WO2004016615A1

WO2004016615A1 - PYRAZOLO (4,3c) CINNOLINE COMPOUNDS AS INHIBITORS OF ITK KINASE ACTIVITY

Info

Publication number: WO2004016615A1
Application number: PCT/SE2003/001278
Authority: WO
Inventors: Jan Dahmen; Martin Hemmerling; Tomas Klingstedt; Peter Sjö
Original assignee: AstraZeneca AB
Current assignee: AstraZeneca AB
Priority date: 2002-08-14
Filing date: 2003-08-13
Publication date: 2004-02-26
Anticipated expiration: 2005-02-14
Also published as: AU2003258922A1; SE0202461D0

Abstract

There are provided novel compounds of formula (I) wherein R and X are as defined in the Specification and optical isomers, racemates and tautomers thereof, and pharmaceutically acceptable salts thereof; together with processes for their preparation, compositions containing them and their use in therapy. The compounds are inhibitors of the kinase Itk.

Description

PYRAZOLO 4,3c CINNOLINE COMPOUNDS AS INHIBITORS OF ITK KINASE ACTIVITY.

Field of the Invention

This invention relates to novel cinnoline derivatives, processes for their preparation, intermediates thereto, pharmaceutical compositions comprising them, and their use in therapy.

Background of the Invention

Inducible T cell Kinase (Itk) is a member of the Tec-family of cytosolic protein tyrosine kinases. In mammalians, this family also includes Btk, Tec, Bmx, and Txk. These kinases regulate various immune cell functions that integrate signals given by the other cytosolic tyrosine kinases as well as serine/threonine kinases, lipid kinases, and small G proteins. Tec-family kinases have the following general structure: a N-terminal pleckstrin-homology (PH) domain, a Tec-homology domain that includes a Btk motif and one or two proline- rich (PR) motifs, a SH3 domain, a SH2 domain and a c-terminal catalytic (SHI) domain. These kinases are expressed exclusively in hematopoietic tissues, with the exception of Tec and Bmx that have also been detected in endothelial cells. The cellular distribution is different for the Tec-family members. For example, Itk is expressed by T cells, NK cells and mast cells, whereas Btk is expressed by all hematopoietic cells except T cells. Thus, hematopoietic cells may express one or several Tec-family kinases. For example, T cells express Itk, Tec and Txk, and mast cells express Btk, Itk and Tec. Btk is by far the most extensively studied among the Tec-family kinases, due to its association with X-linked agammaglobulinemia (XLA), and Btk is currently the only Tec- family kinase with a known human phenotype. XLA patients are virtually devoid of mature B cells and their Ig levels are strongly reduced.

Itk^"'^" mice show defects in T cell activation and differentiation. T helper 2 (Th2) differentiation is disrupted in these mice, whereas Thl differentiation is apparently intact.

In T and B cells, signalling through T cell receptors and B cell receptors leads to activation of Itk and Btk. respectively. Downstream of Itk and Btk a number of different messengers are engaged; scaffolding proteins (SLP-76, LAT, SLP-65), Src kinases, MAP kinases, and PI3-K. These events are followed by PLC-γ activation that leads to IP3 generation and sustained Ca²⁺ flux, and subsequently activation of transcription factors. PLC-γl has been suggested as a direct substrate for Itk. In T cells, Itk (and Tec) may also mediate signalling through the CD28 co-receptor. Furthermore, Itk has in T cells been implicated in the activation of β-integrin. Signalling from Tec-family kinases can also be regulated by PH domain-mediated plasma membrane localization, and by Src-family-mediated phosphorylation of critical tyrosine residues. Interestingly, Itk, Btk and Txk have recently been shown to translocate to the nucleus after activation.

From studies using Itk-/- mice, it has been proposed that Itk is required for Th2 but not Thl cell development. This was demonstrated in the N. brasiliensis and L. major infection models where the Itk-/- animals are protected in the Leishmania model indicating an intact Thl response, whereas they are susceptible to infection with N. Brasiliensis that requires an intact Th2 response for resolution of the infection. This indicates that modulation of Itk activity may prove useful for treatment of Th2-driven disorders and conditions.

We have identified the critical role of Itk in regulating important mast cell and basophil functions and established that the activity of mast cells or basophils may be inhibited through inhibition of Itk. Thus Itk inhibitors may be used as pharmaceutical agents for the treatment of mast cell-driven or basophil-driven conditions or diseases. In particular, we have identified Itk as a target for inhibiting several key events in both acute and late phase allergic reactions common to allergic rhinitis and asthma.

Ames and Byrne (J. Chem. Soc, Perkin Trans. I (1976) 592-4) disclose the preparation of 2,3-dihydro-3-imino-lH-pyrazolo[4,3-c]cinnoline:

There was no disclosure of any specific use for this compound.

The present invention discloses novel substituted 2,3-dihydro-3-imino-lH-pyrazolo[4,3- c]cinnolines and lH-pyrazolo[4,3-c]cinnolin-3-ols that have activity as Itk inhibitors and are thereby useful as pharmaceuticals.

Disclosure of the Invention The present invention provides a compound of formula (I)

(I)

wherein:

X represents O or NH; and

R represents hydrogen, halogen, CI to 6 alkyl, aryl or heteroaryl; said aryl or heteroaryl group each being optionally substituted with one or more groups selected from halogen, CI to 6 alkyl, CI ft) 6 alkoxy, cyano and acetamido (-NHCOCH3);

and pharmaceutically acceptable salts thereof; with the proviso that the compound wherein X represents NH and R represents hydrogen is excluded.

The compounds of formula (I) may exist in enantiomeric forms. It is to be understood that all enantiomers, diastereomers, racemates and mixtures thereof are included within the scope of the invention.

Compounds of formula (I) may also exist in various tautomeric forms. Examples of such tautomeric forms are illustrated in formulae (la) and (lb):

(la) (lb)

All possible tautomeric forms and mixtures thereof are included within the scope of the invention.

In one embodiment, R represents halogen, CI to 6 alkyl, aryl or heteroaryl; said aryl or heteroaryl group each being optionally substituted with one or more groups selected from halogen, CI to 6 alkyl, CI to 6 alkoxy, cyano and acetamido (-NHCOCH3).

