WO2005103036A1

WO2005103036A1 - Pyrimidin-4-yl-1h-indazol-5yl-amines as chk1 kinases inhibitors

Info

Publication number: WO2005103036A1
Application number: PCT/GB2005/001566
Authority: WO
Inventors: Véronique Birault; Christopher Andrew Woodland
Original assignee: Biofocus Discovery Ltd
Priority date: 2004-04-23
Filing date: 2005-04-22
Publication date: 2005-11-03
Also published as: GB0409080D0

Abstract

Compounds of Formula (I) wherein, R1 can be hydrogen, optionally substituted alkyl, optionally substituted alkoxy, OCF3, CF3, amino, optionally substituted aryloxy, halogen, hydroxy, CN, CO2H, NR4R5, CO2R4, CONR4R5, NR4(CO)R5 or S(O)PR4; wherein R4 and R5, which can be the same or different, can be hydrogen, optionally substituted alkyl, optionally substituted aryl, optionally substituted heteroarylalkyl or optionally substituted arylalkyl; and wherein p is 1 or 2; R1 can be in the 3-, 4-, 6- or 7-position of the indazol ring; and R2 is optionally substituted aryl or optionally substituted heteroaryl are useful in the inhibition of protein kinases, in particular serine/threonine kinases, more particularly Chk1 kinase. The compounds can be used in medicine and particularly in the prevention and/or treatment of a wide variety of diseases including cancer, and disease states associated with angiogenesis and/or cellular proliferation.

Description

PYRIMIDIN-4-YL-1H-INDAZ0L-5YL-AMINES AS CH 1 KINASE INHIBITORS

TECHNICAL FIELD

The present invention relates to novel compounds, pharmaceutical compositions comprising the compounds, to processes for their preparation, as well as to the use of the compounds in the inhibition of protein kinases, in particular serine/threonine kinases, more particularly Chkl kinase. The invention also relates to the compounds for use in medicine and particularly in the prevention and/or treatment of a wide variety of diseases including cancer, and disease states associated with angiogenesis and/or cellular proliferation.

BACKGROUND OF THE INVENTION

Protein kinases are a family of enzymes that catalyze phosphorylation of the hydroxy group of specific tyrosine, serine or threonine residues in proteins. The reversible phosphorylation of a specific tyrosine, serine, or threonine residue on a target protein can dramatically alter its function in several ways including activating or inhibiting enzymatic activity, creating or blocking binding sites for other proteins, altering subcellular localization or controlling protein stability. Of the many different cellular functions in which the activity of a protein kinase is known to be required, some represent attractive targets for therapeutic intervention for certain disease states. One example is cell cycle control, where protein kinases play a pivotal role.

Cell cycle checkpoints are mechanisms whereby cells can delay progression through the cell cycle if DNA damage occurs. This delay can then provide an opportunity to allow the cells to repair before re-entering the cell cycle. Checkpoints occur at several stages of the cell cycle and most of these are regulated through p53. However, the G2 checkpoint, which is regulated through the protein kinase Chkl, can act independently of p53. p53 is frequently inactivated in cancers, making the tumour cells more reliant on the G2 checkpoint. Inactivation of the G2 checkpoint by the inhibition of Chkl is therefore an attractive proposition for cancer therapies, as it would increase the sensitivity of tumour cells to DNA damage caused by radiotherapy or chemotherapy. Healthy cells would be less affected, as their p53-dependent checkpoints would still be activated. The present invention focused towards the identification of potent and selective Chkl inhibitors.

The compounds of the invention, including but not limited to those specified in the examples, possess the ability to inhibit Chkl kinase. Such compounds may be useful in the treatment of both primary and metastatic tumours, including carcinomas of the breast, colon, rectum, lung, oropharynx, hypopharynx, esophagus, stomach, pancreas, liver, gallbladder and bile ducts, small intestine, urinary tract (including kidney, bladder and urethra), female genital tract (including cervix, uterus and ovaries as well as choriocarcinoma and gestational trophobalstic disease), male genital tract, endocrine glands, and skin, as well as melanoma, sarcomas, hemangiomas and tumours of the brain, nerves, eyes and meninges. Such compounds may be useful in treating solid tumors arising from hematopoietic malignancies such as leukaemias, as well as in the treatment of lymphomas. In addition these compound may be useful in the prevention of metastase from the tumors described above either when used alone or in combination with radiotherapy and/or other chemotherapeutic agents.

However, despite indications that Chkl inhibitors may be useful in the treatment of a variety of cancers, the development of inhibitors with good activity, selectivity and pharmacokinetic profiles is needed to fully exploit the clinical potential of this target.

