BRPI0920840B1 - MAPK P38 Kinase Inhibitor - Google Patents

Abstract

MAPK P38 KINASE INHIBITOR. The present invention relates to a compound of formula (I) (I) or a pharmaceutically acceptable salt or solvate thereof, including all its tautomers, compositions comprising the same, use of said compound and compositions for treatment, particularly for the treatment of asthma and COPD, and processes for the preparation of said compound.

Description

Field of Invention

The present invention relates to compounds that are inhibitors of p38 mitogen-activated protein kinase enzymes (hereinafter referred to as p38 MAPK kinase inhibitors), particularly their alpha and gamma kinase subtypes, and their use in therapy, especially in the treatment of inflammatory diseases, including inflammatory lung diseases.

Background of the Invention

Four p38 MAPK isoforms (alpha, beta, gamma, and delta, respectively) have been identified, each exhibiting a tissue expression pattern. The alpha and beta p38 MAPK isoforms are ubiquitously expressed throughout the body and are found in many different cell types. The alpha and beta p38 MAPK isoforms are inhibited by certain known small p38 MAPK inhibitor molecules. Early generations of compounds were highly toxic due to the ubiquitous expression pattern of these isoforms and off-target effects of the compounds. More recent inhibitors have been improved to be highly selective for the alpha and beta p38 MAPK isoforms and have a wider safety margin. Little is known about the gamma and delta p38 MAPK isoforms. These isoforms are expressed in specific tissues/cells (unlike the alpha and beta p38 isoforms). The delta isoform of MAPK p38 is expressed more in the pancreas, testes, lung, small intestine, and kidney. It is also abundant in macrophages (Smith, S.J. (2006) Br. J. Pharmacol. 149:393-404) and detectable in neutrophils, CD4+ T cells, and endothelial cells (www.qenecard.org, Karin, K. (1999) J. Immunol.). Very little is known about the expression of MAPK p38 gamma, but it is expressed more in the brain, skeletal muscle, and heart, as well as in lymphocytes and macrophages (www.qenecard.org).

Small, selective inhibitory molecules of p38 gamma and delta MAPKs are currently available, but an existing inhibitor has pan-isoform inhibitory actions. BIRB 796 inhibits all isoforms, but inhibits p38 gamma and p38 delta at higher concentrations than those that inhibit p38 alpha and p38 beta (Kuma, Y. (2005) J. Biol. Chem. 280:19472-19479). BIRB 796 also decreases the phosphorylation of p38 MAPKs or JNKs by the upstream MKK6 or MKK4 kinase. The authors discussed the possibility that the conformational change caused by the inhibitor's binding to MARK may affect the structure of its phosphorylation site and also the docking site for the upstream activator, thus decreasing the phosphorylation of p38 MAPKs or JNKs.

It is believed that p38 MAP kinase plays a fundamental role in many signaling pathways involved in the initiation and maintenance of persistent chronic inflammation in human disease, for example, severe asthma and COPD. There is now abundant literature demonstrating that p38 MAP kinase is activated by a number of pro-inflammatory cytokines and that its activation results in the production and release of other pro-inflammatory cytokines. Indeed, data from some clinical studies demonstrate beneficial changes in disease activity in patients during treatment with p38 MAP kinase inhibitors. For example, Smith, S. J. (2006) Br. J. Pharmacol. 149:393-404 describes the inhibitory effect of p38 MAP kinase inhibitors on cytokine release from human macrophages. The use of p38 MAP kinase inhibitors in the treatment of chronic obstructive pulmonary disease (COPD) is proposed. Small inhibitory molecules targeting MAPK p38 α/β have been shown to be effective in reducing several parameters of inflammation in cells and tissues, obtained from COPD patients who are generally insensitive to corticosteroids (Smith, S. J. (2006) Br. J. Pharmacol. 149:393-404) and in vivo animal models (Underwood, D. C. et al. (2000) 279:895-902; Nath, P. et al. (2006) Eur. J. Pharmacol. 544:160-167). Irusen and others have also suggested the possibility of MAPK p38 α/β involvement in corticosteroid insensitivity through reduced glucocorticoid receptor (GR) binding affinity in the nuclei (Irusen, E. et al., (2002) J. Allergy Clin. Immunol., 109:649-657). Clinical experiences with a series of MAP p38 kinase inhibitors, including AMG548, BIRB 796, VX702, SCIO469, and SCIO323 are described in Lee et al. (2005) Current Med. Chem. 12/2979-2994.

