WO2014193696A2 - Benzimidazole carboxylic acid derivatives, compositions, and methods of use as aurora kinase inhibitors - Google Patents

Abstract

The invention provides benzimidazole carboxylic acid derivatives that inhibit Aurora kinase, pharmaceutical compositions comprising compounds that inhibit Aurora kinase, and methods of treatment using the compounds of the present invention or pharmaceutical compositions comprising compounds of the present invention.

Description

TITLE

BENZIMIDAZOLE CARBOXYLIC ACID DERIVATIVES, COMPOSITIONS, AND METHODS OF USE AS AURORA KINASE INHIBITORS

FIELD OF THE INVENTION

The present invention relates to benzimidazole carboxylic acid derivatives useful as inhibitors of Aurora kinase, and methods of use to treat cancer.

BACKGROUND OF THE INVENTION

A better understanding of the signal transduction pathways and enzymes underlying disease etiology and pathophysiology has greatly facilitated the search for new therapeutic agents. One important class of enzymes that has been the subject of intensive investigation for targeting disease processes is protein kinases.

Protein kinases are key regulators of cell growth, differentiation, metabolism and function. Protein kinases are a family of structurally related enzymes that are responsible for control of a variety of signal transduction processes within the cell. Almost all protein kinases contain a catalytic domain consisting of approximately 250 to 300 amino acids. In general, protein kinases mediate their intracellular signaling by catalytic transfer of a γ- phosphoryl group from ATP to target protein substrates. Protein kinases are classified into families by the substrates they phosphorylate. Sequence motifs have been identified that correspond to each of these kinase families such as protein-tyrosine, protein-serine/threonine, and lipids. In response to a variety of stimuli, protein kinases allow the cell to make decisions by acting as a molecular "on/off switch that can either perturb or regulate target protein function.

Abnormal protein kinase-mediated signal transduction in a cell is the underlying cause of many pathophysiological states. These disease states include, but are not limited to, autoimmune disease, allergy and asthma diseases, neurological and neurodegenerative diseases, metabolic diseases, Alzheimer's disease, cardiovascular disease, and cancer.

Accordingly, protein kinases are considered rational drug targets for therapeutic intervention and protein kinase inhibitors are thought to be effective therapeutic agents.

The aurora family of serine/threonine protein kinases is essential for cell proliferation. The human aurora kinase family consists of three highly homologous kinases (A or "2", B or "1" and C or "3"). During normal cell proliferation, these proteins are involved in chromosome segregation, mitotic spindle function, and cytokinesis. Aurora kinase expression is low in resting cells and peaks during the G2 and mitosis phases of the cell cycle. Several proposed mammalian substrates for Aurora kinases that are important for cell division include histone H3, TPX2, myosin II regulatory light chain, CENP-A, and protein phosphatase 1.

Since the elucidation of their key role in mitotic progression and cell division, Aurora kinases have been closely linked to tumorigenesis. For example, Aurora kinase gene amplification and overexpression has been reported in many cancers. A coding single nucleotide polymorphism (SNP) has been identified that is significantly more frequent in advanced gastric cancer relative to early stage gastric cancer, and this SNP correlates with elevated kinase activity. Overexpression of Aurora A induces centrosome amplification, aneuploidy and transformation in rodent fibroblasts. This oncogenic activity is likely due to the generation of chromosome instability. Indeed, there is a strong correlation between Aurora A overexpression and chromosome aneuploidy in breast and gastric cancer. Aurora B expression is elevated in cell lines derived from tumors of the colon, breast, lung, melanoma, kidney, ovary, pancreas, CNS, gastric tract and leukemias. In prostate cancer, increased nuclear expression of Aurora B was observed in high Gleason grade anaplastic prostate cancer tissues relative to low and intermediate grades, and Aurora B expression was accompanied by the phosphorylation of the histone H3 substrate. Aurora C is overexpressed in primary colorectal cancer.

Because Aurora kinase inhibition in tumor cells can result in mitotic arrest and apoptosis, these kinases are important targets for cancer therapy. Given the central role of mitosis in the progression of virtually all malignancies, inhibitors of the Aurora kinases therefore are expected to have the potential to block growth of cancers or tumors and have application across a broad range of human cancers or tumors.

SUMMARY OF THE INVENTION

This invention provides substituted benzazole derivatives and compositions that inhibit Aurora kinase. In an embodiment, the present invention provides compounds of Formula (I) as depicted below and pharmaceutically acceptable salts thereof. In another embodiment, the present invention provides methods of preparation of compounds of

Formula (I) and pharmaceutically acceptable salts thereof. In another embodiment, the present invention provides pharmaceutical compositions comprising the compounds of Formula (I) and pharmaceutically acceptable salts thereof. In another embodiment, the present invention provides methods of using the compounds and pharmaceutical

compositions comprising a compound of Formula (I) or a pharmaceutically acceptable salt thereof in treating human or animal disorders. The compounds of the invention are useful as inhibitors of Aurora kinase and thus may be useful for the management, treatment, control and adjunct treatment of diseases mediated by Aurora kinase activity such as cell proliferative disorders, including cancer.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows MiaPaCa-2 tumor growth curves, where · represents Vehicle;□ represents Example 1 at a dose of 30 mg/kg i.v.bolus, q.d. daily for 21 days;■ represents

Paclitaxel at a dose of 30 mg/kg i.v. bolus, q.d. daily for 5 days; o represents Inhibitor 1 at a dose of 30 mg/kg i.p., bid. daily for three days, then two days off, for a total of 3 cycles.

FIG. 2 shows the one way analysis of tumor volume (mm³) by treatment group on study day 32. All treatment groups displayed a significant reduction in tumor volume relative to vehicle group on study day 32. (Paclitaxel and Example 1, P-values <0.001).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides compounds that inhibit Aurora kinase. These compounds are useful for inhibiting Aurora kinase in vitro, and may be useful for the treatment of cell proliferative disorders, including cancer in a patient.

In a first embodiment, the present invention provides a compound of Formula (I) or a pharmaceutically salt thereof:

G¹ is -C(R³)(R⁴)-, -C₂_4 alkylene- , or

wherein

R³ and R⁴ are independently selected from the group consisting of hydrogen, methyl, ethyl, propyl, isopropyl, and isobutyl; and the C₂-4 alkylene group may be substituted once or twice with -OH; and

R¹ and R² are independently selected from the group consisting of hydrogen and benzyl.

Embodiment 2: A compound according to embodiment 1, wherein L¹ is -C(O)-.

Embodiment 3: A compound according to embodiment 1, wherein L¹ is -C(0)-0-.

Embodiment 4: A compound according to any one of embodiments 1 to 3, wherein G¹ is -C(R³)(R⁴)-.

Embodiment 5 : A compound according to embodiment 4, wherein R³ and R⁴ are

independently selected from the group consisting of hydrogen and methyl.

Embodiment 6: A compound according to embodiment 4, wherein R³ and R⁴ are hydrogen.

Embodiment 7: A compound according to any one of embodiments 1 to 3, wherein G¹

Embodiment 8: A compound according to any one of embodiments 1 to 3, wherein G¹ is -CH₂-CH₂-CH₂- .

Embodiment 9: A compound according to embodiment 1 having the formula (II) or a

pharmaceutically acceptable salt thereof,

(II).

Embodiment 10: A compound of embodiment 1 having the formula (III) or a

pharmaceutically acceptable salt thereof,

Embodiment 1 1 : A compound according to embodiment 9 or 10, wherein R³ and R⁴ are

independently selected from the group consisting of hydrogen and methyl.

Embodiment 12: A compound according to embodiment 10, wherein R³ and R⁴ are hydrogen.