Remarkably, the present invention provides a class of compounds which interact selectively with Chkl kinase. DISCLOSURE OF THE INVENTION

In a first aspect the invention provides a compound of Formula I:

Formula I

wherein, Rl can be hydrogen, optionally substituted alkyl, optionally substituted alkoxy, OCF₃, CF₃, amino, optionally substituted aryloxy, halogen, hydroxy, CN, CO₂H, NR⁴R⁵, CO₂R⁴, CONR⁴R⁵, NR⁴(CO)R⁵ or S(O)_pR⁴; wherein R⁴ and R⁵, which can be the same or different, can be hydrogen, optionally substituted alkyl, optionally substituted aryl, optionally substituted heteroarylalkyl or optionally substituted arylalkyl; and wherein p is 1 or 2;

Rl can be in the 3-, 4-, 6- or 7-position of the indazol ring; and

R2 is optionally substituted aryl or optionally substituted heteroaryl;

or a pharmaceutically acceptable salt, hydrate, solvate, geometrical isomer, tautomer, optical isomer, or prodrug form thereof. In a preferred embodiment suitable substituents include alkyl, cycloalkyl, OCF₃, CF₃, heterocyclyl, alkenyl, alkynyl, alkoxy, aryloxy, halogen, hydroxy, amino, NO₂, CN, NR³R⁴, CO₂R³, CONR³R⁴, NR³(CO) R⁴, S(O)_pR³; wherein R³ and R⁴, which may be the same or different, are hydrogen, optionally substituted alkyl, optionally substituted aryl, optionally substituted heteroarylalkyl, optionally substituted arylalkyl; heterocyclyl or optionally substituted heteroaryl; and p = 1 or 2.

More preferably Rl is hydrogen and R2 is optionally substituted aryl or heteroaryl. Preferred substituents are substituted alkyl, heterocyclyl, substituted alkoxy, amino, hydroxy, halogen, CONR³R⁴ or NR³(CO)R⁴, wherein R³ and R⁴, which may be the same or different, are hydrogen, optionally substituted alkyl, optionally substituted aryl, optionally substituted heteroarylalkyl, optionally substituted arylalkyl, heterocyclyl or optionally substituted heteroaryl.

It is especially preferred that R2 is a substituted aryl with a substituent in the meta or para position of the aryl ring. Preferred substituents are heterocyclyl or substituted alkyl wherein the substituents are NR³R⁴ or heterocyclyl and wherein R³ and R⁴, which may be the same or different, are hydrogen or optionally substituted alkyl, CONR⁵R⁶ where R⁵ and R⁶, which may be the same or different, are optionally substituted alkyl or heterocyclyl.

Preferred compounds are given in the Examples and listed in Tables 1 and 2.

Any known compound having a structural formula identical to any one of the compounds covered by Formula (I) is hereby explicitly disclaimed per se. In a second aspect the invention provides a pharmaceutical formulation comprising a compound of the invention and a pharmaceutically acceptable diluent or carrier

In a third aspect the invention provides a process for the preparation of a compound of the invention, which comprises at least one of the steps of: a) reacting a first intermediate compound with an optionally substituted 5-aminoindazol to arrive at a second intermediate compound; b) reacting a second intermediate compound with a boronic acid; c) obtaining a compound according to the invention.

In a preferred embodiment, the process comprises all of steps a) to c). Preferably, the first intermediate compound is 4, 6-chloropyridine and preferably the boronic acid has the formula R2B(OH)₂, wherein R2 is as defined above.

In a fourth aspect the invention provides a method for the prophylaxis or treatment of a Chkl kinase related disorder which comprises administering to a subject in need of such treatment an effective amount of a compound or a pharmaceutical formulation of the invention.

In a preferred embodiment the disorder is cancer or a disease state associated with angiogenesis and/or cell proliferation. Preferably, the disorder is selected from the group consisting of primary and metastatic tumours, including carcinomas of the breast, colon, rectum, lung, oropharynx, hypopharynx, oesophagus, stomach, pancreas, liver, gallbladder and bile ducts, small intestine, urinary tract (including kidney, bladder and urethra), female genital tract (including cervix, uterus and ovaries as well as choriocarcinoma and gestational trophobalstic disease), male genital tract, endocrine glands, and skin; melanoma, sarcomas, hemangiomas and tumours of the brain, nerves, eyes and meninges; solid tumours arising from haematopoietic malignancies such as leukaemias; and lymphomas.

In a preferred embodiment of the method the compound or the pharmaceutical formulation of the invention is administered in combination with radiotherapy and/or other chemotherapeutic agents.

In a fifth aspect the invention provides a method for modulating Chkl kinase activity which comprises administering to a subject in need of such treatment an effective amount of a compound or a pharmaceutical formulation of the invention.

In a sixth aspect the invention provides a compound of the invention for use in therapy, especially for use in the prophylaxis or treatment of a Chkl-related disorder.

A further aspect of the invention is the use of a compound as mentioned above for the manufacture of a medicament for use in the prophylaxis or treatment of a Chkl-related disorder. The compound may be administered in combination with radiotherapy and/or other chemotherapeutic agents.

