetrahydrofuran (38.2mL of a 1.0M solution) after 16 hours stirring at room temperature, the reaction was complete and quenched by careful addition of 2% HCI (100mL). The mixture was stirred for 1 h, brine was added and the organic layer was separated which was dried and evaporated to give a colorless solid (7.13g 74%) that was recrystalized from hot dichloromethane/hexane to give an analytical sample. MS (APCI+)380 (M+1+); 1H NMR(400mHz, DMSO) 7.62(s,1 H), 7.50(d,1 H), 7.24(m,3H), 6.29(d of t, 1 H), 5.35(t,1 H), 4.42(d,1 H). Step B: Synthesis of 3,4-D.fluoro-2-(2-fluoro-4-iodod-phenylamino) benzaldehyde.
Figure imgf000024_0003
To [3,4-Difluoro-2-(2-fluoro-4-iodod-phenylamino)-phenyl]-methanol (17.1g, 45 mmol) in dichloromethane was added manganese dioxide (δequiv., 31 g) and the reaction was heated to reflux for 16h. after cooling the reaction was filtered and the filtrate evaporated to give a yellow solid ( 14.15g, 83%). MS (APCI-) 376 (M-1-); 1H NMR(400mHz, DMSO) 9.98(s,1 H), 9.22(s,1 H), 7J3(ddd, 1 H), 7.61 (dd,1 H), 7.42(dt, 1 H), 7.22(m,1 H), 6.85(dt,1 H). Step C: Synthesis of (2,3-Difluoro-6-vinyl-phenyl)-(2-fluoro-4-iodo-phenvπ-amine.
Figure imgf000025_0001
To methyl triphenylphosphonium bromide(13.6g 2.2equiv) in tetrahydrofuran(180mL) at 0°C was added n-butyllithium (2.2equiv, 41mL of 1.6M). The reaction was stirred 10 minutes giving a clear orange solution. 3,4-Difluoro-2-(2-fluoro-4-iodod-phenylamino) benzaldehyde(7.0g, 18.6mmol) was added giving a red solution. After 2 hours the reaction was poured into aqueous ammonium chloride solution. The reaction was extracted with ethyl acetate, washed with brine to give an oil after evaporation of the solvent. This oil was triturated with diethyl ether to remove much of the phosphine oxide by product. The crude material was used with out futher purification. Step D: Synthesis of 1-(3,4-difluoro-2-(2-fluoro-4-iodo-phenylamino)-phenyll-ethane- 1.2-diol.
Figure imgf000025_0002
To crude (2,3-Difluoro-6-vinyl-phenyl)-(2fluoro-4-iodo-phenyl)-amine (~4mmol) in acetone water (8:1 , 40mL) was added N-methylmorpholine N-oxtde(936mg, 2equiv.) and osmium tetraoxide (5mol% in tert-butanol). The reaction was stirred at room temperature for 1.5h and the solvent was evaporated and loaded directly onto a flash column. Chromatography with silica gel eluting with 3/2 diethylether/hexane gave product as a colorless oil that solidified on standing (1.53g est 93%). MS (APCI+) 410 (M+1+); 1H NMR(400mHz, CDCI3), 7.39(dd,1 H), 7.28(m,1 H), 7.14(dt,1H), 7.00(dt,1 H), 6.57(s,1 H), 6.33(dt,1H), 4.90(m,1H), 4J2(t,1 H), 2.91 (d,1 H), 1.95(t,1H). Step E: Synthesis of H3.4-difluoro-2-(2-fluoro-4-iodo-phenylamino)-phenvπ-2- triisopropylsilanyloxy-ethanol.
Figure imgf000026_0001
To 1-{3,4-difluoro-2-(2-fluoro-4-iodo-phenylamino)-phenyl]-ethane-1 ,2-diol (409mg, 1.0mmol)in dry dimethylformamide (3mL) was added imidazole(136mg, 2.0 mmol) and then triisopropylsilyl chloride. The reaction was stirred at room temperature for 3h and then poured into water, the reaction was extracted with diethyl ether, the organic layer was washed with water and then brine. After drying over magnesium sulfate the solvent was removed under reduced pressure to provide product as a colorless oil(600mg). MS (APCI+) 566 (M+1 +); 1H NMR(400mHz, CDCI3) 7.37(dd,1 H), 7.26(m,1 H), 7.15(m,1 H), 6.66(s,1 H), 6.32(dt,1 H), 4.87(m,1 H), 3J0(m,2H), 3.04(d,1 H), 1.05-0.90(m, 21 H). Step F: Synthesis of 1-[3.4-difluoro-2-(2-fluoro-4-iodo-phenylamino)-phenyll-2- triisopropylsilanyloxy-ethanone.
