EA040670B1 - CONDENSED IMIDAZOPIPERIDINE COMPOUND THAT IS A JAK INHIBITOR - Google Patents

Description

Background of the invention Field of technology to which the invention relates

The invention is directed to a JAK kinase inhibitor compound useful in the treatment of various diseases, in particular eye, skin and respiratory diseases. The invention is also directed to crystalline forms of the compound, pharmaceutical compositions containing such a compound, methods of using such a compound to treat diseases treatable with a JAK inhibitor, and methods and intermediates useful for preparing such a compound.

Prior Art

Cytokines are intercellular signaling molecules that include chemokines, interferons, interleukins, lymphokines, and tumor necrosis factor. Cytokines are critical for normal cell growth and immunoregulation, but also stimulate immune-mediated diseases and promote the growth of malignant cells. Elevated levels of many cytokines have been implicated in the pathology of a wide variety of diseases or conditions, especially those characterized by inflammation. Many of the disease-associated cytokines act through Janus dependent tyrosine kinase (JAK) family signaling pathways, which signal through the transcription factor family of signal transducers and transcription activators (STAT).

The JAK family includes four members: JAK1, JAK2, JAK3, and tyrosine kinase 2 (TYK2). Binding of the cytokine to the JAK-dependent cytokine receptor induces receptor dimerization, which leads to phosphorylation of tyrosine residues on the JAK kinase, causing JAK activation. Phosphorylated JAKs in turn bind and phosphorylate various STAT proteins, which dimerize, internalize in the cell nucleus, and directly modulate gene transcription, leading to downstream effects associated with inflammatory disease, among other effects. JAKs are usually associated in pairs with cytokine receptors as homodimers or heterodimers. Specific cytokines are associated with specific JAK matings. Each of the four members of the JAK family is involved in the signaling of at least one of the cytokines associated with inflammation.

Inflammation plays a significant role in many eye diseases, including uveitis, diabetic retinopathy, diabetic macular edema, dry eye syndrome, age-related macular degeneration, retinal vein occlusion, and atopic keratoconjunctivitis. Uveitis encompasses a variety of intraocular inflammatory conditions and is often autoimmune, occurring without a known infectious agent. It is estimated that about 2 million patients in the United States suffer from this condition. In some patients, chronic inflammation associated with uveitis leads to tissue destruction and is the fifth leading cause of blindness in the US. Cytokines elevated in the eyes of uveitis patients that signal through the JAK-STAT pathway include IL-2, IL-4, IL-5, IL-6, IL-10, IL-23, and IFN-γ (Horai and Caspi, J. Interferon Cytokine Res, 2011, 31, 733-744; Ooi et al., Clinical Medicine and Research, 2006, 4, 294-309). Existing therapies for uveitis are often not effective enough, and the disease is poorly controlled in many patients. Steroids, although often effective, have been associated with cataracts and increased intraocular pressure/glaucoma.

Diabetic retinopathy (DR) is caused by damage to the blood vessels in the retina. It is the most common cause of vision loss among people with diabetes. Angiogenic as well as inflammatory pathways play an important role in the disease. Often, DR progresses to diabetic macular edema (DME), the most common cause of vision loss in patients with diabetes. It is estimated that about 1.5 million patients suffer from this disease in the United States alone, of which about 20% have both eyes affected by the disease. It is believed that cytokines that signal through the JAK-STAT pathway, such as IL-6, as well as other cytokines, such as IP-10 and MCP-1 (alternatively referred to as CCL2), whose production is partly driven by signaling through the JAK-STAT pathway STATs play a role in DR/DME-associated inflammation (Abcouwer, J. Clin. Cell. Immunol, 2013, Suppl 1, 1-12; Sohn et al., American Journal of Opthalmology, 2011, 152, 686-694; Owen and Hartnett, Curr Diab Rep, 2013, 13, 476-480; Cheung et al., Molecular Vision, 2012, 18, 830-837; Dong et al., Molecular Vision, 2013, 19, 17341746; Funatsu et al. , Ophthalmology, 2009, 116, 73-79). Existing treatments for DME are not effective enough: intravitreal anti-VEGF treatments are only effective in a subset of patients, and steroids are associated with cataracts and increased intraocular pressure.