In one embodiment, R represents aryl, optionally substituted.

In another embodiment, R represents heteroaryl, optionally substituted.

In another embodiment X represents O. Particular compounds according to the present invention include:

6-brorno- lH-pyrazolo[4,3-c]cinnolin-3-ol;

6-bromo- 1 H-pyrazolo[4,3-c]cinnolin-3-amine; 6-phenyl-lH-pyrazolo[4,3-c]cinnolin-3-ol;

6-(4-methoxyphenyl)-lH-pyrazolo[4,3-c]cinnolin-3-ol;

6-(4-methoxyphenyl)-lH-pyrazolo[4,3-c]cinnolin-3-amine;

6-(2-methoxyphenyl)-lH-pyrazolo[4,3-c]cinnolin-3-ol;

6-(2-methoxyphenyl)-lH-pyrazolo[4,3-c]cinnolin-3-amine; 6-(3-methoxyphenyl)-lH-pyrazolo[4,3-c]cinnolin-3-ol;

6-(3-methoxyphenyl)-lH-pyrazolo[4,3-c]cinnolin-3-amine;

6-phenyl-lH-pyrazolo[4,3-c]cinnolin-3-amine;

6-pyridin-3-yl-lH-pyrazolo[4,3-c]cinnolin-3-ol;

6-pyridin-3-yl-lH-pyrazolo[4,3-c]cinnolin-3-amine; 6-(5-chloro-2-methoxyphenyl)-lH-pyrazolo[4,3-c]cinnolin-3-ol;

8-bromo-lH-pyrazolo[4,3-c]cinnolin-3-ol;

8-phenyl-lH-pyrazolo[4,3-c]cinnolin-3-ol;

8-σ-tolyl-lH-pyrazolo[4,3-c]cinnolin-3-ol;

8-thiophen-2-yl-lH-pyrazolo[4,3-c]cinnolin-3-ol; 8-(3-(acetylamino)phenyl)-lH-pyrazolo[4,3-c]cinnolin-3-ol;

8-(4-cyanophenyl)-lH-pyrazolo[4,3-c]cinnolin-3-ol;

7-bromo- lH-pyrazolo[4,3-c]cinnolin-3-ol;

7-phenyl- lH-pyrazolo[4,3-c]cinnolin-3-ol;

7-o-tolyl- lH-pyrazolo[4,3-c]cinnolin-3-ol; 7-thiophen-2-yl-lH-pyrazolo[4,3-c]cinnolin-3-ol;

7-(3-(acetylamino)phenyl)-lH-pyrazolo[4,3-c]cinnolin-3-ol;

8-(4-cyanophenyl)-lH-pyrazolo[4,3-c]cinnolin-3-ol;

and pharmaceutically acceptable salts thereof. Unless otherwise indicated, the term "CI to 6 alkyl" referred to herein denotes a straight or branched chain alkyl group having from 1 to 6 carbon atoms. Examples of such groups include methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, t-butyl, pentyl and hexyl.

Unless otherwise indicated, the term "CI to 6 alkoxy " referred to herein denotes an oxygen substituent bonded to a straight or branched chain alkyl group having from 1 to 6 carbon atoms. Examples of such groups include methoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy, i-butoxy and s-butoxy.

Unless otherwise indicated, the term "halogen" referred to herein denotes fluorine, chlorine, bromine and iodine.

Unless otherwise indicated, the term "aryl" referred to herein denotes a C6 to 10 carbocyclic aromatic ring system. Examples include phenyl, naphthyl and indanyl.

In one particular embodiment, aryl represents phenyl.

Unless otherwise indicated, the term "heteroaryl" referred to herein denotes a mono- or bi- cyclic aromatic ring system incorporating one or two heteroatoms independently selected from N, O and S. Examples include furyl, thienyl, thiazolyl, pyridyl, pyrimidinyl, pyrazinyl and quinolyl.

In one particular embodiment, heteroaryl represents thienyl or pyridyl.

The present invention includes compounds of formula (I) in the form of salts, in particular acid addition salts. Suitable salts include those formed with both organic and inorganic acids. Such acid addition salts will normally be pharmaceutically acceptable although salts of non-pharmaceutically acceptable acids may be of utility in the preparation and purification of the compound in question. Thus, preferred salts include those formed from hydrochloric, hydrobromic, sulphuric, phosphoric, citric, tartaric, lactic, pyruvic, acetic, succinic, fumaric, maleic, methanesulphonic and benzenesulphonic acids.

In a further aspect the invention provides a process for the preparation of a compound of formula (I) which comprises:

a) preparing a compound of formula (I) wherein X is O, by reaction of a compound of formula (II)

(II)

wherein R is as defined in formula (I), with hydrazine hydrate; or

b) preparing a compound of formula (I) wherein X is NH, by reaction of a compound of formula (III)

wherein R is as defined in formula (I), with hydrazine hydrate;

and where desired or necessary converting the resultant compound of formula (I), or another salt thereof, into a pharmaceutically acceptable salt thereof; or converting one compound of formula (I) into another compound of formula (I); and where desired converting the resultant compound of formula (I) into an optical isomer thereof.

In processes (a) and (b), the reaction with hydrazine hydrate is carried out in a suitable protic solvent, for example, ethanol, at a suitable elevated temperature, for example, at about 80 °C.

Compounds of formula (II) can be prepared from compounds of formula (IN):

(IV)

wherein R is as defined in formula (I), using methods that will in general be well known to the man skilled in the art. For example, by treatment of a compound of formula (TV) with thionyl chloride, optionally in the presence of an inert organic solvent, at a suitable temperature, for example, at about 80 °C, followed by treatment of the resulting acyl chloride with aqueous ammonia at a temperature of about 0 °C.

Compounds of the general formula (IV) can be prepared by reacting a compound of formula (V)

(V) wherein R is as defined in formula (I), with a suitable nucleophilic aqueous base, for example, aqueous potassium hydroxide, in the presence of a suitable inert organic solvent such as dioxane, at a suitable elevated temperature, for example, at about 100 °C.

Compounds of the general formula (V) may be prepared by the method outlined by Stanczak, A. et al. (Pharmazie, 1994, 49, 884).