Embodiment 13: A compound according to any one of embodiments 1 to 12, wherein R¹ is hydrogen and R² is benzyl.

Embodiment 14: A compound according to any one of embodiments 1 to 12, wherein R¹ and R² are hydrogen.

In another aspect, the present invention provides a method of making chloromethyl sec-butylsulfanylformate, wherein the method comprises in a first step addition of sec- butanethiol, triethylamine, and chloromethyl chloroformate to a solution of THF. In a further embodiment, the solution comprises a second step of filtering the solution to remove solids such as triethylammonium chloride.

The term "Aurora kinase inhibitor" or "inhibitor of Aurora kinase" is used to signify a compound having a structure as defined herein, which is capable of interacting with an Aurora kinase and inhibiting its enzymatic activity. Inhibiting Aurora kinase enzymatic activity means reducing the ability of an Aurora kinase to phosphorylate a substrate peptide or protein. In various embodiments, such reduction of Aurora kinase activity is at least about 50%, at least about 75%, at least about 90%, at least about 95%, or at least about 99%. In various embodiments, the concentration of Aurora kinase inhibitor required to reduce an Aurora kinase enzymatic activity is less than about 1 μΜ, less than about 500 nM, or less than about 100 nM.

In some embodiments, such inhibition is selective, i.e., the Aurora kinase inhibitor reduces the ability of an Aurora kinase to phosphorylate a substrate peptide or protein at a concentration that is lower than the concentration of the inhibitor that is required to produce another, unrelated biological effect, e.g., reduction of the enzymatic activity of a different kinase.

As used herein, the term "comprises" means "includes, but is not limited to."

Also included within the scope of the invention are the individual enantiomers of the compounds represented by Formula (I) above as well as any wholly or partially racemic mixtures thereof. The present invention also covers the individual enantiomers of the compounds represented by Formula (I) above as mixtures with diastereoisomers thereof in which one or more stereocenters are inverted. Unless otherwise stated, structures depicted herein are also meant to include compounds which differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the present structure except for the replacement of a hydrogen atom by a deuterium or tritium, or the replacement of a carbon atom by a ¹³C- or ¹⁴C-enriched carbon are within the scope of the invention.

Examples of compounds of the present invention having potentially useful biological activity are listed by name below in Table 1. The ability of compounds to inhibit Aurora kinase activity may be established with representative compounds listed in Table 1 using the peptide phosphorylation assay described below. The compounds of Formula (I) in Table 1 may inhibit Aurora Kinase with an IC₅₀ of less than or equal to 1 microMolar (μΜ; 10^"6 M). Compounds that inhibit Aurora kinase activity are potentially useful in treating cell proliferative disorders. The compounds of the present invention may therefore be particularly useful in the treatment of certain types of cancer.

Table 1.

In another aspect, the present invention comprises a pharmaceutical composition comprising a compound of Formula (I) or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier. The present invention further provides uses of the compound of Formula (I) or a pharmaceutically acceptable salt thereof for inhibiting Aurora kinase activity and for treating an Aurora kinase-mediated disorder.

The present invention also provides a method for the synthesis of compounds useful as intermediates in the preparation of compounds of Formula (I) along with methods for the preparation of compounds of Formula (I).

The compounds of this invention may be useful as inhibitors of Aurora kinase. The compounds can be assayed in vitro for their ability to inhibit an Aurora kinase. In vitro assays include assays to determine inhibition of the ability of an Aurora kinase to

phosphorylate a substrate protein or peptide. Alternate in vitro assays quantitate the ability of the compound to bind to an Aurora kinase. Inhibitor binding may be measured by radiolabelling the inhibitor prior to binding, isolating the inhibitor/Aurora kinase complex and determining the amount of radiolabel bound. Alternatively, inhibitor binding may be determined by running a competition experiment in which new inhibitors are incubated with Aurora kinase bound to a known radioligand. The compounds also can be assayed for their ability to affect cellular or physiological functions mediated by Aurora kinase activity.

Assays for each of these activities are described in the Examples and/or are known in the art.

In another embodiment, the present invention provides a pharmaceutical composition comprising a compound of Formula (I) or a pharmaceutically acceptable salt thereof wherein the compound of Formula (I) or a pharmaceutically acceptable salt thereof is administered in a dose of less than 1,000 mg/kg of body weight per day. In another embodiment, the present invention provides a pharmaceutical composition comprising a compound of Formula (I) or a pharmaceutically acceptable salt thereof wherein the compound of Formula (I) or a pharmaceutically acceptable salt thereof is administered in a dose of less than 100 mg/kg of body weight per day. In another embodiment, the present invention provides a

pharmaceutical composition comprising a compound of Formula (I) or a pharmaceutically acceptable salt thereof wherein the compound of Formula (I) or a pharmaceutically acceptable salt thereof is administered in a dose of less than 10 mg/kg of body weight per day.

Embodiments of the compounds of the present invention demonstrate utility as inhibitors of Aurora kinase activity or as inhibitors of cell proliferation. Embodiments of the invention described herein are additionally directed to pharmaceutical compositions and methods of inhibiting Aurora kinase in a subject, which methods comprise administering to a subject in need of inhibition of Aurora kinase activity a therapeutically effective amount of a compound of Formula (I) or a pharmaceutically acceptable salt thereof, defined above, as a single enantiomer, a racemic mixture, a single stereoisomer, a mixture of stereoisomers, a single diastereoisomer, or a mixture of diastereoisomers.

In an embodiment, the invention provides a method for inhibiting Aurora kinase activity comprising contacting a cell in which inhibition of Aurora kinase is desired with an Aurora kinase inhibitor of Formula (I) or a pharmaceutically acceptable salt thereof. In an embodiment, the Aurora kinase inhibitor interacts with and reduces the activity of fewer than all Aurora kinase enzymes in the cell. Where a compound of the present invention selectively acts as an inhibitor of Aurora kinase in preference to one or more other kinases, treatment of a subject with such a selective compound may possess advantage in the treatment of cancer in the subject over non-specific kinase inhibitors. Thus, in another embodiment, the present invention provides a method for selectively inhibiting Aurora kinase activity in the presence of one or more other kinases comprising contacting a cell in which inhibition of Aurora kinase is desired with an Aurora kinase inhibitor of Formula (I) or a pharmaceutically acceptable salt thereof. The method according to this aspect of the invention causes an inhibition of cell proliferation of the contacted cells. The phrase "inhibiting cell proliferation" is used to denote an ability of an inhibitor of Aurora kinase to inhibit cell number or cell growth in contacted cells as compared to cells not contacted with the inhibitor. An assessment of cell proliferation can be made by counting cells using a cell counter, by measuring uptake of a labeled nucleotide or nucleotide analog, or by an assay of cell viability. Where the cells are in a solid growth (e.g., a solid tumor or organ), such an assessment of cell proliferation can be made by measuring the growth, e.g., with calipers, and comparing the size of the growth of contacted cells with non-contacted cells.

The growth of cells contacted with an inhibitor may be retarded by at least about 50% as compared to growth of non-contacted cells. In various embodiments, cell proliferation of contacted cells is inhibited by at least about 75% as compared to non-contacted cells. In some embodiments, the phrase "inhibiting cell proliferation" includes a reduction in the number of contacted cells, as compared to non-contacted cells. Thus, an inhibitor of Aurora kinase that inhibits cell proliferation in a contacted cell may induce the contacted cell to undergo growth retardation, to undergo growth arrest, to undergo programmed cell death (i.e., apoptosis), or to undergo necrotic cell death.