COPD is a condition in which the underlying inflammation has been reported to be substantially resistant to the anti-inflammatory effects of inhaled corticosteroids. Consequently, an effective strategy for treating COPD may certainly be the development of an intervention that has inherent anti-inflammatory effects and is also capable of increasing the sensitivity of lung tissues in COPD patients to inhaled corticosteroids. The recent publication by Mercado et al. (2007; American - Thoracic Society Abstract A56) demonstrates that silencing gamma p38 has the potential to restore sensitivity to corticosteroids.

However, the main obstacle preventing the definition and exploration of the potential uses of p38 MAP kinase inhibitors in the treatment of chronic human inflammatory diseases has been the toxicity observed in patients. This was severe enough to result in the cancellation of the clinical development of many of the compounds developed.

The compounds developed so far were typically intended for oral administration. This strategy involves optimizing compounds that have achieved their duration of action through an appropriate pharmacokinetic profile. This ensures that there is a sufficient concentration of the drug established and maintained after and between doses to produce a clinical benefit. The inevitable consequence of this approach is that all tissues of the body, especially the liver and intestine, are possibly exposed to therapeutically active concentrations of the drug, whether or not they are adversely affected by the disease being treated. An alternative strategy is to design treatment approaches in which the drug is dosed directly to the inflamed organ (topical therapy). Although this approach is not appropriate for treating all chronic inflammatory diseases, it has been extensively explored in pulmonary diseases (asthma, COPD), skin diseases (atopic dermatitis and type II psoriasis), nasal diseases (allergic finitis), and gastrointestinal diseases (ulcerative colitis). In topical therapy, efficacy is achieved by (i) ensuring that the drug has a prolonged duration of action and remains in the relevant organ to minimize the risks of systemic toxicity, or (ii) producing a formulation that generates a "reservoir" of the active drug that remains available to prolong the desired effects of the drug. Approach (i) is exemplified by the anticholinergic drug tiotropium (Spiriva), which is administered topically to the lung as a treatment for COPD, and which has an exceptionally high affinity for its target receptor, resulting in a very slow excretion rate and a consequent prolonged duration of action. There remains a need to identify and develop new compounds that are p38 MAP kinase inhibitors, which have better therapeutic potential, and particularly, which are more effective, act for a longer time and/or are less toxic. One objective of the present invention is to provide compounds that inhibit the MAP p38 kinase with some subtype specificity, and that exhibit good anti-inflammatory potential.

Summary of the Invention

According to the invention, a compound of formula (I) is provided. or a pharmaceutically acceptable salt or solvate thereof, including all its tautomers.

Brief Description of the Figures

Figure 1 illustrates the pre-dose time versus the number of neutrophils in BALF for the compound of formula (I) in the LPS-induced neutrophil accumulation test.

Figure 2 illustrates the pre-dose time versus % neutrophil inhibition for the compound of formula (I) in the LPS-induced neutrophil accumulation test.

Figure 3 illustrates the dose effects for the compound of formula (I) on the number of activated macrophages in the BAL of mice exposed to cigarette smoke.

Figure 4 illustrates the dose effects for the compound of formula (I) on neutrophil numbers in the BAL of mice exposed to cigarette smoke.

Figure 5 illustrates the effect of the compound of formula (I) on the pulmonary function of parainfluenza-inoculated guinea pigs sensitized with ovalbumin challenged with ovalbumin.

Detailed Description of the Invention

Examples of salts of compound (I) include acid addition salts of strong mineral acids such as the salts of HCl and HBr and acid addition salts of strong organic acids, such as the methanesulfonic acid salt.

This also extends to solvates of compounds of formula (I). Examples of solvates include hydrates.

The description also extends to compounds of formula (I) in which the atom specified in the formula is a naturally occurring or non-naturally occurring isotope. In one embodiment, the isotope is a stable isotope. Thus, the compounds of the invention include, for example, compounds containing deuterium, and the like.