Compounds of the general formula (III) can be prepared from compounds of formula (II) using methods that will in general be well known to the man skilled in the art. For example, by reaction with phosphoryl chloride at a suitable elevated temperature, for example, at about 100 °C.

Compounds of formula (I) wherein R represents optionally substituted aryl or heteroaryl can be conveniently prepared by reaction of a compound of formula (I) wherein R is halogen, particularly bromo, that is a compound of formula (VI),

with a boronic acid derivative of formula (VII):

R-B(OH)₂ (VII)

wherein R represents optionally substituted aryl or heteroaryl. The reaction can be carried out in any suitable inert solvent or solvent mixture, such as a mixture of dioxane and water. The reaction is preferably carried out at a suitable elevated temperature, for example, at about 90 °C, and in the presence of a suitable base, for example, sodium carbonate, and in the presence of a suitable palladium catalyst. Salts of compounds of formula (I) may be formed by reacting the free base or a salt, enantiomer, tautomer or protected derivative thereof, with one or more equivalents of the appropriate acid. The reaction may be carried out in a solvent or medium in which the salt is insoluble, or in a solvent in which the salt is soluble followed by subsequent removal of the solvent in vacuo or by freeze drying. Suitable solvents include, for example, water, dioxan, ethanol, 2-propanol, tetrahydrofuran or diethyl ether, or mixtures thereof. The reaction may be a metathetical process or it may be carried out on an ion exchange resin.

Compounds of formula (I) and intermediate compounds thereto may be prepared as such or in protected form. The protection and deprotection of functional groups is, for example, described in 'Protective Groups in Organic Chemistry', edited by J. W. F. McOmie, Plenum Press (1973), and 'Protective Groups in Organic Synthesis', 3rd edition, T. W. Greene & P. G. M. Wuts, Wiley-Interscience (1999).

The compounds of the invention and intermediates may be isolated from their reaction mixtures, and if necessary further purified, by using standard techniques.

The compounds of formula (I) may exist in enantiomeric or diastereoisomeric forms or mixtures thereof, all of which are included within the scope of the invention. The various optical isomers may be isolated by separation of a racemic mixture of the compounds using conventional techniques, for example, fractional crystallisation or HPLC. Alternatively, the individual enantiomers may be made by reaction of the appropriate optically active starting materials under reaction conditions that will not cause racemisation.

Intermediate compounds may also exist in enantiomeric forms and may be used as purified enantiomers, diastereomers, racemates or mixtures thereof.

According to a further aspect of the invention we provide a compound of formula (I) or a pharmaceutically acceptable salts thereof, for use as a medicament. The compounds of formula (I), and their pharmaceutically acceptable salts, are useful because they possess pharmacological activity in animals. The compounds of formula (I) have activity as pharmaceuticals, in particular as modulators of kinase activity, especially Itk kinase activity, and as such are predicted to be useful in therapy. They may be used in the treatment or prophylaxis of allergic, autoimmune, inflammatory, proliferative and hyperproliferative diseases and immune-mediated diseases including rejection of transplanted organs or tissues and Acquired Immunodeficiency Syndrome (AIDS).

Thus, another aspect of the invention provides the use of a compound of formula (I) or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment or prophylaxis of diseases or conditions in which inhibition of Itk activity is beneficial; and a method of treating, or reducing the risk of, diseases or conditions in which inhibition of Itk activity is beneficial which comprises administering to a person suffering from or at risk of, said disease or condition, a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof.

Examples of these conditions are:

( 1 ) (the respiratory tract) airways diseases including chronic obstructive pulmonary disease (COPD) such as irreversible COPD; asthma, such as bronchial, allergic, intrinsic, extrinsic and dust asthma, particularly chronic or inveterate asthma (for example, late asthma and airways hyper-responsiveness); bronchitis; acute, allergic, atrophic rhinitis and chronic rhinitis including rhinitis caseosa, hypertrophic rhinitis, rhinitis purulenta, rhinitis sicca and rhinitis medicamentosa; membranous rhinitis including croupous, fibrinous and pseudomembranous rhinitis and scrofoulous rhinitis; seasonal rhinitis including rhinitis nervosa (hay fever) and vasomotor rhinitis; sarcoidosis, farmer's lung and related diseases, fibroid lung and idiopathic interstitial pneumonia; sinusitis, chronic rhinosinusitis, nasosinusal polyposis; pulmonary fibrosis; (2) (bone and joints) rheumatoid arthritis, seronegative spondyloarthropathies (including ankylosing spondylitis, psoriatic arthritis and Reiter's disease), Behcet's disease, Sjogren's syndrome and systemic sclerosis;

(3) (skin) psoriasis, atopical dermatitis, contact dermatitis and other eczmatous dermitides, seborrhoetic dermatitis, Lichen planus, Pemphigus, bullous Pemphigus, Epidermolysis bullosa, urticaria, angiodermas, vasculitides, erythemas, cutaneous eosinophilias, uveitis, Alopecia areata and vernal conjunctivitis;

(4) (gastrointestinal tract) Coeliac disease, proctitis, eosinopilic gastro-enteritis, mastocytosis, Crohn's disease, ulcerative colitis, food-related allergies which have effects remote from the gut, for example, migraine, rhinitis and eczema;

(5) (other tissues and systemic disease) multiple sclerosis, atherosclerosis, Acquired Immunodeficiency Syndrome (AIDS), lupus erythematosus, systemic lupus, erythematosus, Hashimoto's thyroiditis, myasthenia gravis, type I diabetes, nephrotic syndrome, eosinophilia fascitis, hyper IgE syndrome, lepromatous leprosy, sezary syndrome and idiopathic thrombocytopenia pupura; tuberculosis;

(6) (allograft rejection) acute and chronic following, for example, transplantation of kidney, heart, liver, lung, bone marrow, skin and cornea; and chronic graft versus host disease.

We are particularly interested in Th2-driven and/or mast cell-driven and/or basophil-driven conditions or diseases.

Thus, a more particular aspect of the invention provides the use of a compound of formula (I) or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment or prophylaxis of Th2-driven and/or mast cell-driven and/or basophil driven diseases or conditions; and a method of treating, or reducing the risk of, Th2-driven and/or mast cell-driven and/or basophil driven diseases or conditions which comprises administering to a person suffering from or at risk of, said disease or condition, a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof.