Subjects may include, but are not limited to, horses, cows, sheep, pigs, mice, dogs, cats, primates such as chimpanzees, gorillas, rhesus monkeys, and, humans. In an embodiment, a subject is a human in need of inhibition of Aurora kinase activity.

The pharmaceutical compositions containing a compound of the invention may be in a form suitable for oral use, for example, as tablets, troches, lozenges, aqueous, or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs. Compositions intended for oral use may be prepared according to any known method, and such compositions may contain one or more agents selected from the group consisting of sweetening agents, flavoring agents, coloring agents, and preserving agents in order to provide pharmaceutically elegant and palatable preparations. Tablets may contain the active ingredient in admixture with non-toxic pharmaceutically-acceptable excipients which are suitable for the manufacture of tablets. These excipients may be for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example corn starch or alginic acid; binding agents, for example, starch, gelatin or acacia; and lubricating agents, for example magnesium stearate, stearic acid or talc. The tablets may be uncoated or they may be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate may be employed. They may also be coated by the techniques to form osmotic therapeutic tablets for controlled release.

Formulations for oral use may also be presented as hard gelatin capsules where the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or a soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example peanut oil, liquid paraffin, or olive oil.

Aqueous suspensions may contain the active compounds in an admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients are suspending agents, for example sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents may be a naturally-occurring phosphatide such as lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example, heptadecaethyl-eneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as

polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate. The aqueous suspensions may also contain one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose or saccharin.

Oily suspensions may be formulated by suspending the active ingredient in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as a liquid paraffin. The oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents such as those set forth above, and flavoring agents may be added to provide a palatable oral preparation. These compositions may be preserved by the addition of an anti-oxidant such as ascorbic acid.

Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active compound in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above.

Additional excipients, for example, sweetening, flavoring, and coloring agents may also be present.

The pharmaceutical compositions of the invention may also be in the form of oil-in- water emulsions. The oily phase may be a vegetable oil, for example, olive oil or arachis oil, or a mineral oil, for example a liquid paraffin, or a mixture thereof. Suitable emulsifying agents may be naturally-occurring gums, for example gum acacia or gum tragacanth, naturally-occurring phosphatides, for example soy bean, lecithin, and esters or partial esters derived from fatty acids and hexitol anhydrides, for example sorbitan monooleate, and condensation products of said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate. The emulsions may also contain sweetening and flavoring agents.

Syrups and elixirs may be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol or sucrose. Such formulations may also contain a demulcent, a preservative and flavoring and coloring agents.

The pharmaceutical compositions may be in the form of a sterile injectable aqueous or oleaginous suspension. This suspension may be formulated according to the known methods using suitable dispersing or wetting agents and suspending agents described above. The sterile injectable preparation may also be a sterile injectable solution or suspension in a nontoxic parenterally-acceptable diluent or solvent, for example as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, sterile water for injection (SWFI), Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conveniently employed as solvent or suspending medium. For this purpose, any bland fixed oil may be employed using synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.

Thus, in another embodiment, the present invention provides a pharmaceutical formulation solution comprising a compound of Formula (I) or a pharmaceutically acceptable salt thereof.

A solution of the invention may be provided in a sealed container, especially one made of glass, either in a unit dosage form or in a multiple dosage form.

Any pharmaceutically acceptable salt of a compound of Formula (I) or a

pharmaceutically acceptable salt thereof may be used for preparing a solution of the invention. Examples of suitable salts may be, for instance, the salts with mineral inorganic acids such as hydrochloric, hydrobromic, sulfuric, phosphoric, nitric and the like, and the salts with certain organic acids such as acetic, succinic, tartaric, ascorbic, citric, glutamic, benzoic,

methanesulfonic, ethanesulfonic and the like. In an embodiment, the compound is a hydrochloric acid salt.

Any solvent which is pharmaceutically acceptable and which is able to dissolve the compound of Formula (I) or a pharmaceutically acceptable salt thereof may be used. The solution of the invention may also contain one or more additional components such as a co- solubilizing agent (which may be the same as a solvent), a tonicity adjustment agent, a stabilizing agent, a preservative, or mixtures thereof. Examples of solvents, co-solubilizing agents, tonicity adjustment agents, stabilizing agents and preservatives which may suitable for a solution formulation are described below.

Suitable solvents and co-solubilizing agents may include, but are not limited to, water; sterile water for injection (SWFI); physiological saline; alcohols, e.g. ethanol, benzyl alcohol and the like; glycols and polyalcohols, e.g. propyleneglycol, glycerin and the like; esters of polyalcohols, e.g. diacetine, triacetine and the like; polyglycols and polyethers, e.g.

polyethyleneglycol 400, propyleneglycol methylethers and the like; dioxolanes, e.g.

isopropylidenglycerin and the like; dimethylisosorbide; pyrrolidone derivatives, e.g. 2- pyrrolidone, N-methyl-2-pyrrolidone, polyvinylpyrrolidone (co-solubilizing agent only) and the like; polyoxyethylenated fatty alcohols; esters of polyoxyethylenated fatty acids;

polysorbates, e.g., Tween™, polyoxyethylene derivatives of polypropyleneglycols, e.g., Pluronics™.

Suitable tonicity adjustment agents may include, but are not limited to,

pharmaceutically acceptable inorganic chlorides, e.g. sodium chloride; dextrose; lactose; mannitol; sorbitol and the like.

Preservatives suitable for physiological administration may be, for instance, esters of parahydroxybenzoic acid (e.g., methyl, ethyl, propyl and butyl esters, or mixtures of them), chlorocresol and the like.

Suitable stabilizing agents include, but are not limited to, monosaccharides (e.g., galactose, fructose, and fucose), disaccharides (e.g., lactose), polysaccharides (e.g., dextran), cyclic oligosaccharides (e.g., alpha-, beta-, gamma-cyclodextrin), aliphatic polyols (e.g., mannitol, sorbitol, and thioglycerol), cyclic polyols (e.g. inositol) and organic solvents (e.g., ethyl alcohol and glycerol).

The above mentioned solvents and co-solubilizing agents, tonicity adjustment agents, stabilizing agents and preservatives can be used alone or as a mixture of two or more of them in a solution formulation.

In an embodiment, a pharmaceutical solution formulation may comprise a compound of Formula (I) or a pharmaceutically acceptable salt thereof, SWFI, and an agent selected from the group consisting of sodium chloride solution (i.e., physiological saline), dextrose, mannitol, or sorbitol, wherein the agent is in an amount of less than or equal to 5%. The pH of such a formulation may also be adjusted to improve the storage stability using a pharmaceutically acceptable acid or base. In the solutions of the invention the concentration of the compound of Formula (I) or a pharmaceutically acceptable salt thereof may be less than 100 mg/mL, or less than 50 mg/mL, or less than 10 mg/mL, or less than 10 mg/mL and greater than 0.01 mg/mL, or between 0.5 mg/mL and 5 mg/mL, or between 0.1 mg/mL and 3 mg/mL. Further, compounds of Formula (I), such as the compound of Example 1, may have improved solubility in aqueous solutions in the pH range acceptable for human use or a pH range between 6.0 - 9.0 as compared to the compound of Example 88 in US Patent No. 7,820,821. Example 1 may have a solubility of about 0.1 mg/mL in an aqueous solution at pH 6.4 or a solubility of about 0.7 mg/mL in an aqueous solution at a pH of about 8.9.

Suitable packaging for the pharmaceutical solution formulations may be all approved containers intended for parenteral use, such as plastic and glass containers, ready-to-use syringes and the like. In an embodiment, the container is a sealed glass container, e.g. a vial or an ampoule. A hermetically sealed glass vial is particularly preferred.