Figure imgf000026_0002
To 1-[3,4-difluoro-2-(2-fluoro-4-iodo-phenylamino)-phenyl]-2-triisopropylsilanyloxy- ethanol ( 300mg, 0.50mmol) in dichloromethane(10mL), was added pyridinium dichromate (282mg, 1 ,5equiv). The reaction was heated to reflux for 4h, then cooled and loaded directly onto a flash column. Chromatography on silica gel eluting with 2:1 hexane/dichloromethane provided product as a yellow oil(122mg, 40%). MS (APCI+) 564 (M+1+); 1H NMR(400mHz, CDCI3) 9J5(s,1H), 7,67(ddd,1 H), 7.40(dd,1 H), 7.35(m,1H), 6.66(m,1 H), 6.58(m,1 H), 4.83(s,2H), 1.10-1.00(m,21 H). Step G: Synthesis of 1-r3.4-difluoro-2-(2-fluoro-4-vinyl-phenylamino)-phenyll-2- hydroxy-ethanone.
Figure imgf000027_0001
To 1-[3,4-difluoro-2-(2-fluoro-4-iodo-phenylamino)-phenyl]-2-triisopropylsilanyloxy- ethanone in dimethoxyethane/water ( 2:1 , 3mL)was added tetrakistriphenylphosphine Pd ( 5%, 18mg). This solution was stirred for 20min, potassium carbonate and 2,4,6-trivinyl- cyclotriboroxane pyridine complex (77mg, lequiv) was added. The reaction was heated at reflux for 3h, thin layer analysis shows that the reaction was complete. The reaction was cooled and tetrabutyl ammonium fluoride (0.45mL of 1.0M solution, 1.5equiv.) and (10%HCI, 2mL) was added. The mixture was heated at reflux for 4h, then cooled and poured into ethyl acetate, washed with water and then saturated sodium bicarbonate to give a brown oil. Chromatography of the crude product on silica gel eluting with 1 :4 hexane/dichloromethane gave product as a yellow solid (34mg, 35%). MS (APCI+) 308 (M+1+); 1H NMR(400mHz, CDCI3) 9.90(s,1 H), 7.43(m,1 H), 7.18(dd, 1H), 7.10(d,1H), 6.98(m,1 H), 6J5(m,1H), 6.65(dd,1 H), 5.65(d,1 H), 5.23(d,1 H), 4.83(d,2H), 3.44(t,1 H). EXAMPLE 7
Figure imgf000027_0002
1-r2-(4-ethyl-2-fluoro-phenylamino)-3,4-difluoro-phenvn-2-hydroxy-ethanone Step A: Synthesis of 1-r3.4-difluoro-2-(2-fluoro-4-vinyl-phenylamino)-phenv-l-2- triisopropylsilanyloxy-ethanol.
Figure imgf000028_0001
To 1-[3,4-difluoro-2-(2-fluoro-4-iodo-phenylamino)-phenyl]-2-triisopropylsilanyloxy- ethanol from Example 6 step E (1.94g, 3.4mmol) using the palladium coupling procedure of Exampe 6 step G provided product after silica gel chromatography 1 :1 hexane/diethyl ether as a colorless oil (1.13g, 72%). MS (APCI+) 466 (M+1+). Step B: Synthesis of 1-r2-(4-ethyl-2-fluoro-phenylamino)-3.4-difluoro-phenvπ-2- triisopropylsilanyloxy-ethanol.
Figure imgf000028_0002
1-[3,4-Difluoro-2-(2-fluoro-4-vinyl-phenylamino)-phenyl]-2-triisopropylsiianyloxy- ethanol (500mg, 1.07mmol) was reduced employing 5%Pt on C in tetrahydrofuran at 55 psi hydrogen for 2h. The reaction was judged complete by hydrogen uptake, the catalyst was removed by filtration and the product was isolated by evaporation of the solvent. The product (445mg, 89%) was employed directly in the next reaction. MS (APCI+) 468 (M+1+). Step C: Synthesis of 1-f2-(4-ethyl-2-fluoro-phenylamino)-3,4-difluoro-phenvn-2- triisopropylsilanyloxy-ethanone.
