NZ621440B2 - 1-pyrazolyl-3-(4-((2-anilinopyrimidin-4-yl)oxy) napththalen-1-yl) ureas as p38 map kinase inhibitors - Google Patents

Abstract

Disclosed is a compound of formula (I) which is an inhibitor of the family of p38 mitogen-activated protein kinase enzymes, and to its use in therapy, including in pharmaceutical combinations, especially in the treatment of inflammatory diseases, including inflammatory diseases of the lung, such as asthma and COPD. The compound of formula (I) is: 1-(3-tert-butyl-1-p-tolyl-1 H-pyrazol-5-yl)-3-(4-(2-(phenylamino)pyrimidin-4-yloxy)naphthalen-1-yl)urea asthma and COPD. The compound of formula (I) is: 1-(3-tert-butyl-1-p-tolyl-1 H-pyrazol-5-yl)-3-(4-(2-(phenylamino)pyrimidin-4-yloxy)naphthalen-1-yl)urea

Description

-PYRAZOLYL 4- 2-ANILINOPYRIMIDINYL OXY NAPTHTHALENYL UREAS AS 938 MAP KINASE TORS Field of the invention The invention relates to compounds which are inhibitors of the family of p38 mitogen- activated protein kinase enzymes (referred to herein as p38 MAP kinase inhibitors), for example the alpha and gamma kinase sub-types thereof, and of Syk kinase and the Src family of ne kinases, and to their use in y, ing in pharmaceutical 1O combinations, especially in the treatment of inflammatory diseases, in particular inflammatory diseases of the lung, such as asthma and COPD, as well as those of the gastrointestinal tract, such as ulcerative colitis and Crohn’s disease and of the eye, such as uveitis.

Background of the invention Four p38 MAPK isoforms (alpha, beta, gamma and delta respectively), have been identified each displaying different patterns of tissue expression in man. The p38 MAPK alpha and beta isoforms are found ubiquitously in the body, being present in many different cell types.

The alpha isoform is well terized in terms of its role in inflammation. Although studies using a chemical genetic approach in mice indicate that the p38 MAPK beta isoform does not play a role in inflammation (O’Keefe, S.J. et al., J. Biol. Chem, 2007, 282(48):34663-71.), it may be ed in pain mechanisms through the regulation of COX2 expression (Fitzsimmons, BL. 92‘ al., Neuroreport, 2010, 21(4):313-7). These isoforms are inhibited by a number of previously described small molecular weight compounds. Early classes of inhibitors were highly toxic due to the broad tissue distribution of these isoforms which resulted in multiple off-target effects of the compounds. Furthermore, development of a substantial number of inhibitors has been discontinued due to unacceptable safety profiles in clinical studies (Pettus, L.H. and Wurz, R.P., Curr. Top. Med. Chem, 2008, 8(16):1452—67.).

As these adverse effects vary with chemotype, and the compounds have distinct kinase selectivity ns, the observed toxicities may be structure-related rather than p38 mechanism-based.

Less is known about the p38 MAPK gamma and delta isoforms, which, unlike the alpha and beta isozymes are sed in specific tissues and cells. The p38 MAPK-delta isoform is expressed more highly in the pancreas, testes, lung, small intestine and the kidney. It is also abundant in macrophages and able in neutrophils, CD4+ T cells and in elial cells (Shmueli, O. et al., s Rendus Biologies, 2003, 326(10-11):1067-1072; Smith, S.

J. Br. J. Pharmacol., 2006, 3-404; Hale, K. K., J. lmmuno/., 1999, 162(7):4246—52; Wang, X. S. et al., J. Biol. Chem, 1997, 272(38):23668-23674.) Very little is known about the 4o distribution of p38 MAPK gamma although it is expressed more highly in brain, al muscle and heart, as well as in lymphocytes and macrophages (Shmueli, O. et al., Comptes Rendus Biologies, 2003, 326(10-11):1067-1072; Hale, K. K., J. Immunol., 1999, :4246- 52; Court, N. W. et al., J. Mol. Cell. Cardiol., 2002, 34(4):413-26; Mertens, S. et al., FEBS Lett., 1996, 383(3):273—6.).

Selective small molecule inhibitors of p38 MAPK gamma and p38 MAPK delta are not currently available, although one previously disclosed compound, BIRB 796, is known to possess pan-isoform inhibitory activity. The inhibition of p38 MAPK gamma and delta isoforms is observed at higher concentrations of the compound than those required to inhibit p38 MAPK alpha and p38 beta (Kuma, Y., J. Biol. Chem., 2005, 280:19472-19479.). in addition BIRB 796 also impaired the phosphorylation of p38 MAPKs or JNKs by the upstream kinase MKK6 or MKK4. Kuma discussed the ility that the conformational change caused by the binding of the inhibitor to the MAPK protein may affect the structure of both its orylation site and the docking site for the upstream activator, thereby impairing the phosphorylation of p38 MAPKs or JNKs. p38 MAP kinase is believed to play a pivotal role in many of the signalling pathways that are involved in initiating and maintaining chronic, persistent inflammation in human disease, for example, in severe asthma and in COPD (Chung, F., Chest, 2011, 139(6):1470—i479.).

There is now an nt literature which trates that p38 MAP kinase is activated by a range of pro-inflammatory cytokines and that its activation results in the recruitment and release of additional pro—inflammatory cytokines. indeed, data from some clinical studies demonstrate beneficial changes in e activity in patients during treatment with p38 MAP kinase inhibitors. For instance Smith bes the inhibitory effect of p38 MAP kinase inhibitors on TNFo (but not iL-8) release from human PBMCs.

The use of inhibitors of p38 MAP kinase in the treatment of chronic obstructive pulmonary disease (COPD) has also been proposed. Small molecule inhibitors targeted to p38 MAPK o/B have proved to be effective in reducing various parameters of inflammation in cells and in tissues obtained from ts with COPD, who are generally corticosteroid insensitive, (Smith, S.J., Br. J. Pharmacol., 2006, 149:393—404.) as well as in various in vivo animal models (Underwood, D.C. ez‘ al., Am. J. Physiol., 2000, 279:L895-902; Nath, P. et al., Eur. J.

Pharmacoi., 2006, 544:160—167.). Irusen and gues have also suggested the possible involvement of p38 MAPK (1/6 with corticosteroid insensitivity via the reduction of binding affinity of the giucocorticoid receptor (GR) in nuclei (irusen, E. et al., J. Allergy Clin. lmmunol., 2002, 9657.). Clinical experience with a range of p38 MAP kinase inhibitors, ing , BIRB 796, VX702, SClO469 and SCIO323 has been described (Lee, MR. and uez, C., CurrentMed. Chem, 2005, 12:2979-2994.).

COPD is a condition in which the underlying mation is reported to be ntially resistant to the anti-inflammatory effects of inhaled osteroids. Consequently, a superior strategy for treating COPD would be to develop an intervention which has both inherent anti- inflammatory effects and the ability to increase the sensitivity of the lung tissues of COPD patients to inhaled corticosteroids. A recent publication of Mercado (Mercado, N., et a/., Mol. 40 Pharmacol., 2011, 80(6):1128-1135.) demonstrates that silencing p38 MAPK v has the potential to e sensitivity to osteroids. Consequently there may be a dual benefit for patients in the use of a p38 MAP kinase tor for the treatment of COPD and severe asthma. However, the major obstacle hindering the utility of p38 MAP kinase inhibitors in the treatment of human chronic inflammatory diseases has been the severe toxicity observed in patients resulting in the withdrawal from clinical development of many compounds including all those specifically mentioned above.

Many patients diagnosed with asthma or with COPD continue to suffer from rolled symptoms and from exacerbations of their medical condition that can result in hospitalisation.

This occurs despite the use of the most advanced, currently available treatment regimens, comprising of combination products of an inhaled corticosteroid and a long acting B-agonist.

Data accumulated over the test decade indicates that a failure to manage effectively the underlying inflammatory component of the disease in the lung is the most likely reason that 1O exacerbations occur. Given the established efficacy of corticosteroids as nflammatory agents and, in particular, of d corticosteroids in the treatment of asthma, these gs have provoked e investigation. Resulting studies have identified that some environmental insults invoke corticosteroid—insensitive inflammatory changes in patients' lungs. An example is the se arising from virally-mediated upper respiratory tract infections , which have ular significance in increasing morbidity associated with asthma and COPD.

Epidemiological investigations have revealed a strong association between viral ions of the upper respiratory tract and a substantial percentage of the exacerbations suffered by patients already diagnosed with chronic respiratory diseases. Some of the most compelling data in this regard derives from longitudinal studies of en suffering from asthma (Papadopoulos, N.G., Papi, A., s, S. and Johnston, S.L., Paediatr. Respir. Rev,. 2004, (3):255-260.). A variety of onal studies support the conclusion that a viral infection can precipitate exacerbations and increase disease severity. For example, experimental clinical infections with rhinovirus have been reported to cause ial hyper—responsiveness to histamine in asthmatics that is unresponsive to treatment with corticosteroids (Grunberg, K., Sharon, R.F., et al., Am. J. Respir. Crit. Care Med, 2001, 164(10):1816—1822.). Further evidence derives from the association observed between disease exacerbations in patients with cystic fibrosis and HRV infections (Wat, D., Gelder, C., et al., J. Cyst. Fibros,. 2008, 72320-328.) Also consistent with this body of data is the finding that respiratory viral infections, including irus, represent an independent risk factor that correlates negatively with the 12 month al rate in paediatric, lung transplant recipients (Liu, M., Worley, 8., etal., Transpl. . 01's,. 2009, 11(4):304-312.).

Clinical research indicates that the viral load is proportionate to the observed symptoms and complications and, by implication, to the severity of inflammation. For example, following mental rhinovirus infection, lower respiratory tract symptoms and ial hyper- responsiveness correlated significantly with virus load (Message, S.D., Laza-Stanca, V., et al., PNAS, 2008; 105(36):13562-13567.). rly, in the absence of other viral agents, 40 rhinovirus infections were commonly associated with lower atory tract ions and wheezing, when the viral load was high in immunocompetent paediatric patients (Gerna, G., Piralla, A., etal., J. Med. Virol,. 2009, 81(8):1498—1507.). interestingly, it has been reported recently that prior exposure to rhinovirus reduced the 45 cytokine responses evoked by bacterial products in human alveolar macrophages (Oliver, B.G., Lim, 8., et al., Thorax, 2008, 63:519-525.). Additionally, infection of nasal epithelial cells with rhinovirus has been documented to promote the adhesion of bacteria, including 8. aureus and H. influenzae (Wang, J.H., Kwon, H.J. and Yong, J.J., The Laryngoscope, 2009, 119(7):1406-1411.). Such cellular effects may contribute to the increased probability of ts suffering a lower respiratory tract infection following an infection in the upper respiratory tract. ingly, it is therapeutically relevant to focus on the ability of novel interventions to decrease viral load in a variety of in vitro s, as a surrogate predictor of their benefit in a clinical setting.

High risk groups, for whom a rhinovirus infection in the upper respiratory tract can lead to severe secondary complications, are not limited to patients with chronic respiratory disease.

They include, for example, the immune compromised who are prone to lower respiratory tract infection, as well as patients undergoing chemotherapy, who face acute, hreatening fever. it has also been suggested that other chronic diseases, such as diabetes, are associated with a compromised immuno-defence response. This increases both the likelihood of acquiring a respiratory tract infection and of being alised as a result ’15 (Peleg, A.Y., Weerarathna, T., et al., Diabetes Metab. Res. Rev., 2007, 23(1):3-13; Kornum, J.B., , W., eta/., Diabetes Care, 2008, 31(8):1541-1545.).

Whilst upper respiratory tract viral infections are a cause of erable morbidity and mortality in those patients with underlying disease or other risk factors; they also represent a significant healthcare burden in the general population and are a major cause of missed days at school and lost time in the ace (Rollinger, J.M. and Schmidtke, M., Med. Res. Rev., 2010, Doi 10.1002/med.20176.). These considerations make it clear that novel medicines, that possess improved efficacy over current therapies, are urgently required to prevent and treat rhinovirus—mediated upper atory tract infections. In general the strategies adopted for the discovery of improved antiviral agents have targeted various proteins produced by the virus, as the point of therapeutic intervention. However, the wide range of rhinovirus pes makes this a particularly challenging approach to pursue and may explain why, at the t time, a medicine for the prophylaxis and treatment of rhinovirus ions has yet to be approved by any regulatory agency.

