This application claims the benefit of U. S. Provisional Application No. 60/690,620 filed on June 14, 2005, the contents of which is hereby incorporated by reference in its entirety.

Field of the Invention

This invention relates to methods of preparing MEK inhibitor Λ/-[(R)-2,3-dihydroxy-propoxy]-3,4- difluoro-2-(2-fluoro-4-iodo-phenylamino)-benzamide that is useful for treating diseases mediated by MEK activity in mammals.

Background of the Invention

The compound Λ/-[(R)-2,3-dihydroxy-propoxy]-3,4-difluoro-2-(2-fluoro-4-i odo-phenylamino)- benzamide represented by formula 1

i is a highly specific non-ATP-competitive inhibitor of MEK1 and MEK2. The compound of formula ± (Compound I) is also known as the compound PD 0325901. Compound I is disclosed in WO 02/06213; WO 04/045617; WO 2005/040098; EP 1262176; U.S. Patent Application Pub. No. 2003/0055095 A1 ; U.S. Patent Application Pub. No. 2004/0054172 A1; U.S. Patent Application Pub. No. 2004/0147478 A1 ; and U.S. Patent Application No. 10/969,681, the disclosures of which are incorporated herein by reference in their entireties.

Numerous mitogen-activated protein kinase (MAPK) signaling cascades are involved in controlling cellular processes including proliferation, differentiation, apoptosis, and stress responses. Each MAPK module consists of 3 cytoplasmic kinases: a mitogen-activated protein kinase (MAPK), a mitogen-activated protein kinase kinase (MAPKK), and a mitogen-activated protein kinase kinase kinase (MAPKKK). MEK occupies a strategic downstream position in this intracellular signaling cascade catalyzing the phosphorylation of its MAP kinase substrates, ERK1 and ERK2. Anderson et al. "Requirement for integration of signals from two distinct phosphorylation pathways for activation of MAP kinase." Nature 1990, v.343, pp. 651-653. In the ERK pathway, MAPKK corresponds with MEK (MAP kinase ERK Kinase) and the MAPK corresponds with ERK (Extracellular Regulated Kinase). No substrates for MEK have been identified other than ERK1 and ERK2. Seger et al. "Purification and characterization of mitogen-activated protein kinase activator(s) from epidermal growth factor-stimulated A431 cells." J. Biol. Chem., 1992, v. 267, pp. 14373-14381. This tight selectivity in addition to the unique

ability to act as a dual-specificity kinase is consistent with MEK's central role in integration of signals into the MAPK pathway. The RAF-MEK-ERK pathway mediates proliferative and anti-apoptotic signaling from growth factors and oncogenic factors such as Ras and Raf mutant phenotypes that promote tumor growth, progression, and metastasis. By virtue of its central role in mediating the transmission of growth- promoting signals from multiple growth factor receptors, the Ras-MAP kinase cascade provides molecular targets with potentially broad therapeutic applications.

One method of synthesizing Compound I is disclosed in the above-referenced WO 02/06213 and

U.S. Patent Application Pub. No. 2004/0054172 A1. This method begins with the reaction of 2-fluoro-4- iodo-phenylamine and 2,3,4-trifluoro-benzoic acid in the presence of an organic base, such as lithium diisopropylamide, to form 3,4-difluoro-2-(2-fluoro-4-iodo-phenylamino)-benzoic acid, which is then reacted with (R)-0-(2,2-dimethyl-[1,3]dioxolan-4-ylmethyl)-hydroxylamine in the presence of a peptide coupling agent (e.g., diphenylphosphinic chloride) and a tertiary amine base (e.g., diisopropylethylamine). The resulting product is hydrolyzed under standard acidic hydrolysis conditions (e.g., p-TsOH in MeOH) to provide Compound 1. (R)-O-(2,2-dimethyl-[1,3]dioxolan-4-ylmethyl)-hydroxylamine is prepared by reaction of [(4S)-2,2-dimethyl-1,3-dioxolan-4-yl]methanol with N-hydroxyphthalimide in the presence of Ph ₃ P and diethyl azodicarboxylate.

Another method of synthesizing Compound I, which is disclosed in the above-referenced U.S.

Patent Application No. 10/969,681, comprises reaction of 3,4-difluoro-2-(2-fluoro-4-iodo-phenylamino)- benzoic acid with (R)-O-(2,2-dimethyl-[1,3]dioxolan-4-ylmethyl)-hydroxylamine in the presence of N ₁ N ¹ - carbonyldiimidazole. The resulting product is hydrolyzed with aqueous acid and crystallized to provide polymorphic form IV of Compound I.

Although the described methods are effective synthetic routes for small-scale synthesis of Compound I, there remains a need in the art for new synthetic routes that are safe, efficient and cost effective when carried out on a commercial scale.

Summary of the Invention

In one embodiment, the present invention provides a method of preparing Λ/-[(R)-2,3-dihydroxy- propoxy]-3,4-difluoro-2-(2-fluoro-4-iodo-phenylamino)-benzam ide, the method comprising: a) treating [(4S)-2,2-dimethyl-1,3-dioxolan-4-yl]methanol with trifluoromethansulfonic anhydride to form [(4R)-2,2-dimethyl-1 ,3-dioxolan-4-yl]methyl trifluoromethanesulfonate;