One or more additional components such as co-solubilizing agents, tonicity adjustment agents, stabilizing agents and preservatives, for instance of the kind previously specified, may be added to the solution prior to passing the solution through the sterilizing filter.

Specific pharmaceutical solution formulations with different pH's and concentrations are illustrated in the Examples which follow.

Thus, according to the invention there is also provided a method of inhibiting the growth of a tumor or cancer, which comprises administering to a host suffering from said tumor or cancer an injectable solution according to the invention containing the active drug substance in an amount sufficient to inhibit the growth of said tumor.

The injectable solutions of the invention may be administered by rapid intravenous injection or infusion according to a variety of possible dose schedules.

The compositions may also be in the form of suppositories for rectal administration of the compounds of the invention. These compositions can be prepared by mixing the drug with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at the rectal temperature and will thus melt in the rectum to release the drug. Such materials include cocoa butter and polyethylene glycols, for example.

For topical use, creams, ointments, jellies, solutions of suspensions, etc., containing the compounds of the invention are contemplated. For the purpose of this application, topical applications shall include mouth washes and gargles. The compounds of the present invention may also be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles, and multilamellar vesicles. Liposomes may be formed from a variety of phospholipids, such as cholesterol, stearylamine, or phosphatidylcholines.

Pharmaceutically-acceptable salts of the compounds of the present invention, where a basic or acidic group is present in the structure, are also included within the scope of the invention. The term "pharmaceutically acceptable salts" refers to non-toxic salts of the compounds of this invention which are generally prepared by reacting the free base with a suitable organic or inorganic acid or by reacting the acid with a suitable organic or inorganic base. Representative salts include the following salts: Acetate, Benzenesulfonate, Benzoate, Bicarbonate, Bisulfate, Bitartrate, Borate, Bromide, Calcium Edetate, Camsylate, Carbonate, Chloride, Clavulanate, Citrate, Dihydrochloride, Edetate, Edisylate, Estolate, Esylate, Fumarate, Gluceptate, Gluconate, Glutamate, Glycollylarsanilate, Hexylresorcinate,

Hydrabamine, Hydrobromide, Hydrocloride, Hydroxynaphthoate, Iodide, Isethionate, Lactate, Lactobionate, Laurate, Malate, Maleate, Mandelate, Mesylate, Methylbromide, Methylnitrate, Methylsulfate, Monopotassium Maleate, Mucate, Napsylate, Nitrate, N-methylglucamine, Oxalate, Pamoate (Embonate), Palmitate, Pantothenate, Phosphate/diphosphate,

Polygalacturonate, Potassium, Salicylate, Sodium, Stearate, Subacetate, Succinate, Tannate, Tartrate, Teoclate, Tosylate, Triethiodide, Trimethylammonium and Valerate. When an acidic substituent is present, such as— P(0)(OH)₂ , there can be formed the ammonium, tris(hydroxymethyl)aminomethane, meglumine ((2R,3R,4R,5S)-6-(methylamino)hexane- 1,2,3,4,5-pentol), morpholinium, sodium, potassium, barium, or calcium salt, and the like, for use as the dosage form. When a basic group is present, such as amino or a basic heteroaryl radical, such as pyridyl, an acidic salt, such as hydrochloride, hydrobromide, phosphate, sulfate, trifluoroacetate, trichloroacetate, acetate, oxlate, maleate, pyruvate, malonate, succinate, citrate, tartarate, fumarate, mandelate, benzoate, cinnamate, methanesulfonate, ethanesulfonate, picrate and the like, and include acids related to the pharmaceutically- acceptable salts listed in the Journal of Pharmaceutical Science, 66, 2 (1977) p. 1-19. In an embodiment, a compound of the present invention is a sodium, potassium, ammonium, tris(hydroxymethyl)aminomethane, or meglumine salt of a compound of Formula (I).

Other salts which are not pharmaceutically acceptable may be useful in the

preparation of compounds of the invention and these form a further aspect of the invention. Thus, in a further embodiment, there is provided a pharmaceutical composition comprising a compound of the present invention, or a pharmaceutically acceptable salt, thereof, and a pharmaceutically acceptable carrier.

The pharmaceutical compositions of the present invention may be useful in therapeutic applications relating to an Aurora kinase-mediated disorder. As used herein, the term "Aurora kinase-mediated disorder" includes any disorder, disease or condition which is caused or characterized by an increase in Aurora kinase expression or activity, or which requires Aurora kinase activity. The term "Aurora kinase-mediated disorder" also includes any disorder, disease or condition in which inhibition of Aurora kinase activity is beneficial. Aurora kinase-mediated disorders include proliferative disorders. Non-limiting examples of proliferative disorders include chronic inflammatory proliferative disorders, e.g., psoriasis and rheumatoid arthritis and chronic pulmonary disease; proliferative ocular disorders, e.g., diabetic retinopathy; benign proliferative disorders, e.g., hemangiomas; restenosis, artherosclerosis, angiogenisis, and cancer.

In an embodiment, the composition is formulated for administration to a subject having or at risk of developing or experiencing a recurrence of an Aurora kinase-mediated disorder. In an embodiment, the pharmaceutical compositions of the invention are those formulated for oral, intravenous, or subcutaneous administration. However, any of the above dosage forms containing a therapeutically effective amount of a compound of the invention are within the bounds of routine experimentation and therefore, within the scope of the instant invention. In some embodiments, the pharmaceutical composition of the invention may further comprise another therapeutic agent. In an embodiment, such other therapeutic agent is one normally administered to a subject with the disease or condition being treated.

As used herein, "therapeutically effective amount" is an amount of the compound of Formula (I) or a pharmaceutically acceptable salt thereof sufficient to cause a detectable decrease in Aurora kinase activity or the severity of an Aurora kinase-mediated disorder. The amount of Aurora kinase inhibitor needed will depend on the effectiveness of the inhibitor for the given cell type and the length of time required to treat the disorder. It should also be understood that a specific dosage and treatment regimen for any particular subject will depend upon a variety of factors, including the activity of the specific compound employed, the age, body weight, general health, sex, and diet of the patient, time of administration, rate of excretion, drug combinations, the judgment of the treating physician, and the severity of the particular disease being treated. In another aspect, the invention provides a method for treating a subject having or at risk of developing or experiencing a recurrence of an Aurora kinase-mediated disorder. The method comprises the step of administering to the subject a compound or pharmaceutical composition according to the invention. The compounds and pharmaceutical compositions of the invention may be used to achieve a beneficial therapeutic or prophylactic effect, for example, in a subject with a proliferative disorder, as discussed above, such as cancer.

As used herein, the term "cancer" refers to a cellular disorder characterized by uncontrolled or dysregulated cell proliferation, decreased cellular differentiation,

inappropriate ability to invade surrounding tissue, and/or ability to establish new growth at ectopic sites. The term "cancer" includes, but is not limited to, solid tumors and bloodborne tumors. The term "cancer" encompasses diseases of skin, tissues, organs, bone, cartilage, blood, and vessels. The term "cancer" further encompasses primary and metastatic cancers.

Non-limiting examples of solid tumors that can be treated by the methods of the invention include pancreatic cancer; bladder cancer; colorectal cancer; breast cancer, including metastatic breast cancer; prostate cancer, including androgen-dependent and androgen- independent prostate cancer; renal cancer, including, e.g., metastatic renal cell carcinoma; hepatocellular cancer; lung cancer, including, e.g., non-small cell lung cancer (NSCLC), bronchioloalveolar carcinoma (BAC), and adenocarcinoma of the lung; ovarian cancer, including, e.g., progressive epithelial or primary peritoneal cancer; cervical cancer; gastric cancer; esophageal cancer; head and neck cancer, including, e.g., squamous cell carcinoma of the head and neck; melanoma; neuroendocrine cancer, including metastatic neuroendocrine tumors; brain tumors, including, e.g., glioma, anaplastic oligodendroglioma, adult glioblastoma multiforme, and adult anaplastic astrocytoma; bone cancer; and soft tissue sarcoma.