Examples of Chkl-related disorders include cancer or a disease state associated with angiogenesis and/or cell proliferation. Preferably, the disorder is selected from the group consisting of primary and metastatic tumours, including carcinomas of the breast, colon, rectum, lung, oropharynx, hypopharynx, oesophagus, stomach, pancreas, liver, gallbladder and bile ducts, small intestine, urinary tract (including kidney, bladder and urethra), female genital tract (including cervix, uterus and ovaries as well as choriocarcinoma and gestational trophobalstic disease), male genital tract, endocrine glands, and skin; melanoma, sarcomas, hemangiomas and tumours of the brain, nerves, eyes and meninges; solid tumours arising from haematopoietic malignancies such as leukaemias; and lymphomas.

Definitions

The following definitions shall apply throughout the specification and the appended claims.

Within the context of the present application, the term "comprises" is taken to mean "includes among other things", and is not taken to mean "consists of only".

Unless otherwise stated or indicated, the term "alkyl" denotes a straight or branched alkyl group. Preferably said alkyl group is a "lower alkyl" having from 1 to 6 carbon atoms ("C^-alky!"). Examples of said lower alkyl groups include methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl, t-butyl and straight- and branched-chain pentyl and hexyl. For parts of the range "Cι-₆-alkyl" all subgroups thereof are contemplated such as Cι-₅-alkyl, Cι-₄-alkyl, Cι-₃-alkyl, C^-alkyl, C₂-₆-alkyl, C₂-₅-alkyl, C₂. ₄-alkyl, C₂-₃-alkyl, C₃.₆-alkyl, C₄.₅-alkyl, etc. "Halo-Cι-₆-alkyl" means a C_halky I group substituted with one or more halogen atoms. Likewise, "aryl- Cι-₆-alkyl" means a C^-alky! group substituted with one or more aryl groups.

Unless otherwise stated or indicated, the term "cycloalkyl" denotes a cyclic alkyl group preferably having a ring size from 3 to 8 carbon atoms ("C₃.₈-cycloalkyl"). Examples of said cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, methylcyclohexyl, cycloheptyl, and cyclooctyl. For parts of the range "C₃-₈-cycloalkyl" all subgroups thereof are contemplated such as C₃.₇-cycloalkyl, C₃.₆-cycloalkyl, C₃.₅-cycloalkyl, C₃-₄-cycloaIkyl, C₄.₈-cycloalkyl, C₄-₇-cycloalkyl, C₄-₆-cycloalkyl, C₄-₅- cycloalkyl, C₅-₇-cycloalkyl, C₆-₇-cycloalkyl, etc. Unless otherwise stated or indicated, the term "alkoxy" denotes a straight or branched alkoxy group. Preferably said alkoxy group is a "lower alkoxy" group having from 1 to 6 carbon atoms ("C^ alkoxy"). Examples of said lower alkoxy include methoxy, ethoxy, n-propoxy, iso-propoxy, n- butoxy, iso-butoxy, sec-butoxy, t-butoxy and straight- and branched- chain pentoxy and hexoxy. For parts of the range "Cι-₆-alkoxy" all subgroups thereof are contemplated such as Cι-₅-alkoxy, Cι-₄-alkoxy, Cι-₃- alkoxy, C^-alkoxy, C₂.₆-alkoxy, C₂.₅-alkoxy, C₂-₄-alkoxy, C₂.₃-alkoxy, C₃-₆- alkoxy, C₄-₅-alkoxy, etc.

Unless otherwise stated or indicated, the term "alkenyl" means a straight chain or branched alkenyl radical preferably of 2 to 6 carbon atoms and containing one or more carbon-carbon double bonds and includes but is not limited to ethylene, π-propyl-1-ene, n-propyl-2-ene, isopropylene, etc.

Unless otherwise stated or indicated, the term "alkynyl" means a straight chain or branched alkynyl radical preferably of 2 to 6 carbon atoms and containing one or more carbon-carbon triple bonds and includes but is not limited to ethynyl, 2-methylethynyl etc.

Unless otherwise stated or indicated, the term "aryl" refers to a hydrocarbon ring system, which is preferably a 3-10 membered ring system, having at least one aromatic ring or being fused to one or more saturated or unsaturated rings including, but not limited to phenyl, pentalenyl, indenyl, indanyl, isoindolinyl, chromanyl, naphthyl, fluorenyl, anthryl, phenanthryl and pyrenyl. The aryl rings may optionally be substituted with Cι_₆-alkyl. Examples of substituted aryl groups are benzyl and 2-methylphenyl. Likewise, aryloxy refers to an aryl group bonded to an oxygen atom.