A process for preparing a compound of formula (I) involves the reaction of a compound of formula (II): with a compound of formula (III): where LG represents a leaving group (e.g., chlorine),

The reaction is carried out properly in the presence of a base (e.g., diisopropylethylamine). The reaction is carried out properly in an aprotic solvent or solvent mixture, e.g., DCM and DMF.

A compound of formula (II) can be prepared by reacting a compound of formula (IV): with a compound of the formula (V): and a compound of formula (VI): where LG2 and LG3 each independently represent leaving groups (e.g., LG2 and LG3 both represent imidazolyl groups). The reaction is suitably conducted in an aprotic solvent (e.g., dichloromethane).

A compound of formula (V) can be prepared by reducing a compound of formula (VII): For example, by hydrogenation in the presence of a catalyst, such as platinum on a carbon support.

The reaction is carried out appropriately in a polar protic solvent (e.g., methanol and acetic acid, 1:1). A compound of formula (VII) can be prepared by the reaction of a compound of formula (VIII): with a compound of formula (IX): under Mitsunobu conditions, such as in the presence of triphenylphosphine and diisopropyl azodicarboxylate. The reaction is suitably carried out in a polar aprotic solvent (e.g., tetrahydrofuran). Alternatively, a compound of formula (I) can be prepared by the reaction of a compound of formula (X): with a compound of formula (IV) defined above and a compound of formula (XI): where LG4 and LG5 each independently represent leaving groups (e.g., LG4 and LG5 both represent imidazolyl groups). The reaction is conducted appropriately in a polar aprotic solvent.

A compound of formula (X) can be prepared by reducing a compound of formula (XII): For example, by hydrogenation in the presence of a catalyst, such as platinum on a carbon support. A compound of formula (XII) can be prepared by reacting a compound of formula (XIII): where LGe represents a leaving group (e.g., chlorine) and a compound of formula (VII) defined above. The reaction is suitably conducted in the presence of a base (e.g., diisopropylethylamine). The reaction is suitably conducted in a polar solvent, e.g., a mixture of DCM and DMF. The compounds of formulas (III), (IV), (VI), (VIII), (IX), (XI), and (XIII) are commercially available or are known and can be prepared by conventional methods. See, for example, Regan, J. et al., J. Med. Chem., 2003, 46:4676-4686, WO00/043384, W02007/087448, and W02007/089512.

If desired or necessary, the intermediate compounds may be protected by the use of conventional protecting groups. The protecting groups and the means for their removal are described in "Protective Groups in Organic Synthesis" by Theodora W. Greene and Peter G.M. Wuts, published by John Wiley & Sons, Inc; 4th Revised Edition, 2006, ISBN-10: 0471697540. The unusual intermediates are claimed as an aspect of the invention.

Furthermore, the present invention provides a pharmaceutical composition comprising a compound of formula (I) optionally in combination with one or more pharmaceutically acceptable diluents or carriers. The diluents and vehicles may include those suitable for parenteral, oral, topical, mucosal and rectal administration.

As mentioned above, such compositions can be prepared, for example, for parenteral, subcutaneous, intramuscular, intravenous, intra-articular or periarticular administration, particularly in the form of liquid solutions or suspensions; for oral administration, particularly in the form of tablets or capsules; for topical administration, for example, pulmonary or intranasal, particularly in the form of powders, nasal drops or aerosols, and transdermal administration; for mucosal administration, for example, buccal, sublingual or vaginal mucosa, and for rectal administration, for example in the form of a suppository. The compositions can be conveniently administered in unit dosage form and can be prepared by any methods well-known in pharmaceutical science, for example, as described in "Remington's Pharmaceutical Sciences", 17th edition, Mack Publishing Company, Easton, PA, (1985). Formulations for parenteral administration may contain excipients such as water or sterile saline solution, alkylene glycols such as propylene glycol, polyalkylene glycols such as polyethylene glycol, vegetable oils, hydrogenated naphthalenes, and the like. Formulations for nasal administration may be solid and may contain excipients, for example, lactose or dextran, or may be aqueous or oily solutions for use as nasal drops or metered-dose spray. For oral administration, typical excipients include sugars, calcium stearate, magnesium stearate, pregelatinized starch, and the like.