Viral entry into the host cell is associated with the activation of a number of intracellular signalling pathways which are believed to play a prominent role in the initiation of inflammatory processes (reviewed by , S, 2007; Signal Transduction, 7:81—88.) and of viral propagation and subsequent release. One such mechanism, which has been ined to play a role in influenza virus propagation in vitro, is activation of the phosphoinositide 3—kinase /Akt pathway. it has been reported that this signalling pathway is activated by the N81 protein of the virus (Shin, Y.K., Liu, Q. at al., J. Gen. Virol., 2007, 88:13-18.) and that its inhibition reduces the titres of progeny virus (Ehrhardt, C., Marjuki, H. et a/., Cell Microbiol., 2006, 8:1336—1348.). rmore, the MEK inhibitor U0126 has been documented to inhibit viral ation without eliciting the emergence of ant variants of the virus (Ludwig, S., Wolff, T. at al., FEBS Lett, 2004, 561(1-3):37—43.). More recently, studies targeting inhibition of Syk kinase have trated that the enzyme plays an ant role in mediating rhinovirus entry into cells and also virus—induced inflammatory responses, including [CAM-1 up—regulation (Sanderson, M.P., Lau, C.W. et al., Inflamm. Allergy Drug Targets, 2009, 8287-95.). Syk activity is reported to be controlled by c-Src as an upstream kinase in HRV infection (Lau, C. et al., J. l., 2008, 180(2):870-880.). A small number of studies have appeared that link the activation of cellular Src (Src1 or p60-Src) or Src family kinases to ion with s. These e a report that irus elicits a PI3 kinase mediated activation of Akt through a c-Src dependent ism. it has also been suggested that Rhinovirus—39 induced lL—8 production in epithelial cells depends upon Src kinase tion (Bentley, J.K., Newcomb, D.C., J. Virof., 2007, 81:1186—1194.). Finally, it has been proposed that activation of Src kinase is involved in the induction of mucin production by rhinovirus-14 in epithelial cells and sub—mucosa! glands (lnoue, D. and Yamaya, M., Resp/r. l. Neurobiol., 2006, 154(3):484—499.).

It has been disclosed previously that compounds that inhbit the activity of both c—Src and Syk kinases are effective agents against rhinovirus replication (Charron, C.E. at at, WO 2011/158042.) and that compounds that inhibit p59—HCK are effective against influenza virus ation (Charron, C.E. et al., .). For the reasons ised above, compounds designed to treat chronic respiratory diseases that combine these nt ties with the inhibition of p38 MAPKs, are ed to be particularly efficacious.

Certain p38 MAPK inhibitors have also been described as inhibitors of the replication of respiratory syncitial virus (Cass, L. et al., W0 20111158039.) Furthermore, it is noteworthy that a p38 MAPK inhibitor was found to deliver benefit for patients with IBD after one week’s treatment which was not sustained over a four week course of treatment (Schreiber, S. etal., Clin. Gastro. Hepatology, 2006, 42325—334).

In addition to playing key roles in cell signalling events which control the activity of pro— inflammatory pathways, kinase enzymes are now also recognised to regulate the activity of a range of cellular ons. Among those which have been discussed recently are the maintenance of DNA integrity (Shilo, Y. Nature Reviews Cancer, 2003, 3:155—168.) and co— ordination of the complex processes of cell division. An illustration of recent findings is a publication describing the impact of a set of inhibitors acting upon the so-called “Olaharsky kinases” on the frequency of micronucleus formation in vitro (Olaharsky, A.J. et al., PLoS Comput. Bio/., 2009, 5(7):e1000446.). Micronucleus formation is implicated in, or associated with, disruption of mitotic processes and is therefore an undesirable station of potential toxicity. Inhibition of glycogen synthase kinase 3d (GSK3or) was found to be a particularly significant factor that ses the likelihood of a kinase inhibitor promoting ucleus formation. Recently, inhibition of the kinase GSKBB with RNAi was also 40 reported to promote micronucleus formation (Tighe, A. et al., BMC Cell Biology, 2007, 8:34.).

It may be possible to attenuate the adverse effects arising from drug interactions with Olaharsky kinases, such as GSKSd, by optimisation of the dose and/or by ng the route of administration. However, it would be more advantageous to identify therapeutically useful molecules that demonstrate low or undectable activity t these off-target enzymes and consequently elicit little or no disruption of c processes, as measured in mitosis assays.

It is evident from consideration of the literature cited hereinabove that there remains a need to identify and develop new p38 MAP kinase inhibitors that have improved eutic potential over currently available ents. Desirable compounds are those that exhibit a superior therapeutic index by exerting, at the least, an equally efficacious effect as us agents but, in one or more respects, are less toxic at the relevant therapeutic dose. An objective of the present invention therefore, is to provide such novel compounds that inhibit the enzyme activity of p38 MAP kinase, for example with certain sub—type icities, together with Syk kinase and ne kinases within the Src family (particularly c-Src) thereby possessing good anti-inflammatory properties, and suitable for use in therapy, or to at least provide a useful ative.

The Compound (I) exhibits a longer duration of action and/or persistence of action in comparison to the previously disclosed allosteric p38 MAP kinase inhibitor BIRB 796 (Pargellis, C. et al., Nature Struct. Bio/., 2002, 68—272.). An additional embodiment provides such novel compound in one or more solid, lline forms that s high chemical and physical ity suitable for formulation as inhaled medicaments.

Summary of the invention Thus in one aspect of the invention there is provided a compound of formula (l): \ / \N %WN/N Compound (I) or a pharmaceutically acceptable salt or solvate thereof, including all stereoisomers and tautomers thereof.

“Compound of formula (l)" may also be referred to herein as “Compound (l)”. in another aspect of the invention there is provided Compound (l) as defined above as the free base.

In another aspect of the invention there is provided Compound (I) as defined above as the anhydrous free base.

In another aspect of the invention there is provided Compound (I) as defined above as the anhydrous free base in solid crystalline form.

In a further aspect of the ion there is provided Compound (I) as defined above as the anhydrous free base in solid crystalline polymorphic form A.

In a further aspect of the invention there is provided Compound (I) as defined above as the anhydrous free base in solid crystalline polymorphic form B.

Brief description of figures Figure 1 shows an X-ray powder diffraction (XRPD) pattern obtained from a sample of nd (I) as the anhydrous free base in solid crystalline polymorphic form A.

Figure 2 displays an XRPD pattern acquired from a sample of Compound (I) as the anhydrous free base in solid crystalline polymorphic form B, post micronization.

Figure 3 reveals the results of thermogravimetric analysis of a sample of Compound (I) as the anhydrous free base in solid crystalline polymorphic form B, post micronization.

Figure 4 represents dynamic vapour sorption (DVS) isotherm plots d from samples of Compound (I) as the ous free base in solid lline rphic form B post micronization.

Figure 5 represents the results of a hysterisis experiment conducted on Compound (I) as the anhydrous free base in solid, crystalline, polymorphic form B, post micronization, to determine the degree and rate of moisture tion/desorption with time against changes in relative humidity.

Figure 6 is the infrared (IR) spectrum obtained from a sample of Compound (I) as the ous free base in solid crystalline polymorphic form B post micronization.

Figure 7 shows l analysis of a sample of Compound (I) as the anhydrous free base in solid crystalline polymorphic form B (micronized) by differential scanning calorimetry (DSC).

Detailed description of the invention The compound of formula (I) disclosed herein is: 1-(3-tert-butyI—1-p—tolyl-1H—pyrazoIyI)-3— (4-(2-(phenylamino)pyrimidin—4-yloxy)naphthalen-t—yl)urea. es of salts of Compound (I) include all pharmaceutically acceptable salts, such as, without limitation, acid addition saIts of strong mineral acids such as HCI and HBr salts and addition salts of strong organic acids such as methanesulfonic acid.

As employed herein below the definition of a compound of formula (I) is intended to e salts, solvates, and all tautomers of said compound, unless the context specifically indicates otherwise. es of solvates include hydrates.

The invention provided herein extends to prodrugs of the compound of formula (I), that is to say compounds which break down and/or are metabolised in vivo to provide an active compound of formula (I). General examples of prodrugs include simple esters, and other esters such as mixed carbonate esters, carbamates, glycosides, ethers, acetals and ketals.

The ion embraces all isotopic derivatives of Compound (I). Thus the invention embraces compounds which are compounds of Compound (I) having one or more atoms that have been replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number most commonly found in nature, or in which the tion ‘IO of an atom having an atomic mass or mass number found less commonly in nature has been increased (the latter concept being referred to as pic enrichment”). Thus the compounds of the disclosure include those where the atom ied is a naturally occurring or non-naturally occurring isotope. In one embodiment the isotope is a stable isotope. Thus the nds of the sure include, for example deuterium containing compounds and the like. Thus, in one embodiment of the invention Compound (I) contains an enriched level of deuterium in one or more hydrogen atoms (eg. for a given hydrogen atom the level of the deuterium e exceeds 20%, 50%, 75%, 90%, 95% or 99% by number). Examples of other isotopes that can be incorporated into nd (I) or enriched in Compound (I) include isotopes of en, carbon, nitrogen, oxygen, fluorine, iodine and chlorine such as 3H, “C, 13C, 14C, 15N, 18F, 123I or 125I, which may be naturally occurring or non-naturally occurring isotopes.

In a further aspect of the invention there is ed one or more metabolites of the compound of formula (I), in particular a metabolite that retains one or more of the therapeutic activities of the compound of formula (I). A metabolite, as employed herein, is a compound that is produced in vivo from the metabolism of the compound of formula (I), such as, without limitation, oxidative lites and/or metabolites generated, for example, from O- dealkylation.

The disclosure also extends to all rphic forms of the compounds herein defined.

A route suitable for the preparation of the compound of formula (I) is shown below (Scheme W0 2013;’050757 PCT/G32012/052445 Scheme 1 fINC/ 1. PhNHzl H N Cw/N 2 DBU MeCN HzN NY 2 33“ .HCI Step2 Step 1 Cl PhOC(O)C|/ / \ Na2C03 N‘N NH2 kOPh + OON/N Step3 H2N NIY H\©N EtaN , N/ N\ 22/ 00Wl Step 4 0% Compound (I) Protective groups may be required to protect chemically sensitive groups during one or more of the reactions described above, to ensure that the process can be carried out and/or is efficient. Thus if desired or necessary, intermediate compounds may be protected by the use of conventional protective groups. Protective groups and the means for their l are described in “Protective Groups in Organic Synthesis”, by Theodora W. Greene and Peter G.

M. Wuts, published by John Wiley & Sons Inc; 4th Rev Ed., 2006, 0: 0471697540.

A detailed preparation of Compound (I) is provided in e 1.

Novel intermediates as described herein form an aspect of the invention.

In another aspect of the ion, there is provided Compound (I) as the anhydrous free base in solid, crystalline form. In a further aspect of the invention, there is ed Compound (I) as the anhydrous free base in solid, crystalline, rphic form A which may be obtained, for example, by crystallising Compound (I) from pyl acetate. In a particular aspect of the invention, there is provided Compound (I) as the anhydrous free base in solid, crystalline, polymorphic form B, which may be obtained, for example, by crystallising Compound (I) from acetone and water. A typical ratio of acetone to water that is suitable for this process is between 5:1 and 200:1 e.g. around 10:1. atively, form B may be obtained by llising Compound (I) from acetone alone. Detailed preparations of Compound (I) as the anhydrous free base in solid crystalline polymorphic forms A and B are provided in Examples 1 and 3 of the Experimental Section, respectively.

In a further aspect of the invention, the solid state properties of Compound (I) may be improved by further slurrying or recrystallization steps to e, for example, material with improved morphology and/0r containing a reduced level of residual solvent. For example, residual solvent may be removed from Compound (I) as the anhydrous free base in solid, lline, polymorphic form B by slurrying Compound (I) in polymorphic form B, in water, or alternatively by further tallization from acetone. A detailed description of an exemplary slurrying procedure is ed in Example 33 of the Experimental Section.