In some other embodiments, the cancer is a hematologic malignancy. Non-limiting examples of hematologic malignancy include acute myeloid leukemia (AML); chronic myelogenous leukemia (CML), including accelerated CML and CML blast phase (CML-BP); acute lymphoblastic leukemia (ALL); chronic lymphocytic leukemia (CLL); Hodgkin's disease (HD); non-Hodgkin's lymphoma (NHL), including follicular lymphoma and mantle cell lymphoma; B-cell lymphoma; T-cell lymphoma; multiple myeloma (MN);

Waldenstrom's macroglobulinemia; myelodysplastic syndromes (MDS), including refractory anemia (RA), refractory anemia with ringed siderblasts (RARS), (refractory anemia with excess blasts (RAEB), and RAEB in transformation (RAEB-T); and myeloproliferative syndromes. In some embodiments, the compound or composition of the invention is used to treat a cancer in which the activity of an Aurora kinase is amplified. In some embodiments, the compound or composition of the invention is used to treat a patient having or at risk of developing or experiencing a recurrence in a cancer selected from the group consisting of colorectal cancer, ovarian cancer, breast cancer, gastric cancer, prostate cancer, and pancreatic cancer. In certain embodiments, the cancer is selected from the group consisting of breast cancer, colorectal cancer, and pancreatic cancer.

In some embodiments, the Aurora kinase inhibitor of the invention is administered in conjunction with another therapeutic agent. The other therapeutic agent may also inhibit Aurora kinase or may operate by a different mechanism. In some embodiments, the other therapeutic agent is one that is normally administered to subject with the disease or condition being treated. The Aurora kinase inhibitor of the invention may be administered with the other therapeutic agent in a single dosage form or as a separate dosage form. When administered as a separate dosage form, the other therapeutic agent may be administered prior to, at the same time as, or following administration of the Aurora kinase inhibitor of the invention.

In some embodiments, the Aurora kinase inhibitor of the invention is administered in conjunction with a therapeutic agent selected from the group consisting of cytotoxic agents, radiotherapy, immunotherapy, or other kinase inhibitors. Non-limiting examples of cytotoxic agents that may be suitable for use in combination with the Aurora kinase inhibitors of the invention include: antimetabolites, including, e.g., capecitibine, gemcitabine, 5-fluorouracil or 5-fluorouracil/leucovorin, fludarabine, cytarabine, mercaptopurine, thioguanine, pentostatin, and methotrexate; topoisomerase inhibitors, including, e.g., etoposide, teniposide, camptothecin, topotecan, irinotecan, doxorubicin, and daunorubicin; vinca alkaloids, including, e.g., vincristine and vinblastin; taxanes, including, e.g., paclitaxel and docetaxel; platinum agents, including, e.g., cisplatin, carboplatin, and oxaliplatin; antibiotics, including, e.g., actinomycin D, bleomycin, mitomycin C, adriamycin, daunorubicin, idarubicin, doxorubicin and pegylated liposomal doxorubicin; alkylating agents such as melphalan, chlorambucil, busulfan, thiotepa, ifosfamide, carmustine, lomustine, semustine, streptozocin, decarbazine, and cyclophosphamide; thalidomide; protein tyrosine kinase inhibitors, including, e.g., imatinib mesylate, erlotinib, and gefitinib; antibodies, including, e.g., trastuzumab, rituximab, cetuximab, and bevacizumab; mitoxantrone; dexamethasone;

prednisone; and temozolomide. EXAMPLES

The present invention may be further understood by reference to the following non- limiting examples. Examples of compounds of the present invention and procedures that may be used to prepare and identify useful compounds of the present invention are described below.

LC-MS data are obtained using gradient elution on a parallel MUX system, running four Waters 1525 binary HPLC pumps, equipped with a MUX-UV 2488 multichannel UV- Vis detector (recording at 215 and 254 nM) and a Leap Technologies HTS PAL autosampler using a Sepax GP-C18, 4.6 x 50 mm; 5 micron particle-size column. In general, a three minute gradient is run from 25% B (97.5%acetonitrile, 2.5% water, 0.05% TFA) and 75% A (97.5% water, 2.5% acetonitrile, 0.05% TFA) to 100% B. The system is interfaced with a Waters Micromass ZQ mass spectrometer using electrospray ionization. MassLynx software is employed. All MS data were obtained in the positive mode unless otherwise noted. The reported m/z data are generally accurate within about ±1 for the M+ ion.

1H NMR data were obtained on a Varian Mercury 400 MHz spectrometer and chemical shifts were referenced using either the residual solvent proton signal (e.g., residual CHCI₃ in CDCI₃) or the TMS signal as an internal reference. Medium pressure liquid chromatography (MPLC) was performed using Teledyne Isco CombiFlash Companion and Combiflash Rf instruments, monitoring elution by UV absorption at 215 and 254 nM.

All reagents and solvents including anhydrous solvents were commercially available and were used as received unless described otherwise. Any solutions of Grignard reagents and organolithium reagents were commercially available and were used as received and at the concentrations listed on their labels. Reactions are stirred using a magnetic stirring apparatus and magnetic stir bar in most cases. All reactions using air-sensitive reagents were run under inert gas. For reactions not heated using a microwave-generating apparatus, the reaction temperatures reported in the experimental section refer to the temperatures of an oil bath or cooling bath placed around a reaction vessel. For reactions performed using a microwave- generating apparatus, the temperatures refer to the temperatures reported by the microwave apparatus.

Abbreviations Below are definitions of some common abbreviations that are used in the specification. The specification may also employ other abbreviations whose meanings are well known in the relevant art.

DCM = dichloromethane

DIC = N,N'-diisopropylcarbodiimide

DIEA = diisopropylethylamine

DME = 1,2-dimethoxyethane

DMF = N,N'-dimethylformamide

DMSO = dimethylsulfoxide

EtOAc = ethyl acetate

EtOH = ethanol

lH NMR = proton NMR analysis

HBTU = 2-(lH-benzotriazol-l-yl)-l ,l ,3,3-tetramethyluronium hexafluorophosphate

HC1 = hydrochloric acid

LC/MS, LCMS = liquid chromatography- mass spectrometry analysis

MeOH = methanol

NMP = N-methylpyrrolidinone

rt = room temperature

h or hr = hours

min = minutes

TEA = triethylamine

THF = tetrahydrofuran

TLC = thin layer chromatography

M = molar concentration

N = normal concentration

Example 1 - Preparation of phosphonooxymethyl 4-[6-(lH-Indazol-6-ylcarbamoyl)-2-[[2- (trifluoromethyl)phenyl]amino]-3H-benzimidazol-5-yl]piperazine-l-carboxylate

1. Chloromethyl seobutylsulfanylformate

To a solution of sec-butanethiol (30.1 mL, 277 mmol) and TEA (43.0 mL, 308 mmol) in THF (300 mL) at 0 °C was added chloromethyl chloroformate (27.4 mL, 308 mmol) over 10 min. The reaction was allowed to warm to rt and was stirred for 14 h. The reaction mixture was filtered through filter aid and the cake was washed with THF (2 x 100 mL). The filtrate was concentrated in vacuo at 30 - 35 °C to give 48.6 g of the product which was used in the next step without further purification. ^XH NMR (400 MHz, CDC1₃) δ 5.76 (s, 2 H), 3.42 (m, 1 H), 1.66 (m, 2 H), 1.38 (m, 3 H), 1.00 (t, 3 H) ppm.