Orally administered compositions may comprise one or more physiologically compatible vehicles and/or excipients, and may be in solid or liquid form. Tablets and capsules may be prepared with binding agents, for example, molasses, acacia, gelatin, sorbitol, tragacanth, or poly(vinylpyrrolidone); fillers, such as lactose, sucrose, corn starch, calcium phosphate, sorbitol, or glycine; lubricants, such as magnesium stearate, talc, polyethylene glycol, or silica; and surfactants, such as sodium lauryl sulfate. Liquid compositions may contain conventional additives such as suspending agents, for example, sorbitol syrup, methylcellulose, sugar molasses, gelatin, carboxymethylcellulose, or edible fats; emulsifying agents such as lecithin or acacia; Vegetable oils such as almond oil, coconut oil, cod liver oil, or peanut oil; preservatives such as butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT). Liquid compositions may be encapsulated, for example, in gelatin, to produce a unit dosage form.

Oral solid dosage forms include tablets, split hard-shell capsules, and elastic soft gelatin capsules (ESCs).

A dry shell formulation typically comprises a concentration of about 40% to 60% gelatin, a concentration of about 20% to 30% plasticizer (such as glycerin, sorbitol, or propylene glycol), and a concentration of about 30% to 40% water. Other materials such as preservatives, colorants, opacifiers, and flavors may also be present. The liquid filler material comprises a solid drug that has been dissolved, solubilized, or dispersed (with suspending aids such as beeswax, hydrogenated castor oil, or polyethylene glycol 4000) or a liquid drug in vehicles or combinations of vehicles such as mineral oil, vegetable oils, triglycerides, glycols, polyols, and surfactants.

The compound of formula (I) is suitably administered topically to the lung. Therefore, according to the invention, a pharmaceutical composition is provided comprising a compound of formula (I) optionally in combination with one or more topically acceptable diluents or vehicles. Topical administration to the lung can be achieved using an aerosol formulation. Aerosol formulations typically comprise the active ingredient suspended or dissolved in a suitable aerosol propellant, such as chlorofluorocarbons (CFCs) or hydrofluorocarbons (HFCs). Suitable CFC propellants include trichloromonofluoromethane (propellant 11), dichlorotetrafluoromethane (propellant 114), and dichlorodifluoromethane (propellant 12). Suitable HFC propellants include tetrafluoroethane (HFC-134a) and heptafluoropropane (HFC-227). The propellant typically comprises 40% to 99.5%, for example, 40% to 90% by weight of the total inhalation composition. The formulation may include excipients, including cosolvents (e.g., ethanol) and surfactants (e.g., lecithin, sorbitan trioleate, and the like). Aerosol formulations are packaged in tubes, and an appropriate dose is dispensed via a dosing valve (e.g., as supplied by Bespak, Valois, or 3M).

Topical administration to the lung can also be performed using a non-pressurized formulation, such as an aqueous solution or suspension. This can be administered via a nebulizer. Topical administration to the lung can also be performed using a dry powder formulation. A dry powder formulation should contain the compound of formula (I) in finely divided form, typically with a mass average diameter (MMAD) of 1-10 microns. The formulation should typically contain a topically acceptable compound such as lactose, usually with a large particle average size, for example, a mass average diameter (MMAD) of 100 µm or more. Exemplary powder delivery systems include SPINHALER, DISKHALER, TURBOHALER, DISKUS and CLICKHALER.

The compounds of formula (I) are intended to have therapeutic activity. In another aspect, the present invention provides a compound of formula (I) for use as a medicament.

The compounds of formula (I) are expected to be useful in the treatment of respiratory disorders including COPD (including chronic bronchitis and emphysema), asthma, pediatric asthma, cystic fibrosis, sarcoidosis, idiopathic pulmonary fibrosis, allergic rhinitis, rhinitis, sinusitis, especially asthma, chronic bronchitis and COPD.

It is also expected that the compounds of formula (I) will be useful in the treatment of certain conditions that can be treated by topical or local therapy, including allergic conjunctivitis, conjunctivitis, allergic dermatitis, contact dermatitis, psoriasis, ulcerative colitis, inflamed joints secondary to rheumatoid arthritis or osteoarthritis.

It is also expected that the compounds of formula (I) will be useful in the treatment of certain other conditions including rheumatoid arthritis, pancreatitis, cachexia, inhibition of growth and metastasis of tumors, including non-small cell lung carcinoma, breast carcinoma, gastric carcinoma, colorectal carcinomas and malignant melanoma.