Unless otherwise stated or indicated, the term "heteroaryl" refers to a hydrocarbon ring system having at least one aromatic ring which contains at least one heteroatom such as O, N, or S. Preferably the hydrocarbon ring system is a 3-10 membered ring system. Examples of heteroaryl groups include furyl, pyrrolyl, thienyl, oxazolyl, isoxazolyl, imidazolyl, thiazolyl, isothiazolyl, pyridinyl, pyrimidinyl, quinazolinyl, indolyl, pyrazolyl, pyridazinyl, quinolinyl, benzofuranyl, dihydrobenzofuranyl, benzodioxolyl, benzodioxinyl, benzothiazolyl, benzothiadiazolyl, and benzotriazolyl groups.

"Heterocyclyl" means a preferably 3-10 membered ring system containing one or more heteroatoms selected from N, O or S and includes heteroaryl. The heterocyclyl system can contain one ring or may be fused to one or more saturated or unsaturated rings; the heterocyclyl can be fully saturated, partially saturated or unsaturated and includes but is not limited to heteroaryl and heterocarbocyclyl. Examples of carbocyclyl or heterocyclyl groups include but are not limited to cyclohexyl, phenyl, acridine, benzimidazole, benzofuran, benzothiophene, benzoxazole, benzothiazole, carbazole, cinnoline, dioxin, dioxane, dioxolane, dithiane, dithiazine, dithiazole, dithiolane, furan, imidazole, imidazoline, imidazolidine, indole, indoline, indolizine, indazole, isoindole, isoquinoline, isoxazole, isothiazole, morpholine, napthyridine, oxazole, oxadiazole, oxathiazole, oxathiazolidine, oxazine, oxadiazine, phenazine, phenothiazine, phenoxazine, phthalazine, piperazine, piperidine, pteridine, purine, pyran, pyrazine, pyrazole, pyrazoline, pyrazolidine, pyridazine, pyridine, pyrimidine, pyrrole, pyrrolidine, pyrroline, quinoline, quinoxaline, quinazoline, quinolizine, tetrahydrofuran, tetrazine, tetrazole, thiophene, thiadiazine, thiadiazole, thiatriazole, thiazine, thiazole, thiomorpholine, thianaphthalene, thiopyran, triazine, triazole, and trithiane.

Unless otherwise stated or indicated, the term "halogen" shall mean fluorine, chlorine, bromine or iodine.

The term "leaving group" refers to a group to be displaced from a molecule during a nucleophilic displacement reaction. Examples of leaving groups are bromide, chloride and methanesulfonate, especially bromide and methanesulfonate

"Pharmaceutically acceptable" means being useful in preparing a pharmaceutical composition that is generally safe, non-toxic and neither biologically nor otherwise undesirable and includes being useful for veterinary use as well as human pharmaceutical use.

"Treatment" as used herein includes prophylaxis of the named disorder or condition, or amelioration or elimination of the disorder once it has been established.

"An effective amount" refers to an amount of a compound that confers a therapeutic effect on the treated subject. The therapeutic effect may be objective (i.e., measurable by some test or marker) or subjective (i.e., subject gives an indication of or feels an effect).

The term "prodrug forms" means a pharmacologically acceptable derivative, such as an ester or an amide, which derivative is biotransformed in the body to form the active drug. Reference is made to

Goodman and Gilman ^'s, The Pharmacological basis of Therapeutics, 8^th ed., Mc-Graw-Hill, Int. Ed. 1992, "Biotransformation of Drugs", p. 13-15.

The following abbreviations have been used:

ACN means acetonitrile,

DEA means diethylamine,

DEPT means distortion enhancement polarisation transfer,

DMSO means dimethyl sulfoxide,

ELS means electron light scattering,

HPLC means high performance liquid chromatography,

Rt means retention time,

TFA means trifiuoroacetic acid,

THF means tetrahydrofuran,

TLC means thin layer chromatography. All diastereomeric forms possible (pure enantiomers, tautomers, racemic mixtures and unequal mixtures of two or more enantiomers) are within the scope of the invention. Such compounds can also occur as cis- or trans-, E- or Z- double bond isomer forms. All isomeric forms and mixtures thereof are contemplated.

The compounds of the formula (I) may be used as such or, where appropriate, as pharmacologically acceptable salts (acid or base addition salts) thereof. The pharmacologically acceptable addition salts mentioned above are meant to comprise the therapeutically active non-toxic acid and base addition salt forms that the compounds are able to form. Compounds that have basic properties can be converted to their pharmaceutically acceptable acid addition salts by treating the base form with an appropriate acid. Exemplary acids include inorganic acids, such as hydrogen chloride, hydrogen bromide, hydrogen iodide, sulfuric acid, phosphoric acid; and organic acids such as formic acid, acetic acid, propanoic acid, hγdroxyacetic acid, lactic acid, pyruvic acid, glycolic acid, maleic acid, malonic acid, oxalic acid, benzenesulfonic acid, toluenesulfonic acid, methanesulfonic acid, trifluoroacetic acid, fumaric acid, succinic acid, malic acid, tartaric acid, citric acid, salicylic acid, p-aminosalicylic acid, pamoic acid, benzoic acid, ascorbic acid and the like. Exemplary base addition salt forms are the sodium, potassium, calcium salts, and salts with pharmaceutically acceptable amines such as, for example, ammonia, alkylamines, benzathine, and amino acids, such as, e.g. arginine and lysine. The term addition salt as used herein also comprises solvates which the compounds and salts thereof are able to form, such as, for example, hydrates, alcoholates and the like.