2. Dibenzyloxyphosphoryloxymethyl sgc-butylsulfanylformate

To a solution of dibenzylphosphate (1 11 g, 399 mmol) in NMP (400 mL) at rt was added potassium t-butoxide (44.8 g, 399 mmol) with stirring. After 20 min chloromethyl sec- butylsulfanylformate (48.6 g, 266 mmol) was added and the reaction was stirred at 65 °C for 16 hr. After cooling to rt, the reaction was divided in two equal portions, each poured into 2 L of water and extracted with ethyl acetate (3 x 300 mL). The combined ethyl acetate extracts were washed with brine (300 mL), dried over Na₂S0₄, and concentrated in vacuo. The crude product was purified by flash chromatography on silica gel using a 0 - 40% ethyl acetate/hexanes gradient to afford 52.2 g of the desired product. LCMS: m/z 424.8 [M+l].

1H NMR (400 MHz, CDC1₃) δ 7.36 (m, 10 H), 5.65 (d, 2 H), 5.07 (d, 4 H), 3.37 (m, 1 H), 1.63 (m, 2 H), 1.33 (m, 3 H), 0.96 (t, 3 H) ppm.

A solution of dibenzyloxyphosphoryloxymethyl sec -butylsulfanylformate (14.9 g, 35.0 mmol) in dry DCM (200 mL) was cooled in a dry ice/acetonitrile bath to -40 °C. To the cold reaction mixture was added sulfuryl chloride (4.25 mL, 52.5 mmol) at a rapid dropwise rate. After 20 minutes at -40 °C, the cooling bath was removed. The mixture was allowed to warm to rt and was stirred at rt for 3 hr. The mixture was concentrated in vacuo and the crude chloroformate was used directly without purification. ^XH NMR (400 MHz, CDCI₃) δ 7.36 (m, 10 H), 5.62 (d, 2 H), 5.08 (d, 4 H) ppm.

4. Dibenzyloxyphosphoryloxymethyl 4-r6-(lH-Indazol-6-ylcarbamoyl)-2-rr2- (trifluoromethyl phenyl1amino1-3H-benzimidazol-5-yl1piperazine-l-carboxylate

N-( 1 H-Indazol-6-yl)-6-piperazin- 1 -yl-2- [[2-(trifluoromethyl)phenyl] amino] - 1 H- benzimidazole-5-carboxamide hydrochloride (See Example 88, US Patent No. 7,820,821) was stirred in methanol (1 ml per 100 mg of HCl salt) and was warmed to 40 °C. TEA (1 mL per g of HCl salt) was added dropwise and the mixture was allowed to cool to rt, was stirred at rt for 15 min, then was chilled to 5 °C. The resulting solid was collected, washed with cold water and methanol, and dried in vacuo to afford the free base N-(lH-indazol-6-yl)-6- piperazin- 1 -yl-2- [ [2-(trifluoromethyl)phenyl] amino] - 1 H-benzimidazole-5-carboxamide (Inhibitor 1).

To a solution of N-(lH-indazol-6-yl)-6-piperazin-l-yl-2-[[2- (trifluoromethyl)phenyl]amino]-lH-benzimidazole-5-carboxamide (8.0 g, 15.4 mmol) in dry DMF (80 mL) was added K₂C0₃ (10.63 g, 76.9 mmol). The mixture was stirred at rt for 20 min, then was treated with neat, freshly prepared dibenzyloxyphosphoryloxymethyl carbonochloridate (23.0 mmol). The mixture was stirred at rt for 30 min. The residual solids were removed by filtration through a bed of filter aid and the cake was washed with THF (200 mL). The filtrate was concentrated in vacuo and the crude residue was suspended in water (250 mL) and extracted with ethyl acetate (3 x 300 mL). The combined organics were washed with brine (100 mL), dried over Na₂S0₄, and concentrated in vacuo. The crude residue was purified by flash chromatography on silica gel using a 0 - 10%

ammonia/methanol in DCM gradient to afford 3.7 g of the phosphate. LCMS: m/z 854.9 [M+l]. ^XH NMR (400 MHz, DMSO-i/₆) δ 12.92 (br s, 1 H), 12.12 (m, 1 H), 11.20 (br s, 1 H), 8.85 (br s, 1 H), 8.44 (s, 1 H), 7.99 (m, 2 H), 7.70 (t, 3 H), 7.30 (m, 12 H), 5.64 (d, 2 H), 5.07 (d, 4 H), 3.58 (m, 4 H), 2.94 (m, 4 H) ppm. 5. Final Product

To a solution of dibenzyloxyphosphoryloxymethyl 4-[6-(lH-indazol-6-ylcarbamoyl)- 2-[[2-(trifluoromethyl)phenyl]amino]-3H-benzimidazol-5-yl]piperazine-l-carboxylate (0.1 g, 0.117 mmol) in anhydrous THF (10 mL) was added fresh N,0-bis(trimethylsilyl)acetamide (0.43 mL, 1.76 mmol) and 10% palladium on carbon (0.1 g, 0.0468 mmol). The reaction was alternately degassed under vacuum and purged with hydrogen through five cycles. The reaction was stirred under balloon pressure of hydrogen for 3 hr. The mixture was filtered through filter paper and a fine fritted funnel, followed by washing the filter with THF (10 mL). The filtrate was concentrated in vacuo at 25 °C and MeOH (10 mL) was added and concentrated in vacuo at 25 °C. The crude phosphate was triturated with 5% MeOH in ethyl acetate (3 x 5 mL) to yield 0.03 g of the title compound. LCMS: m/z 674.8 [M+l]. ^XH NMR (400 MHz, DMSO-i/₆) δ 12.88 (br s, 1 H), 12.12, (br s, 1 H), 11.20 (br s, 1 H), 8.37 (m, 2 H), 7.99 (br s, 2 H), 7.72 (d, 3 H), 7.28 (m, 3 H), 5.49 (d, 2 H), 3.65 (br s, 4 H), 3.01 (br s, 4 H) ppm. Example 2 - Preparation of Benzyloxy(hydroxy)phosphoryl)oxymethyl 4-[6-(lH-indazol-6- ylcarbamoyl)-2-[[2-(trifluoromethyl)phenyl]amino]-3H-benzimidazol-5-yl]piperazine-l- carboxylate

A 100 mL pressure bottle was charged with a solution of

dibenzyloxyphosphoryloxymethyl 4-[6-(lH-indazol-6-ylcarbamoyl)-2-[[2- (trifluoromethyl)phenyl]amino]-3H-benzimidazol-5-yl]piperazine-l-carboxylate (0.9 g, 1.05 mmol) in MeOH (10 mL), THF (5 mL) and acetic acid (1 mL). The solution was degassed with nitrogen bubbling. To the magnetically stirred solution was added 10% palladium on carbon (0.5 g). The reaction was pressurized with H₂ to 60 psi and stirred vigorously for 16 hr. The palladium was removed by filtration through a bed of filter aid, washing with

DCM/MeOH mixture (1: 1, 50 mL). All volatiles were removed in vacuo and the product was purified by reverse phase MPLC chromatography on a CI 8 column using a gradient of water/acetonitrile with 0.1% TFA to afford 0.08 g of the product. LCMS: m/z 764.8 [M+l]. ¾ NMR (400 MHz, DMSO-i¾) δ 12.94 (br s, 1 H), 12.09 (m, 1 H), 11.25 (br s, 1 H), 8.39 (m, 1 H), 8.18 (br s, 1 H), 7.96 (m, 2 H), 7.75 (m, 3 H), 7.32 (m, 8 H), 5.50 (m, 2 H), 4.87 (d, 2 H), 3.54 (br s, 4 H), 2.98 (m, 4 H) ppm.