. b) reacting [(4R)-2,2-dimethyl-1 ,3-dioxolan-4-yl]methyl trifluoromethanesulfonate with N- hydroxyphthalamide to afford (R)-2-(2,2-dimethyI-[1,3]dioxolan-4-ylmethoxy)-isoindole-1,3 -dione; c) converting (R)-2-(2,2-dimethyl-[1 ,3]dioxolan-4-ylmethoxy)-isoindole-1 ,3-dione into 0-{[(4R)-2,2- dimethyl-1,3-dioxolan-4-yl]methyl}hydroxylamine; d) coupling 0-{[(4R)-2,2-dimethyl-1 ,3-dioxolan-4-yl]methyl}hydroxylamine with 3,4-difluoro-2-(2- fluoro-4-iodophenylamino)-benzoic acid using a carboxylic acid activating reagent to form N-{[(4R)-2,2- dimethyI-1,3-dioxolan-4-yl]methoxy}-3,4-difluoro-2-[(2-fluor o-4-iodophenyl)amino]benzamide; and e) deprotecting N-{[(4R)-2,2-dimethyl-1 ,3-dioxolan-4-yl]methoxy}-3,4-difluoro-2-[(2-fluoro-4- iodophenyl)amino]benzamide to produce Λ/-[(R)-2 ₁ 3-dihydroxy-propoxy]-3,4-difluoro-2-(2-fluoro-4-iodo- phenylamino)-benzamide.

In another embodiment, the present invention provides a method of preparing Λ/-[(R)-2,3- dihydroxy-propoxy]-3,4-difluoro-2-(2-fluoro-4-iodo-phenyIami no)-benzamide, wherein steps a) through c) are carried out as a one-pot reaction.

In another embodiment, the present invention provides a method of preparing Λ/-[(R)-2,3- dihydroxy-propoxy]-3,4-difluoro-2-(2-fluoro-4-iodo-phenylami no)-benzamide, wherein step c) comprises use of aqueous ammonia for converting (R)-2-(2,2-dimethyl-[1,3]dioxolan-4-ylmethoxy)-isoindole-1,3 - dione into 0-{[(4R)-2,2-dimethyl-1 ,3-dioxolan-4-yl]methyl}hydroxylamine.

In another embodiment, the present invention provides a method of preparing Λ/-[(R)-2,3- dihydroxy-propoxy]-3,4-difluoro-2-(2-fluoro-4-iodo-phenylami no)-benzamide, wherein 3,4-difluoro-2-(2- fluoro-4-iodophenylamino)-benzoic acid is prepared by a reaction comprising coupling a compound of formula 2

2 wherein X is halogen with 2-fluoro-4-iodoaniline in the presence of a Group I metal cation amide.

In another embodiment, the present invention provides a method of preparing Λ/-[(R)-2,3- dihydroxy-propoxy]-3,4-difluoro-2-(2-fluoro-4-iodo-phenylami no)-benzamide, wherein X is fluorine and the Group I metal cation amide is lithium amide.

In another embodiment, the present invention provides a method of preparing Λ/-[(R)-2,3- dihydroxy-propoxy]-3,4-difluoro-2-(2-fluoro-4-iodo-phenylami no)-benzamide, wherein a small amount of the compound of formula 2 and 2-fluoro-4-iodoaniline is initially added to lithium amide in an aprotic solvent followed by slow continuous addition of a remaining portion. In another embodiment, the present invention provides a method of preparing Λ/-[(R)-2,3- dihydroxy-propoxy]-3,4-difluoro-2-(2-fluoro-4-iodo-phenylami no)-benzamide, wherein the aprotic solvent is tetrahydrofuran.

In another embodiment, the present invention provides a method of preparing Λ/-[(R)-2,3- dihydroxy-propoxy]-3,4-difluoro-2-(2-fluoro-4-iodo-phenylami no)-benzamide, wherein the carboxylic acid activating reagent in step d) is 1 ,1 '-carbonyldiimidazole.

In another embodiment, the present invention provides a method of preparing Λ/-[(R)-2,3- dihydroxy-propoxy]-3,4-difluoro-2-(2-fluoro-4-iodo-phenylami no)-benzamide, wherein the carboxylic acid activating reagent in step d) is thionyl chloride.

In another embodiment, the present invention provides a method of preparing /V-[(R)-2,3- dihydroxy-propoxy]-3,4-difluoro-2-(2-fluoro-4-iodo-phenylami no)-benzamide comprising at least one single-phase system crystallizing of Λ/-[(R)-2,3-dihydroxy-propoxy]-3,4-difluoro-2-(2-fluoro-4-i odo- phenylamino)-benzamide.

- A -

In another embodiment, the present invention provides a method of preparing Λ/-[(R)-2,3- dihydroxy-propoxy]-3,4-difluoro-2-(2-fIuoro-4-iodo-phenylami no)-benzamide, wherein a first single-phase system crystallizing of Λ/-[(R)-2,3-dihydroxy-propoxy]-3,4-difluoro-2-(2-fluoro-4-i odo-phenylamino)- benzamide is carried out in toluene with from 1 to 20 % v/v of acetonitrile and a second single-phase system crystallizing of A/-[(R)-2,3-dihydroxy-propoxy]-3,4-difluoro-2-(2-fluoro-4-io do-phenylamino)- benzamide is carried out in a mixture of ethanol and toluene.

Definitions and Abbreviations of Terms

Compound I is Λ/-[(R)-2,3-dihydroxy-propoxy]-3,4-difluoro-2-(2-fluoro-4-i odo-phenylamino)- benzamide

The phrase "mediated by MEK activity" refers to biological or molecular processes that are regulated, modulated, or inhibited by MEK protein kinase activity. For certain applications, inhibition of the protein kinase activity associated with MAPK signaling cascades, among others, and those which inhibit abnormal cell growth or inflammation are preferred. As used herein, the term "alkyl" means a branched- or straight-chained (linear) paraffinic hydrocarbon group (saturated aliphatic group) having from 1 to 10 carbon atoms in its chain, which may be generally represented by the formula C _k H _2k+ i, where k is an integer of from 1 to 10. Examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, pentyl, n-pentyl, isopentyl, neopentyl, and hexyl, and the simple aliphatic isomers thereof. The term "aryl" means an aromatic monocyclic or fused polycyclic ring structure having a total of from 4 to 18, preferably 6 to 18, ring carbon atoms (no heteroatoms). Exemplary aryl groups include phenyl, naphthyl, anthracenyl, and the like.