Thus, in another aspect, the present invention provides a compound of formula (I) for use in treating the conditions mentioned above, for example, by administering a therapeutically acceptable amount of said compound to a patient in need of it.

In another aspect, the present invention provides the use of a compound of formula (I) for the manufacture of a medicament for the treatment of the conditions mentioned above.

In another aspect, the present invention provides a method of treating the conditions mentioned above, comprising administering to an individual an effective amount of a compound of formula (I) or a pharmaceutical composition thereof.

The invention also extends to the use of pharmaceutical compositions/formulations in the treatment of one or more of said conditions.

The term "treatment" is intended to encompass both prophylaxis and therapeutic treatment.

A compound of formula (I) may also be administered in combination with one or more other active ingredients, for example, active ingredients suitable for treating the conditions mentioned above. For example, possible combinations for the treatment of respiratory disorders include combinations with steroids (e.g., budesonide, beclomethasone dipropionate, fluticasone propionate, mometasone furoate, fluticasone furoate), beta-agonists (e.g., terbutaline, salbutamol, salmeterol, formoterol) and/or xanthines (e.g., theophylline). examples

Diisopropyl azodicarboxylate (DIAD) (8.07 mL, 41.0 mmol) was added dropwise to a solution of 4-nitronaphthol (5.17 g, 27.3 mmol), triphenylphosphine (10.75 g, 41.0 mmol) and 2-aminopyridine-4-methanol (2) (5.09 g, 41.0 mmol) in THF (50 mL) at -15°C. The mixture was stirred overnight at room temperature and the volatiles were then removed under vacuum. The crude product was ground in EtOAc (150 mL), removed by filtration and washed with EtOAc (100 mL). A second trituration in MeOH (100 mL) gave 2-amino-4-[(4-nitronafthalen-1-yloxy)methyl]-pyridine (3) (4.54 g, 56%) as a yellow solid: m/z 296 (M+H)+ (ES+). 2-Amino-4-[(4-aminoπafthalenπ-1-yloxy)methyl]-pyridine (4)

A solution of 2-amino-4-[(4-nitronaphthalen-1-yloxy)methyl]-pyridine (3) (4.50 g, 15.24 mmol) in methanol (200 mL) and glacial acetic acid (200 mL) was passed through a Thales 'H-cube' flow reactor (2 mL min⁻¹, 40°C, 55 mm 10% Pt/C Cat-Cart®, filled with H₂) and the volatiles were then removed under vacuum. The crude product was subjected to an SCX capture and released by eluting with 1% ammonia solution in MeOH and the solvent was removed under vacuum to give 2-amino-4-[(4-aminonaphthalen-1-yloxy)methyl]-pyridine (4) (3.82 g, 94%) as a mauve solid: m/z 266 (M+H)+ (ES+). 1-{4-[(2-Aminopyridin-4-yl)methoxy1naphthalen-1-yl}-3-(3-f-butyl-1-p-tolyl-1/7-pyrazol-5-yl)-urea (5)

A solution of 3-t-butyl-1-p-15-tolyl-1H-pyrazol-5-amine (6) (5.91 g, 25.80 mmol) in DCM (15 mL) was added dropwise to a solution of 1,T-carbonyldi-imidazole (CDI) (4.18 g, 25.80 mmol) in DCM (15 mL) under nitrogen for 40 min. The resulting solution was stirred at room temperature for one hour and then added dropwise, under nitrogen, to a solution of 2-amino-4-[(4-aminonaphthalen-1-yloxy)methyl]-pyridine (4) (3.80 g, 12.89 mmol). The mixture was stirred overnight and the volatiles were then removed under vacuum. The crude material was purified by fast chromatography (Biotage 120 g); eluting with 0 to 6% MeOH in DCM to give 1-{4-[(2-aminopyridin-4-yl)methoxy]naphthalen-1-yl}-3-(3-t-butyl-1 - 5 p-tolyl-1H-pyrazol-5-yl)-urea (5) (4.27 g, 63%): m/z 521 (M+H)+ (ES+). /V-[4-({4-[3-(3-/-Butyl-1-p-tolyl-1H-pyrazol-5-yl)ureido]naphthalen-1-yloxymethyl) pyridin-2-yl]-2-methoxy-acetamide (1)