Biological Data

The compounds of the present invention elicit measurable pharmacological responses.

The compounds of the present invention in Table 1 have a binding affinity (IC₅₀ < 1 μΜ) for one or more aurora kinase. In addition to the binding to aurora kinases, compounds of the present invention may also measurably inhibit the proliferation of tumor cells. Example A

Aurora A Enzyme

Aurora kinase A assays utilize the peptide substrate STK2 (Upstate) as a phosphoryl group acceptor.

Assays are performed in 96-well U-bottom plates. Aurora A is purchased from PanVera. Compounds are diluted in DMSO prior to addition in the assay. Typically, assays are performed by incubating enzyme (0.2 - 10 nM) with or without inhibitor, 0.1-100 μΜ ATP, 0.1-10 mM MnCl₂, 1- 10 μΜ sodium orthovanadate, 1-10 mM DTT, and 1 -100 μΜ peptide together for the time range of 5-120 min at 37 °C in a final assay volume of 60 μϊ_^. The buffer used to bring the final assay volume up to 60 μί is 50 mM MOPS, pH 7.0, containing 1-5% DMSO and 0.05% BSA.

A 5 μϊ_^ aliquot of the enzyme incubation is transferred to a black 384 well plate, 5 μϊ_^ of ADP-Glo reagent (Promega) in 0.5mM MgCl₂ is added to each well, and the well is incubated for 40 min. Kinase Detect (Promega) (ΙΟμί) is added and the mixture is incubated for 1 hr then read for luminescence on an Envision plate reader (Perkin Elmer). Incubations in the 384 well plate are at room temp.

ADP produced in the assay is calculated from a standard curve. The std curve is linear and IC5₀ values are determined from % enzyme inhibition versus compound concentration curve plots using GraphPad Prism™ according to the 4 parameter logistic equation Y=Bottom+(Top-Bottom)/l+10^A((LogEC50-X)*HillSlope)) where X is the logarithm of compound concentration and Y is percent inhibition.

The compounds in Table 1 exhibited an IC5₀ value of less than or equal to 1.0 μΜ for Aurora A kinase in the above assay as shown in the table below. Example Aurora A IC₅₀, nM

1 6.1

2 2.2

Example B

In Vitro Cell Proliferation

Compounds were tested for their ability to inhibit cell proliferation and viability.

Dual Hoechst 33342 dye/ propidium iodide staining was used to measure cell number and cell viability.

The antiproliferative activity of compounds was studied using a panel of human tumors cells obtained from ATCC: MIA Paca-2 (human pancreatic carcinoma cell line). These adherent cells (1,000 - 20,000) were plated in complete media (RPMI-1640, DMEM, F12K, or McCoy's 5A) containing 10% dialyzed fetal bovine serum (Gibco) and glucose (1- 25 mM) in tissue-culturetreated Optilux 96-well black plates (Becton Dickinson) and placed in a humidified incubator at 37 °C, 95% 0₂, 5% CO2 for 18-24 hours. Media was removed and replaced with 90 uL fresh media. Compound (0.00001-100 μΜ) is diluted in media containing 3% DMSO and added to 30 cells. Untreated cells or cells containing compound are incubated for 24-96 hours. During the last 30 minutes of the incubation period, 10 μϊ_^ of a propidium iodide (10 μg/mL)/Hoescht 33342 dye reagent (32 μΜ) in PBS was added to each well and incubated in a humidified incubator at 37 °C, 95% O2, 5% CO2.

Propidium iodide/ Hoechst 33342 fluorescent staining of cells was measured using an InCell 2000 analyzer instrument with 10X objective. The instrument setting for the Hoechst channel was excitation at 350 nm and emission at 455 nm. The setting for the propidium iodide channel was excitation at 550 nm and emission at 605 nm. The nuclei were counted in the Hoechst 33342 channel; the dead cells were counted in the propidium iodide channel. Compound IC5₀ values were determined from the cell number (Hoechst nuclear dye) versus compound concentration curve plots using GraphPad PRISM according to the 4 parameter logistic equation 10 Y=Bottom+(Top-Bottom)/l+10^A((Log(IC50)-X)*Hill Slope)). For cell death analysis, compound IC5₀ values were determined from percentage of cells positive for PI dye versus compound concentration curve plots using GraphPad PRISM according to the 4 parameter logistic equation Y=Bottom+(Top-Bottom)/l+10^A((Log(IC50)-X)*Hill Slope)). The compounds in Table 1 exhibited an IC5₀ values, in inhibiting cell proliferation or viability, of less than or equal to 10 μΜ against MIA Paca-2 and/or SKOV tumor cells, as shown in the table below.

percentage cell death at 1 μΜ compound concentration

ND = not determined

Example C

Materials and Methods

Female nude mice (Crl:NU(NCr)- ox«7«w, Charles River Laboratories) (approx 56 days old). Mice were provided ad libitum water (reverse osmosis, 1 ppm CI) and fed ad libitum Irradiated Teklad Global Rodent Lab Diet® consisting of Crude Protein 18.9%, Fat (acid hydrolysis) 6.5%, Crude Fiber 2.7%, Energy Density 3.1 kcal/g, 13.0 kJ/g Calories from Protein 24%, Calories from Fat 16% Calories from Carbohydrate 60% (Harlan). The mice were group-housed in Innovive sterile isolators (5 mice per cage) on a 12-hour light cycle at 69-72°F with positive airflow setting of 56.

Tumor cells from human MIAPaCa2 pancreatic carcinoma cells (obtained from ATCC, and determined to be free of mouse and rat pathogens by Charles River) grown in cell culture media in DMEM containing 10% heat-inactivated fetal bovine serum and 10 mM glucose. MiaPaca2 (2xl0⁷ cells/mL) were suspended 1 : 1 in PBS: Matrigel (Becton

Dickinson). Each mouse received 2xl0⁶ (passage 7) cells injected sc in the right flank in 0.1 mL using a 27G1/2 needle lcc syringe. 1 1 days later designated as Day 1, mice were randomized by random cage placement and mice were further distributed by tumor size to an equal tumor size distribution per cage. The mean tumor weight was approximately 74 mg at the start of the study.

Tumor Measurement Tumor volume was recorded 2 times per week or more during the study period.

Tumor volume was calculated using the formula:

Tumor Volume (mm³) = w²*H2

Where w=width and /= length in mm of the subcutaneous (sc) tumor is measured using an electronic caliper. Tumor weight may be estimated with the assumption that 1 mg is equivalent to 1 mm³ of tumor volume. Body weights were captured in the database along with tumor measurements 2 to 3 times a week or more, but taken daily for dose

administration.

Endpoint

Each animal exited the study when its tumor reaches the predetermined endpoint size (2000 mm³). Each animal was be observed frequently for overt signs of any adverse, tumor- related side effects, and clinical signs of toxicity are recorded and observed, or an animal may exit the study if there is tumor-related toxicity defined as difficulty in animal breathing, difficulty in moving due to tumor burden, or bleeding due necrotic tumor mass, or if animal is moribund.