The phrase "Group I metal cation" means Li ⁺ , Na ⁺ , K ⁺ , Rb ⁺ , Cs ⁺ , or Fr ⁺ .

The phrase "Group Il metal cation" means Be ⁺² , Mg ⁺² , Ca ⁺² , Sr ⁺² , Ba ⁺² , or Ra ⁺² . The phrase "Group I metal cation amide" means a compound in which a hydrogen atom on nitrogen from ammonia or an amine is replaced by a metal cation which is Li ⁺ , Na ⁺ , K ⁺ , Rb ⁺ , Cs ⁺ , or Fr ⁺ . The phrase "Group Il metal cation amide" means a compound in which a hydrogen atom on nitrogen from ammonia or an amine is replaced by a metal cation which is Be ⁺² , Mg ⁺² , Ca ⁺² , Sr ⁺² ,

Ba ⁺² , or Ra ⁺² . The phrase "Group I metal cation dialkylamide" means a compound in which a hydrogen atom on nitrogen from a dialkylamine, which comprises two independent unsubstituted alkyl groups as defined above, is replaced by a metal cation which is Li ⁺ , Na ⁺ , K ⁺ , Rb ⁺ , Cs ⁺ , or Fr ⁺ . Illustrative examples of a Group I metal cation diaikylamide includes lithium diisopropylamide.

The phrase "Group Il metal cation dialkylamide" means a compound in which a hydrogen atom on nitrogen from a dialkylamine, which comprises two independent unsubstituted alkyl groups as defined above, is replaced by a metal cation which is Be ⁺² , Mg ⁺² , Ca ⁺² , Sr ⁺² , Ba ⁺² , or Ra ⁺² . Illustrative examples of a Group Il metal cation dialkylamide includes magnesium bis(diisopropylamide).

The phrase "Group I metal cation bis(trialkylsilyl)amide" means a compound in which a hydrogen atom on nitrogen from a bis(trialkylsilyl)amine, which comprises two independent trialkylsilyl groups

wherein each alkyl is independently unsubstituted alkyl as defined above, is replaced by a metal cation which is Li ⁺ , Na ⁺ , K ⁺ , Rb ⁺ , Cs ⁺ , or Fr ⁺ . Illustrative examples of a Group I metal cation bis(trialkylsilyl)amide includes lithium bis(trimethylsilyl)amide ("LiHDMS" or "lithium hexamethyldisilazane"). The phrase "Group Il metal cation bis(trialkylsilyl)amide" means a compound in which a hydrogen atom on nitrogen from a bis(trialkylsilyl)amine, which comprises two independent trialkylsilyl groups wherein each alkyl is independently unsubstituted alkyl as defined above, is replaced by a metal cation which is Be ⁺² , Mg ⁺² , Ca ⁺² , Sr ⁺² , Ba ⁺² , or Ra ⁺² . Illustrative examples of a Group Il metal cation bis(trialkylsilyl)amide includes magnesium di[bis(trimethylsilyl)amide]. The phrase "Group I metal cation alkoxide" means a compound in which a hydrogen atom on oxygen from an alcohol, which comprises an unsubstituted alkyl group as defined above, is replaced by a metal cation which is Li ⁺ , Na ⁺ , K ⁺ , Rb ⁺ , Cs ⁺ , or Fr ⁺ . Illustrative examples of a Group I metal cation alkoxide includes lithium methoxide, sodium ethoxide, and potassium terf-butoxide.

The phrase "Group Il metal cation alkoxide" means a compound in which a hydrogen atom on oxygen from an alcohol, which comprises an unsubstituted alkyl group as defined above, is replaced by a metal cation which is Be ⁺² , Mg ⁺² , Ca ⁺² , Sr ⁺² , Ba ⁺² , or Ra ⁺² . Illustrative examples of a Group Il metal cation alkoxide includes magnesium bismethoxide and calcium bisethoxide.

Of the above mentioned bases, preferred are bases which comprise a salt of a Group I metal cation. More preferred are bases which comprise a salt of Li ⁺ , Na ⁺ , or K ⁺ . Still more preferred are bases which comprise a salt of Li ⁺ . However, any base whereof the conjugate acid has a pKa ≥ 16 is suitable for the invention process.

The phrase "carboxylic acid activating reagent" means a reagent which activates a -C(=O)OH group, or the corresponding conjugate base (ie, -C(=O)O~), towards a coupling reaction that involves displacement of the OH or O~, respectively. Illustrative examples of carboxylic acid activating reagents include lipase enzymes, mineral acids, including HCI and sulfuric acid, boron trifluoride etherate, 2,4,6-trichloro-1,3,5-triazine, 3-nitro-2-pyridinesulfenyl chloride, trifluoroacetic anhydride, mesyl chloride, S(O)CI ₂ , S(O) ₂ CI ₂ , P(O)CI ₃ , oxalyl chloride, (phenyl) ₂ P(=O)CI ("DPPCI"), 1,1'-carbonyldiimidazole ("CDI"), triphenylphosphine/diethylazodicarboxylate, N.N'-dicyclohexylcarbodiimide ("DCC"), the water soluble carbodiimides, including 1-(3-dirnethylaminopropyl)-3-ethylcarbodiimide hydrochloride ("EDC") and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide methiodide, 2-ethoxy-1-ethoxycarbonyl- 1 ,2-dihydroquinoline ("EEDQ"), benzotriazol-1-yloxy-tris(dimethylamino)-phosphonium hexafluorophosphate ("BOP"), and bromo-tris(pyrrolidino)-phosphonium hexafluorophosphate ("PyBrOP"). Additional carboxylic acid activating reagents may be found in Comprehensive Organic Transformations, by Richard C. Larock, VCH Publishers, Inc, New York, 1989. Preferred carboxylic acid activating reagents are selected from: (COCI) ₂ , S(O)CI ₂ , S(O) ₂ CI ₂ ,

P(O)Cl3, (phenyl) ₂ P(=O)CI, 1,1 -carbonyldiimidazole, triphenylphosphine/diethylazodicarboxylate, EDC, EDCI, and N,N'-dicyclohexylcarbodiimide.