In a preferred aspect of the invention, we provide a method for the treatment or prevention of a reversible obstructive airway disease, especially asthma, which comprises administering a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof to a human that is suffering from or susceptible to the disease. We also provide the use of a compound of formula (I) or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment or prevention of a reversible obstructive airway disease, especially asthma.

In another preferred aspect of the invention, we provide a method for the treatment or prevention of rhinitis which comprises administering a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof to a human that is suffering from or susceptible to rhinitis, especially allergic rhinitis. We also provide the use of a compound of formula (I) or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment or prevention of rhinitis, especially allergic rhinitis.

Prophylaxis is expected to be particularly relevant to the treatment of persons who have suffered a previous episode of, or are otherwise considered to be at increased risk of, the disease or condition in question. Persons at risk of developing a particular disease or condition generally include those having a family history of the disease or condition, or those who have been identified by genetic testing or screening to be particularly susceptible to developing the disease or condition.

For the above mentioned therapeutic indications, the dose of the compound to be administered will depend on the compound employed, the disease being treated, the mode of administration, the age, weight and sex of the patient. Such factors may be determined by the attending physician. However, in general, satisfactory results are obtained when the compounds are administered to a human at a daily dosage of between 0.1 mg/kg to 100 mg/kg (measured as the active ingredient).

The compounds of formula (I) may be used on their own, or in the form of appropriate pharmaceutical formulations comprising the compound of the invention in combination with a pharmaceutically acceptable diluent, adjuvant or carrier. Particularly preferred are compositions not containing material capable of causing an adverse reaction, for example, an allergic reaction. Conventional procedures for the selection and preparation of suitable pharmaceutical formulations are described in, for example, "Pharmaceuticals - The Science of Dosage Form Designs", M. E. Aulton, Churchill Livingstone, 1988.

According to the invention, there is provided a pharmaceutical formulation comprising preferably less than 95% by weight and more preferably less than 50% by weight of a compound of formula (I) in admixture with a pharmaceutically acceptable diluent or carrier.

We also provide a method of preparation of such pharmaceutical formulations that comprises mixing the ingredients.

The compounds may be administered topically, for example, to the lungs and/or the airways, in the form of solutions, suspensions, HFA aerosols or dry powder formulations, for example, formulations in the inhaler device known as the Turbuhaler^® ; or systemically, for example, by oral administration in the form of tablets, pills, capsules, syrups, powders or granules; or by parenteral administration, for example, in the form of sterile parenteral solutions or suspensions; or by rectal administration, for example, in the form of suppositories.

Dry powder formulations and pressurized HFA aerosols of the compounds of the invention may be administered by oral or nasal inhalation. For inhalation, the compound is desirably finely divided. The finely divided compound preferably has a mass median diameter of less than 10 μm, and may be suspended in a propellant mixture with the assistance of a dispersant, such as a C₈-C₂o fatty acid or salt thereof, (for example, oleic acid), a bile salt, a phospholipid, an alkyl saccharide, a perfluorinated or polyethoxylated surfactant, or other pharmaceutically acceptable dispersant.

The compounds of the invention may also be administered by means of a dry powder inhaler. The inhaler may be a single or a multi dose inhaler, and may be a breath actuated dry powder inhaler.

One possibility is to mix the finely divided compound with a carrier substance, for example, a mono-, di- or polysaccharide, a sugar alcohol, or an other polyol. Suitable carriers are sugars, for example, lactose, glucose, raffinose, melezitose, lactitol, maltitol, trehalose, sucrose, mannitol; and starch. Alternatively the finely divided compound may be coated by another substance. The powder mixture may also be dispensed into hard gelatine capsules, each containing the desired dose of the active compound.

Another possibility is to process the finely divided powder into spheres which break up during the inhalation procedure. This spheronized powder may be filled into the drug reservoir of a multidose inhaler, for example, that known as the Turbuhaler^® in which a dosing unit meters the desired dose which is then inhaled by the patient. With this system the active compound, with or without a carrier substance, is delivered to the patient.

For oral administration the active compound may be admixed with an adjuvant or a carrier, for example, lactose, saccharose, sorbitol, mannitol; a starch, for example, potato starch, corn starch or amylopectin; a cellulose derivative; a binder, for example, gelatine or polyvinylpyrrolidone; and/or a lubricant, for example, magnesium stearate, calcium stearate, polyethylene glycol, a wax, paraffin, and the like, and then compressed into tablets. If coated tablets are required, the cores, prepared as described above, may be coated w ith a concentrated sugar solution which may contain, for example, gum arabic. gelatine, talcum, titanium dioxide, and the like. Alternatively, the tablet may be coated with a suitable polymer dissolved in a readily volatile organic solvent.

For the preparation of soft gelatine capsules, the compound may be admixed with, for example, a vegetable oil or polyethylene glycol. Hard gelatine capsules may contain granules of the compound using either the above mentioned excipients for tablets. Also liquid or semisolid formulations of the drug may be filled into hard gelatine capsules.

Liquid preparations for oral application may be in the form of syrups or suspensions, for example, solutions containing the compound, the balance being sugar and a mixture of ethanol, water, glycerol and propylene glycol. Optionally such liquid preparations may contain colouring agents, flavouring agents, saccharine and/or carboxymethylcellulose as a thickening agent or other excipients known to those skilled in art.

The compounds of the invention may also be administered in conjunction with other compounds used for the treatment of the above conditions.

The following Examples are intended to illustrate, but in no way limit the scope of the invention.

General methods AU reactions were performed in dried glassware in an argon atmosphere at room temperature, unless otherwise noted. All reagents and solvents were used as received. Merck Silica gel 60 (0.040-0.063 mm) was used for preparative silica gel chromatography. A Kromasil KR-100-5-C18 column (250 x 20 mm, Akzo Nobel) and mixtures of acetonitrile/water at a flow rate of 10 ml/min were used for preparative HPLC. Reactions were monitored at 254 nm by analytical HPLC, using a Kromasil C-18 column (150 x 4.6 mm) and a gradient (containing 0.1 % trifluoroacetic acid) of 5 to 100% of acetonitrile in water at a flow rate of 1 ml/min. Evaporations of solvents were performed under reduced pressure using a rotary evaporator at a maximum temperature of 60 °C. Products were dried under reduced pressure at about 40 °C. Η-NMR spectra were recorded on a Varian Inova 400 MHz or Varian Mercury 300 MHz instrument. The central solvent peak of chloroform-d (5_H 7.27 ppm), dimethylsulfoxide-ck (δ_H 2.50 ppm) or methanol-J₄ (δ_H 3.35 ppm) were used as internal references. Low resolution mass spectra were obtained oh a Hewlett Packard 1100 LC-MS system equipped with a APCI ionisation chamber.