Figure imgf000029_0001
1-[2-(4-Ethyl-2-fluoro-phenylamino)-3,4-difluoro-phenyl]-2-triisopropylsilanyloxy- ethanol (445mg, 0.95mmol) was oxidized as in Example 6 step F, to provide product as a yellow oil(270mg, 60%). MS (APCI+) 466 (M+1 +); 1H NMR(400mHz, CDCI3) 9.87(s,1 H), 7.64(dt, 1H), 6.8-7.0(m,3H), 6.64(m,1 H), 4.86(s,2H), 2.61 (q,2H), 1.22(t,3H), 1.2-1.0(m,21 H). Step D: Synthesis of 1-r2-(4-ethyl-2-fluoro-phenylamino)-3.4-difluoro-phenyll-2- hydroxy-ethanone.
Figure imgf000029_0002
To 1-[2-(4-ethyl-2-fluoro-phenylamino)-3,4-difluoro-phenyl]-2-triisopropylsilanyloxy- ethanone (270mg) in tetrahydrofuran (10mL) was added 10%HCI (1.5mL). the reaction was heated at reflux for 3h and then poured into ethylacetate, washed with water and then saturated sodium bicarbonate to provide a yellow oil. Chromatography of the crude product in silica gel (1:4 hexane/dichloromethane provided product (48mg, 27%). MS (APCI+) 310 (M+1 +); 1H NMR(400mHz, CDCI3) 9.93(s,1H), 7.42(m,1 H), 7.00(dt,1 H), 6.92(m,2H), 6.66(m,1 H), 4.83(s,2H), 3.52(br s, 1 H), 2.63(q,2H), 1.22(t,3H). EXAMPLE 8
Figure imgf000030_0001
1-{3,4-difluoro-2-.2-fluoro-4-(2-hvdroxy-ethylVphenylaminol-phenylV2-hvdroxyl- ethanone. Step A: Synthesis of 1-(3,4-difluoro-2-r2-fluoro-4-(2-hvdroxy-ethyl)-phenylamino1- phenyll-2-triisopropylsilanyloxy-ethanol.
Figure imgf000030_0002
To 1-[3,4-difluoro-2-(2-fluoro-4-vinyl-phenylamino)-phenyl]-2-triisopropylsilanyloxy- ethanol (634mg, 1.35mmol) in dry tetrahydrofuran (15mL) at 0°C was added borane in tetrahydrofuran ( 2JmL, of 1.0M solution). After 1.5h sodium perborate hydrate (805mg, 6equiv.) in water (15mL) was added. The reaction was stirred for 3 hours, poured into water and extracted with ethyl acetate to give a colorless oil that was used without further purification. MS (APCI-) 310 (M-1-) 482. Step B: Synthesis of 1-(3.4-difluoro-2-[2-fluoro-4-(2-triisopropylsilanyloxy-ethyl)- phenylaminol-phenyll-2-triisopropylsilanyloxy-ethanol.
Figure imgf000031_0001
1-{3,4-Difluoro-2-[2-fluoro-4-(2-hydroxy-ethyI)-phenylamino]-phenyl}-2- triisopropylsilanyloxy-ethanol (1.35mmol) was protected as previously described in Example 6 step E. Product (520mg, 60%, 2steps) was obtained as a colorless oil after chromatography on silica gel eluting with 1 :1 hexane/diethylether. MS (APCI+) 640 (M+1+); 1H NMR(400mHz, CDCI3) 7.15(m,1 H), 6.94(m,2H), 6,78(m,1 H), 6.47(m,1 H), 6.44(s,1 H), 4.87(m,1 H), 3.82(t,2H), 3.72(m,2H), 2J6(t,2H), 1.1-0.90(m,42H). Step C: Synthesis of 1-{3,4-difluoro-2-[2-fluoro-4-(2-triisopropylsilanyloxy-ethyl)- phenylaminol-phenyll-2-triisopropylsilanyloxy-ethanone.
Figure imgf000031_0002
1-{3,4-Difluoro-2-[2-fluoro-4-(2-triisopropylsilanyloxy-ethyl)-phenylamino]-phenyl}-2- triisopropylsilanyloxy-ethanol ( 520mg, 0.81 mmol) was oxidized employing the procedure in Example 6 step F to give an orange oil (364mg) that was used directly in the next step to produce final product. Step D: Synthesis of 1-(3.4-difluoro-2-.2-fluoro-4-(2-hvdroxy-ethv0-phenylaminoy phenyl)-2-hvdroxyl-ethanone.