The term "hydrazinolysis" means a chemical method that uses hydrazine or its derivatives to cleave amide bonds.

The phrase "reactive functional group" means a group that is expected to react with certain solvents, reagents, catalysts, reaction starting materials, reaction intermediates, or reaction products under the particular reaction conditions employed. Examples of reactive functional groups include, but are not limited to, NH ₂ , OH, SH, CO ₂ H, N=C=O, C(O)CI, and the like.

The phrase "non-nucleophilic base" means a base that is slow to act as a nucleophile in a substitution reaction such as, for example, a nucieophilic aromatic substitution reaction. Examples of non- nucleophilic bases include tertiary organic amines, which are defined below, Group I metal cation hydrides, Group Il metal cation hydrides, Group I metal cation dialkylamides, Group Il metal cation dialkylamides, Group I metal cation bis(trialkylsilyl)amides, Group Il metal cation bis(trialkylsilyl)amides, Group I metal cation terf/a/y-alkoxides, and Group Il metal cation terf/a/y-alkoxides.

The phrase "acid catalyst" means a Brønsted acid or Lewis acid which may be present in catalytic, stoichiometric, or greater than stoichiometric amounts. The phrase "aprotic solvent" means a solvent that does not yield a proton (i.e., acts as a Brønsted acid) under the particular conditions employed. This means that the pKa (relative to water or, optionally, DMSO) of an aprotic solvent is greater than the pKa of the conjugate acid of the strongest base employed. Typical aprotic solvents with high pKa's (i.e., >30) include diethyl ether, tetrahydrofuran, dioxane, dimethylsulfoxide, hexane, heptane, dimethylformamide, toluene, and benzene. Typical aprotic solvents with lower pKa's (ie, 19<pKa<30) include ethyl acetate, acetone, and acetonitrile. Solvents with pKa's less than 19 such as, for example, tert-butyl alcohol, usually are not aprotic, although nitromethane is an aprotic solvent. Solvents that contain a functional group selected from OH, NH, and SH are typically not aprotic.

The phrase "protic solvent" or "protic contaminant" mean a solvent or contaminant, respectively, that does yield a proton under the particular conditions employed.

The phrase "tertiary organic amine" means a trisubstituted nitrogen group wherein the three substituents are independently selected from Ci-Ci ₂ alkyl, 03-C-J ₂ cycloalkyl, and benzyl, or wherein two of the three substituents are taken together with the nitrogen atom to which they are attached to form a

5-membered or 6-membered, monocyclic heterocycle containing one nitrogen atom and carbon atoms and the third substituent is selected from C-|-Ci ₂ alkyl, C3-Ci ₂ cycloalkyl, and benzyl, or wherein the three substituents are taken together with the nitrogen atom to which they are attached to form a 7-membered to 12-membered bicyclic heterocycle containing 1 or 2 nitrogen atoms total and carbon atoms, and optionally having a carbon-nitrogen double bond ("C=N") when 2 nitrogen atoms are present. Illustrative examples of a tertiary organic amine include triethylamine, diisopropylethylamine, benzyldiethylamine, dicyclohexyl-methyl-amine, 1,8-diazabicyclo[5.4.0]undec-7-ene ("DBU"), 1,4-diazabicyclo[2.2.2]octane ("TED"), and 1,5-diazabicyclo[4.3.0]non-5-ene.

The term "purifying" means separating a desired compound from undesired components of a mixture which contains both by methods which include distillation, chromatography, including column chromatography, thin layer chromatography, normal phase chromatography, reverse phase chromatography, gas phase chromatography, and ion exchange chromatography, precipitation,

extraction, rotary evaporation, chemical-based trapping by reaction with an incompatible functional group, including quenching with polymer-bound quenching reagents, filtration, centrifugation, physical separation, and fractional crystallization.

The phrase "carried out on a commercial scale" means a process, which employs more than 1 kilogram of a reagent.

The term "polymorph" refers to different crystalline forms of the same compound and includes, but is not limited to, other solid state molecular forms including hydrates (e.g., bound water present in the crystalline structure) and solvates (e.g., bound solvents other than water) of the same compound.

The term "peak intensities" refers to relative signal intensities within a given X-ray diffraction pattern. Factors which can affect the relative peak intensities are sample thickness and preferred orientation (i.e. the crystalline particles are not distributed randomly).

The term "peak positions" as used herein refers to X-ray reflection positions as measured and observed in X-ray powder diffraction experiments. Peak positions are directly related to the dimensions of the unit cell. The term "slurry" refers to a solid substance suspended in a liquid medium, typically water or an organic solvent.

The term "separating from" refers to a step in a synthesis in which the desired agent is isolated from other non-desired agents, including, but not limited to any of the following steps: filtering, washing with extra solvent or water, drying with heat and or under vacuum. The term "X-ray powder diffraction pattern" or PXRD refers to the experimentally observed diffractogram or parameters derived therefrom. X-Ray powder diffraction patterns are characterized by peak position (abscissa) and peak intensities (ordinate).