For clinical use, the compounds of the invention are formulated into pharmaceutical formulations for oral, rectal, parenteral or other mode of administration. Pharmaceutical formulations are usually prepared by mixing the active substance, or a pharmaceutically acceptable salt thereof, with a conventional pharmaceutical excipient (a pharmaceutically acceptable diluent or carrier). Examples of excipients are water, gelatin, gum arabicum, lactose, microcrystalline cellulose, starch, sodium starch glycolate, calcium hydrogen phosphate, magnesium stearate, talcum, colloidal silicon dioxide, and the like. Such formulations may also contain other pharmacologically active agents, and conventional additives, such as stabilizers, wetting agents, emulsifiers, flavouring agents, buffers, and the like. The use of such excipients for pharmaceutically active substances is well known in the art. Except in so far as conventional excipient is incompatible with the active compound, use thereof in the pharmaceutical formulation of the invention is contemplated.

Such formulations may also contain other pharmacologically active agents, and conventional additives, such as stabilizers, wetting agents, emulsifiers, flavouring agents, buffers, and the like.

The formulations can be further prepared by known methods such as granulation, compression, microencapsulation, spray coating, etc. The formulations may be prepared by conventional methods in the dosage form of tablets, capsules, granules, powders, syrups, suspensions, suppositories or injections. Liquid formulations may be prepared by dissolving or suspending the active substance in water or other suitable vehicles. Tablets and granules may be coated in a conventional manner.

Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, 'chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum mono stearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the active compound (e.g., a compound according to an embodiment of the invention) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile- filtered solution thereof.

Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.

Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art.

It is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.

Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds which exhibit large therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.

The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration. In a further aspect the invention relates to methods of making compounds of any of the formulae herein comprising reacting any one or more of the compounds of the formulae delineated herein, including any processes delineated herein. The compounds of formula (I) may be prepared by, or in analogy with, conventional methods.

The processes described above may be carried out to give a compound of the invention in the form of a free base or as an acid addition salt. A pharmaceutically acceptable acid addition salt may be obtained by dissolving the free base in a suitable organic solvent and treating the solution with an acid, in accordance with conventional procedures for preparing acid addition slats from base compounds. Examples of addition salt forming acids are mentioned above.

The compounds of formula (I) may possess one or more chiral carbon atoms, and they may therefore be obtained in the form of optical isomers, e.g. as a pure enantiomer, or as a mixture of enantiomers (racemate) or as a mixture containing diastereomers. The separation of mixtures of optical isomers to obtain pure enantiomers is well known in the art and may, for example, be achieved by fractional crystallization of salts with optically active (chiral) acids or by chromatographic separation on chiral columns.

The necessary starting materials for preparing the compounds of formula (I) are either known or may be prepared in analogy with the preparation of known compounds. The dose level and frequency of dosage of the specific compound will vary depending on a variety of factors including the potency of the specific compound employed, the metabolic stability and length of action of that compound, the patient's age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the condition to be treated, and the patient undergoing therapy. The daily dosage may, for example, range from about 0.001 mg to about 100 mg per kilo of body weight, administered singly or multiply in doses, e.g. from about 0.01 mg to about 25 mg each. Normally, such a dosage is given orally but parenteral administration may also be chosen.

In accordance with methods of treatment and pharmaceutical compositions of the invention, the compounds can be administered alone or in combination with other anticancer agents

Within this specification embodiments have been described in a way that enables a clear and concise specification to be written, but it will be appreciated that embodiments may be variously combined or separated without parting from the invention.

The invention will now be further illustrated by the following non-limiting examples.

EXAMPLES

Experimental Methods

All reagents were commercial grade and were used as received without further purification, unless otherwise specified. Commercially available anhydrous solvents were used for reactions conducted under inert atmosphere. Reagent grade solvents were used in all other cases, unless otherwise specified. Column chromatography was performed on Matrex® silica gel 60 (35-70 micron). TLC was carried out using pre-coated silica gel F-254 plates (thickness 0.25 mm). ¹H NMR spectra were recorded on a Bruker Avance250 at 400 MHz. Chemical shifts for ¹H NMR spectra are given in part per million and either tetramethylsilane (0.00 ppm) or residual solvent peaks were used as internal reference. Splitting patterns are designated as follows: s, singlet; d, doublet; t, triplet; q, quartet; p, pentet; m, multiplet; br, broad. Coupling constants are given in Hertz (Hz). Only selected data are reported. Electrospray MS spectra were obtained on a Micromass platform LCMS spectrometer. Compounds were named using AutoNom 2000.