In one embodiment, there is provided solid, crystalline polymorphic form A of nd (I) as the anhydrous free base having an XRPD pattern substantially as shown in Figure 1. The method of obtaining the XRPD data is described in ical Methods and the data discussed in Example 5.

Thus, there is provided Compound (I) as the anhydrous free base in solid, crystalline, polymorphic form A having an XRPD pattern with at least one (for example one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen or all sixteen) peak(s) at 7.8, 8.7, 10.3, 11.2, 12.4, 15.2, 16.2, 17.5, 19.7, 20.8, 22.6, 23.1, 24.6, 25.5, 26.7, 27.4 (i 0.2 degrees, 2-theta values), these peaks being teristic of the solid, crystalline, polymorphic form A. The peaks at 10.3, 15.2, 17.5, 23.1, 24.6, 26.7 and 27.4 are particularly characteristic for the solid, crystalline, polymorphic form A and therefore it is preferable that at least one (for example one, two, three, four, five, six or all seven) of these peaks is observable in the XRPD pattern.

In another embodiment, there is ed solid, crystalline, polymorphic form B of Compound (I) as the anhydrous free base (micronized) having an XRPD pattern substantially as shown in Figure 2. The method of micronization is described in Example 4 and the method of obtaining the XRPD data is bed in Analytical s and the data discussed in Example 5.

Thus, there is provided Compound (I) as the anhydrous free base in solid crystalline polymorphic form B (micronized) having an XRPD pattern with at least one (for e one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen or all eighteen) peaks at 3.9, 6.1, 7.7, 8.6, 10.9, 11.8, 12.7, 14.3, 15.9, 16.7, 18.3, 18.7, 19.9, 20.9, 22.0, 22.6, 25.2, 28.9 (i 0.2 degrees, 2-theta values), these peaks being characteristic of the solid crystalline polymorphic form B. The peaks at 3.9, 6.1, 11.8, 14.3, 16.7, 18.3, 18.7 and 28.9 are particularly characteristic for the solid, crystalline, polymorphic form B and ore it is preferable that at least one (for example one, two, three, four, five, six, seven or all eight) of these peaks is observable in the XRPD pattern.

The melting points of Compound (I) as the ous free base in solid, crystalline, polymorphic forms A and B were ined using differential scanning calorimetry as described in Example 6. Compound (I) as the anhydrous free base in solid, crystalline, polymorphic form A was found to have a melting point of 191.6 °C, and Compound (I) as the anhydrous free base in solid, crystalline, polymorphic form B was found to have a melting point of 214 °C. Polymorphic form B was also found to have a higher heat of fusion than polymorphic form A. As explained in Example 6, these results suggest that polymorphic form B is thermodynamically more stable than polymorphic form A.

The al and chemical stabilities of Compound (I) as the ous free base in solid, lline, polymorphic form B, were investigated, the results of which are disclosed herein.

In order to assess physical stability, nd (I) as the anhydrous free base in solid, crystalline, polymorphic form B was micronized following the procedure described in Example 4, and samples of the resulting material were stored in open containers and subjected to different ambient temperatures and relative humidities. The physical properties and stabilities of the samples were investigated using TGA, DSC, DVS, IR spectroscopy and XRPD analysis. Full experimental procedures are provided in the General Procedures section and the results are summarised in Example 7 (Table 8). As discussed in Example 7, Compound (I) as the anhydrous free base in solid, crystalline, polymorphic form B (micronized) was found to have good physical stability. The same experimental procedures were also carried out using Compound (I) as the anhydrous free base in solid, crystalline, polymorphic form B in cronized form and the results were found to be ntially similar to those obtained for the micronized material i.e. Compound (I) as the anhydrous free base in solid, lline, polymorphic form B in both micronized and unmicronized forms was found to have good physical stability.

In order to assess chemical stability, Compound (I) as the ous free base in solid, crystalline, polymorphic form B was micronized following the procedure described in Example 4. Micronized samples were stored in open containers and ted to different ambient temperatures and relative humidities. The al stabilities of the samples were analysed by HPLC. The results are summarised in Example 8 (Table 9) where it is indicated that Compound (I) as the anhydrous free base in solid, crystalline, polymorphic form B, post 3O microniastion was found to be ally stable, although some sensitivity towards light was detected.

As a result of the solid state s sed herein, it is concluded that nd (I) as the anhydrous free base in solid, crystalline, polymorphic form B can be ized and that the resulting material has good physical and chemical stability.

The compound of formula (I) is a p38 MAP kinase inhibitor (especially of the alpha subtype) and in one aspect the compound is useful in the treatment of inflammatory diseases, for example COPD and/or asthma.

Surprisingly, the compound exhibits a long duration of action and/or tence of action in comparison to the previously disclosed p38 MAP kinase inhibitor BIRBYQG.

WO 50757 Persistence of action as used herein is related to the dissociation rate or dissociation constant of the compound from the target (such as a receptor). A low dissociation rate may lead to persistence.

A low dissociation rate in combination with a high association rate tends to e potent therapeutic entities.

The compound of a (l) is expected to be potent in vivo. lly, the prior art compounds developed to date have been intended for oral administration. This gy es optimizing nds which achieve their duration of action by an appropriate pharmacokinetic profile,.thereby ng that a sufficiently high drug concentration is established and maintained between doses to provide clinical benefit.

The inevitable consequence of this approach is that all bodily tissues, and especially the liver and the gut, are exposed to supra-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 paradigms in which the drug is dosed ly to the inflamed organ (topical therapy). While this approach is not suitable for treating all chronic inflammatory diseases, it has been extensively exploited in lung diseases (asthma, COPD), skin conditions (atopic dermatitis and psoriasis), nasal diseases (allergic rhinitis) and gastrointestinal disorders (ulcerative colitis). ln topical y, efficacy can be achieved either by ensuring that the drug has a sustained on of action and is ed in the relevant organ to ze the risks of systemic toxicity or by producing a formulation which generates a “reservoir” of the active drug. which is available to sustai its desired effects. The first approach is exemplified by the anticholinergic drug tiotropium (Spiriva).,This compound is administered topically to the lung as a treatment for COPD, and has an exceptionally high affinity for its target or, 3O resulting in a very slow off rate and a consequent sustained duration of action. in one aspect of the disclosure the compound of formula (I) is particularly suitable for topical delivery, such as topical delivery to the lungs, in particular for the treatment of respiratory disease, for example chronic respiratory diseases such as COPD and/or asthma.

In one embodiment the compound of formula (I) is suitable for sensitizing patients to treatment with a corticosteroid who have become refractory to such treatment regimens.

The compound of formula (I) may also be useful for the ent of rheumatoid arthritis.

The compound of formula (I) may have antiviral properties, for example the y to prevent infection of cells (such as respiratory epithelial cells) with a avirus, in particular a rhinovirus, influenza or respiratory syncytial virus.

PCT/G32012/052445 Thus the compound is thought to be an antiviral agent, in particular le for the prevention, treatment or ration of picornavirus infections, such as rhinovirus infection, influenza or respiratory syncytial virus.

In one embodiment the nd of formula (I) is able to reduce inflammation induced by viral ion, such as rhinovirus infection and in particular viral ions that result in the release of cytokines such as IL—8, especially in vivo. This activity may, for example, be tested in vitro employing a rhinovirus induced lL-8 assay as described in the Examples herein. 1O In one embodiment the compound of formula (I) is able to reduce ICAM1 expression induced by rhinovirus, especially in vivo. ICAM1 is the receptor mechanism used by so-called major groove rhinovirus serotypes to infect cells. This ty may be measured, for example by a method described in the Examples herein.

It is expected that the above properties render the compound of formula (I) particularly suitable for use in the treatment and/or prophylaxis of exacerbations of inflamatory diseases, in particular viral exacerbations, in patients with one or more of the following c conditions such as congestive heart failure, COPD, , diabetes, cancer and/or in immunosuppressed patients, for example post-organ transplant.

In particular, the compound of formula (I) may be useful in the treatment of one or more respiratory disorders including COPD (including chronic bronchitis and emphysema), asthma, paediatric asthma, cystic fibrosis, sarcoidosis, idiopathic pulmonary fibrosis, allergic rhinitis, rhinitis, sinusitis, especially asthma, and COPD (including chronic bronchitis and emphysema).

The compound of formula (I) may also be useful in the treatment of one or more conditions which may be treated by topical or local therapy including allergic conjunctivitis, conjunctivitis, allergic dermatitis, contact dermatitis, psoriasis, tive colitis, ed joints secondary to rheumatoid tis or to osteoarthritis.

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

The compound of a (I) may be useful in the treatment of eye diseases or disorders including allergic ctivitis, ctivitis, diabetic pathy, macular oedema (including wet macular oedema and dry macular oedema), post-operative cataract 40 inflammation or, particularly, uveitis (including posterior, anterior and pan uveitis).

The compound of formula (I) may be useful in the treatment of gastrointestinal diseases or disorders ing tive colitis or Crohn’s disease.

The nd of formula (I) may also re-sensitise the t’s condition to treatment with a corticosteroid, when the patient’s condition has become refractory to the same.

Furthermore, the present invention provides a pharmaceutical composition comprising a compound according to the disclosure optionally in combination with one or more pharmaceutically acceptable diluents or carriers.

The present invention also provides a process for preparing such a pharmaceutical composition which comprising mixing the ingredients.

Diluents and rs may include those suitable for parenteral, oral, topical, mucosal and rectal administration.

As mentioned above, such compositions may be prepared e.g. for parenteral, aneous, intramuscular, intravenous, intra—articular or pen-articular administration, particularly in the form of liquid solutions or suspensions; for oral administration, particularly in the form of tablets or capsules; for l e.g. pulmonary or intranasal administration, particularly in the form of powders, nasal drops or aerosols and transdermal administration; for mucosal administration e.g. to buccal, gual or vaginal , and for rectal administration e.g. in the form of a itory.

The compositions may conveniently be stered in unit dosage form and may be prepared by any of the methods well-known in the pharmaceutical art, for example as described in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, , PA., (1985). Formulations for parenteral administration may contain as excipients sterile water or saline, ne glycols such as propylene glycol, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, 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 in the form of nasal drops or metered sprays. For buccal administration typical excipients include sugars, calcium stearate, magnesium stearate, pregelatinated starch, and the like.

Compositions suitable for oral administration may comprise one or more physiologically compatible carriers and/or excipients and may be in solid or liquid form. Tablets and capsules may be prepared with binding agents, for example, syrup, acacia, gelatin, sorbitol, tragacanth, or poly—vinylpyrollidone; fillers, such as lactose, sucrose, corn starch, calcium phosphate, sorbitol, or glycine; lubricants, such as magnesium stearate, talc, polyethylene glycol, or ; and surfactants, such as sodium lauryl sulfate. Liquid compositions may 40 contain tional additives such as suspending , for example sorbitol syrup, methyl cellulose, sugar syrup, gelatin, ymethyl—cellulose, or edible fats; emulsifying agents such as in, or acacia; ble oils such as almond oil, t oil, cod liver oil, or peanut oil; preservatives such as butylated hydroxyanisole (BHA) and butylated ytoluene (BHT). Liquid compositions may be encapsulated in, for example, gelatin to provide a unit dosage form.

Solid oral dosage forms include tablets, two-piece hard shell capsules and soft elastic gelatin (SEG) capsules.

A dry shell formulation typically ses of about 40% to 60% w/w tration of gelatin, about a 20% to 30% concentration of plasticizer (such as glycerin, sorbitol or propylene glycol) and about a 30% to 40% concentration of water. Other materials such as 1O preservatives, dyes, opacifiers and flavours also may be present. The liquid fill material comprises a solid drug that has been dissolved, solubilized or sed (with suspending agents such as x, 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 e—active agents.

Suitably the nd of formula (I) is administered topically to the lung. Hence we provide according to the invention a pharmaceutical ition comprising Compound (I) of the disclosure optionally in combination with one or more topically acceptable diluents or rs. Topical administration to the lung may be achieved by use of an aerosol formulation.