Figure imgf000031_0003
1-{3,4-Difluoro-2-[2-fluoro-4-(2-triisopropylsilanyloxy-ethyl)-phenylamino]-phenyl}-2- triisopropylsilanyloxy-ethanone (364mg) was deprotected employing the procedure described in Example 7 step D to give product as a yellow solid ( 29mg). MS (APCI-) 324 (M-1-); 1H NMR(400mHz, CDCI3) 9.90(s,1 H), 7.43(m,1 H), 7.07-6.92(m,3H), 6J0(m,1 H), 4.83(s,2H), 3.86(t,2H), 2.84(t,2H).
Figure imgf000032_0001
1-r3.4-difluoro-2-(2-fluoro-4-iodo-phenylamino)-phenvn-ethanone. Step A: synthesis of 1-f3.4-difluoro-2-(2-fluoro-4-iodo-phenylamino)-ρhenvπ-ethanol.
Figure imgf000032_0002
Hydroboration and oxidation of (2,3-Difluoro-6-vinyl-phenyl)-(2-fluoro-4-iodo-phenyl)- amine from Example 6 step C using the procedure described in Example 8 step A gave substantial amounts of the secondary alcohol that could be isolated by chromatography on silica gel eluting with 3:1 hexane/ethyl acetate. MS (APCI+) 394 (M+1 +); 1H NMR(400mHz, CDCI3) 7.39(dd,1H), 7.28(m,1 H), 7.08(dt,1 H), 6.97(m,1 H), 6.36(dt,1 H), 4.98(q, 1 H), 1.47(d,3H). Step B synthesis of 1-.3,4-difluoro-2-(2-fluoro-4-iodo-phenylamino)-phenvπ-ethanone.
Figure imgf000032_0003
To 1-[3,4-difluoro-2-(2-fluoro-4-iodo-phenylamino)-phenyl]-ethanol (540mg, 1.1mmol) in acetone (10mL) at 0°C was added Jones reagent until the brown color persisted for δminutes. The reaction was then poured into water , extracted with ethyl acetate. The organic layer was washed with water and then brine to give a yellow oil. Chromatography on silica gel eluting with 2:1 hexane/dichloromethane provided product (120mg, 22%). MS (APC1+) 392 (M+1 +); 1H NMR(400mHz, CDCI3) 10.06(s,1 H), 7.64(dq,1 H), 7.42(dd,1 H), 7.35(m,1 H), 6J5(m,2H), 2.63(s,3H). EXAMPLE 10 1-r2-(4-Ethvnyl-2-fluoro-phenylamino)-3.4-difluoro-phenviyethanone
Figure imgf000033_0001
Step A: Preparation of 1-r3,4-Difluoro-2-(2-fluoro-4-trimethylsilanylethynyl- phenylaminoVphenvn-ethanone 3,4-Difluoro-2-(2-fluoro-4-trimethylsilanylethynyl-phenylamino)-N-methoxy-N-methyl- benzamide (1 g, 2.46 mmol) was dissolved in tetrahydrofuran (12 mL). Methylmagnesium bromide (4.01 mL, 1.4M in toluene/THF, 5.61 mmol) was added and the reaction stirred at room temperature for 1 hour. The reaction was quenched with saturated ammonium chloride solution, extracted with ethyl acetate, dried with MgS04ι and concentrated. The product was taken on crude. Step B: Preparation of 1-r2-(4-Ethvnyl-2-fluoro-phenylamino)-3.4-difluoro-phenyll- ethanone 1-[3,4-Difluoro-2-(2-fluoro-4-trimethylsilanylethynyl-phenylamino)-phenyl]-ethanone (0.89g, 2.46 mmol) was dissolved in tetrahydrofuran (10 mL). TBAF (2J1 mL, 1.0M in THF) was added and the reaction stirred at room temperature for 1 hour. Water was added and the mixture was extracted with ethyl acetate, dried with MgS0 , and concentrated. The residue was purified on silica gel using a gradient of 18%-26% ethyl acetate in hexanes to yield the pure product (500mg, 70% two steps). M.P. = 78-80°C. 400 MHz 1H-NMR (DMSO-cfe) δ 9.55 (s, 1 H), 7.82 (m, 1 H), 7.33 (dd, 1 H, J - 12.0, 1.7 Hz), 7.18 (m, 2H), 6.90 (m, 1 H), 4.13 (s, 1H), 2.54 (s, 3H). MS (APCI+) 289.1 [M+1]. Anal. Calcd/Found for C16H10F3NO: C, 66.44/66.29; H, 3.48/3.56; N, 4.84/4.76. EXAMPLE 11