Preferred compounds of the present invention are shown in Table 1. Table 1

An in-vitro assay of Chkl activity was developed using recombinant Chkl kinase, full length in the form of a GST-Chkl fusion protein supplied by the MRC Protein Phosphorylation Unit in Dundee. Assays were performed in polypropylene 96 or 384 well plates in a buffer containing lOOmM Tris - HCI (pH 7.5), 0.2mM EGTA, 20mM Mg Acetate, 0.2% β-mercaptoethanol. Chkl enzyme was diluted in 50mM Tris -HCI (pH 7.5), O.lmM EGTA,lmg/mL BSA, 0.1% β-mercaptoethanol. Compounds were incubated with enzyme (3mU) and a mixture containing substrate (biotin- KKKVSRSGLYRSPSMPENLNRPR, 3μM), cold ATP(lμM), and ^33_P-γ- ATP(0.2μCi) to start the reaction. Reactions were allowed to proceed for the 30min and stopped by addition of 50mM EDTA. 90% of the total reaction mixture was transferred to 96 or 384 well streptavidin coated flashplates and incubated for lhr. Flashplates were then washed 3x with lOOμl PBS containing 0.01% Tween 20. The biotinylated phosphorylated substrate bound to the flashplates was detected using a Packard topcount scintillation reader. Potency determinations for each compound were conducted across a 1.69nM to 100μM concentration range using duplicate data points and the protocol described above.

The activity of exemplary compounds is presented in Table 2, wherein "+" indicates active compounds, "++" indicates very active compounds and "+++" indicates even more active compounds. In Table 2, "+++" represents an IC₅₀<lμM, "++" represents an IC₅₀<10μM, "+" represents 10μM <IC₅₀ <30μM.

Table 2

Remarkably, the present invention provides a class of compounds which interact selectively with the Chkl kinase and show selectivity over other kinases , more specifically the key selectivity target CDKl. For example [6-(4-Dimethylaminomethyl-phenyl)-pyrimidin-4-yl]-(lH-indazol-5-yl)- amine shows good selectivity over the key selectivity target CDKl with an IC₅₀ value of 37.2μM (>50x selectivity).

PREPARATION OF COMPOUNDS General Scheme for synthesising compounds of the invention

4,6-chloropyridine (A) can be aminated with a 5-aminoindazol. The resultant compounds (B) can then be reacted with the boronic acids to yield the final compounds of formula (C). Formula (C) corresponds to Formula I. The 5-aminoindazol used can optionally be substituted.

Preparation of a compound of formula (B)

General procedure (an amine displacement reaction) :

A mixture of 4,6-dichloro-pyrimidine (lg, 6.7 mmol) and lH-Indazol-5- ylamine (0.9g, 6.6 mmol) was prepared in 10 ml of iPrOH. To this was added ethyldiisopropylamine (1.5mL, 8.7 mmol). The reaction was heated at 80 °C for 12 to 18 hrs, with stirring. The reaction was then cooled and the solvent evaporated. Ethyl acetate and water were added.

The product was extracted with ethyl acetate and the combined organics then washed with brine and dried over MgSO₄. The crude compound was used without further purification.

Example:

(6-Chloro-pyrimidin-4-yl)-(lH-indazol-5-yl)-amine

LCMS (ES⁺): 246 [100 %, (M+H)⁺]

^XH NMR (400 MHz, </₆-DMSO): δ/ppm 12.95 (s, 1H), 10.1 (s, 1H), 8.6

(s, 1H), 8.2 (s, 1H), 8.05 (s, 1H), 7.75 (m, 1H), 7.15 (m, 1H,), 6.95 (s,

1H).

Preparation of a compound of formula (C) General procedure (boronic acid coupling):

A mixture of (6-Chloro-pyrimidin-4-yl)-(lH-indazol-5-yl)-amine (B) (0.3 mmol) and boronic acid (0.4 mmol) was prepared in a microwave reaction tube with magnetic stirrer. To this was added potassium hydrogenocarbonate (110 mg, 1.1 mmol), triphenylphosphine (22 mg, 0.09 mmol) and palladium acetate (7 mg, 0.03 mmol) followed by dimethoxyethane: water solution (2.5ml 3: 1)(1.5 mL). The reaction tube was flushed with nitrogen, then sealed and placed in a microwave reactor and heated at 150 °C for 1 hr, with stirring. The reaction was then cooled and ethylacetate (15 mL) and water (10 mL) were added. The dark mixture was filtered free of palladium residues and the organics extracted with a mixture of ethylacetate/methanol. Purification of the crude material was achieved by preparative HPLC or by silica chromatography using a Biotage system.