Tumor Regression and Tumor Response Criteria

Treatment efficacy was also determined from the tumor volumes of animals remaining in the study on the last day, and from the number of regression responses. The MTV(n) is defined as the median tumor volume on day 45 in the number of animals remaining, n, whose tumors have not attained the endpoint volume. Treatment may cause partial regression (PR) or complete regression (CR) of the tumor in an animal. In a PR response, the tumor volume is 50% or less of its Day 1 volume for three consecutive measurements during the course of the study, and equal to or greater than 13.5 mm³ for one or more of these three measurements. In a CR response, the tumor volume is less than 13.5 mm³ for three consecutive measurements during the course of the study. An animal with a CR response at the termination of a study is additionally classified as a tumor-free survivor (TFS). Regression responses were monitored and recorded. Tumor growth curves show the group median tumor volume as a function of time. When an animal exits the study due to tumor size or treatment-related death, the final tumor volume recorded for the animal is included with the data at subsequent time points when used to calculate the mean volume. Tumor growth curves are truncated when the tumors in more than 50% of the animals in a group have grown to the endpoint volume.

Statistical and Graphical Analysis

Prism (GraphPad) was used for graphic presentation of mean tumor volume as a function of time for individual treatment groups. Statistical and graphical analysis was performed using JMP 10.0.0 software (SAS Institute, Inc.). Meaningful differences in tumor size for drug treatment groups was assessed by a one way analysis of variance (ANOVA) for mean tumor volumes of treatment groups using the Dunnett's method in comparison to vehicle control group.

The antitumor activity of Example 1 administered alone was evaluated against MiaPaCa-2 pancreatic xenograft tumors grown subcutaneously in athymic mice (a preclinical model of pancreatic cancer). Compounds were dosed on the following schedule and in the specified vehicles:

Example 1 was dosed i.v.bolus, q.d. daily for 21 days via tail vein injection.

Example 1 was formulated in 10% PEG300 containing 3.1 molar equivalents of NaOH in phosphate buffered saline, pH 7.4 at 3 mg/mL, respectively. These solutions were prepared fresh each day of dosing. Dosing was 30 mg/kg at a dosing volume of 10 mL/Kg.

Paclitaxel was dosed i.v. bolus, q.d. daily for 5 days via tail vein injection. Paclitaxel (Phyton Biotech) at 30 mg/mL was prepared by adding 90 mg of drug powder into 1.5 mL of ethanol and 1.5 mL of Cremophor EL. This stock was stored at room temperature and protected from light. Prior to dosing, a 0.33 mL aliquot of stock was diluted into 3 mL sterile 5% dextrose in water (D5W), final Paclitaxel cone, of 3 mg/mL. Dosing was 30 mg/kg at a dosing volume of 10 mL/Kg.

Inhibitor 1 (trihydrochloride( -(lH-indazol-6-yl)-6-piperazin-l-yl-2-[[2- (trifluoromethyl)phenyl] amino]- lH-benzimidazole-5-carboxamide hydrochloride), See Example 88, US Patent No. 7,820,821) was dosed i.p., bid. daily for three days, then two days off, for a total of three such cycles. Inhibitor 1 as the tri-HCl salt was formulated as a 3 mg/mL dosing solution in 2% Tween 80 in water, and prepared fresh prior to dosing. 30 mg was dissolved in 10 mL 2% Tween80 in water. This solution was prepared fresh each day of dosing. Dosing was 30 mg/kg at a dosing volume of 10 mL/Kg.

Drug treatment started when mean tumor sizes reached approximately 74 mg (day 11). Mice bearing sc tumors were randomized, and treatment groups consisted often mice. The mean tumor size for each group at various points in the study are listed below in Table A.

Table A. MiaPaCa-2 xenograft model

In Table A, Vehichle is 10% PEG300 in sodium phosphate buffer, pH 7.4, iv.

The MiaPaCa-2 tumor growth curves are shown in Figure 1. Example 1 produced 68.3% inhibition of tumor growth relative to vehicle - treated tumors growth on day 32.

Paclitaxel (30 mg/kg) produced 83% inhibition of tumor growth on day 32. Inhibitor 1 (30 mg/kg) produced 51% inhibition of tumor growth on day 32.

Further, all treatment groups displayed a significant reduction in tumor volume relative to vehicle group on study day 32. (Paclitaxel and Example 1 having P-values <0.001). See Figure 2. While the invention has been described and illustrated with reference to certain embodiments thereof, those skilled in the art will appreciate that various changes, modifications and substitutions can be made therein without departing from the spirit and scope of the invention. For example, effective dosages other than the dosages as set forth herein may be applicable as a consequence of variations in the responsiveness of the subject being treated for an Aurora kinase mediated disorder. Likewise, the specific pharmacological responses observed may vary according to and depending on the particular active compound selected or whether there are present pharmaceutical carriers, as well as the type of formulation and mode of administration employed, and such expected variations or differences in the results are contemplated in accordance with the objects and practices of the present invention.

Claims

We claim:

1. A compound of Formula (I) or a pharmaceutically salt thereof:

(I)

wherein

L¹ is -C(O)- or -C(0)-0-;

-C(R³)(R⁴)-, -C₂_4 alkylene-, or

wherein

R³ and R⁴ are independently selected from the group consisting of hydrogen, methyl, ethyl, propyl, isopropyl, and isobutyl; and the C2-4 alkylene group may be substituted once or twice with -OH; and

R¹ and R² are independently selected from the group consisting of hydrogen and benzyl.

2. A compound according to claim 1, wherein L¹ is -C(O)-.

3. A compound according to claim 1, wherein L¹ is -C(0)-0-.

4. A compound according to any one of claims 1 to 3, wherein G¹ is -C(R³)(R⁴)-.

5. A compound according to claim 4, wherein R³ and R⁴ are independently selected from the group consisting of hydrogen and methyl.

6. A compound according to claim 4, wherein R³ and R⁴ are hydrogen.

7. A compound according to any one of claims 1 to 3, wherein G¹ is -CH₂-CH₂-.

8. A compound according to any one of claims 1 to 3, wherein G¹ is -CH₂-CH₂-CH₂- .

9. A compound according to claim 1 having the formula (II) or a pharmaceutically acceptable salt thereof,

(II).

A compound of claim 1 having the formula (III) or a pharmaceutically acceptable salt thereof,

11. A compound according to claim 9 or 10, wherein R³ and R⁴ are independently

selected from the group consisting of hydrogen and methyl.

12. A compound according to claim 10, wherein R³ and R⁴ are hydrogen.

13. A compound according to any one of claims 1 to 12, wherein R¹ is hydrogen and R² is benzyl.

14. A compound according to any one of claim 1 to 12, wherein R¹ and R² are hydrogen.

15. A pharmaceutical composition comprising a compound of any one of claims 1 to 14 and a pharmaceutically acceptable carrier.

16. The pharmaceutical composition of claim 15 further comprising an additional

therapeutic agent selected from the group consisting of antimetabolites, protein tyrosine kinase inhibitors, and antibodies.

17. A method for inhibiting Aurora kinase activity comprising contacting a cell in a

subject in which inhibition of Aurora kinase A or B is desired with a compound of claim 1.

A method for treating an Aurora kinase-mediated disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound in claim 1.

19. The method according to claim 18, wherein the Aurora kinase-mediated disorder is a cancer.

20. The method according to claim 19, wherein the cancer is selected from the group consisting of colorectal cancer, ovarian cancer, breast cancer, gastric cancer, prostate cancer, brain cancer, bone cancer, bladder cancer, head and neck cancer, lung cancer, renal cancer, pancreatic cancer, sarcoma, leukemia, and lymphoma.

21. The method according to claim 20, wherein the cancer is selected from the group consisting of breast cancer, colorectal cancer, and pancreatic cancer.

22. The method of claim 18, further comprising the step of administering to a subject an additional therapeutic agent selected from the group consisting of: antimetabolites, protein tyrosine kinase inhibitors, and antibodies.

23. A method of making chloromethyl sec-butylsulfanylformate, wherein the method comprises addition of seobutanethiol, triethylamine, and chloromethyl chloroformate to a solution of THF.