Figure imgf000033_0002
1-r2-(4-Ethyl-2-fluoro-phenylamino)-3,4-difluoro-phenvπ-5-hvdroxy-pentan-1-one Step A: Preparation of 2-(4-Ethyl-2-fluoro-phenylamino)-3.4-difluoro-N-methoxy-N- methyl-benzamide 2-(4-Ethynyl-2-fluoro-phenylamino)-3,4-difluoro-N-methoxy-N-methyl-benzamide (4.11g, 12.2 mmol) and 10% Pd/C (1.01g) were taken up in tetrahydrofuran (100 mL) and stirred under and atmosphere of hydrogen for 16 hours. The mixture was filtered. The filtrate was concentrated to yield the product in near quantitative yield. 400 MHz 1H-NMR (CDCI3) δ 7.58 (s, 1 H), 7.16 (m, 1H), 7.06 (m, 1 H), 6.94 (dd, 1 H, J = 12.7, 1.7 Hz), 6.80 (d, 1 H, J = 8.3 Hz), 6.72 (m, 1 H), 3.35 (s, 3H), 3.03 (s, 3H), 2.47 (q, 2H, J = 7.6 Hz), 1.08 (t, 3H, J = 7.6 Hz). Step B: Preparation of 1-r2-(4-Ethyl-2-fluoro-phenylamino)-3.4-difluoro-phenvn-pent- 4-en-1-one 2-(4-Ethyl-2-fluoro-phenylamino)-3,4-difluoro-N-methoxy-N-methyl-benzamide (1.5g, 4.43 mmol) was dissolved in THF. 3-Butenylmagnesium chloride (23.3 mL, 11.7 mmol) was added and the reaction stirred at room temperature for 30 minutes. Water was added and the mixture was extracted with ethyl acetate, dried with MgS04, and concentrated. The residue was purified on silica gel using a gradient of 0%-15% ethyl acetate in hexanes to yield the pure product (1.04g, 70%). 400 MHz 1H-NMR (CDCI3) δ 9.66 (s, 1 H), 7.86 (m, 1 H), 7.02 (m, 2H), 6.92 (m, 2H), 5J8 (m, 1 H), 4.99 (m, 1 H), 4.90 (m, 1 H), 3.10 (t, 2H, J = 7.2 Hz), 2.52 (q, 2H, J = 7.6 Hz), 2.28 (m, 2H), 1.11 (t, 3H, J = 7.6 Hz). Step C: Preparation of 1-r2-(4-Ethyl-2-fluoro-phenylamino)-3.4-difluoro-phenyll-5- hydroxy-pentan-1 -one 1-[2-(4-Ethyl-2-fluoro-phenylamino)-3,4-difluoro-phenyl]-pent-4-en-1-one (0.83g, 2.49 mmol) was cooled to 0°C. BH3-THF (2.12 mL, 1.0 M in THF) was added and the reaction warmed to room temperature over 2 hours. Water (2 mL) and NaB03-H20 (750 μL, 7.47 mmol) were added and the reaction continued to stir at room temperature for 1 hour. More water was added and the mixture was extracted with ethyl acetate, dried with MgS0 , and concentrated. The residue was purified on silica gel using a gradient of 45%-50% ethyl acetate in hexanes to yield the pure product (240mg, 27%). 400 MHz 1H NMR (CDCI3) δ 7.64
(m, 1 H), 6.92 (m, 3H), 6.66 (m, 1 H), 3.68 (t, 2H, J = 6.4 Hz), 3.02 (t, 2H, J = 7.1 Hz), 2.61 (q,
2H, J = 7.6 Hz), 1.82 (m, 2H), 1.65 (m, 2H), 1.22 (t, 3H, J = 7.6 Hz). MS (APCI+) 351.1
[M+1]. EXAMPLE 12
Figure imgf000034_0001
1-r2-(4-Ethvnyl-2-fluoro-phenylamino)-3.4-difluoro-phenvn-pent-4-en-1-one 2-(4-Ethynyl-2-fluoro-phenylamino)-3,4-difluoro-N-methoxy-N-methyl-benzamide
(3.0g, 8.97 mmol) was dissolved in tetrahydrofuran (50 mL) and cooled to -78°C.