Dry eye syndrome (DED) is a multifactorial disease affecting approximately 5 million patients in the United States. Inflammation of the ocular surface is believed to play an important role in the development and spread of this disease. Elevated levels of cytokines such as IL-1, IL-2, IL 4, IL-5, IL-6 and IFN-γ have been noted in the eye fluids of DED patients. (Stevenson et al., Arch Ophthalmol, 2012, 130, 90-100) and levels often correlate with disease severity. Age-related macular degeneration and atopic keratoconjunctivitis are believed to be associated with JAK-dependent cytokines.

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Retinal vein occlusion (RVO) is a widespread ocular disabling disease. Obstruction of retinal blood flow can lead to damage to the retinal vasculature, hemorrhage, and tissue ischemia. Although the causes of RVO are multifactorial, both vascular and inflammatory mediators have been shown to be important (Deobhakta et al., International Journal of Inflammation, 2013, article ID 438412). Elevated levels of cytokines that signal through the JAK-STAT pathway, such as IL-6 and IL-13, as well as other cytokines, such as MCP-1, whose production is partly driven by JAK-STAT signaling, have been found in patients' eye tissues. with PBO (Shchuko et al., Indian Journal of Ophthalmology, 2015, 63(12), 905-911). Although many patients with RVO are treated with photocoagulation, this is an inherently destructive therapy. Anti-VEGF agents are also used, but they are effective only in a subset of patients. Steroid drugs that reduce inflammation in the eye (triamcinolone acetonide and dexamethasone implants) have also been shown to provide beneficial results for patients with some forms of RVO, but have also been shown to cause cataracts and increased intraocular pressure/glaucoma.

Atopic dermatitis (AD) is a common chronic inflammatory skin disease that affects approximately 14 million people in the United States alone. In developed countries, AD is estimated to affect 10-20% of children and 1-3% of adults (Bao et al., JAK-STAT, 2013, 2, e24137), and the prevalence of the disease is increasing. Increases in pro-inflammatory cytokines that depend on the JAK-STAT pathway, specifically IL-4, IL-5, IL-10, IL-13, and IFNy, are associated with AD (Bao et al., Leung et al., The Journal of Clinical Investigation, 2004, 113, 651-657). In addition, activation of IL-31, another cytokine that signals through JAK mating, has been shown to play a role in pruritus associated with the chronic AD condition (Sunkoly et al., Journal of Allergy and Clinical Immunology, 2006, 117, 411 -417).

Asthma is a chronic respiratory disease for which there is no prevention or cure. The disease is characterized by inflammation, fibrosis, hypersensitivity, and airway remodeling, all contributing to airflow limitation. An estimated 300 million people worldwide have asthma and it is estimated that by 2025 the number of people with asthma will increase by more than 100 million. Although most patients can control their asthma symptoms with inhaled corticosteroids, which can be combined with a leukotriene modifier and/or a long-acting β-agonist, there remains a subset of patients with severe asthma who are not controlled by conventional therapies. Cytokines involved in inflammation in asthma that signal through the JAKSTAT pathway include IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-11, IL-13, IL- 23, IL-31, IL-27, thymic stromal lymphopoietin (TSLP), interferon-γ (IFNy) and granulocyte-macrophage colony stimulating factor (GM-CSF). Inflammation of the airways is characteristic of other respiratory diseases besides asthma. Chronic obstructive pulmonary disease (COPD), cystic fibrosis (CF), pneumonitis, interstitial lung diseases (including idiopathic pulmonary fibrosis), acute lung injury, acute respiratory distress syndrome, bronchitis, emphysema, bronchiolitis obliterans, and sarcoidosis are also respiratory diseases, the pathophysiology of which is believed to be associated with JAK-signaling cytokines.

Given the number of cytokines that are elevated in inflammatory diseases and that each cytokine is associated with a particular JAK pairing, a chemical inhibitor with broad activity against all members of the JAK family could be of wide use in the treatment of ocular, skin and respiratory diseases. There remains a need for a strong inhibitor of various JAKs.