As used in the present application, "DSC" means Differential Scanning Calorimetry.

The term "one-pot reaction" (process, procedure, etc.) refers to a reaction or series of reaction steps (process, procedure, etc.) carried out with no isolation of intermediates.

As used in the present application, "Et" means ethyl, "Ac" means acetyl, "Me" means methyl, "Ms" means methanesulfonyl (CH ₃ SO ₂ ), "iPr" means isopropyl, "Ph" means phenyl, "EtOAc" means ethyl acetate, "HOAc" means acetic acid, "NEt ₃ " or "Et ₃ N" means triethylamine, "Tf means trifluoromethanesulfonyl, "THF" means tetrahydrofuran, "GDI" means 1,1'-carbonyldiimidazole, "HOBt" means hydroxy benzotriazole, "MeOH" means methanol, "i-PrOAc" means isopropyl acetate, "KOAc" means potassium acetate, "DMSO" means dimethylsulfoxide, "AcCI" means acetyl chloride, "CDCI ₃ " means deuterated chloroform, "MTBE" means methyl t-butyl ether, "DMF" means dimethyl formamide, and "Ac ₂ O" means acetic anhydride.

Brief Description of the Drawings

Figure 1 is an X-ray powder diffraction diagram of polymorphic Form IV of Λ/-[(R)-2,3-dihydroxy-propoxy]- 3,4-difluoro-2-(2-fluoro-4-iodo-phenylamino)-benzamide.

Detailed Description of the Invention

The present invention provides a new synthetic route including Steps I through Step III to the MEK inhibitor Λ/-[(R)-2,3-dihydroxy-propoxy]-3,4-difluoro-2-(2-fluoro-4-i odo-phenylamino)-benzamide (Compound I).

Step I: Preparation of 0-{r(4RV2.2-dimethyl-1.3-dioxolan-4-ynmethyl}hydroxylanπine (6) The method of the present invention comprises a novel Step I of preparing of 0-{[(4R)-2,2- dimethyl-1 ,3-dioxolan-4-yl]methyl}hydroxylamine (6) from [(4S)-2,2-dimethyl-1 ,3-dioxoIan-4-yl]methanol (1) through the formation of [(4R)-2,2-dimethyl-1 ,3-dioxolan-4-yl]methyl trifluoromethanesulfonate (3) and its coupling with N-hydroxyphthalimide (4) to afford 2-{[(4R)-2,2-dimethyl-1 ,3-dioxolan-4-yl]methoxy}-1 H- isoindole-1 ,3(2H)-dione (5), which is subsequently de-protected to give 6 as shown in Scheme 1.

Scheme 1

The reaction of compound (1) with trifluoromethanesulfonic anhydride (2) is carried out in the presence of a non-nucleophilic base, such as, for example, a tertiary organic amine, in an aprotic solvent at a temperature of from -5O ⁰ C to 5 ⁰ C, preferably, at a temperature less than -15 ⁰ C, to form triflate (3). A preferred tertiary organic amine is triethylamine, and a preferred solvent is toluene. Treatment of triflate (3) with N-hydroxyphthalimide (4) furnishes phthalimide (5), which can be isolated if desired. However, in order to minimize processing time and increase overall yield, 0-{[(4R)- 2,2-dimethyl-1,3-dioxolan-4-yl]methyl}hydroxylamine (6) can be prepared in a one-pot process with no phthalimide (S) isolation.

Cleavage of the phthalimide function could be achieved by methods known in the art, for example, by hydrazinolysis. However, the use of less hazardous aqueous or anhydrous ammonia instead of methyl hydrazine (CH ₃ NHNH ₂ ) is preferred.

Step II: Preparation of 3.4-difluoro-2-(2-fluoro-4-iodophenylamino)-benzoic acid (9) As shown in Scheme 2, Step Il of the method of the present invention provides 3,4-difluoro-2-(2- fluoro-4-iodophenylamino)-benzoic acid (9).

Scheme 2

Preparation of compound (9) can be carried out by reacting compound (7), wherein X is halogen, or O-SC^R^ or 0-P( ³ O)(OR^, wherein R^ is alkyl or aryl, with compound (8) optionally in a solvent, and in the presence of from about 1 mol equivalent to about 10 mol equivalents of at least one base, wherein the base is selected from: a Group I metal cation hydride or a Group 2 metal cation hydride, including lithium hydride, sodium hydride, potassium hydride, and calcium hydride, a Group I metal cation dialkylamide or a Group 2 metal cation dialkylamide, including lithium diisopropylamide, a Group I metal cation amide or a Group 2 metal cation amide, including lithium amide, sodium amide, potassium amide, a Group I metal cation alkoxide or a Group 2 metal cation alkoxide, including sodium ethoxide, potassium terf-butoxide, and magnesium ethoxide, and a Group I metal cation hexamethyldisilazide, including lithium hexamethyldisilazide; for a time, and at a temperature, sufficient to yield compound (9).

Preferably, preparation of compound (9) is carried out by reacting compound (7), wherein X is halogen, more preferably, X is fluorine, in an aprotic solvent with compound (8) in the presence of from about 3 mol equivalents to about 5 mol equivalents of a Group I metal cation amide at a temperature of from 2O C to 55 ^° C, more preferably, at a temperature from 45 ^° C to 55 ^° C. A catalytic amount of Group I metal cation dialkylamide can be added if necessary. A preferred Group I metal cation amide is lithium amide, a preferred Group I metal cation dialkylamide is lithium diisopropylamide, and a preferred solvent is tetrahydrofuran. Preferably, the reaction is performed by adding a small amount of compound (7) and compound (8) to lithium amide in tetrahydrofuran followed by slow continuous addition of the remaining

portion. This procedure minimizes the risk of reactor over-pressurization due to gas side product (ammonia) generation.