3-Butenylmagnesium bromide (35.9 mL, 0.5 M in THF). The mixture stirred at -78°C for 30 minutes, then warmed to room temperature overnight. The reaction was quenched with saturated ammonium chloride solution. The mixture was extracted with dichloromethane, dried with MgS04, and concentrated. The residue was purified on silica gel using a gradient of 5%-15% ethyl acetate in hexanes to yield the pure product (800mg, 27%). 400 MHz 1H- NMR (DMSO-cf6) δ 7.88 (m, 1 H), 7.38 (dd, 1H, J = 12.0, 2.0 Hz), 7.23 (m, 2H), 6.92 (m, 1 H), 5.81 (m, 1 H), 5.01 (dd, 1 H, J = 7.3, 2.0 Hz), 4.93 (dd, 1 H, J = 10.2, 2.0 Hz), 3.34 (s, 1 H), 3.11 (t, 2H, J = 7.3 Hz), 2.31 (m, 3H). MS (APCI+) 329.1 [M+1]. EXAMPLE 13 Cellular Assay for Measuring MEK Inhibition MEK inhibitors were evaluated by determining their ability to inhibit phosphorylation of
MAP kinase (ERK) in murine colon 26 (C26) carcinoma cells. Since ERK1 and ERK2 represent the only known substrates for MEKIand MEK2, the measurement of inhibition of ERK phosphorylation in cells provides direct read out of cellular MEK inhibition by the compounds of the invention. Detection of phosphorylation of ERK was carried out either by Western blot or ELISA format. Briefly, the assays involve treatment of exponentially growing C26 cells with varying concentrations of the test compound (or vehicle control) for one hour at 37° C. For Western blot assay, cells were rinsed free of compound/vehicle and lysed in a solution containing 70 mM NaCI, 50 mM glycerol phosphate, 10 mM HEPES, pH 7.4, 1% Triton X-100, 1 mM Na3V04, 100 μM PMSF, 10 μM leupeptin and 10 μM pepstatin. Supernatants were then subjected to gel electrophoresis and hybridized to a primary antibody recognizing dually phosphorylated ERK1 and ERK2. To evaluate total MAPK levels, blots were subsequently 'stripped' and re-probed with a 1 :1 mixture of polyclonal antibodies recognizing unphosphorylated ERK1 and ERK2. For pERK ELISA assay, pERK TiterZyme Enzyme immunometric Assay kits were acquired from Assay Designs, Inc (Ann Arbor, Ml). Briefly, cells were harvested in lysis solution containing 50mM β-glycerophosphate, 10mM HEPES, pH7.4, 70mM NaCI, 2mM EDTA and 1 %SDS and protein lysates were diluted 1 :15 with supplied Assay buffer prior to the execution of the assay. The subsequent steps were carried out essentially as recommended by the manufacturer. The inhibition data generated by the above protocols is disclosed in Table I. If several concentrations of inhibitor were tested, IC50 values (the concentration which gives 50% inhibition) were determined graphically from the dose response curve for % inhibition. Otherwise, percent inhibitions at measured concentrations are reported. Table I. Cellular Inhibition of ERK Phosphorylation by Compounds of the Invention
Figure imgf000035_0001
Figure imgf000036_0001
EXAMPLE 14 Carraqeenan-induced Footpad Edema (CFE) Rat Model Male outbred Wistar rats (135-150 g, Charles River Labs) are dosed orally with 10 mL/kg vehicle or test compound 1 hour prior to administration of a sonicated suspension of carrageenan (1 mg/0.1 mL saline). Carrageenan is injected into the subplantar region of the right hind paw. Paw volume is determined by mercury plethysmography immediately after injection and again five hours after carrageenan injection. Percent inhibition of edema is determined, and the ID40 calculated by linear regression. Differences in swelling compared to control animals are assessed by a 1-way ANOVA, followed by Dunnett's test. EXAMPLE 15 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 μg 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 Cll. EXAMPLE 16 SCW-induced monoarticular arthritis Arthritis is induced as described by Schwab et al., Infection and Immunity, 1991;59:4436-4442 with minor modifications. Rats receive 6 μg sonicated SCW [in 10 μL 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 μg of SCW (250 μL) administered IV. 