Preparation 1 4-Amino-8-bromo-3-cinnolinecarboxamide a) (2E)-2-r(2-Bromophenyl)hvdrazono1-2-cvanoacetamide

Sodium nitrite (8.05 g, 1 16 mmol) dissolved in water (25 ml) was added dropwise to an ice-cooled (<5 °C) suspension of o-bromoaniline (20.6 g, 1 16 mmol) in concentrated hydrochloric acid (29 ml). After 15 minutes stirring, sodium acetate (28.7 g, 0.35 mol) in water (120 ml) was added.

Sodium acetate (9.56 g, 1 16 mmol) in water (35 ml) was added to cyanacetamide (9.81 g,

1 16 mmol) which was dissolved in a mixture of water (170 ml) and ethanol (120 ml), cooled to <5 °C, and the previously prepared diazonium salt was added portionwise under vigorous mechanical stirring.

Stirring was continued for 2 h at room temperature. The precipitate was filtered off, washed twice with water, then ethanol (3 x 200 ml), then diethyl ether (3 x 300 ml) and finally dried at 50 °C under vacuum to afford the title compound (24 g, 77%). Η NMR (400 MHz, DMSO-d₆): δ 10.01 (1 H, s); 7.97 (2H, brs); 7.65 (2H, m); 7.42 (1H, t); 7.09 (lH, t).

APCI-MS ^m/z: 266.9/268.9 [MH⁺]. b) 4-Amino-8-bromocinnoline-3-carboxamide hydrochloride

A suspension of (2E)-2-[(2-bromophenyl)hydrazono]-2-cyanoacetamide (24 g, 90 mmol) and anhydrous aluminum chloride (49 g, 360 mmol) in dry chlorobenzene (200 ml) was heated to 1 10-1 15 °C under vigorous stirring for 1 h. After cooling, 2 M hydrochloric acid

(200 ml) was added slowly to the homogenous reaction mixture which was then heated to

90-100 °C for 1 h.

The precipitate was filtered off, washed with ethanol and recrystallized from a mixture of 2N hydrochloric acid and ethanol to afford the title compound ( 19.5 g. 71 %). Η NMR (400 MHz, DMSO-d₆) 6 8 61 (1H, dd), 8 35 (1H, dd), 7 69 (1H, t), 4 18 (5H, brs)

APCI-MS ^m/z 266 9/268 9 [MH⁺]

Preparation 2 8-Bromo-4-hydroxy-3-cιnnolιnecarboxylιc acid

4-Ammo-8-bromo-3-cιnnohnecarboxamιde (2 70 g, 10 1 mmol) was suspended in a mixture of dioxane (25 ml) and 25% aqueous potassium hydroxide (40 ml) in a polyethylene reaction vessel and heated to reflux for 32 h After cooling down to room temperatuie the mixture was acidified with acetic acid in order to precipitate the product The solid was filtered off, washed twice with water and dried in a vacuum desiccator The crude title compound was obtained as a brown powder (1 99 g, 73%) APCI-MS ^m/z 268 9/270 9 [MH⁺]

Preparation 3 8-Bromo-4-chloro-3-cιnnolιnecarboxamιde 8-Bromo-4-hydroxy-3-cιnnolιnecarboxyhc acid (1 99 g, 7 40 mmol) was suspended in thionyl chloride (50 ml) and the mixture was heated to reflux overnight After cooling, the thionyl chloride was evaporated off The residue was suspended in acetone (20 ml) and cone ammonia (5 0 ml, 75 mol) was added dropwise at 0 °C Stirring was continued for 30 minutes and the precipitate was filtered off, washed twice with water and dried in a vacuum desiccator The title compound was obtained as brown solid (1 74 g, 82%) APCI-MS ^m/z 286 0/287 9 [MH⁺]

Preparation 4 8-Bromo-4-chloro-3-cιnno]ιnecarbonιtrιle

8-Bromo-4-chloro-3-cιnnolιnecarboxamιde (1 17 g, 4 08 mmol) was dissolved in phosphory] chloride (15 ml) and stirred at 100 °C overnight The phosphoryl chloride was removed in vacuo and the residue was suspended in tert-butylmethylether, filtered off and washed with diluted ammonia, water and tert-butylmethylether The title compound was obtained as a biow solid after drying in a vacuum desiccator (0 99 g, 91 %) APCI-MS /z 267 9/269 8 [MH⁺] Example 1 6-BiOmo-lH-pyrazolo[4,3-clcinnolin-3-ol

A suspension of 8-bromo-4-chlorocinnoline-3-carboxamide (0.26 g, 0.91 mmol) and hydrazine hydrate (0.14 g, 2.7 mmol) in ethanol (1.5 ml) was heated to reflux for 2 h. After cooling, water (3.0 ml) was added. The title compound was obtained as a red solid after drying in a vacuum desiccator (0.21 g, 85%).

Η NMR (400 MHz, DMSO-d₆): δ 1 1.36 (1H, brs); 8.00 (1H, d); 7.88 (1H, d); 7.45 (1H, brs); 7.36 (lH, t).

APCI-MS ^m/z: 264.9, 266.9 [MH⁺].

Example 2 6-Bromo- 1 H-pyrazolo^r4,3-L cinnolin-3-amine

A suspension of 8-bromo-4-chlorocinnoline-3-carbonitrile (0.25 g, 0.93 mmol) and hydrazine hydrate (0.14 g, 2.79 mmol) in ethanol (1.5 ml) was heated to reflux for 2 h.

After cooling down and addition of water (3.0 ml), a precipitate could be filtered off.

Drying overnight in a vacuum desiccator afforded the title compound (0.21 g, 85%) as a red solid.