The essence of the invention

In one aspect, the invention provides a JAK inhibitor compound useful in the treatment of an inflammatory disease.

In particular, in one aspect, the invention provides 1-(2-(6-(2-ethyl-5-fluoro-4-hydroxyphenyl)4-fluoro-1H-indazol-3-yl)-1,4,6,7- tetrahydro-5H-imidazo[4,5-c]pyridin-5-yl)-2-morpholinoethan-1-one formula

hereinafter referred to as compound 1, or a pharmaceutically acceptable salt thereof.

The invention also provides crystalline forms of the compound, Form 1 and Form 2.

The invention also provides a pharmaceutical composition containing Compound 1 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

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In one aspect, the invention provides a method of treating an ocular disease in a mammal, comprising administering Compound 1 or a pharmaceutical composition of the invention to the mammal. In one aspect, the ocular disease is uveitis, diabetic retinopathy, diabetic macular edema, dry eye syndrome, age-related macular degeneration, retinal vein occlusion, and atopic keratoconjunctivitis. In particular, the eye disease is diabetic macular edema or uveitis.

In yet another method aspect, the invention provides a method for treating an inflammatory skin disease, in particular atopic dermatitis, comprising applying Compound 1 or a pharmaceutical composition of the invention to the skin of a mammal.

In another aspect, the invention provides a method for treating a respiratory disease in a mammal, comprising administering Compound 1, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of the invention, to the mammal.

In separate and different aspects, the invention also provides synthetic methods and intermediates described in this application, which are useful for obtaining compound 1.

The invention also provides compound 1 described herein for use in medical treatment, as well as the use of a compound of the invention for the preparation of a composition or medicament for the treatment of an ocular disease, skin disease, or respiratory disease in a mammal.

Brief description of the drawings

Various aspects of the present invention are illustrated with reference to the accompanying drawings.

Fig. 1 is a powder X-ray diffraction (PXRD) pattern of crystalline Form 2 of Compound 1 (hereinafter Form 2);

fig. 2 is a differential scanning calorimetry (DSC) thermogram of crystalline form 2;

fig. 3 is a thermal gravimetric analysis (TGA) thermogram of crystalline form 2;

fig. 4 is a dynamic moisture sorption (DMS) isotherm of crystalline form 2 observed at a temperature of about 25°C;

fig. 5 is a polarized light microscopy image of form 2;

fig. 6 is a powder X-ray diffraction pattern (PXRD) of the crystalline Form 1 of Compound 1 (hereinafter Form 1);

fig. 7 is a differential scanning calorimetry (DSC) thermogram of crystalline form 1;

fig. 8 is a thermal gravimetric analysis (TGA) thermogram of crystalline form 1;

fig. 9 is a dynamic moisture sorption (DMS) isotherm of crystalline form 1 observed at a temperature of about 25°C;

fig. 10 is a polarized light microscopy image of Form 1.

Detailed description of the invention

The chemical structures are named in this application according to the IUPAC nomenclature used in the ChemDraw program (PerkinElmer, Inc., Cambridge, MA).

In addition, the imidazole portion of the tetrahydroimidazopyridine group in the structure of the compound of the invention exists in tautomeric forms. The compound can also be represented in the equivalent form

In accordance with the IUPAC nomenclature, the presented structures lead to different numbering of atoms of the tetrahydroimidazopyridine moiety. Accordingly, this structure is given as 1-(2-(6-(2-ethyl5-fluoro-4-hydroxyphenyl)-4-fluoro-1H-indazol-3-yl)-3,4,6,7-tetrahydro-5H- imidazo[4,5-c]pyridin-5-yl)2-morpholinoethan-1-one. It may also be listed as 1-(2-(6-(2-ethyl-5-fluoro-4-hydroxyphenyl)-4fluoro-1H-indazol-3-yl)-1,4,6,7-tetrahydro-5H -imidazo[4,5-c]pyridin-5-yl)-2-morpholinoethan-1-one. It should be understood that although the structures are shown or named in a specific form, the invention also includes their tautomer.