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 hour 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 measurements are obtained by determining the baseline volumes of the sensitized hindpaw before reactivation on Day 21 , and comparing them with volumes at subsequent time points such as Day 22, 23, 24, and 25. Paw volume is determined by mercury plethysmography. EXAMPLE 17 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., 1998;39:9-17). Compounds are dissolved in solutions containing combinations of absolute ethanol, 0.2% hydroxypropyl methylcellulose in water, propylene glycol, cremophor, and dextrose, or other solvent or suspending vehicle. Mice are dosed orally or intraperitoneally once, twice or three times daily from the day of transplant (Day 0) through Day 13 or until grafts have been rejected. Rats are dosed once, twice, or three times daily from Day 0 through Day 13. Each animal is anesthetized and an incision is made at the base of the recipient ear, cutting only the dorsal epidermis and dermis. The incision is spread open and down to the cartilage parallel to the head, and sufficiently wide to accommodate the appropriate tunneling for a rat or insertion tool for a mouse. A neonatal mouse or rat pup less than 60 hours old is anesthetized and cervically dislocated. The heart is removed from the chest, rinsed with saline, bisected longitudinally with a scalpel, and rinsed with sterile saline. The donor heart fragment is placed into the preformed tunnel with the insertion tool and air or residual fluid is gently expressed from the tunnel with light pressure. No suturing, adhesive bonding, bandaging, or treatment with antibiotics is required. Implants are examined at 10- to 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 to 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. EXAMPLE 18 The analgesic activity of the compounds of the present invention is assessed by a test with rats. Rats weighing from 175 to 200 g are injected with carrageenan (2% in 0.9% sodium chloride aqueous solution, 100 μL injection volume) into the footpad of one hind limb. The rats are placed on a glass plate with illumination from a halogen lamp placed directly under the injected paw. The time (in seconds) from beginning illumination until the hindlimb was withdrawn from the glass was measured and scored as Paw Withdrawal Latency (PWL). Drug substances were given by oral gavage injection 2% hours after carrageenan injection to the footpad. PWL was measured prior to carrageenan injection, just prior to drug injection, and 1 , 2 (and sometimes 3) hours after drug injection. Carrageenan (a polysaccharide extracted from seaweed) causes a sterile inflammation when injected under the skin. Injection into the rat footpad causes little or no spontaneous pain-related behavior but induces hyperalgesia (pain-related behavioral responses of greater intensity than expected) to peripheral thermal or mechanical stimuli. This hyperalgesia is maximal 2 to 3 hours after injection. Treatment of rats with various analgesic drugs reduces hyperalgesia measured in this way and is a conventional test for detection of analgesic activity in rats. (Hargreaves K, Dubner R, Brown F, Flores C, Joris J. A new and sensitive method for measuring thermal nociception in cutaneous hyperalgesia. Pain, 1988;32:77-88 and Kayser V, Guilbaud G. Local and remote modifications of nociceptive sensitivity during carrageenan-induced inflammation in the rat. Pain, 1987;28:99-108). Untreated rats have a PWL of approximately 10 seconds. Carrageenan injection reduces PWL to approximately 3 seconds for at least 4 hours, indicating thermal hyperalgesia. Inhibition of the carrageenan thermal hyperalgesia response is determined by the difference between reduced PWL prior to drug and subsequent to drug treatment, and was expressed as percent inhibition of the response. Administration of MEK inhibitors dose-dependently reduced thermal hyperalgesia.

Claims

CLAIMS What is claimed is: 1. A compound of Formula I
Figure imgf000039_0001