Η-NMR (300 MHz, DMSO-d₆): δ 13.22 (1H, s); 8.28 (1H, d, J 8.1Hz); 8.23 (1H, d, J

7.4Hz); 7.77 (1 H, t, I 7.6Hz); 6.27 (2H, s).

APCI-MS ^m/z: 263.9/265.9 [MH⁺].

Example 3

General protocol for Suzuki couplings

A solution of the respective bromo-lH-pyrazolo[4,3-c]cinnoline (0.075 mmol), boronic acid (0.225 mmol, 3.0 equiv.) and { l,l'-bis(diphenylphosphino)ferrocene}- dichloropalladium(II) (3.1 mg, 5 mol%) in a mixture of dioxane (400 μL), ethanol (40 μL) and aqueous 2M sodium carbonate (300 μL) was heated in a sealed vial to 90 °C for 3 h. After cooling, ethyl acetate (5 ml) was added and the resulting suspension was filtered through a pad of celite. The solid was washed thoroughly with acetonitrile and 50% aqueous acetonitrile. The collected filtrates were evaporated by freeze-drying. The crude residue was dissolved in 50% aqueous acetonitrile, acidified by 2-3 drops of trifluoroacetic acid and purified by reversed phase HPLC. After freeze-drying, the product was isolated as its trifluoroacetic acid salt, typically as a red powder.

Example 3a 6-Phenyl-lH-pyrazolor4,3-clcinnolin-3-ol

5 Prepared as described in Example 3 starting from 6-bromo-lΗ-pyrazolo[4,3-c]cinnolin-3- ol and phenyl boronic acid.

Η NMR (400 MHz, DMSO-d₆+D₂O): δ 8.06 (1H, bd); 7.80-7.43 (7H, m).

Η NMR (400 MHz, DMSO-d₆+NaOH): δ 8.14 (1 H, dd); 7.62 (2H, dd); 7.48-7.35 (3H, m); 7.35-7.31 (2H, m). lo APCI-MS ^m/z: 263.1 [MH⁺].

Example 3b 6-(4-Methoxyphenyl)-lH-pyrazolof4.3-clcinnolin-3-ol trifluoroacetic acid salt

Prepared as described in Example 3 starting from 6-bromo-lΗ-pyrazolo[4,3-c]cinnolin-3- 15 ol and 4-methoxyphenyl boronic acid.

Η-NMR (400 MHz, DMSO-d₆): δ 10.89 (1H, s); 7.92 (1H, dd); 7.61 (2H, d); 7.43-7.42 (2H, m); 7.00 (2H, d); 3.81 (3H, s). APCI-MS ^m/z: 293.0 [MH⁺].

20 Example 3c 6-(4-Methoxyphenyl)-lH-pyrazolor4,3-clcinnolin-3-amine trifluoroacetic acid salt

Prepared as described in Example 3 starting from 7-bromo-lΗ-pyrazolo[4,3-c]cinnolin-3- amine and 4-methoxyphenyl boronic acid.

Η-NMR (400 MHz, DMSO-d₆): δ 13.05 (1H, s); 8.23 (1H, d, J 8.1 ); 7.91 (1H, t); 7.81 25 (1 H, d); 7.69 (2H, d); 7.07 (2H, d); 6.1 1 (2H, s); 3.84 (3H, s).

APCI-MS ^m/z: 292.2 [MH⁺].

Example 3d 6-(2-Methoxyphenyl)-lH-pyrazolof4,3-clcinnolin-3-ol

Prepared as described in Example 3 starting from 6-bromo-l Η-pyrazolo[4,3-c]cinnolin-3- i() ol and 2-methoxyphenyl boronic acid. H-NMR (400 MHz, DMSO-d₆): δ 12.96 (1H, brs ); 1 1.90 (1H, s); 8.05 (1H, d); 7.61 (1H, t); 7.52-7.45 (2H, m); 7.30 (1H, dd); 7.18 (1H, d); 7.1 1 (1H, t); 3.71 (3H, s).

Example 3e 6-(2-Methoxyphenyl)-lH-pyrazolor4.3-clcinnolin-3-amine

Prepared as described in Example 3 starting from 6-bromo-lΗ-pyrazolo[4,3-c]cinnolin-3- amine and 2-methoxyphenyl boronic acid. APCI-MS ^m/z: 292.0 [MH⁺],

Example 3f 6-(3-Methoxyphenyl)-lH-pyrazolo[4.3-clcinnolin-3-ol

Prepared as described in Example 3 starting from 6-bromo-lΗ-pyrazolo[4,3-c]cinnolin-3- ol and 3-methoxyphenyl boronic acid. APCI-MS ^m/z: 293.0 [MH⁺].

Example 3g 6-(3-Methoxyphenyl)-lH-pyrazolor4,3-clcinnolin-3-amine

Prepared as described in Example 3 starting from 6-bromo-lΗ-pyrazolo[4,3-c]cinnolin-3- amine and 3-methoxyphenyl boronic acid.

Η-NMR (400 MHz, DMSO-d₆): δ 13.05 (1H, brs ); 8.28 (1H, dd); 7.93 (1H, t); 7.84 (1H, d); 7.42 (1H, m); 7.28-7.25 (2H, m); 7.02 ( 1H, m); 6.17 (2H, brs); 3.81 (3H, s). APCI-MS ^m/z: 292.0 [MH⁺].

Example 3h 6-Phenyl-lH-pyrazolor4.3-clcinnolin-3-amine

Prepared as described in Example 3 starting from 6-bromo-lΗ-pyrazolo[4,3-c]cinnolin-3- amine and phenyl boronic acid. Η-NMR (400 MHz, DMSO-d₆): δ 13.09 (1H, brs); 8.28 (1H, dd); 7.94 (1 H, t); 7.84 (1H, d); 7.74-7.71 (2H, m); 7.52-7.42 (3H, m); 6.14 (2H, brs ). APCI-MS /z: 262.0 [MH⁺].

Example 3i 6-Pyridin-3-yl-lH-pyrazolo[4,3-clcinnolin-3-ol Prepared as described in Example 3 starting from 6-bromo-lH-pyrazolo[4,3-c]cinnolin-3- ol and 3-pyridyl boronic acid.