The compounds of the invention also contain several basic groups and therefore such compounds may exist as a free base or in various salt forms such as a monoprotonated salt form, a diprotonated salt form, or mixtures thereof. All such forms are included within the scope of the present invention unless otherwise indicated.

The present invention also includes isotopically labeled compounds of formula 1, i.e. compounds

- 3 040670 formula 1, where one or more atoms have been replaced or enriched with an atom having the same atomic number, but an atomic mass different from the atomic mass that prevails in nature. Examples of isotopes that may be included in a compound of Formula 1 include, but are not limited to, ² H, ³ H, ^P C, ¹³ C, ¹⁴ C, ¹³ N, 15 N, 15 O, ¹⁷ O, ¹⁸ O, and ^{18 F} Of particular interest are compounds of formula 1 enriched in tritium or carbon-14, which can be used, for example, in tissue distribution studies. Also of particular interest are compounds of formula 1 enriched in deuterium, especially at the site of metabolism, which are expected to have greater metabolic stability. Also of particular interest are compounds of formula 1 enriched in a positron emitting isotope such as ⁿ C, ¹⁸ F, ¹⁵ O and ¹³ N, such compounds can be used, for example, in positron emission tomography (PET) studies.

Definitions.

In describing the present invention, including its various aspects and embodiments, the following terms have the following meanings.

The term "therapeutically effective amount" means an amount sufficient to effect treatment when administered to a patient in need of treatment.

The term treating or treating means preventing, ameliorating, or suppressing a medical condition, disease, or disorder being treated (eg, a respiratory disease) in a patient (particularly, a human); or alleviating symptoms of a medical condition, disease, or disorder.

The term pharmaceutically acceptable salt means a salt that is acceptable for administration to a patient or a mammal, such as a human (eg, salts that have an acceptable mammalian safety for the dosage regimen used). Typical pharmaceutically acceptable salts include acetic, ascorbic, benzenesulfonic, benzoic, camphorsulfonic, citric, ethanesulfonic, 1,2-ethanedisulfonic, fumaric, gentisic, gluconic, glucuronic, glutamic, hippuric, hydrobromic, hydrochloric, isethionic, lactic, lactobionic, maleic, malic, mandelic, methanesulfonic, mucinic, naphthalenesulfonic, naphthalene-1,5-disulfonic, naphthalene-2,6-disulfonic, nicotinic, nitric, orotic, pamoic, pantothenic, phosphoric, succinic, sulfuric, tartaric, p-toluenesulfonic and xinaphic acids and so on.

The term salt thereof means a compound obtained by replacing the hydrogen of an acid with a cation such as a metal cation or an organic cation and the like. For example, the cation may be the protonated form of the compound of Formula 1, i. e. a form in which one or more amino groups are protonated with an acid. Typically, the salt is a pharmaceutically acceptable salt, although this is not required for salts of intermediates that are not intended to be administered to a patient.

The term amino protecting group means a protecting group suitable for preventing undesired reactions at the nitrogen of the amino group. Exemplary amino protecting groups include, but are not limited to, formyl; acyl groups, for example alkanoyl groups such as acetyl and trifluoroacetyl; alkoxycarbonyl groups such as t-butoxycarbonyl (Boc); arylmethoxycarbonyl groups such as benzyloxycarbonyl (Cbz) and 9-fluorenylmethoxycarbonyl (Fmoc); arylmethyl groups such as benzyl (Bn), trityl (Tr) and 1,1-di-(4'-methoxyphenyl)methyl; silyl groups such as trimethylsilyl (TMS), t-butyldimethylsilyl (TBDMS), [2-(trimethylsilyl)ethoxy]methyl (SEM), and the like.

The term hydroxy protecting group means a protecting group suitable for preventing undesired reactions at the hydroxy group. Exemplary hydroxy protecting groups include, but are not limited to, alkyl groups such as methyl, ethyl, and tert-butyl; acyl groups, for example alkanoyl groups such as acetyl; arylmethyl groups such as benzyl (Bn), p-methoxybenzyl (PMB), 9-fluorenylmethyl (Fm) and diphenylmethyl (benzhydryl, DPM); silyl groups such as trimethylsilyl (TMS) and tert-butyldimethylsilyl (TBS); and so on.