Aerosol formulations typically comprise the active ient suspended or dissolved in a suitable aerosol propellant, such as a chlorofluorocarbon (CFC) or a hydrofluorocarbon (HFC). Suitable CFC propellants include oromonofluoromethane (propellant 11), rotetrafluoro methane (propellant 114), and dichlorodifluoromethane (propellant 12).

Suitable HFC propellants e tetrafluoroethane (HFC—134a) and heptafluoropropane (HFC-227). The propellant lly comprises 40% to 99.5% e.g. 40% to 90% by weight of the total inhalation composition. The formulation may comprise excipients including co— solvents (e.g. ethanol) and surfactants (e.g. lecithin, an trioleate and the like). Aerosol formulations are packaged in canisters and a suitable dose is delivered by means of a metering valve (e.g. as supplied by Bespak, Valois or 3M).

Topical stration to the lung may also be achieved by use of a non-pressurised formulation such as an aqueous solution or suspension. This may be administered by means of a nebuliser. Nebulisers may be portable or non—portable Topical administration to the lung may also be achieved by use of a wder formulation. A dry powder formulation will contain the compound of the disclosure in finely divided form, typically with a mass mean aerodynamic diameter (MMAD) of 1-10 pm. The formulation will typically contain a topically able diluent such as lactose, usually of larger particle size e.g. an MMAD of 50 pm or more, e.g. 100 pm or more. An alternative topically acceptable diluent is mannitol. Examples of dry powder delivery systems include SPlNHALER, DlSKHALER, TURBOHALER, DISKUS 40 and CLlCKHALER. Further examples of dry powder inhaler s include ECLIPSE, ROTAHALER, HANDIHALER, AEROLlSER, CYCLOHALER, BREEZHALER/NEOHALER, FLOWCAPS, TWINCAPS, X—CAPS, TURBOSPlN, ELPENHALER, TURBUHALER, MlATHALER, TWISTHALER, NOVOLlZER, SKYEHALER, ORIEL dry powder inhaler, MICRODOSE, ACCUHALER, PULVINAL, LER, ULTRAHALER, TAIFUN, PULMOJET, LER, GYROHALER, TAPER, CONIX, XCELOVAIR and PROHALER.

One aspect of the invention relates to a dry powder pharmaceutical formulation for inhalation comprising: (i) Compound (I) ,w N/N \N gmGMT) nd (l) that is 1-(3-ten‘-butyI—i-p~tonI—1H-pyrazoI-S-yl)-3—(4-(2-(phenyiamino)pyrimidin-4— yloxy)naphthalen-i-yI)urea or a pharmaceutically acceptable salt thereof, including all stereoisomers and tautomers thereof, in particulate form (e.g. solid crystalline Form B) as active ingredient; (ii) particulate lactose as carrier; and (iii) a particulate metal salt of stearic acid, such as magnesium stearate.

The invention also provides for an inhalation device comprising one or more doses of said formulation.

The nd of formula (I) has therapeutic ty. In a further aspect, the present invention provides a compound of the sure for use as a medicament. Thus, in a r aspect, the present ion provides a compound as bed herein for use in the treatment of one or more of the above mentioned conditions.

In a further aspect, the present invention es use of Compound (I) as described herein for the manufacture of a medicament for the treatment of one or more of the above mentioned conditions.

In a further aspect, the present invention provides a method of treatment of one or more of the above mentioned conditions which comprises administering to a subject an effective amount of Compound (I) of the disclosure or a pharmaceutical composition comprising the compound.

The word “treatment” is intended to embrace prophylaxis as well as therapeutic treatment.

Compound (I) of the invention may also be administered in ation with one or more other active ingredients e.g. active ingredients suitable for treating the above mentioned conditions. For example possible combinations for treatment of respiratory disorders include combinations with steroids (e.g. nide, beclomethasone dipropionate, fluticasone nate, mometasone furoate, fluticasone furoate), beta agonists (e.g. terbutaline, amol, salmeterol, formoterol) and/or xanthines (e.g. theophylline). Other le actives include anticholinergics, such as tiotropium and anti-viral agents such as, but not limited to, vir or oseltamivir, for example as the phosphate. Other anti-viral agents include peramivir and laninamivir. Further possible combinations for ent of respiratory disorders include combinations with steroids such as flunisolide, ciclesonide and triamcinolone; beta agonists such bambuterol, levalbuterol, clenbuterol, fenoterol, broxaterol, indacaterol, reproterol, procaterol and vilanterol; muscarinic antagonists, (e.g. ipratropium, tiotropium, oxitropium, glycopyrronium, glycopyrrolate, aclidinium, trospium) and leukotriene antagonists (e.g. zafirlukast, kast, zileuton, montelukast). It will be understood that any of the aforementioned active ingredients may be employed in the form of a pharmaceutically acceptable salt. in one embodiment the compound of formula (l) and the other active ingredient(s) are co- formulated in the same pharmaceutical formulation. In another embodiment the other active ingredient(s) are administered in one or more separate ceutical formulations.

Hence, another aspect of the ion provides a combination product sing: (A) a compound of the present invention (i.e. a compound of formula (l) as defined above, or a pharmaceutically acceptable salt thereof); and (B) another therapeutic agent, wherein each of components (A) and (B) is formulated in admixture with a pharmaceutically- acceptable diluent or carrier.

In this aspect of the invention, the combination product may be either a single (combination) ceutical formulation or a kit—of—parts.

Thus, this aspect of the ion encompasses a pharmaceutical formulation including a compound of the present invention and another eutic agent, in admixture with a pharmaceutically acceptable diluent or carrier (which formulation is hereinafter referred to as a “combined preparation”). it also encompasses a kit of parts comprising components: (i) a ceutical formulation including a compound of the present invention in admixture with a pharmaceutically acceptable diluent or carrier; and (ii) a pharmaceutical formulation including another therapeutic agent, in admixture with a ceutically—acceptable diluent or carrier, which components (i) and (ii) are each provided in a form that is suitable for administration in conjunction with the other.

Component (i) of the kit of parts is thus ent (A) above in admixture with a pharmaceutically acceptable diluent or carrier. Similarly, component (ii) is component (B) above in admixture with a ceutically acceptable diluent or carrier.

The other therapeutic agent (i.e. component (B) above) may be, for example, any of the active ingredients mentioned above in connection with the treatment of respiratory disorders.

The data ed hereinbelow in relation to the antiviral ties of the nd of formula (I) provides evidence that other antiviral therapies in combination with a compound of formula (I) would be useful in the treatment or prevention of virally-induced exacerbations (for example respiratory viral infections) suffered by patients with respiratory disease such as COPD andIor asthma and/or one or more of the indications listed above. Thus, in one aspect there is provided the use of Compound (I) in combination with an anti—viral therapy such as, but not limited to, zanamavir or oseltamivir (for example oseltamivir phosphate) in the 1O treatment or tion of respiratory viral infections suffered by patients with atory disease such as COPD and/or .

The inventors also believe that other antiviral therapies in combination with Compound (l) would be useful in the ent or prevention of y induced exacerbations (for example respiratory viral infections) in patients with chronic conditions other than respiratory diseases, for example conditions such as congestive heart e, diabetes, cancer, or ions suffered by immunosuppressed patients, for example post-organ transplant. Thus, in a further aspect there is provided the use of a compound of the invention in combination with an anti-viral therapy, such as, but not limited to, zanamavir or oseltamivir (for example oseltamivir ate), in the treatment or prevention of respiratory viral infections in patients with chronic conditions such as congestive heart failure, diabetes, cancer, or in conditions suffered by immunosuppressed patients, for example post-organ transplant.

EXPERIMENTAL SECTION Abbreviations used herein are defined below (Table 1). Any abbreviations not defined are intended to convey their generally accepted g.

Table 1: Abbreviations AcOH glacial acetic acid Aq aqueous ATP adenosine~5'-triphosphate BALF bronchoalveolae lavage fluid BEGM bronchial lial cell growth media br broad BSA bovine serum albumin t® catalytic cartridge CDl 1,1 -carbony|—diimidazole COPD chronic obstructive pulmonary disease CXCL1 chemokine (C-X—C motif) ligand “i d doublet DCM dichloromethane DMSO dimethyl sulfoxide DSC differential scanning calorimetry d-U937 cells PMA differentiated U-937 cells DVS c vapour sorption (ESi) electrospray ionization, positive mode Et ethyl EtOAc ethyl e FCS foetal calf serum FRET fluorescence resonance energy transfer GSKSa glycogen synthase kinase 30 HBEC primary human bronchial epithelial cells hr hour(s) HRP horseradish peroxidise HRV human irus lCAM-1 inter-cellular adhesion molecule 1 IR infrared JNK c-Jun N-terminal kinase KC keratinocyte chemoattractant Kd dissociation constant LPS Lipopolysaccharide (M+H)+ protonated molecular ion MAPK mitogen protein activated n kinase —K2 mitogen-activated protein kinase—activated protein kinase-2 Me methyl MeCN acetonitrile MeOH methanol MHZ megahertz min minute(s) MlP1a macrophage inflammatory protein 1 alpha MMAD mass median aerodynamic diameter MOI multiplicity of infection m.p. melting point MTT —dimethylthiazol-2—yl)-2,5-dlphenyltetrazolium bromide m/z: mass-to—charge ratio NMR nuclear magnetic nce (spectroscopy) PBMC peripheral blood mononuclear cell PBS phosphate buffered saline Ph phenyl PHA phytohaemagglutinin PMA phorbol myristate acetate pTSA 4—methylbenzenesulfonic acid PCT/G32012/052445 q t RT room temperature RP HPLC e phase high performance liquid chromatography RSV respiratory syncytical virus 8 singlet sat saturated SCX solid supported cation exchange (resin) SDS sodium dodecyl sulphate SNAr nucleophilic aromatic substitution t triplet TCleo 50% tissue culture infectious dose TGA thermogravimetric analysis THF tetrahydrofuran TNFd tumor necrosis factor alpha XRPD X-ray powder diffraction General Procedures All ng materials and solvents were obtained either from commercial s or prepared ing to the ture citation. Unless otherwise stated all reactions were stirred. Organic solutions were ely dried over anhydrous magnesium sulfate. Hydrogenations were performed on a Thales H—cube flow reactor under the conditions stated. Column chromatography was performed on pre-packed silica (230-400 mesh, 40—63 pm) cartridges 1O using the amount indicated. SCX was purchased from Supelco and treated with 1M hydrochloric acid prior to use. Unless stated otherwise the reaction mixture to be purified was first diluted with MeOH and made acidic with a few drops of AcOH. This solution was loaded directly onto the SCX and washed with MeOH. The desired material was then eluted by washing with 1% NH3 in MeOH.

Preparative Reverse Phase High Performance Liquid Chromatography: Agilent Scalar column C18, 5 pm (21.2 X 50 mm), flow rate 28 mL min‘1 eluting with a HgO-MeCN gradient containing 0.1% v/v formic acid over 10 min using UV detection at 215 and 254 nm. Gradient information: 0.0—0.5 min; 95% H20-5% MeCN; 0.5-7.0 min; ramped from 95% H20—5% MeCN to 5% HgO-95% MeCN; 7.0-7.9 min; held at 5% % MeCN; 7.9-8.0 min; ed to 95% H20-5% MeCN; .0 min; held at 95% H20—5% MeCN.

Analytical Methods Reverse Phase High Performance Liquid Chromatography: (Method 1): Agilent Scalar column C18, 5 pm (4.6 X 50 mm) or Waters XBridge C18, 5 pm (4.6 x 50 mm) flow rate 2.5 mL min‘1 eluting with a HZO-MeCN gradient containing either 0.1% v/v formic acid (Method 1 acidic) or NH3 d 1 basic) over 7 min employing UV detection at 215 and 254 nm.