Step III: Preparation of N-((RV2.3-dihydroxypropoxy)-3.4-difluoro-2-(2-fluoro-4-iodo- phenylamino)- benzamide (Compound I)

Compound I can be obtained by coupling 0-{[(4R)-2,2-dimethyl-1,3-dioxolan-4- yl]methyl}hydroxylamine (6) with 3,4-difluoro-2-(2-fluoro-4-iodophenylamino)-benzoic acid (9) using a carboxylic acid activating reagent such as, for example, COCI2, S(O)C^, S(O)2Cl2, P(O)Cl3, triphenylphosphine/diethylazodicarboxylate, diphenylphosphinic chloride, N, N'-dicyclohexylcarbodiimide, (benzotriazol-1 -yloxy)tripyrolidinophosphonium hexafluorophosphate, (benzotriazol-1 - yloxy)tris(dimethylamino)phosphonium hexafluorophosphate, N-ethyl-N'-(3- dimethylaminopropyl)carbodiimide hydrochloride, or 1,1'-carbonyldiimidazole (CDI).

A preferred carboxylic acid activating reagent is 1,1'-carbonyldimidazole (CDI) shown in Scheme 3. Preparation of the desirable polymorphic Form IV of Compound I using CDI is described in the above- referenced U.S. Patent Application No. 10/969,681.

Scheme 3

10

10 11 Compound I

In according to the present invention, the method was modified to include the advantageous procedure for product purification and isolation, which procedure is performed in single-phase systems such as, for example, toluene/acetonitrile for the first isolation/crystallization and ethanol/toluene for the second recrystallization. Water addition, implemented in the previous procedure, was omitted to avoid the two-phase crystallization from the immiscible water-toluene system that caused inconsistent product purity. The one-phase procedure of the present invention provides consistent control and removal of un- reacted starting material and side products.

Alternatively, Compound I can be obtained by coupling 0-{[(4R)-2,2-dimethyl-1,3-dioxolan-4- yl]methyl}hydroxylamine (6) with 3,4-difluoro-2-(2-fluoro-4-iodophenylamino)-benzoic acid (9) using thionyl chloride (SOCI ₂ ) as shown in Scheme 4.

Scheme 4

Compound I

Examples

The reagents and conditions of the reactions described herein are merely illustrative of the wide variety of starting materials, their amounts and conditions which may be suitably employed in the present invention as would be appreciated by those skilled in the art, and are not intended to be limiting in any way.

HPLC (Conditions A): 10 μL injection volume onto Agilent Zorbax RX-C18 150 mm x 4.6 mm x 3.5 μm column at 30 ^° C column temperature, 1.0 mL/min flow rate and detection at 246 nm. Mobile phase A (v/v): 25 mM Acetate Buffer, pH 6.0; Mobile phase B (v/v): Acetonitrile, and Linear Gradient Table:

Sample Preparation: Dilute 100 μL reaction mixture to 10 mL with acetonitrile. Mix in a vial 200 μL of this sample solution with 300 μL carbonate buffer pH 10.0 and 300 μL solution of 2-mercaptopyridine in acetonitrile (18 mM), heat the vial for 10 minutes at 50 ⁰ C and dilute to 1:1 ratio in mobile phase A.

GC (Conditions B): 1 μL injection onto an RTX-5 column (30 m x 0.25 mm x 0.25 μm) with initial oven temperature of 120 ^° C for 2 min. to final temperature of 250 ^° C in 15 ^° C/minute ramping and a final time of 2.33 min; Flow rate: 1 mL/min.

HPLC (Conditions C): 5 μL injection onto Phenomenex Luna C18(2) 150 mm x 4.6 mm x 3μm column ; flow rate : 1.0 mL/min; detection at 225 nm; mobile phase A: 95/5 v/v Water/Acetonitrile with 0.1% Trifluoroacetic acid (TFA), mobile phase B: 5/95 v/v Water/Acetonitriie with 0.1% TFA; Linear Gradient Table:

Sample preparation: Dilute 1 ml_ reaction mixture to 100 mL with acetonitrile and dilute 1 mL of this solution to 10 mL with 50:50 Water/Acetonitrile.

HPLC (Conditions D): 5 μL injection onto Waters SymmetryShield RP 18, 150 mm x 4.6 mm x 3.5 μm column; flow rate: 1.0 mL/min; detection at 235 nm; mobile phase A: 25 mM Acetate Buffer adjusted to pH 5.5, mobile phase B: Acetonitrile; Linear Gradient Table:

Sample preparation: Dilute 40 μL of reaction mixture in 20 mL acetonitrile.

HPLC (Conditions E): 10 μL sample injection onto YMC ODS-AQ 5 μm, 250 mm x 4.6 mm column; flow rate: 1.0 ml_/min; detection at 280 nm; temperature 30 ^° C; mobile phase : 75/25 v/v Acetonitrile/Water with 0.1% Formic acid.

Sample preparation: Quench reaction mixture sample with dipropylamine and stir for about 5 minutes before further dilution with mobile phase.