Methoxyacetyl chloride (92 μL, 1.01 mmol) was added to a solution of 1-{4-[(2-aminopyridin-4-yl)methoxy]naphthalen-1-yl}-3-(3-β-butyl-1-p-tolyl-10 1/7-pyrazol-5-yl)-urea (5) (526 mg, 0.96 mmol) and DIPEA (184 μL, 1.06 mmol) in a mixture of DCM and DMF (10:1, 11 mL) under stirring. After one hour at room temperature, further fractions of DIPEA (184 μL, 1.06 mmol) and methoxyacetyl chloride (92 μL, 1.01 mmol) were added sequentially and stirring was continued for one hour. A 1% solution of 15 ammonia in MeOH (40 ml) was added and the mixture was stirred for 15 minutes and then concentrated under vacuum. The crude product was purified by fast column chromatography (Biotage 40 g); eluting with 0 to 6% MeOH in DCM to yield /V-[4-({4-[3-(3-f-butyl-1-p-tolyl-1/-/-pyrazol-5-yl)ureido]natalen-1-yloxy}methyl)pyridin-2-yl]-2-methoxyacetamide (1) (286 mg, 20 49%). m/z 593 (M+H)+ (ES+).1H NMR (400 MHz, DMSO-d6) δ: 1..27 (9 H, s), 2.39 (3 H, s), 3.32 (3 H, s), 4.08 (2H, s), 5.39 (2H, s), 6.36 (1H, s), 7.03 (1H, d), 7.28 (1H, dd), 7.36 (2H, m), 7.44 (2H, m), 7.56-7.64 (3H, m), 7.93 (1H, m), 8.30-8.35 (3H, m), 8.58 (1H, s), 8.79 (1H, s) and 1002 (1H, s). Biological Test In vitro test *1: Cell-based assay for MAPK alpha p38 by detection of MAPKAP-K2 phosphorylation 5 *2: No significant toxic effect observed in the MTT assay. A description of these assays is as follows:

Enzyme Inhibition Assay

The enzyme inhibitory activity of the compound was determined by fluorescence resonance energy transfer (FRET) using synthetic peptides labeled with donor and acceptor fluorophores (Z-LYTE, Invitrogen). Briefly, recombinant phosphorylated p38 gamma MAPK (MAPK12, Millipore) was diluted in HEPES buffer, mixed with the compound at the desired final concentrations, and incubated for two hours at room temperature. The FRET peptide (2 µM) and ATP (100 µM) were then added to the enzyme/compound mixture and incubated for one hour. A revealing reagent (protease) was added for one hour before detection in a fluorescence microplate reader. The site-specific protease cleaves only non-phosphorylated peptide and eliminates the FRET signal. The phosphorylation levels of each reaction were calculated using the ratio of coumarin (donor) emission to fluorescein (acceptor) emission, with high ratios indicating high phosphorylation and low ratios indicating low phosphorylation levels. The percentage inhibition of each reaction was calculated relative to the uninhibited control, and the 50% inhibitory concentration (Cl5o value) was then calculated from the concentration-response curve. In the case of MAPK p38 alpha (MAPK14: Invitrogen), enzyme activity was indirectly assessed by determining the activation/phosphorylation of the downstream molecule, MAPKAP-K2. The MAPK p38-a protein was mixed with its inactive target MAPKAP-K2 (Invitrogen) and the compound was left for two hours at room temperature. The FRET peptide (2 µM), which is a phosphorylation target for MAPKAP-K2, and ATP (10 µM) were then added to the enzyme/compound mixture and incubated for one hour. The developing reagent was then added and the mixture was incubated for one hour before fluorescence detection completed the assay protocol.

LPS-induced TNF-alpha release: potency

U937 cells, a human monocytic cell line, were differentiated into macrophage-like cells by incubation with phorbol myristate-acetate (PMA; 100 ng/ml) for 48 to 72 hours. When appropriate, cells were pre-incubated with final concentrations of the compound for two hours. Cells were then stimulated with 0.1 ug/mL of LPS (from E. coli: O111:B4, Sigma) for 4 hours, and the supernatant was collected for determination of TNFα concentration by sandwich ELISA (Duo-set, R&D Systems). THP-1, a human monocytic cell line, was also used for this assay. THP-1 cells were stimulated with 1 ug/mL of LPS (from E. coli: O111:B4, Sigma) for 4 hours, and the supernatant was collected for determination of TNFα concentration. The percentage inhibition of TNFα production was calculated at each concentration of the test compound by comparison with the vehicle control, and the 50% inhibitory concentration (Cl5o) value was determined from the resulting concentration-response curve.