Gradient ation: 0.0~0.1 min, 95% H20-5% MeCN; 0.1—5.0 min, ramped from 95% H20- % MeCN to 5% H20—95% MeCN; 5.0-5.5 min, held at 5% H20-95% MeCN; 5.5—5.6 min, held at 5% H20-95% MeCN, flow rate increased to 3.5 mL min'1; 5.6-6.6 min, held at 5% H20-95% MeCN, flow rate 3.5 mL min'1; 6.6-6.75 min, returned to 95% H20—5% MeCN, flow rate 3.5 mL min'1; 6.75-6.9 min, held at 95% H20-5% MeCN, flow rate 3.5 mL.min'1; 0 min, held at 95% HZO-5% MeCN, flow rate reduced to 2.5 mL min'1.

Reverse Phase High Performance Liquid Chromatography: (Method 2): Agilent Extend C18 column, 1.8 pm (4.6 x 30 mm) at 40°C; flow rate 2.5—4.5 mL min"1 eluting with a HZO—MeCN 1O gradient containing 0.1% v/v formic acid over 4 min employing UV detection at 254 nm.

Gradient information: 0-3.00 min, ramped from 95% H20~5% MeCN to 5% H20—95% MeCN; 3.00-3.01 min, held at 5% H20~95% MeCN, flow rate increased to 4.5 mL min“; 3.01 3.50 min, held at 5% H20-95% MeCN; 3.50360 min, ed to 95% H20-5% MeCN, flow rate reduced to 3.50 mL min"; 3.60-3.90 min, held at 95% H20-5% MeCN; 3.90-4.00 min, held at 95% H20-5% MeCN, flow rate reduced to 2.5 mL min". 1H NMR Spectroscopy: Spectra were acquired on a Bruker Avance ||l spectrometer at 400 MHz using residual undeuterated solvent as reference.

Dynamic Vapour Sorption: Plots were obtained using a Surface Measurement Systems dynamic vapor sorption model DVS—1 using about 10 mg of the sample. The weight change was recorded with respect to atmospheric humidity at 25°C and was determined using the following parameters: drying: 60 min under dry nitrogen; equilibrium: 60 min/step; data interval: 0.05% or 2.0 min. The relative humidity [RH %] ement points were as follows: first set: 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 90, 80, 70, 60, 50, 40, 30, 20, 10, 5 second set: 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 90, 80, 70, 60, 50, 40, 30, 20, 10, 5, 0.

X-Ray Powder Diffraction: Patterns were obtained on a PANalytical (Philips) X’PertPRO MPD diffractometer equipped with a Cu LFF X—ray tube (45 kV; 40 mA; Bragg-Brentano; spinner stage) and were acquired using Cu Ker radiation under the ing measurement conditions: scan mode: continuous; scan range: 3 to 50° 29; step size: 0.02°/step; counting time: 30 sec/step; r revolution time: 1 sec; nt beam path: program. divergence slit: 15 mm; Soller slit: 0.04 rad; beam mask: 15 mm; anti scatter slit: 1°; beam knife: +; Diffracted beam path: long anti scatter shield: +; Soller slit: 0.04 rad; Ni filter: +; detector: X'Celerator. s were ed by spreading on a zero background sample holder.

Infrared Spectroscopy: Micro attenuated total reflectance (microATR) was used and the sample was analyzed using a suitable TR accessory and the following measurement 40 conditions: apparatus: Thermo Nexus 670 FTIR ometer; number of scans: 32; tion: 1 cm“; wavelength range:4000 to 400 cm“; detector: DTGS with KBr windows; beamsplitter: Ge on KBr; micro ATR accessory: Harrick Split Pea with Si crystal.

WO 50757 Differential Scanning Calorimetry: Data were collected on a TA—lnstruments Q1000 MTDSC equipped with R08 cooling unit. Typically 3 mg of each compound, in a standard aluminium trument sample pan, was heated at 10 °C/min from 25 C to 300°C. A nitrogen purge at 50 mL/min was maintained over the sample.

Thermogravimetric is: Data were collected on a TA—lnstruments Q500 thermogravimeter Typically 10 mg of each sample was transferred into a ore-weighed aluminium pan and was heated at 20 °C/min from ambient temperature to 300 °C or < 80[(w/w)%] unless otherwise stated. al Stability by HPLC: Analyses were carried out on a Waters Xbridge C18 column (3.0 x 150 mm; 3.5 pm) using the following operating conditions: column temperature: 40°C; sample temperature: 5°C; flow rate: 0.45 mL/min; ion volume: 7 uL; UV detection at 260 nm; mobile phase composition comprised of Phase A: 10 mM ammonium acetate + 0.1%, v/v trifluoroacetic acid in water and Phase B: acetonitrile, using the gradient defined by the parameters below (Table 2).

Table 2: Gradient Conditions for Chemical Stability Studies by HPLC.

% Composition at Run Time (min) Eluent O 20 25 26 32 Phase A 70 0 O 70 7O PhaseB 30 100 100 30 30 Experimental methods for biological testing Enzyme Inhibition Assays The kinase enzyme g activities of compounds sed herein were determined using a proprietary assay which measures active site-directed competition binding to an immobilized ligand (Fabian, MA. et al., Nature Biotechnol, 2005, 23:329—336). These assays were conducted by DiscoverX rly Ambit; San Diego, CA). The Kd value (Dissociation constant value) was calculated as the index of affinity of the compounds to each kinase.

Enzyme Inhibition Assays The enzyme tory activities of compounds sed herein were determined by FRET using synthetic peptides labelled with both donor and acceptor fluorophores (Z-LYTE, lnvitrogen Ltd., Paisley, UK). p38 MAPKa Enzyme Inhibition The inhibitory activities of test compounds against the p38 MAPKG isoform (MAPK14: ogen), were evaluated indirectly by determining the level of activation 1' phosphorylation of the down-stream molecule, MAPKAP-KZ. The p38 MAPKa protein (80 ng/mL, 2.5 uL) was mixed with the test compound (2.5 uL of either 4 ug/mL, 0.4 ug/mL, 0.04 ug/mL or 0.004 ug/mL) for 2 hr at RT. The mix solution (2.5 uL) of the p38o inactive target MAPKAP—K2 (lnvitrogen, 600 ng/mL) and FRET peptide (8 uM; a phosphorylation target for MAPKAP—KZ) was then added and the kinase reaction was initiated by adding ATP (40 mm, 2.5pL). The 1O mixture was incubated for 1 hr at RT. Development reagent (protease, 5 uL) was added for 1 hr prior to detection in a fluorescence microplate reader (Varioskan® Flash, ThermoFisher ific). p38 MAPKv Enzyme Inhibition The inhibitory activities of compounds of the invention t p38MAPKv (MAPK12: Invitrogen), were evaluated in a similar fashion to that described hereinabove. The enzyme (800 ng/mL, 2.5 uL) was incubated with the test compound (2.5uL at either 4 ug/mL, 0.4 ug/mL, 0.04 ug/mL, or 0.004 ug/mL) for 2 hr at RT. The FRET peptides (8 uM, 2.5 uL), and 2O appropriate ATP on (2.5 uL, 400 uM) was then added to the enzymes / nd mixtures and incubated for 1 hr. pment reagent (protease, 5 uL) was added for 1 hr prior to detection in a fluorescence microplate reader (Varioskan® Flash, Thermo Scientific).

C—Src and Syk Enzyme Inhibition The inhibitory activities of compounds of the invention against c-Src and Syk enzymes (Invitrogen), were evaluated in a similar fashion to that described hereinabove. The relevant enzyme (3000 ng/mL or 2000 ng/mL respectively, 2.5 uL) was incubated with the test compound (either 4 ug/mL, 0.4 ug/mL, 0.04 ug/mL, or 0.004 ug/mL, 2.5 uL each) for 2 hr at RT. The FRET peptides (8 uM, 2.5 uL), and appropriate ATP solutions (2.5 uL, 800 uM for c— Src, and 60 uM ATP for Syk) were then added to the enzyme / compound mixtures and incubated for 1 hr. Development reagent (protease, 5 0L) was added for 1 hr prior to detection in a fluorescence microplate reader skan® Flash, ThermoFisher Scientific).

GSK 3a Enzyme tion The inhibitory activities of test compounds t the GSK 3o enzyme isoform (lnvitrogen), were ted by determining the level of activation / phosphorylation of the target e.

The GSK3-o protein (500 ng/mL, 2.5 uL) was mixed with the test compound (2.5 uL at either 40 4 pg/mL, 0.4 ug/mL, 0.04 ug/mL, or 0.004 ug/mL) for 2 hr at RT. The FRET peptide (8 uM, 2.5 uL), which is a phosphorylation target for GSK3c, and ATP (40 uM, 2.5 pL) were then added to the enzyme / compound mixture and the resulting mixture incubated for 1 hr.

Development reagent (protease, 5 uL) was added for 1 hr prior to detection in a fluorescence microplate reader (Varioskan® Flash, Fisher Scientific). in all cases, the site-specific se cleaves non-phosphorylated peptide only and eliminates the FRET signal. Phosphorylation levels of each reaction were calculated using the ratio of coumarin emission (donor) over fluorescein emission (acceptor), for which low ratios indicate high phosphorylation and high ratios indicate low phosphorylation levels. The percentage inhibition of each reaction was calculated relative to non—inhibited control and the 50% inhibitory concentration (K350 value) was then calculated from the concentration- response curve. 1O Cellular Assays LPS-induced TNFa / IL-8 Release in d-U937Cells U937 cells, a human tic cell line, were differentiated into macrophage—type cells by incubation with PMA (100 ng/mL) for 48 to 72 hr. Cells were pre—incubated with final concentrations of test compound for 2 hr and were then stimulated with LPS (0.1 pg/mL; from E. Coli: O111:B4, Sigma) for 4 hr. The supernatant was collected for determination of TNFa and lL—8 concentrations by sandwich ELISA (Duo-set, R&D systems). The inhibition of TNFor production was calculated as a tage of that achieved by 10 pg/mL of BIRBTQS at each concentration of test compound by comparison t vehicle control. The relative 50% effective tration (REC50) was determined from the resultant concentration—response curve. The inhibition of IL-8 production was calculated at each concentration of test compound by comparison with vehicle control. The 50% inhibitory concentration (ICso) was determined from the resultant tration—response curve.

LPS—induced TNFa Release in THP-‘l Cells THP-1 cells, a human monocytic cell line, were ated with 3 ug/mL of LPS (from E. Coli; 0111284, Sigma) for 4 hr and the supernatant collected for ination of the TNFoc concentration by sandwich ELISA (Duo-set, R&D systems). The inhibition of TNch production was calculated at each tration by comparison with vehicle control. The 50% inhibitory concentration (ICSO) was determined from the resultant concentration- response curve.

Poly l:C-induced [CAM-1 Expression in BEASZB Cells Poly l:C was used in these studies as a , RNA virus mimic. Poly l:C-Oligofectamine mixture (1 pg/mL Poly l:C, i 2% Oligofectamine, 25 pL; lnvivogen Ltd., San Diego, CA, and lnvitrogen, Carlsbad, CA, tively) was transfected into BEASZB cells (human bronchial 40 lial cells, ATCC). Cells were pre-incubated with final trations of test compounds for 2 hr and the level of lCAlVl—1 expression on the cell e was determined by cell-based ELISA. At a time point 18 hr after poly l:C transfection, cells were fixed with 4% formaldehyde in PBS (100 pL) and then endogenous peroxidase was quenched by the addition of washing buffer (100 uL, 0.05% Tween in PBS: PBS—Tween) containing 0.1% PCT/G32012/052445 sodium azide and 1% hydrogen peroxide. Cells were washed with wash-buffer (3 x 200 uL). and after blocking the wells with 5% milk in PBS-Tween (100 uL) for 1 hr, the cells were ted with uman ICAM—1 antibody (50 uL; Cell Signaling Technology, Danvers, MA) in 1% BSA PBS overnight at 4°C.

The cells were washed with PBS-Tween (3 X 200 uL) and incubated with the secondary antibody (100 uL; HRP-conjugated anti—rabbit lgG, Dako Ltd., Glostrup, Denmark). The cells were then ted with of substrate (50 uL) for 2~20min, followed by the addition of stop solution (50 uL, 1N H2804).The ICAM-1 signal was detected by reading and reading the absorbance at 450 nm against a reference wavelength of 655 nm using a spectrophotometer. The cells were then washed with PBS—Tween (3 x 200 uL) and total cell numbers in each well were determined by reading absorbance at 595 nm after Crystal Violet staining (50 LL of a 2% on in PBS) and elution by 1% SDS solution (100 pL) in distilled water. The measured OD 450—655 readings were corrected for cell number by dividing with the OD595 reading in each well. The inhibition of lCAM-1 expression was calculated at each concentration of test compound by comparison with vehicle control. The 50% inhibitory concentration (le0) was determined from the resultant concentration-response curve.