wherein Q is d-β alkyl, C2-6 alkenyl, C2.s alkynyl, or C2.5 heteroaryl, wherein the d_6 alkyl, C2.6 alkenyl, and C2.6 alkynyl groups are optionally substituted with between 1 and 2 hydroxy substituents, and the C2.5 heteroaryl is optionally substituted with d^ alkyl, and further wherein the d.4 alkyl is optionally substituted with between 1 and 3 substituents independently selected from hydroxy and amino; R1 and R5 are each independently hydrogen or fluorine; R2 is hydrogen, chlorine, fluorine or methyl; R4 is bromine, chlorine, fluorine, iodine, C-|.6 alkyl, C2.6 alkenyl, C2.6 alkynyl, C3.6 cycloalkyl, -(CH2)-C3.6 cycloalkyl, cyano, -0-(CM alkyl), -S-(d-.2 alkyl), -SOCH3, -S02CH3, - S02NR6R7, -C≡C-(CH2)nNH2, -C≡C-(CH2)nNHCH3, -C=C-(CH2)nN(CH3)2, -C≡C-CH2OCH3, -C=C(CH2)nOH, -C=C-(CH2)nNH2, (Z)-CHCHCH2OCH3, -(Z)-CHCH-(CH2)nNHCH3, (Z)-CHCH- (CH2)nN(CH3)2, -(CH2)pC02R6, C(0)d-3 alkyl, C(0)NHCH3, -(CH2)mNH2, -(CH2)mNHCH3, -(CH2)mN(CH3)2, -(CH2)mOR6, -CH2S(CH2)t(CH3), -(CH2)PCF3, -C≡CCF3 , -CH=CHCF3, - CH2CHCF2, -CH=CF2, -(CF2)VCF3, -CH2(CF2)nCF3, -(CH2)tCF(CF3)2, -CH(CF3)2, -CF2CF(CF3)2, or -C(CF3)3, wherein the Cι.6 alkyl and C2.6 alkynyl are optionally substituted with between 1 and 3 substituents independently selected from hydroxy and alkyl; R6 and R7 are each independently hydrogen, methyl, or ethyl; m is 1 to 4; n is 1 to 2; p is 0 to 2; t is 0 to 1 ; v is 1 to 5; and pharmaceutically acceptable salts, d.6 amides and Cι-6 esters thereof. 2. The compound of Claim 1 wherein Q is Cι-6 alkyl, C2.6 alkenyl, or C2.5 heteroaryl, wherein the Cι_6 alkyl group is optionally substituted with between 1 and 2 hydroxy substituents and the C2.5 heteroaryl is optionally substituted with methyl. 3. The compound of Claim 1 , wherein Q is C2.5 heteroaryl containing from one to two heteroatoms independently selected from the group consisting of nitrogen, oxygen and sulfur, and further wherein the C2.5 heteroaryl is optionally substituted with d- alkyl, and further wherein the d^ alkyl is optionally substituted with between 1 and 3 substituents independently selected from hydroxy and amino. 4. The compound of Claim 1, wherein Q is methyl, -CH2OH, -(CH2) OH, butenyl, methyl-substituted oxazolyl, thiazolyl, methyl-substituted imidazolyl, or pyridinyl. 5. The compound of Claim 1 wherein ^ is fluorine. 6. The compound of Claim 1 wherein R5 is fluorine. 7. The compound of Claim 1 wherein R2 is fluorine. 8. The compound of Claim 1 wherein R4 is iodine, d-e alkyl, C2.6 alkenyl, or C2.6 alkynyl, and further wherein the d.6 alkyl, C2.6 alkenyl, and C2.6 alkynyl are optionally substituted with hydroxy. 9. The compound of Claim 1 which is 1-[3,4-Difluoro-2-(2-fluoro-4-iodo-phenylamino)-phenyl]-2-hydroxy-ethanone; [2-(4-Ethynyl-2-fluoro-phenylamino)-3,4-difluoro-phenyl]-pyridin-2-yl-methanone; [2-(4-Ethynyl-2-fluoro-phenylamino)-3,4-difluoro-phenyl]-thiazol-2-yl-methanone; [2-(4-Ethynyl-2-fluoro-phenylamino)-3,4-difluoro-phenyl]-(1-methyl-1 H-imidazol-2-yl)- methanone; [3,4-Difluoro-2-(2-fluoro-4-iodo-phenylamino)-phenyl]-(4-methyl-oxazol-2-yl)- methanone; 1-[3,4-Difluoro-2-(2-fluoro-4-iodo-phenylamino)-phenyl]-ethanone; 1-{3,4-Difluoro-2-[2-fluoro-4-(2-hydroxy-ethyl)-phenylamino]-pheny)}-2-hydroxy- ethanone; 1-[2-(4-Ethyl-2-fluoro-phenylamino)-3,4-difluoro-phenyl]-2-hydroxy-ethanone; 1-[2-(4-Ethynyl-2-fluoro-phenylamino)-3,4-difluoro-phenyl]-ethanone; 1-[2-(4-Ethyl-2-fluoro-phenylamino)-3,4-difluoro-phenyl]-5-hydroxy-pentan-1-one; 1-[3,4-Difluoro-2-(2-fluoro-4-vinyl-phenylamino)-phenyl]-2-hydroxy-ethanone; or 1-[2-(4-Ethynyl-2-fluoro-phenylamino)-3,4-difluoro-phenyl]-pent-4-en-1-one. 10. A pharmaceutical composition comprising a compound of Claim 1 and a pharmaceutically acceptable carrier. 11. 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. 12. A method of treating cancer in a patient in need thereof comprising administering a therapeutically effective amount of a compound of Claim 1. 13. A method of treating psoriasis in a patient in need thereof comprising administering a therapeutically effective amount of a compound of Claim 1. 14. A method of treating restenosis, autoimmune disease, atherosclerosis, rheumatoid arthritis, heart failure, chronic pain, neuropathic pain, or osteoarthritis in a patient in need thereof comprising administering a therapeutically effective amount of a compound of Claim 1. 15. 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 or at least one chemotherapeutic agent.
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