DSC measurement was performed using a Mettler-Toledo DSC 822, temperature range 25 ^° to 150 ^° C with 5 ^° C/min heating rate in a 40 μL aluminum pan. Experimental Conditions for Powder X-Rav Diffraction (XRD):

A Rigaku Miniflex+ X-ray diffractometer was used for the acquisition of the powder XRD patterns. The instrument operates using the Cu Ka ₁ emission with a nickel filter at 1.50451 units. The major instrumental parameters are set or fixed at:

X-ray: Cu / 30 kV (fixed) / 15 mA (fixed)

Divergence Slit: Variable Scattering Slit: 4.2° (fixed) Receiving Slit: 0.3 mm (fixed) Scan Mode: FT Preset Time: 2.0 s Scan Width: 0.050° Scan Axis: 2Theta/Theta Scan Range: 3.000° to 40.000°

Jade Software Version: 5.0.36(SP1) 01/05/01 (Materials Data, Inc.) Rigaku Software: Rigaku Standard Measurement for Windows 3.1 Version 3.6

(1994-1995)

Example 1. Preparation of 0-ffl4R)-2.2-dimethyl-1.3-dioxolan-4-vπmethyl}hvdroxylamine (6)

A solution containing [(4S)-2,2-dimethyl-1,3-dioxolan-4-yl]methanol (1) (13.54 ml_, 0.109 mol) (DAISO Co., Ltd., CAS# 22323-82-6) and triethylamine (18.2 ml_, 0.131 mol) in 115 mL toluene was cooled to -15 C, then trifluoromethanesulfonic anhydride (2) (18.34 mL, 30.75 g, 0.109 mol) (Aldrich, Catalog # 17,617-6 ) was added drop wise while maintaining the temperature at less than -15 ^° C. The mixture was then stirred for 2 hours, and transferred to a separate flask containing a mixture (slurry) of N- hydroxyphthalimide (4) (18.99 g, 0.116 mol) (Aldrich, Catalog # H5.370-4) and 18.2 mL (0.13 mol) triethylamine in 95 mL toluene. The resulting mixture was warmed to 20-25 ^° C and stirred for at least 5 hours or until reaction completion (determined by HPLC (Conditions A)). Water (93 mL) was then added to quench the reaction mixture, the phases were separated, and the bottom aqueous layer was discarded. The water quench was repeated two more times resulting in a pale yellow organic layer. The organic layer was heated to 35 C and treated with 36.7 mL ammonium hydroxide solution (contains about 28-29% wt/wt ammonia). The mixture was stirred for at least 12 hours or until the reaction was deemed complete as determined by GC (Conditions B). The water was then removed under reduced pressure by co- distilling it with toluene to about half of the original volume at temperatures around 35-45 C. Toluene (170 mL) was added to the concentrated solution and the distillation was repeated. A sample was drawn for water content determination by Karl Fisher method (using EM Science Aquastar AQV-2000 Titrator with a sample injected to a pot containing methanol and salicylic acid). The distillation was repeated ifl water content was more than 0.1%. The concentrated solution was filtered to remove the white solid side product, and the filtrate was stored as 112mL (98 g) product solution containing 9.7% w/w compound 6 in toluene. This solution was ready for use in the final coupling step (Example 3). Overall chemical yield was 59%. A small sample was evaporated to yield a sample for NMR identification.

¹ H NMR (400 MHz, CDCI ₃ ): δ 5.5 (bs, 2H), 4.35 (m, 1H), 4.07 (dd, 1H), 3.77 (m, 2H), 3.69 (dd, 1H), 1.44 (s, 3H), 1.37 (s, 3H).

Example 2. Preparation of 3.4-difluoro-2-(2-fluoro-4-iodophenylamino)-benzoic acid (9)

A solution of 2-fluoro-4-iodoaniline (8) (16.4 g, 0.069 mol) (Aldrich, Catalog # 30,660-6) and 2,3,4- trifluorobenzoic acid (7) (11.98 g, 0.068 mol) (Aldrich, Cat # 33,382-4) in 38 mL tetrahydrofuran (THF) was prepared and a portion (about 5%) of this solution was added to a stirring slurry of lithium amide (5 g, 0.22 mol) in 40 mL THF at 50-55 C. After about 15-30 min. an exotherm followed by gas release and color change are observed. The remaining portion of the (8) and (7) solution was added slowly over 1-2 hr while maintaining temperatures within 45-55 ^° C. The mixture was stirred until the reaction was deemed complete (by HPLC (Conditions C). The final mixture was then cooled to 20-25 ^° C and transferred to another reactor containing 6 N hydrochloric acid (47 mL) followed by 25 mL acetonitrile, stirred, and the bottom aqueous phase was discarded after treatment with 40 mL 50% sodium hydroxide solution. The organic phase was concentrated under reduced pressure and 57 mL acetone was added. The mixture was heated to 50 ^° C, stirred, and added with 25 mL warm (40-50 ^° C) water and cooled to 25-30 ^° C to allow crystallization to occur (within 1-4 hours). Once the crystallization occurred, the mixture was further cooled to 0 to -5 ^° C and stirred for about 2 hours. The solid product was filtered and the wet cake was dried in vacuum oven at about 55 ^° C. Overall chemical yield was 21.4 g, 80%.

¹ H NMR (400 MHz, (CD ₃ ) ₂ SO): δ 13.74 (bs, 1H), 9.15 (m, 1 H), 7.80 (dd, 1H), 7.62 (d, 1H), 7.41 (d, 1H), 7.10 (q, 1H), 6.81 (m, 1H).

Example 2B. Preparation of 3.4-difluoro-2-(2-fluoro-4-iodophenylamino)-benzoic acid (9) by the solid addition of lithium amide method

To a stirring solution of 2,3,4-trifluorobenzoic acid (13) (5.0 g, 28.4 mmol) and 2-fluoro-4- iodoaniline (14) (6.73 g, 28.4 mmol) in MeCN (100 mL), under N ₂ atmosphere was added lithium amide (2.61 g, 113.6 mmol) in small portions. The reaction mixture was heated to reflux for 45 minutes, cooled to ambient temperature and quenched with 1 N HCI and then water. The yellowish white precipitate was filtered, washed with water. The solid was triturated in CH ₂ CI ₂ (30 mL) for 1h, filtered and dried in a vacuum oven at 45 ^° C for 14 hours to give 8.Og (72%) of compound (9) as an off-white solid, mp 201.5-203 ^° C.