1H-NMR (400 MHz, DMSO-d₆): δ 13.29 (IH, brs ); 1 1.96 (IH, s); 8.70 (IH, m); 8.1 1 (IH, m); 7.98 (IH, m); 7.66 (IH, m); 7.57 (IH, m); 7.19-7.11 (2H, m); 6.96 (IH, s). APCI-MS ^m/z: 264.0 [MH⁺].

Example 3i 6-Pyridin-3-yl- 1 H-pyrazolor4,3-clcinnolin-3-amine

Prepared as described in Example 3 starting from 6-bromo-l Η-pyrazolo[4,3-c]cinnolin-3- amine and 3-pyridyl boronic acid. Η-NMR (400 MHz, DMSO-d₆): δ 13.12 (IH, brs ); 8.92 (IH, d); 8.64 (IH, dd); 8.34 (IH, dd); 8.16 (IH, dt); 7.98 (IH, t); 7.92 (IH, d); 7.55 (IH, dd); 6.21 (2H, brs ). APCI-MS ^m/z: 263.1 [MH⁺].

Example 3k 6-(5-Chloro-2-methoxypheny])-lH-pyrazolor4.3-c1cinnolin-3-ol Prepared as described in Example 3 starting from 6-bromo-lΗ-pyrazolo[4,3-c]cinnolin-3- ol and 5-chloro-2-methoxyphenyl boronic acid. APCI-MS ^m/z: 327.2 [MH⁺].

Example 4 8-Bromo- 1 H-pyrazolo 4.3-clcinnolin-3-ol The title compound was prepared by a route analogous to that described in Preparations 1 to 3 and Example 1 , but starting with 4-bromoaniline.

Η NMR (400 MHz, DMSO-d₆): δ 1 1.59 (IH, brs ); 8.12 (IH, d); 7.79 (IH, d); 7.76 (IH, dd). APCI-MS ^m/z: 264.9/266.9 [MH⁺].

Example 5a 8-Phenyl-lH-pyrazolo 4,3-c1cinnolin-3-ol trifluoroacetate

The title compound was prepared as described in Example 3 starting from 8-bromo-lΗ- pyrazolo[4,3-c]cinnolin-3-ol and phenyl boronic acid. APCI-MS ^m/z: 263.2 [MH⁺]. Example 5b 8-o-Tolyl- lH-pyrazolor4,3-c1cinnolin-3-ol trifluoroacetate

The title compound was prepared as described in Example 3 starting from 8-bromo-lΗ- pyrazolo[4,3-c]cinnolin-3-ol and 2-methylphenyl boronic acid. APCI-MS ^m/z: 277.2 [MH⁺].

Example 5c 8-Thiophen-2-yl-lH-pyrazolor4.3-clcinnolin-3-ol trifluoroacetate

The title compound was prepared as described in Example 3 starting from 8-bromo-lΗ- pyrazolo[4,3-c]cinnolin-3-ol and 2-thienyl boronic acid. APCI-MS ^m/z: 269.2 [MH⁺].

Example 5d 8-(3-(Acetylamino)phenyl)-lH-pyrazolor4,3-clcinnolin-3-ol trifluoroacetate

The title compound was prepared as described in Example 3 starting from 8-bromo-lΗ- pyrazolo[4,3-c]cinnolin-3-ol and 3-(acetylamino)phenyl boronic acid. APCI-MS ^m/z: 320.2 [MH⁺].

Example 5e 8-(4-Cyanophenyl)-l H-pyrazoloF4,3-c1cinnolin-3-ol. bis trifluoroacetate

The title compound was prepared as described in Example 3 starting from 8-bromo-lΗ- pyrazolo[4,3-c]cinnolin-3-ol and 4-cyanophenyl boronic acid. APCI-MS ^m/z: 288.2 [MH⁺].

Example 6 7-Bromo- 1 H-pyrazolor4,3-clcinnolin-3-ol

The title compound was prepared by a route analogous to that described in Preparations 1 to 3 and Example 1 , but starting with 3-bromoaniline. The intermediate 7-bromo-4- chlorocinnoline-3-carboxamide (0.280 g, 0.98 mmol) was treated with hydrazine hydrate (190 μl, 3.92 mmol) in ethanol ( 13 ml). After cooling, the precipitate was filtered off, washed with water and dried to give the title compound. Yield (201 mg, 77%). Η NMR (400 MHz, DMSO-d₆): δ 1 1.55 (IH, brs); 7.98 (IH, d); 7.95 (IH, d); 7.65 (IH, dd).

APCI-MS ^m/z: 264.9, 266.9 [MH⁺].

Example 7a 7-Phenyl-lH-pyrazoloF4,3-clcinnolin-3-ol trifluoroacetate

The title compound was prepared as described in Example 3 starting from 7-bromo-lΗ- pyrazolo[4,3-c]cinnolin-3-ol and phenyl boronic acid. APCI-MS ^m/z: 263.2 [MH⁺].

Example 7b 7-o-Tolyl-lH-pyrazolol4,3-clcinnolin-3-ol trifluoroacetate

The title compound was prepared as described in Example 3 starting from 7-bromo-lΗ- pyrazolo[4,3-c]cinnolin-3-ol and 2-methylphenyl boronic acid. APCI-MS ^m/z: 277.2 [MH⁺].

Example 7c 7-Thiophen-2-yl- 1 H-pyrazolor4,3-clcinnolin-3-ol trifluoroacetate

The title compound was prepared as described in Example 3 starting from 7-bromo-lΗ- pyrazolo[4,3-c]cinnolin-3-ol and 2-thienyl boronic acid. APCI-MS ^m/z: 269.2 [MH⁺].

Example 7d 7-(3-(Acetylamino)phenyl)-lH-pyrazolof4,3-clcinnolin-3-ol trifluoroacetate

The title compound was prepared as described in Example 3 starting from 7-bromo-lΗ- pyrazolo[4,3-c]cinnolin-3-ol and 3-(acetylamino)phenyl boronic acid. APCI-MS ^m/z: 320.2 [MH⁺].