Cell Mitosis Assay Peripheral blood mononucleocytes (PBMCs) from healthy subjects were ted from whole blood (Quintiles, London, UK) using a y gradient paque®-1077, Sigma— Aldrich, Poole, UK). The PBMCs (3 million cells per ) were subsequently treated with 2% PHA (Sigma—Aldrich, Poole, UK) for 48 hr, followed by a 20 hr exposure to varying concentrations of test compounds. At 2 hr before collection, PBMCs were treated with demecolcine (0.1 ug/mL; lnvitrogen, Paisley, UK,) to arrest cells in metaphase. To observe mitotic cells, PBlVle were permeabilised and fixed by adding Intraprep (50 pL; n Coulter, France), and stained with anti-phospho—histone 3 (0.26 ng/L; #9701; Cell Signalling, Danvers, MA) and propidium iodide (1 mg/mL; Sigma-Aldrich, Poole, UK,) as previously 3O described (Muehlbauer PA. and Schuler M.J., Mutation Research, 2003, 537:117-130). scence was observed using an ATTUNE flow cytometer (lnvitrogen, Paisley, UK), gating for lymphocytes. The percentage tion of mitosis was calculated for each treatment relative to vehicle (0.5% DMSO) treatment.

Rhinovirus-induced lL-8 Release and ICAM-1 sion Human rhinovirus RV16 was ed from the American Type Culture Collection (Manassas, VA). Viral stocks were generated by infecting Hela cells with HRV until 80% of the cells were cytopathic.

BEASZB cells were infected with HRV at an MOl of 5 and incubated for 2 hr at 33°C with gentle shaking for to e absorption. The cells were then washed with PBS, fresh media added and the cells were incubated for a further 72 hr. The supernatant was collected for assay of IL-8 concentrations using a Duoset ELISA development kit (R&D systems, Minneapolis, MN).

The level of cell surface lCAM-1 expression was determined by cell-based ELISA. At 72 hr after infection, cells were fixed with 4% formaldehyde in PBS. After quenching nous peroxidase by adding 0.1% sodium azide and 1% en peroxide, wells were washed with wash—buffer (0.05% Tween in PBS: PBS-Tween). After blocking well with 5% milk in PBS-Tween for 1 hr, the cells were incubated with uman ICAM—i antibody in 5% BSA PBS—Tween ) overnight. Wells were washed with PBS—Tween and incubated with the ary antibody (HRP-conjugated anti-rabbit lgG, Dako Ltd.). The lCAM-1 signal was detected by adding substrate and reading at 450 nm with a reference wavelength of 655 nm using a spectrophotometer. The wells were then washed with PBS—Tween and total cell numbers in each well were determined by reading ance at 595 nm after Crystal Violet staining and elution by 1% SDS solution. The measured OD450-555 readings were corrected for cell number by dividing with the OD595 g in each well. Compounds were added 2 hr before HRV infection and 2 hr after infection when non-infected HRV was washed out.

Assessment of HRV16 induced CPE in MRC5 cells MRC-5 cells were infected with HRV16 at an MOI OH in DMEM containing 5% FCS and 1.5 mM MgCl2, followed by incubation for 1 hr at 33°C to promote adsorption. The supernatants were aspirated, and then fresh media added followed by incubation for 4 days. Where appropriate, cells were pre-incubated with compound or DMSO for 2 hr, and the compounds and DMSO added again after washout of the virus.

Supernatants were aspirated and incubated with methylene blue solution (100 uL, 2% formaldehyde, 10% methanol and 0.175 % Methylene Blue) for 2 hr at RT. After washing, 1% SDS in distilled water (100 uL) was added to each well, and the plates were shaken lightly for 1—2 hr prior to reading the absorbance at 660 nm. The percentage tion for each well 3O was calculated. The IC50 value was calculated from the concentration—response curve generated by the serial dilutions of the test compounds.

In vitro RSV virus load in primary bronchial epithelial cells.

Normal human bronchial epithelial cells (NHBEC) grown in 96 well plates were infected with RSV A2 (Strain A2, HPA, Salisbury, UK) at an MOl of 0.001 in the LHC8 MediazRPMI—164O (50:50) containing 15 mM ium chloride and incubated for 1 hr at 37°C for adsorption.

The cells were then washed with PBS (3 x 200 uL), fresh media (200 uL) was added and tion continued for 4 days. Where appropriate, cells were pre-incubated with the 40 compound or DMSO for 2 hr, and then added again after t of the virus.

The cells were fixed with 4% formaldehyde in PBS solution (50 uL) for 20 min, washed with washing buffer (3 X 200 uL; PBS including 0.5% BSA and 0.05% Tween—20) and incubated with blocking solution (5% condensed milk in PBS) for 1 hr. Cells were then washed with washing buffer (3 x 200 uL) and incubated for 1 hr at RT with anti- RSV (2F7) F-fusion protein antibody (40 pL; mouse monoclonal, lot 798760, Cat. No. ab43812, Abcam) in 5% BSA in PBS-tween). After washing, cells were incubated with an HRP-conjugated secondary antibody solution (50 uL) in 5% BSA in PBS-Tween (lot 70, Cat.No. P0447, Dako) and then TMB substrate (50 pL; substrate reagent pack, lot , Cat. No. DY999, R&D Systems, Inc.) was added. This reaction was stopped by the addition of 2N H2804 (50 uL) and the resultant signal was determined colorimetrically (OD: 450 nm with a reference ngth of 655 nm) in a microplate reader (Varioskan® Flash, ThermoFisher Scientific).

Cells were then washed and a 2.5% l violet solution (50 uL; lot 8656, Cat. No. PL7000, Pro-Lab Diagnostics) was applied for 30 min. After washing with washing buffer, 1% SDS in distilled water (100 uL) was added to each well, and plates were shaken lightly on the shaker for 1 hr prior to reading the absorbance at 595 nm. The measured OD450_555 readings were corrected to the cell number by dividing the OD450-655 by the OD595 gs. The percentage inhibition for each well was calculated and the leO value was calculated from the concentration-response curve generated from the serial dilutions of compound.

The Effect of Test Compounds on Cell Viability: MTT Assay Differentiated U937 cells were pre-incubated with each test compound (final concentration 1 ug/mL or 10 uglmL in 200 uL media indicated below) under two protocols: the first for 4 hr in % FCS RPMI164O media and the second in 10% FCS RPM|1640 media for 24 h. The supernatant was replaced with new media (200 pL) and MTT stock solution (10 uL, 5 mg/mL) was added to each well. After incubation for 1 hr the media were removed, DMSO (200 uL) was added to each well and the plates were shaken lightly for 1 hr prior to reading the absorbance at 550 nm. The percentage loss of cell viability was calculated for each well relative to vehicle (0.5% DMSO) treatment. Consequently an apparent increase in cell viability for drug treatment relative to vehicle is tabulated as a negative tage. 3O ne production in sputum hages from COPD. ts with COPD were d with a nebulised solution of 3% (w/v) onic saline using an onic nebuliser (Devilbiss, Carthage, MO) with tidal breathing for 5 min. This procedure was repeated 3 maximum of three times until enough sputum was obtained. The sputum s were homogenized and mixed vigorously using a vortex mixer in 0.02% v/v dithiothreitol (DTT) solution. The samples were re—suspended in PBS (40 mL) followed by centrifugation at 1500 rpm at 4°C for 10 min to obtain sputum cell pellets. The pellets were washed twice with PBS (40mL). The sputum cells were then re—suspended in macrophage serum-free medium phage-SFM, Life technologies, Paisley, UK; to achieve 2x106/well 40 in a 24 well plate) containing 20 U/mL penicillin, 0.02 mg/mL streptomycin and 5 ug/mL amphotericin B and seeded on high bound 96-well plate, followed by incubation for 2 hr at 37°C and at 5% 002 to allow the macrophages to attach to the bottom of the plate. The cells on the plate were washed with fresh macrophage—SFM (200 l) to remove neutrophils and other contaminated cells. The adherent cells (mainly sputum macrophages) on the plate PCT/G32012/052445 were used for further analysis. Sputum induction and isolation were conducted in Quintiles Drug Research Unit at Guys Hospital and ethics approval and written informed consent was obtained by Quintiles.

Where appropriate, 1 uL of a solution containing either the test compound or reference article at the stated concentrations or alternatively 1 uL of DMSO as the vehicle control was added to each well (200 uL in media) and the cells were incubated for 2 hr. The cells were stimulated with LPS solution (50 uL, final concentration: 1 uglmL) and incubated for 4hr at 37°C and 5% 002. The supernatant was then collected and kept at —80°C. Millipore’s luminex 1O kits were used to measure the four analytes. After thawing the supernatant, the magnetic antibody beads were multiplexed and incubated in a 96—well plate with standard, background solution or the appropriate volume of sample overnight with shaking at 4°C. After washing twice with 200 pL of wash buffer provided by the kit per well using a magnetic plate washer, the beads were incubated for 1 hr at RT with 25 uL of the biotin conjugated antibody solution provided by the kit with shaking. Streptavidin solution was added for 30 min with shaking at RT. After g with 200uL wash buffer per well, the beads were ended in sheath fluid (150 uL) and analyzed immediately. The level of each analyte in the supernatant was calculated using Xcel Fit software with a 4 or meter equation using each standard curve. The inhibitions of each cytokine tion were calculated at each tration by comparison with vehicle control. The ICSO values were determined from tration- inhibition curves using XL—Fit (idbs, Guildford, UK) Cytokine production in primary bronchial lial cells from COPD.

Primary airway epithelial cells obtained from patients with COPD were purchased from Asterand on, UK), and ined in bronchial epithelial cell growth media that was prepared by mixing together LHCS (lnvitrogen) (500 mL), with LHC9 (lnvitrogen) (500 mL) and 3 uL of retinoic acid solution (5 mg/mL in neat DMSO . The media was removed by aspiration and fresh BEGM (200 uL) was added to each well. Where appropriate, 1 pL of a solution of the test compound at the state concentrations or 1 uL of DMSO as the vehicle control was added and the cells were incubated for 2 hr. The cells were ated with TNFoc (50 pL; final concentration 50 ng/mL) and then incubated for 4hr at 37°C and 5% 002.

The supernatant was then collected and kept at —20°C.

The levels of lL-6 and lL-8 were determined by ELlSA using R&D Systems’ Human lL-6 IL-8 Duoset® Elisa Kits. The inhibition of |L-6 and lL-8 production was calculated at each concentration by ison with vehicle control. The 50% inhibitory concentrations (I050) were determined from the resultant tration-response curves using XL-Fit (idbs, Guildford, UK).

WO 2013050757 2012/052445 In Vivo Screening: Pharmacodynamics and Anti-inflammatory Activity LPS-induced neutrophil accumulation in mice sted Balblc mice were dosed by the intra tracheal route with either vehicle, or the test substance at the indicated times (within the range 2—8 hr) before stimulation of the inflammatory response by application of an LPS challenge. At T = 0, mice were placed into an exposure chamber and exposed to LPS (7.0 mL, 0.5 mg/mL solution in PBS) for 30 min).