MTT test

Differentiated U937 cells were pre-incubated with compound for 4 hours in 5% FCS or 10% FCS for 24 hours and 72 hours. The supernatant was replaced with 200 µL of fresh medium and 10 µL of MTT stock solution (5 mg/mL) was added to each well. After one hour of incubation, the media were removed, 200 µL of DMSO was added to each well, and the plates were shaken gently for one hour before reading the absorbance at 550 nm.

The percentage loss of cell viability was calculated for each well relative to the vehicle treatment (0.5% DMSO). Consequently, an apparent increase in cell viability for the drug treatment relative to the vehicle is tabulated as a negative percentage.

In vivo test LPS-induced neutrophils in mice

Non-fasted mice were dosed intratracheally with vehicle or test substance at the same time points ("pre-dose") indicated relative to the start of LPS treatment. At T = 0, mice were placed in an exposure chamber and exposed to LPS. Eight hours after the LPS challenge, the animals were anesthetized, the trachea was cannulated, and BALF was extracted by infusing and withdrawing 1 mL of PBS into the lungs via a tracheal catheter. Total and differential leukocyte counts in the BALF samples were measured using a Neubaur hemocytometer. Cytocentrifugation smears (cytospiri) of the BALF samples were prepared by centrifugation at 200 rpm for 5 minutes at room temperature and stained using the DiffQuik staining system (Dade Behring). Cells were counted using an oil immersion microscope.

The results are illustrated in Figures 1 and 2. Data for neutrophil numbers are reported as total and differential number (test substance relative to vehicle) of cells per milliliter of BALF, mean ± SEM (n = 8).

Cigarette Smoke Pattern

A/J mice (males, 5 weeks old) were exposed to cigarette smoke (4% cigarette smoke diluted with compressed air) for 30 min/day for 11 days using a Small Animal Tobacco Smoke Inhalation Experiment System (Model SIS-CS; Sibata Scientific Technology, Tokyo, Japan). The test substances were administered intranasally (35 μL of 50% DMSO/PBS solution) and therapeutically twice daily for 3 days after the final cigarette smoke exposure. Twelve hours after the last dose, the animals were anesthetized, the trachea was cannulated, and bronchoalveolar lavage fluid (BALF) was collected. Alveolar macrophage and neutrophil counts were determined by FACS analysis (EPICS® ALTRA II, Beckman Coulter, Inc., Fullerton, CA, USA) using MOMA2 anti-mouse antibody (macrophage) or 7/4 anti-mouse antibody (neutrophil).

The results are illustrated in Figure 3 for activated alveolar macrophages and in Figure 4 for neutrophils. Data for cell numbers are indicated as mean ± SEM. The cigarette smoke model used for this study, reported as a corticosteroid-refractory system (Medicherla S. et al., (2008); J. Pharmacol. Exp. Then 324(3):921-9), confirmed that fluticasone propionate did not inhibit neutrophil or macrophage accumulation within the airways at 50 μg/mL (35 μL twice daily (b.i.d.), the same dose that produced >80% inhibition of LPS-induced neutrophil accumulation. In Figure 3: ^Significant difference between air exposure and cigarette smoke exposure. * **P<0.001 versus Control with cigarette smoke (CS) (ANNOVA, Dunnett's multiple comparison), n = 6-11 In Figure 4: * ##Significant difference between air exposure and cigarette smoke exposure. * P<0.05 or ***P<0.001 versus Control with cigarette smoke (CS) (ANNOVA, Dunnett's multiple comparison), n = 6-11

Ovalbumin challenge model / parainfluenza infection (in vivo model for steroid resistance) Male Dunkin-Hartley guinea pigs (300-350 g, n = 6/group) were sensitized with 100 μg of ovalubumin (OVA) + 100 mg AI2(OH)3 in 1 mL of normal saline (intraperitoneal) on Days 2 and 6.