Example:

[6-(4-Aminomethyl-phenyl)-pyrϊmidin-4-yl]-(lH-indazol-5-yl)- amine

LCMS (ES⁺): 317 [100 %, (M+ H)⁺]

*H NMR (400 MHz, </₆-DMSO): δ/ppm 13.1 (s, 1H), 9.95(s, 1H), 8.8 (s,

1H), 8.25 (br s, 3H), 8.2 (m, 3H), 7.6-7.8 (m, 4H), 7.35 (s, 1H), 4.2 (s,

2H).

It will be appreciated by those skilled in the art that the foregoing description is exemplary and explanatory in nature, and is intended to illustrate the invention and its preferred embodiments. Through routine experimentation, an artisan will recognise apparent modifications and variations that may be made without departing from the spirit as scope of the invention as defined in the appended claims.

Claims

1. A compound of Formula I:

Formula I wherein, Rl can be hydrogen, optionally substituted alkyl, optionally substituted alkoxy, OCF₃, CF₃, amino, optionally substituted aryloxy, halogen, hydroxy, CN, CO₂H, NR⁴R⁵, CO₂R⁴, CONR⁴R⁵, NR⁴(CO)R⁵ or S(O)_pR⁴; wherein R⁴ and R⁵, which can be the same or different, can be hydrogen, optionally substituted alkyl, optionally substituted aryl, optionally substituted heteroarylalkyl or optionally substituted arylalkyl; and wherein p is 1 or 2;

Rl can be in the 3, 4, 6 or 7 position of the indazol ring; and

R2 is optionally substituted aryl or optionally substituted heteroaryl; or a pharmaceutically acceptable salt, hydrate, solvate, geometrical isomer, tautomer, optical isomer, or prodrug form thereof.

A compound according to claim 1, wherein the substituents are selected from the group comprising alkyl, cycloalkyl, OCF₃, CF₃, heterocyclyl, alkenyl, alkynyl, alkoxy, aryloxy, halogen, hydroxy, amino, NO₂, CN, NR³R⁴, CO₂R³, CONR³R⁴, NR³(CO) R⁴, S(O)_pR³; wherein R³ and R⁴, which may be the same or different, are hydrogen, optionally substituted alkyl, optionally substituted aryl, optionally substituted heteroarylalkyl, optionally substituted arylalkyl; heterocyclyl or optionally substituted heteroaryl; and p = 1 or

2.

3. A compound according to claim 1 or 2, wherein Rl is hydrogen and R2 is optionally substituted aryl or heteroaryl.

4. A compound according to claim 3, wherein substituents are selected from the group comprising substituted alkyl, heterocyclyl, substituted alkoxy, amino, hydroxy, halogen, CONR³R⁴ or NR³(CO)R⁴, wherein R³ and R⁴, which may be the same or different, are hydrogen, optionally substituted alkyl, optionally substituted aryl, optionally substituted heteroarylalkyl, optionally substituted arylalkyl, heterocyclyl or optionally substituted heteroaryl.

5. A compound according to any preceding claim, wherein R2 is a substituted aryl with a substituent in the meta or para position of the aryl ring.

6. A compound according to claim 5 wherein the substituent is heterocyclyl or substituted alkyl wherein the substituent is NR³R⁴ or heterocyclyl and wherein R³ and R⁴, which may be the same or different, are hydrogen or optionally substituted alkyl, CONR⁵R⁵ wherein R⁵ and R⁶, which may be the same or different, are optionally substituted alkyl or heterocyclyl.

7. A compound according to any preceding claim which is selected from the group consisting of: [6-(5-Dimethylaminomethyl-2-methoxy-phenyl)pyrimidin-4-yl]-(lH- indazol-5-yl)-amine; N-{3-[6-(lH-Indazol-5-ylamino)-pyrimidin-4-yl]phenyl}- methanesulfonamide; N5-[6-(4-Dimethylaminomethyl-phenyl)-pyrimidin-4yl]-lH-indazole- 3,5-diamine; {6-[6-(3-Dimethylamino-propoxy)-pyridin-3-yl]pyrimidin-4-yl}-(lH- indazol-5-yl)-amine; 3-[6-(lH-Indazol-5-ylamino)-pyrimidin-4-yl]benzamide; [6-(4-Dimethylaminomethyl-phenyl)-pyrimidin-4-yl]-(l-methyl-lH- indazol-5-yl)-amine; {4-[6-(lH-Indazol-5-ylamino)-pyrimidin-4-yl]-phenyl}-(4-methyl- piperazin-l-yl)-methanone; {3-[6-(lH-Indazol-5-yIamino)-pyrimidin-4-yl]phenyl}methanol; (lH-Indazol-5-yl)-[6-(lH-pyrazol-4-yl)-pyrimidin-4yl]-amine; {4-[6-(lH-Indazol-5-ylamino)-pyrimidin-4-yl]phenyl}methanol; (lH-IndazoI-5-yl)-{6-[6-(4-methyl-piperazin-l-yl)-pyridin-3-yi]- pyrimidin-4-yl}-amine; 3-[6-(lH-Indazol-5-ylamino)-pyrimidin-4-yl]-phenol; [6-(3-Dimethylaminomethyl-phenyl)-pyrimidin-4-yl]-(lH-indazol-5- yl)-amine; [6-(4-Dimethylaminomethyl-3-fluoro-phenyl)-pyrimidin-4-yl]-(lH- indazol-5-yl)-amine; N-(3-Dimethylamino-propyl)-4-[6-(lH-indazol-5-ylamino)-pyrimidin- 4-yl]-benzamide; N-(2-Dimethylamino-ethyl)-4-[6-(lH-indazol-5-ylamino)-pyrimidin- 4-yl]-benzamide; [6-(4-Dimethylaminomethyl-phenyl)-pyrimidin-4-yl]-(lH-indazol-5- yl)-amine; and [6-(4-Aminomethyl-phenyl)-pyrimidin-4-yl]-(lH-indazol-5-yl)-amine.