After a further 8 hr the animals were etized, their tracheas cannuiated and BALF 1O extracted by infusing and then withdrawing from their lungs 1.0 mL of PBS via the tracheal catheter. Total and differential white cell counts in the BALF samples were measured using a Neubaur haemocytometer. Cytospin smears of the BALF samples were prepared by fugation at 200 rpm for 5 min at RT and stained using a Difouik stain system (Dade Behring). Cells were counted using oil immersion microscopy. Data for neutrophil numbers in BAL are shown as mean i S.E.M. (standard error of the mean). The percentage inhibition of neutrophil accumulation was calculated for each treatment relative to vehicle treatment. tte Smoke Model A/J mice (males, 5 weeks old) were exposed to cigarette smoke (4% cigarette smoke, diluted with air) for 30 min/day for 11 days using a Tobacco Smoke inhalation ment System for small s (Model SIS-CS; Sibata Scientific Technology, Tokyo, Japan). Test substances were administered intra-nasally (35 uL of solution in 50% DMSO/PBS) once daily for 3 days after the final cigarette smoke exposure. At 12 hr after the last dosing, each of the animals was anesthetized, the trachea cannulated and bronchoaiveolar lavage fluid (BALF) was collected. The s of alveolar macrophages and phils were determined by FACS analysis (EPICS® ALTRA ll, Beckman Coulter, |nc., Fullerton, CA, USA) using anti- mouse MOMAZ antibody (macrophage) or anti-mouse 7/4 antibody ophil). BALF was centrifuged and the supernatant was collected. The level of keratinocyte chemoattractant (KC; CXCL1) in BALF was quantitated using a Quentikine® mouse KC ELISA kit (R&D systems, Inc, Minneapolis, MN, USA).

Example 1- Preparation of Compound (I) The following intermediates used to prepare Compound (I) of the invention have been previously described and were prepared using the procedures contained in the references cited below (Table 3).

W0 50757 Table 3: Previously bed intermediates.

Intermediate Structure Name, LCMS Data and Reference m 3-tert-butylp-toiyi-1H-pyrazoiamine.

\ NH N 2 R‘2.46 min (Method 1 ; m/z 230 (M+H)*, (ES+ ).

Cirillo, P. F. eta/., W0 2000I43384, 27 Jul 2000. ow 4-((2—chloropyrimldinyl)oxy)naphthalen-1—amine. ' B Rt 1.80 min (Method 2); m/z 272/274 (M+H)- + + HZN 00 NYN , (ES ).

Cirillo, P. F. at 6]., W0 2002/92576, 21 Nov 2000. intermediate C: 4-((4-Aminonaphthalenyl)oxy)-N-phenylpyrimidinamine.

Aniline 0 intermediate B —-————-> D | Intermediate C pTSA HzN HN\© To a nitrogen purged on of e of intermediate B (50.0 g, 184 mmol) and aniline (42.0 mL, 460 mmol) in THF (200 mL) was added pTSA (17.5 g, 92.0 mmol) in a single portion. The reaction mixture was heated to 70 °C for 1.5 hr during which time which a precipitate formed. The mixture was cooled to RT and diluted with THF (200 mL). The precipitate was collected by filtration, washed with THF (2 x 100mL) and then suspended in a heterogeneous mixture of DCM (600 mL) and aq. NaOH (2M, 200 mL) and stirred vigorously for 1 hr, during which time the suspended solids dissolved. The layers were separated and the aq layer was extracted with DCM (200 mL). The DCM extracts were combined, dried and evaporated in vacuo. The residue was triturated with ether (150 mL) and the resulting solid was washed with ether (2 x 50 mL) to afford intermediate C as an off white solid (26 g, 43%); R‘1.95 min (Method 2); m/z 329 (M+H)+ (ES+).

WO 50757 2012/052445 Compound (I): 1-(3-(tert-Butyl)(p-tolyl)-1H-pyrazoly|)(4-((2-(pheny|amino) pyrimidinyl)oxy)naphthalenyl)urea. o \ 1. PhOC(O)Cl ediateA ———> N/ \ Y} )L O \N N N NYN 2.lntermediateC H H Me Compound 1 A heterogeneous mixture of a solution of Na2003 (3.84 g, 36 mmol) in water (42 mL) and intermediate A (10.5 g, 45.7 mmol) in isopropyl acetate (130 mL, 1.082 mol) was stirred vigorously at RT for 5 min and was then treated with phenyl carbonochloridate (5.77 mL, 45.7 mmol). Stirring of the mixture was continued for a further 4 hr after which the layers were separated. The organic phase was added to a solution of Intermediate C (10.0 g, 30.5 1O mmol) and ylamine (423 uL, 3.05 mmol) in isopropyl acetate (60 mL, 511 mmol). The reaction mixture was warmed to 48 °C for 1 hr, then diluted with isopropyl acetate (190 mL) and cooled to RT for a further 18 hr, during which time a precipitate formed. The precipitate was isolated by filtration, washed with isopropyl e and then dried in vacuo at 40 °C to afford the title compound, Compound (1) as a white solid (anhydrous free base, polymorphic form A) (16.5 g, 92 %); R‘2.74 min (Method 2); m/z 584 (M+H)+ (ESf); 1H NMR (400 MHz, s) 6: 1.30 (9H, s), 2.41 (3H, s), 6.43 (1H, s), 6.58 (1H, d), 6.78 (1H, t), 6.97 (2H, t), 7.28 (2H, br m), 7.39 (2H, d), 7.40 (1H, d), 7.49 (2H, d), 7.56 (1H, m), 7.63 (1H, m), 7.82 (1H, dd), 7.95 (1H, d), 8.10 (1H, d), 8.40 (1H, d), 8.77 (1H, s), 9.16 (1H, br s), 9.50 (1H, br 3).

Example 2 - Summary of in Vitro and In Vivo Screening Results The in Vitro profile of Compound (l) sed herein, as determined using the protocols described above, are presented below (Tables 4a-f) in comparison with a structurally related Reference Compound which is N—(4-(4-(3—(3-terf—butyl—1~p—tolyl-1H-pyrazol—5-yl)ureido) naphthalen—1~yloxy)pyridinyl)methoxyacetamide, which has been previously described as a potent anti-inflammatory agent with anti—viral activity (lto, K. et a/., , 2010/050575, 7 Oct 2010 and Ito, K. et 31., , PCT/GBZOOQ/051702, 17 Jun 2010.

The compound of the present ion demonstrates a very similar inhibitory profile to the Reference Compound in the range of kinase enzyme assays with the marked exception of the inhibitory activity of Compound (I) against the enzyme GSK3G, which is very much weaker than the Reference Compound (Table 4a).

Table 4a: p38 MAPK, c—Src, Syk and GSK3 or Enzyme Profile of Compound (I) IC5o Values for Enzyme Inhibition (nM) Test C°mp°und p38 MAPKa p38 MAPKv c-Src Syk GSKSa Compound (I) 60 3739 22 334 >17000 Reference 12 344 5 42 45 Compound The kinase binding profile of Compound (I) of the t ion was also compared with the Reference Compound against p38 MAPK, HCK, cSrc, Syk, and GSKBG/B. Compound (I) displayed a very different phenotype, demonstrating nd inhibition of binding versus p38MAPK, HCK, cSrc and Syk kinases, t significant effect against GSK3d (Table 4b).

Table 4b: Comparison of the Enzyme Binding Profile of Compound (I) with the Reference Compound.

Kd value for kinase binding (nM) Test Compound p33 p38 HCK cSrc GSK3d MAPKa Syk GSK3B MAPKy Compound (I) 20 43 8 10 14 20000 1200 W—-——-—-—————— 1 5 5 4 9 180 24 compound The compound of the present ion demonstrates a similar profile to the nce Compound in cellular assays that reveal anti—inflammatory ties against endotoxin mediated release of both TN For and lL-8 (Table 4c). The profiles of the compounds are also similar in ar systems measuring their effects on respiratory virus ation (HRV induced ICAM1 and CPE expression and RSV stimulated expression of F—protein) as well as virus—induced inflammation (HRV evoked release of lL~8; Table 4d).

Table 4c: Inhibition of LPS Induced TNFa and lL—8 Release and Polle Induced ICAM- Expression for Compound (I) LPS Induced Release (nM) polylc I ICAM1 (nM) Test “L “:8 ———— Compound |C5o (THP1) RECso (du937) leo (du937) lcso (BEASZB) Compound (I) 3.4 2.3 2.2 10.2 Reference 13 0'13 1'3 2‘1 Compound Table 4d: The Effect of nd (I) on HRV-16 Propagation (CPE) and Inflammation (Expression of ICAM-1 and lL-8 Release) and on RSV Propagation (F-Protein Expression). lC5o Values (nM) for HRV |C50 Values (nM) for RSV Test Stimulated Release/Expression Stimulated Expression Substance lL-8 ICAM1 CPE F-Protein (BEASZB) (BEASZB) (MRCS) (HBEC) Compound (I) 0.036 0.023 17.1 15.4 Reference 0.065 0.37 4.7 22.0 Compound The compound of the present invention demonstrated higher efficacy in pro-inflammatory ne production in sputum hage and bronchial epithelial cells obtained from COPD patients, which were largely insensitive to asone propionate, a corticosteroid.

(Table 4e).

Table 4e: The Effect of nd (I) and asone propionate on pro-inflammatory cytokine release in sputum macrophages and bronchial epithelial cell from COPD patients.

IC50 values (nM) and /or E max (% in parentheses)1 for Test Substance Indicated Cells Type Cytokine Compound (I) Fluticasone Propionate lL-6 43 (79) (26) lL-8 68 (64) Sputum (19) Macmphage TNFd 17 (86) (18) MIP1a 7.5 (89) (20) Epithelial Cell IL-8 0.85 (100) (17) 1. E-max values (maximum inhibiton) were ated as the % tion obtained at 0.1 ug/mL However, advantageously, Compound (I) shows markedly less activity in assay systems that measure its impact on cell viability and cell division (mitosis) indicating that the compound is likely to possess a superior therapeutic index over the Reference Compound (Table 4f).

Table 4f: Effect of Compound (I) on Cellular Viability and Cell Division MTT Assay1 s Assay Test Cell viability at time point % Inhibition at 5 uglmL Substance indicated in d-U937 CeIls in PBMC Cells 4h 24h Compound (I) -ve ~ve 31.3 §::;:::: -ve +ve 87.8 1. Cell viability : —ve and +ve indicate the value is below and above respectively, the no significant effect threshold defined as 30% inhibition at 1 ug/mL at the time point indicated.

Treatment of mice with Compound (I) was found to produce a dose dependent inhibition on LPS—induced neutrophil lation and a time course ment revealed that the drug substance had a long duration of action (Table 5).

Table 5: The Effects of Treatment with Compound (I) on LPS-Induced Airway Neutrophilia in Mice.

Neutrophil numbers in BALF (x105imL) at pre-dose time indicated (% inhibition)‘ Compound (I) (mg/mL) 2 hr 8hr 12hr Vehicle 18.9 i 2.5 - - 0.05 15.6 i 2.1 (18) - - 0.2 9.8 i 1.6 (48) - 1.0 4.4 i 0.89 (77) 9.9 i 1.8 (48) 18.3 i 2.3 (4) 1. N = 8 per group The result of treatment with Compound (I) on macrophage and phil lation in BALF in the mouse cigarette smoke model was investigated (Table 6a). The cigarette smoke model used for this study is reported to be a corticosteroid refractory system, (Medicherla S. et at, J. col. Exp. Ther., 2008, 324(3):921-9.) and it was confirmed that quticasone propionate did not t either neutrophil or macrophage accumulation into airways at 1.75 ug/mouse (35 1.1L, bid, i.n.), the same dose that produced >80% inhibition of LPS—induced neutrophil accumulation.

Treatment of mice with Compound (I) was found to produce a dose—dependent inhibition on both macrophage and neutrophil accumulation in BALF induced by cigarette smoke.

PCT/G32012/052445 Table 6a: The Effects of Treatment with Compound (I) on Tobacco Smoke in Mice.

Cell numbers in BALF x 104/mL (% inhibition) Treatment compound (I) (uglmL) Macrophage phil Vehicle + Air 4.3 i 0.45 2.6 i 0.21 Vehicle + Tobacco Smoke 14.4 i 0.33 13.7 i 0.31 0.32 13.3 i 0.20 (11) 12.4 i 0.32 (12) 1.6 11.6 i: 0.42 (28) 10.5 i 0.06 (29) 8.0 10.1 i 0.42 (43) 9.1 i 0.28 (41) 40 7.9 i 0.20 (64) 7.9 i 0.34 (52) The data for cell numbers are shown as the mean 1* SEM, N=5 Treatment of mice with Compound (I) also inhibited cigarette smoke d CXCL1 (KC) production in BALF in a dose-dependent manner (Table 6b).