Parainfluenza virus (PIV-3; 106 infectious units) or virus-free medium was instilled nasally on Days 11 and 12. Animals were treated with nebulized fluticasone propionate at a dose of 1.5 mg daily. Initial studies established that this dose of fluticasone propionate inhibited ovalbumin-mediated changes in lung function in sensitized animals treated with PIV3 medium. Example 1 (4.5 mg daily) or vehicle (DMSO:ethanol:saline, 30:30:40%) from Days 10–15. All animals were challenged for one hour with nebulized OVA (10 μg/mL) on Day 15, and repeated measurements of specific airway conductance (sGaw) were made over a 24-hour period using whole-body plethysmography. sGaw measurements after OVA challenge are plotted as % change from baseline. See Figure 5. Figure 5 Data are indicated as the average of 6 observations; (•) PIV3 + vehicle treatment; (■) PIV3 + fluticasone propionate treatment; (A) PIV3 + treatment with Example 1 Synopsis

In vitro biological studies demonstrate that the compound of formula (I) is a potent inhibitor of the alpha and gamma subtypes of MAP p38 kinase with good efficacy in an in vitro model of anti-inflammatory activity (LPS-induced TNF-alpha release from differentiated U937 cells and THP-1 cells). MTT assays show that the compound does not exhibit manifest cellular toxicity at the concentrations used.

In vivo biological studies demonstrate that the compound of formula (I) is effective in inhibiting LPS-induced neutrophil accumulation in an animal model, with a long duration of effect, as indicated by significant inhibition even 12 or more hours after pre-dosing. Furthermore, the compound of formula (I) has been shown to be effective in two in vivo models of steroid-resistant inflammation.

In this entire descriptive report and the claims that follow, unless the context requires otherwise, the term "includes," and variations such as "comprises" and "comprising," shall be understood as implying the inclusion of a specified integer, step, group of integers, or group of steps, but not the exclusion of any other integer, step, group of integers, or group of steps. All patents and patent applications referenced herein are incorporated by reference in their entirety. The patent application of which this descriptive report and claims form part may be used as a basis for priority in relation to any subsequent patent application. The claims of such subsequent patent application may be directed to any feature or combination of features described herein. They may take the form of product, composition, process, or use claims and may include, by way of example and without limitation, the claims described herein. Abbreviations Ac acyl ATP 5'-triphosphate adenosine BALF Bronchoalveolar lavage fluid BSA CatCart® bovine serum albumin catalytic cartridge (trademark) CDI carbonyldiimidazole DCM dichloromethane DIAD diisopropyl azodicarboxylate DMF dimethylformamide DMSO dimethyl sulfoxide COPD chronic obstructive pulmonary disease DIAD diisopropyl azodicarboxylate DIBAL-H diisobutylaluminum hydride DIPEA /V-ethyl-/V-isopropylpropan-2-amine Et ethyl FCS fetal yolk serum h hour(s) HRP horseradish peroxidase JNK N-terminal c-Jun kinase MAPK mitogen-activated protein kinase Me methyl PBS phosphate-buffered saline PPh3 triphenylphosphine RT room temperature SCX cation exchange over solid support SDS sodium dodecyl sulfate TFA trifluoroacetic acid THF tetrahydrofuran TN Fee tumor necrosis factor alpha TMB 3,3',5,5'-tetramethylbenzidine MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

Claims (6)

1. Compound, characterized by the fact that it has the formula (I) or a pharmaceutically acceptable salt thereof, including all its tautomers. 2. Pharmaceutical composition, characterized in that it comprises a compound as defined in claim 1, in combination with one or more pharmaceutically acceptable diluents or vehicles. 3. Compound of formula (I), according to claim 1, characterized in that it is for use as a medicament. 4. Process, characterized in that it is for the preparation of a compound of formula (I) or a pharmaceutically acceptable salt thereof, including all its tautomers, comprising the reaction of a compound of formula (II): with a compound of formula (III): where LG1 represents an outgoing group. 5. Process, characterized in that it is for the preparation of a compound of formula (I) or a pharmaceutically acceptable salt thereof, including all its tautomers, comprising the reaction of a compound of formula (X): with a compound of formula (IV): and a compound of formula (XI): where LG4 and LG5 each independently represent exit groups. 6. Compound, characterized by the fact that it has the formula (X): or a protected derivative thereof, or a salt thereof.