Example 3. Preparation of N-((R)-2.3-dihvdroxypropoxy)-3.4-difluoro-2-(2-fluoro-4-iodo -phenylamino)- benzamide (Compound \)

3,4-Difluoro-2-(2-fluoro-4-iodophenylamino)-benzoic acid (9) (20 g, 0.051 mol) in 100 mL acetonitrile was treated with 1,1'-carbonyldiimidazole (CDI) (8.66 g, 0.053 mol) (Aldrich, Cat # 11,553-3) and stirred for about 2 hours at 20-25 ^° C until the reaction was deemed complete by HPLC (Conditions D). 94 mL (84.9 g) of 9.7% w/w solution of O-{[(4R)-2,2-dimethyl-1,3-dioxolan-4-yl]methyl}hydroxylamine (6) in toluene was then added and stirred for about 4 hours or until the reaction was deemed complete by HPLC (Conditions D). To this mixture was added 66 mL of 5.6 % hydrochloric acid solution, and after stirring, the bottom aqueous phase was discarded. Again 66 mL of 5.6 % hydrochloric acid solution was added to the organic phase and stirred at 20-25 ^° C for 12-18 hours or until the reaction was deemed complete by HPLC (Conditions D). The bottom layer was then discarded and the remaining organic layer was concentrated under reduced pressure to remove about 10-20% solvent, and the volume was adjusted to about 9-11 mL/g with toluene (80 mL). Crude product was then crystallized at 10-15 ^° C. The slurry was allowed to stir for about 2 hours and the crude solid product was filtered, and dried. The dried crude product was recharged to the reactor and dissolved into 150 mL of 5% v/v ethanol/toluene mixture at 55- 67 ^° C. The solution was then clarified at this temperature through filter (line filter) to remove any remaining particulate matter. The solution was then cooled slowly to 5 ^° C to crystallize and stirred for at least 2 h, filtered and dried. The dried solid product was redissolved in EtOH (60 mL) at 35 ^° C, and product was precipitated out by adding water (300 mL) at 35 ^° C followed by cooling to 20 ⁰ C. The slurry was stirred for at least 2 hours to transform the crystals to the desired polymorphic Form IV as determined by DSC and Powder X-ray Diffraction pattern (PXRD). The slurry was filtered and dried under vacuum oven at 70- 90 ^° C to yield the final N-((R)-2,3-dihydroxypropoxy)-3,4-difluoro-2-(2-fluoro-4-iodo -phenylamino)- benzamide (Compound I) product. Overall chemical yield was 13 g, 53%. Melting point (DSC): 112+1 ^° C. Appearance: White to off-white crystals.

Shown in Figure 1, PXRD conforms to polymorphic crystal Form IV disclosed in the above mentioned U.S. Patent Application No. 10/969,681

¹ H NMR (400 MHz, (CD ₃ ) ₂ SO): δ 11.89 (bs, 1H), 8.71 (bs, 1H), 7.57 (d, 1H), 7.37 (m, 2H), 7.20 (q, 1H), 6.67 (m, 1H), 4.84 (bs, 1H), 4.60 (m, 1H), 3.87 (m, 1 H), 3.7 (m, 2H), 3.34 (m, 2H).

Example 4. Preparation of N-((R)-2.3-dihydroxypropoxyV3.4-difluoro-2-(2-fluoro-4-iodo- phenylanrιinoV benzamide (Compound \)

To a stirring solution of 3,4-difluoro-2-(2-fluoro-4-iodophenylamino)-benzoic acid (9) (120 g, 0.30 mol) in a mixture of 1 mL N,N-dimethylformamide and 1000 mL toluene was added thionyl chloride (55 g, 0.462 mol). The mixture was heated to 50-65 C and stirred for 2 hours or until reaction completion as determined by HPLC (Conditions E). The final reaction mixture was then cooled and concentrated under reduced pressure to a slurry keeping the temperature below 35 ^° C. Toluene (600 mL) was added to dissolve the slurry and vacuum distillation was repeated. Additional toluene (600 mL) was added to the slurry dissolving all solids and the solution was then cooled to 5 ^° -10 ^° C. The solution was then treated with O-{[(4R)-2,2-dimethyl-1,3-dioxolan-4-yl]methyl}hydroxylamine (6) (63 g, 0.43 mol) solution in 207 mL toluene followed by potassium carbonate (65 g) and water (200 mL), stirred for at least 2 hours at 20- 25 ^° C. The stirring was stopped to allow phase separation and the bottom phase was discarded. The remaining organic layer was treated with hydrochloric acid solution (7.4%, 240 mL) until pH was less than 1 and stirred for 2 hours. The final reaction mixture was slightly concentrated under vacuum collecting about 100 mL distillate and the resulting organic solution was cooled to 5 ^° C to crystallize the product and filtered. The filter cake was washed with toluene (1000 mL) followed by water (100 mL) and the wet cake (crude product Compound I) was charged back to the flask. Toluene (100 mL), ethanol (100 mL) and water (100 mL) are then added, stirred at 30-35 ^° C for about 15 min, and the bottom aqueous phase was discarded. Water (200 mL) was then added to the organic solution and the mixture was stirred at about 3O C to allow for crystallization. The stirring was continued for 2 hours after product crystallized, then it was further cooled to about 0 ^° C and stirred for at least 2 hours. The slurry was filtered and wet cake was dried under reduced pressure at 55-85 ^° C to yield the final product N-((R)-2,3-dihydroxypropoxy)-3,4- difluoro-2-(2-fluoro-4-iodo-phenylamino)-benzamide (Compound I) product. Overall chemical yield was 86 g, 58%.