8. A pharmaceutical formulation comprising a compound according to any preceding claim and a pharmaceutically acceptable diluent or carrier.

9. A process for the preparation of a compound according to any one of claim 1 to 7, which comprises at least one of the steps of: a) reacting a first intermediate compound with an optionally substituted 5-aminoindazol to arrive at a second intermediate compound; b) reacting a second intermediate compound with a boronic acid; c) obtaining a compound according to any one of claims 1 to 7.

10. A process according to claim 9 which comprises all of steps a) to c).

11. A process according to claim 9 or 10 wherein the first intermediate compound is 4, 6-chloropyridine.

12. A process according to any one of claims 9 to 11 wherein the boronic acid has the formula R2B(OH)₂, wherein R2 is as defined in any one of claims 1 to 7.

13. A method for the prophylaxis or treatment of a Chkl kinase related disorder which comprises administering to a subject in need of such treatment an effective amount of a compound according to any one of claims 1 to 7 or a pharmaceutical formulation according to claim 8.

14. A method according to claim 13, wherein the disorder is cancer or a disease state associated with angiogenesis and/or cell proliferation.

15. A method according to claim 13 or 14, wherein the disorder is selected from the group consisting of primary and metastatic tumours, including carcinomas of the breast, colon, rectum, lung, oropharynx, hypopharynx, esophagus, stomach, pancreas, liver, gallbladder and bile ducts, small intestine, urinary tract (including kidney, bladder and urethra), female genital tract (including cervix, uterus and ovaries as well as choriocarcinoma and gestational trophobalstic disease), male genital tract, endocrine glands, and skin; melanoma, sarcomas, hemangiomas and tumours of the brain, nerves, eyes and meninges; solid tumours arising from hematopoietic malignancies such as leukaemias; and lymphomas.

16. A method according to any one of claims 13 to 15, wherein the compound of any one of claims 1 to 7 or the pharmaceutical formulation of claim 8 is administered in combination with radiotherapy and/or other chemotherapeutic agents.

17. A method for modulating Chkl kinase activity which comprises administering to a subject in need of such treatment an effective amount of a compound according to any one of claims 1 to 7 or a pharmaceutical formulation according to claim 8.

18. A compound according to any one of claims 1 to 7 for use in therapy.

19. A compound according to claim 18 for use in the prophylaxis or treatment of a Chkl-related disorder.

20. Use of a compound according to any one of claims 1 to 7 for the manufacture of a medicament for use in the prophylaxis or treatment of a Chkl-related disorder.

21. The use according to claim 20 wherein the disorder is cancer or a disease state associated with angiogenesis and/or cell proliferation.

22. The use according to claim 20 or 21 wherein the disorder is selected from the group consisting of primary and metastatic tumours, including carcinomas of the breast, colon, rectum, lung, oropharynx, hypopharynx, oesophagus, stomach, pancreas, liver, gallbladder and bile ducts, small intestine, urinary tract (including kidney, bladder and urethra), female genital tract (including cervix, uterus and ovaries as well as choriocarcinoma and gestational trophobalstic disease), male genital tract, endocrine glands, and skin; melanoma, sarcomas, hemangiomas and tumours of the brain, nerves, eyes and meninges; solid tumours arising from haematopoietic malignancies such as leukaemias; and lymphomas.

23. The use according to any one of claims 20 to 22, wherein the compound of any one of claims 1 to 7 is administered in combination with radiotherapy and/or other chemotherapeutic agents.

24. An assay comprising a Chkl kinase and a compound according to any one of claims 1 to 7.

25. Use of an assay according to claim 24 for identifying a compound which has therapeutic affect or importation of a compound identified using an assay according to claim 24.

26. A compound, a process for the preparation of a compound, a pharmaceutical formulation, a use, a method of treatment or prophylaxis or an assay substantially as described herein.