Example 7e 8-(4-Cyanophen yl)- 1 H-pyrazolo|^"4,3-clcinnolin-3-ol trifluoroacetate

The title compound was prepared as described in Example 3 starting from 7-bromo-lΗ- pyrazolo[4,3-c]cinnolin-3-ol and 4-cyanophenyl boronic acid. APCI-MS ^m/z: 288.2 [MH⁺]. Screen

Itk LANCE TRF assay

The Itk kinase assay utilized recombinant human Itk kinase domain fused with GST

(Glutathione S-Transferase). The protein was expressed in High five insect cells, purified in one step on an affinity chromatography glutathione column and stored in 50 mM Tris/HCl (pH 7.6), 150 mM NaCl, 5% (w/v) mannitol, 1 mM DTT, 30% glycerol at -70 °C. The kinase substrate used in the assay was a biotinylated peptide derived from the Src- optimal substrate (Nair et al, J. Med. Chem., 38: 4276, 1995; biotin- AEEEIYGEFE AKKKK) .

The assay additions were as follows: Test compounds (or controls; 1 μL in 100% DMSO) were added to black 96-well flat-bottomed plates (Greiner 655076) followed by 20 μL Itk in assay buffer and the reaction was started by adding 20 μL ATP and peptide substrate in assay buffer. The assay buffer constitution during phosphorylation was: 50 mM HEPES (pH 6.8), 10 mM MgCl₂, 0.015% Brij 35, 1 mM DTT, 10% glycerol, 160 ng/well Itk, 2 μM peptide substrate and 50 μM ATP. The assay was stopped after 50 minutes (RT) by adding 150 μL ice-cold Stop solution (50 mM Tris/HCl, pH 7.5, 10 mM EDTA, 0.9% NaCl and 0.1% BSA) together with LANCE reagents (2 nM PT66-Eu³⁺, Wallac AD0069 and 5 μg/ml Streptavidin-APC, Wallac AD0059. Both concentrations were final in stopped assay solution). The plates were measured on a Wallac 1420 Victor 2 instrument with TRF settings after lh incubation, and the ratio (665 signal/615 signal)*10000 was used to calculate the inhibition values. IC50 values were determined using XLfit.

When tested in the above screens, the compounds of Examples 1 to 7 gave IC50 values for inhibition of Itk activity of less than 25 μM, indicating that the compounds of the invention are expected to possess useful therapeutic properties. Representative results are shown in the following Table:

Claims

1. A compound of formula (I)

(I)

wherein:

X represents O or NH; and R represents hydrogen, halogen, CI to 6 alkyl, aryl or heteroaryl; said aryl or heteroaryl group each being optionally substituted with one or more groups selected from halogen, CI to 6 alkyl, CI to 6 alkoxy, cyano and acetamido (-NHCOCH3); and pharmaceutically acceptable salts thereof; with the proviso that the compound wherein X represents NH and R represents hydrogen is excluded.

2. A compound according to Claim 1 wherein X represents O.

3. A compound according to Claim 1 or Claim 2 wherein R represents aryl optionally substituted with one or more groups selected from halogen, CI to 6 alkyl, CI to 6 alkoxy, cyano and acetamido (-NHCOCH3).

4. A compound of formula (I), according to any one of Claims 1 to 3, which is: 6-bromo-lH-pyrazolo[4,3-c]cinnolin-3-ol; 6-bromo-lH-pyrazolo[4,3-c]cinnolin-3-amine; 6-phenyl- l H-pyrazolo[4,3-c]cinnolin-3-ol; 6-(4-methoxyphenyl)-lH-pyrazolo[4,3-c]cinnolin-3-ol;

6-(4-methoxyphenyl)-lH-pyrazolo[4,3-c]cinnolin-3-amine;

6-(2-methoxyphenyl)-lH-pyrazolo[4,3-c]cinnolin-3-ol;

6-(3-methoxyphenyl)-lH-pyrazolo[4,3-c]cinnolin-3-arnine;

6-phenyl- 1 H-pyrazolo[4,3-c]cinnolin-3-amine;

6-pyridin-3-yl-lH-pyrazolo[4,3-c]cinnolin-3-ol;

8-bromo-lH-pyrazolo[4,3-c]cinnolin-3-ol;

8-phenyl-lH-pyrazolo[4,3-c]cinnolin-3-ol;

8-o-tolyI-lH-pyrazolo[4,3-c]cinnolin-3-ol;

8-(4-cyanophenyl)- lH-pyrazolo[4,3-c]cinnolin-3-ol;

7-bromo-lH-pyrazolo[4,3-c]cinnolin-3-ol;

7-phenyl-lH-pyrazolo[4,3-c]cinnolin-3-ol;

7-o-tolyl-lH-pyrazolo[4,3-c]cinnolin-3-ol; 7-thiophen-2-yl-lH-pyrazolo[4,3-c]cinnolin-3-ol;

7-(3-(acetylamino)phenyl)-lH-pyrazolo[4,3-c]cinnolin-3-ol;

8-(4-cyanophenyl)-lH-pyrazolo[4,3-c]cinnolin-3-ol;

or a pharmaceutically acceptable salt of any one thereof.

5. A compound of formula (I), according to Claim 1 , or a pharmaceutically acceptable salt thereof, for use as a medicament.

6. A pharmaceutical formulation comprising a compound of formula (I), as defined in any one of Claims 1 to 4, or a pharmaceutically acceptable salt thereof, optionally in admixture with a pharmaceutically acceptable diluent or carrier.

7. A method of treating, or reducing the risk of, a human disease or condition in which inhibition of Itk kinase activity is beneficial which comprises administering to a person suffering from or susceptible to such a disease or condition, a therapeutically effective amount of a compound of formula (I), as defined in any one of Claims 1 to 4, or a pharmaceutically acceptable salt thereof.

8. The use of a compound of formula (I) as defined in any one of Claims 1 to 4, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment or prophylaxis of human diseases or conditions in which inhibition of Itk kinase activity is beneficial.

9. The use according to Claim 8 wherein the disease is asthma.

10. The use according to Claim 8 wherein the disease is allergic rhinitis.

1 1. A process for the preparation of a compound of formula (I), as defined in any one of Claims 1 to 4, and optical isomers, racemates and tautomers thereof and pharmaceutically acceptable salts thereof, which comprises:

(I")

wherein R is as defined in Claim 1 , with hydrazine hydrate; or b) preparing a compound of formula (I) wherein X is NH, by reaction of a compound of formula (III)

(III)

wherein R is as defined in Claim 1 , with hydrazine hydrate;