Table 6b: The Effects of Treatment with nd (I) on CXCL1 (KC) release in BALF on Tobacco Smoke in Mice.

Treatment CXCL1 in BALF Compound (I) (uglmL) pg/mL (% inhibition) Vehicle + Air 8.2 i 0.30 Vehicle + Tobacco Smoke 13.6 i 1.69 0.32 13.6 i 1.69 (0) 1.6 12.2 i 0.96 (26) 8.0 11.4:0.15(41) 40 9.5 i 0.84 (76) The data for CXCL level are shown as the mean 1- SEM, N=5 In summary, these results suggest that the Compound (I) has r anti-inflammatory properties to the Reference Compound disclosed above and, advantageously, is associated with a superior therapeutic index. e 3: Preparation of nd (I) as the anhydrous free base in solid, crystalline, polymorphic form B Compound (I) (398 g, in polymorphic form A) was taken up in acetone (3.98 L) and the solution heated to 50°C. NORIT A SUPRA (19.9 9, an activated carbon) and diatomaceous PCT/G32012/052445 earth, flux~calcined (3.98 g; a filter agent) were then added and the mixture was heated to reflux (56°C) for 15 min. The mixture was filtered and the resulting solid was washed with acetone (100 mL). The combined filtrate and washing acetone was warmed to reflux (56°C), and 900 mL of solvent was removed via distillation under atmospheric pressure at 56°C. The mixture was cooled to 50°C and water (398 mL) was then added over a period of 1 hr whilst the temperature was ined at 50°C. After an additional 30 min at 50°C the heterogeneous mixture was cooled to 20°C over 6h and then stirred at 20°C for 10 hr. The resulting product was filtered and the cake was washed with acetone (318 mL). The product was dried in vacuo at 45°C for 20 hr to produce nd (I) as the anhydrous free base in 1O solid, crystalline, polymorphic form B (240.9 g; 60.5% yield).

The above method may optionally be adapted to facilitate crystallization with seeding.

Example 3a: Preparation of Compound (I) as the anhydrous free base in solid, crystalline, polymorphic form B containing reduced residual solvent Optionally, Compound (I) as the anhydrous free base in solid, crystalline, polymorphic form B, as prepared ing to the procedure described above (Example 3) or a similar method, may be re-slurried from water in order to reduce al solvent as follows: Compound (I) as the anhydrous free base in solid, lline, polymorphic form B (230 g, as ed according to Example 3) was suspended in deionized water (2.30 L) and was stirred at 20°C for 4hr. The mixture was filtered and the product was washed with deionized water (2 x 115 mL) and was then dried at 45°C in vacuo to e Compound (I) as the anhydrous free base in solid, crystalline, polymorphic form B ning reduced residual solvent (22? g, 98.7%).

Example 4: Micronization of Compound (I) as the anhydrous free base in solid, crystalline, polymorphic form B Micronized crystalline polymorphic form B of Compound (I) as the anhydrous free base was prepared using a jet mill micronization device (1.5 bar using a manual feeder with an injector pressure of 1.5 bar) (manufactured by Hosokawa Alpine). The particle size distribution was measured using laser ction rn Mastersizer 20008 ment). Particle size distributions may be represented using D10, D50 and D90 values. The D50 median value of particle size butions is defined as the particle size in s that divides the distribution in half. The measurement derived from laser diffraction is more accurately described as a volume distribution, and consequently the D50 value obtained using this procedure is more meaningfully referred to as a Dv5o value (median for a volume distribution). As used herein 40 Dv values refer to particle size distributions measured using laser diffraction. rly, D10 and D90 values, used in the context of laser diffraction, are taken to mean Dv1o and DVgo values and refer to the particle size whereby 10% of the distribution lies below the D10 value, and 90% of the distribution lies below the D90 value, respectively. Micronized crystalline W0 20137050757 polymorphic form B of nd (I) as the anhydrous free base had the ing le size distribution: D10 of 0.850 um; D50 of1.941 pm and D90 of 4.563 pm.

Example 5: XRPD analysis of Compound (l) as the anhydrous free base in solid, crystalline, polymorphic forms A and B XRPD analysis of Compound (I) as the anhydrous free base in solid crystalline polymorphic forms A and B (polymorphic form B was micronized following the procedure of Example 4) was undertaken using the method described in General Procedures. The resulting diffraction 1O patterns are shown in Figures 1 and 2 respectively. Both XRPD patterns showed diffraction peaks without the presence of a halo, thereby indicating that both materials are crystalline.

Peaks and their intensities are listed below (Table 7a and Table 7b).

Table 7a: Characteristic XRPD peaks and their intensities for Compound (I) as the anhydrous free base in solid, crystalline, form A XRPD Peaks 2-Theta 1 Intensities 7.8 19.7 8.7 20.8 .3 22.6 11.2 23.1 12.4 24.6 .2 25.5 16.2 26.7 17.5 27.4 1. Values are :r 0.2 degrees Table 7b: Characteristic XRPD peaks for Compound (l) as the ous free base in solid lline form B, post micronization.

XRPD peaks 2-Theta Values1 Intensities 3.9 16.7 6.1 18.3 7.7 18.7 8.6 19.9 .9 20.9 11.8 22.0 12.7 22.6 14.3 25.2 .9 28.9 1. Values are i 0.2 degrees Example 6: Melting point determination of Compound (l) as the anhydrous free base in solid, crystalline, polymorphic forms A and B The melting points of Compound (I) as the anhydrous free base in solid crystalline polymorphic forms A and B (the latter post micronization) were obtained using ential scanning calorimetry (DSC), as bed in the General Procedures. Polymorphic form A melted at 191.6°C and rphic form B melted at 214.0°C. From the DSC data it was calculated that form B had a higher heat of fusion than form A. Since form B also has a higher melting point than form A, this tes that polymorphic forms A and B are monotropic related, meaning that higher melting polymorphic form B will be more stable than lower melting polymorphic form A, at all temperatures. As such, it can be expected that polymorphic form B is thermodynamically more stable than polymorphic form A.

Example 7: Thermal analysis of Compound (l) as the anhydrous free base in solid, crystalline, polymorphic form B, post micronization.

Thermal analysis of Compound (I) as the anhydrous free base in lline polymorphic form B (micronized) was undertaken using TGA, DVS, XRPD analysis, lR spectroscopy and DSC as described in General ures. Where appropriate, a sample at ambient ature and relative humidity (reference sample/ ”0 days”) was compared with samples stored at s temperatures and relative humidities (comparative samples).

Thermogravimetric Analysis: The reference sample (t = 0) and the comparative samples that were exposed prior to analysis to different storage conditions, were heated at a rate of °C/min from RT to 300°C. The TGA curve of the reference sample (t = 0) is illustrated in Figure 3 and the results for all samples are ised below (Table 8). As can be seen from Figure 3, a weight loss of 0.6% was observed in the temperature range from RT to 180°C, which was due to solvent evaporation. The weight loss that occurred above 180°C was due to evaporation and decomposition of the product. ing this weight loss profile with those of the comparative samples in Table 8, no icant differences were observed.

Dynamic vapour sorption: The DVS isotherm plot for the micronized reference sample is illustrated in Figure 4 and the DVS change in mass plot for the micronized reference sample is illustrated in Figure 5. During the l drying step, no weight loss was registered and the product showed no hygroscopic behavior. The product adsorbed up to 0.4% moisture depending on the atmospheric humidity. The product was found to dry out tely and ed in the same lline solid state (form B) during the test, as evidenced by the R spectrum and XRPD pattern being substantially the same before and after the DVS analysis.

XRPD Analysis and JR Spectroscopy: The XPRD diffraction pattern of the reference sample (t = 0) is illustrated in Figure 2 and the IR trace is illustrated in Figure 6. The ction pattern and IR trace were compared with those of the comparative samples (exposed to different storage conditions) and the results are summarised in Table 8. The diffraction patterns and IR traces were identical for all samples. ential ng Calorimetry. The reference sample (t = 0) and comparative samples, previously exposed to different storage ions, were heated at a rate of 10°C/min from °C to 300°C. The DSC curve of the reference sample is illustrated in Figure 7 and the results for all samples are summarised below (Table 8). From Figure 7, it is evident that the reference sample melted with decomposition at 214.0°C.

Table 8: Thermal analysis of Compound (I) as the anhydrous free base in solid crystalline polymorphic form B post micronization.

Stota'ge W 2 IR DSC3 Appearance Conditions <100°C <180°C _ Cryst. Cryst.

T - zero 0.6 214 off-white Ref Ref 1 week i 80°C NT NT ~Ref ~Ref NT off—white NT NT Ref Ref NT off—white. _81'L__ 4 weeks l RT/ 0.8 0.5 ~Ref Ref 214 off—white' <5 % RH 4 weeks / RT/ 0-5 0.4 Ref ~Ref 214 off—whlte‘ 56% RH 4 weeks / RT / 0.9 0.5 Ref Ref 21 4 off—white_ 4 weeks / 50°C 1.3 0.5 ~Ref ~Ref 214 off-white 0.8 0.3 ~Ref ~Ref 214 off—white‘ 75% RH 1. Cryst.: crystalline; 2..~Ref: pattern identical with reference sample, 3. max (°C); NT: Not tested in this assay In summary, it is evident that Compound (I) as the anhydrous free base in solid, crystalline, polymorphic form B has good physical stability.

PCT/G32012/052445 Example 8: HPLC analysis of Compound (l) as the anhydrous free base in solid, crystalline, polymorphic form B post micronization.

The chemical stability of Compound (l) as the anhydrous free base in solid, crystalline, polymorphic form B, following ization, was determined by comparing a sample maintained at t temperature and relative humidity (reference sample) with samples stored at various temperatures and relative humidities as set out hereinabove (the comparative s, Table 8). The reference and comparative samples were then analyzed by HPLC using the method described in General ures and by visual inspection. The 1O results from this study (data summarised in Table 9) reveal that Compound (I), prepared as the anhydrous free base in solid, crystalline, polymorphic form B is chemically stable gh some sensitivity to light was observed.

Table 9: Chemical stability of Compound (l) as the anhydrous free base in solid, crystalline, polymorphic form B, post ization.

Sum of impurities by HPLC (%)1 ance1 Storage Conditions 1 week 4 weeks 1 week 4 weeks T = zero 0.66 NT off-white NT 0.3 day, lCH light2 1.44 NT ite NT 80°C 0.73 NT Off-white NT 70°C I 75% RH 0.73 NT Off—White NT 40°C / 75% RH 0.66 0.68 Off—White Off—White 50°C 0.69 0.66 Off-White Off-White RT/ <5% RH NT 0.67 NT Off-White RT / 56% RH NT 0.67 NT Off-White RT / 75% RH NT 0.67 NT Off-White 1. NT in this assay ; 2. Stimulated daylight: light t 700 W/mz.

Example 9 — Preparation of pharmaceutical formulations An exemplary pharmaceutical formulation of the invention would consist of 0.4 wt.% of Compound (I) (as the anhydrous free base in solid crystalline polymorphic form B), 98.6 wt.% lactose drate (inhalation grade) and 1.0 wt.% magnesium stearate, wherein the wt.% of all components is based on the weight of the dry pharmaceutical formulation.

Throughout the specification and the claims which follow, unless the context requires otherwise, the word ‘comprise’, and variations such as ‘comprises’ and ‘comprising’, will be understood to imply the ion of a stated integer, step, group of integers or group of steps but not to the exclusion of any other integer, step, group of integers or group of steps.

All patents and patent applications referred to herein are incorporated by reference in their entirety.

Claims (4)

What is claimed is:

1. A compound of formula (I) mtowO \ N\ N /N i ~ N H H O Y HN\© Me (I) or a pharmaceutically acceptable salt thereof, including all stereoisomers and tautomers thereof.

2. A compound according to claim 1, as the free base.

3. A compound according to claim 2, as the ous free base in solid crystalline form.

4. A compound according to claim 3, wherein the compound of formula (I) as the anhydrous free base is in solid crystalline form having the X-ray powder ction pattern substantially as shown in