Various protecting groups, as well as their introduction and removal, are described in T.W. Greene and P.G.M. Wuts, Protecting Groups in Organic Synthesis, Third Edition, Wiley, New York.

General synthesis procedures.

Compound 1 and its intermediates can be prepared according to the following general methods and procedures using commercially available or routinely prepared starting materials and reagents. In addition, compounds having an acidic or basic atom or functional group can be used or can be obtained in the form of a salt, unless otherwise indicated (in some cases, the use of a salt in a particular reaction will require the conversion of the salt form to a non-salt form, for example, to the free base form using the usual procedures before carrying out the reaction).

Although a particular embodiment of the present invention may be shown or described in the following procedures, those skilled in the art will appreciate that other embodiments or aspects of the present invention may also be obtained using such procedures or

- 4 040670 using other methods, reagents and starting materials known to specialists in this field. In particular, it should be understood that Compound 1 can be prepared in a variety of ways in which reactants are combined in a different order to provide different intermediates on the way to the final products.

Preparation of 1-(2-(6-(2-ethyl-5-fluoro-4-hydroxyphenyl)-4-fluoro-III-indazol-3-yl)-1,4,6,7-tetrahydro5H-imidazo[4, 5-c]pyridin-5-yl)-2-morpholinoethan-1-one (compound 1) is described in detail in the accompanying examples. The key stages are generally presented in Scheme 1, Scheme 1

where reagent 7 is 2,5-dioxopyrrolidin-1-yl-2-morpholinoacetate, i.e. variable R ^A is the 2,5-dioxopyrrolidinyl activating agent described in Example 1. Alternatively, morpholine-4-ylacetic acid, ie. R ^A is hydrogen, used as reagent 7 under typical amide bond formation conditions as described in Example 4.

Intermediate 3 can be prepared as described in Preparations 1 and 3 below. An alternative way to obtain scheme 2.

key secure intermediate connection is illustrated in

Scheme 2

with benzyl-protected imine cobromindazole aldehyde 8 can be reacted by union 2 to give intermediate 9. The reaction is typically carried out in the presence of sodium bisulfite at a temperature of about 130 to about 140° C. for about 1 to about 6 hours or up to until the reaction is essentially complete. Compound 9 is reduced using a reducing agent such as sodium borohydride to give compound 10 which is combined with protected phenyl trifluoroborate 11 under typical Suzuki Miyaura coupling reaction conditions to give intermediate 5. The reaction is typically carried out at elevated temperature in the presence of a palladium catalyst. The Suzuki reaction partner 11, shown in Scheme 2 as the potassium trifluoroborate salt, can be prepared by reacting the corresponding boronate (intermediate 1-5 in Preparation 1 below) with potassium hydrogen difluoride to give intermediate 11. Alternatively, a boronate intermediate can be used in place of trifluoroborate 11.

Accordingly, in a method aspect, the invention provides a process for preparing a compound of formula 1 or a pharmaceutically acceptable salt thereof, comprising reacting a compound of formula 6 with a compound of formula 7 as illustrated in Scheme 1 to obtain a compound of formula 1 or a pharmaceutically acceptable salt thereof.

In an additional method aspect, the invention provides a compound of formula 5 and a compound of formula 6 useful in the preparation of a compound of formula 1.

crystalline forms.

In another aspect, the invention provides 1-(2-(6-(2-ethyl-5-fluoro-4-hydroxyphenyl)-4-fluoro-1Hindazol-3-yl)-1,4,6,7-tetrahydro-5H- imidazo[4,5-c]pyridin-5-yl)-2-morpholinoethan-1-one (1) in crystalline form.

Form 1.

The crystalline form 1 of the invention is the crystalline free form of compound 1. In one aspect, form 1 is characterized by a powder x-ray diffraction (PXRD) pattern having characteristic diffraction peaks, among other peaks, at 2θ values of 8.16 ± 0.20, 8.97 ±0.20, 15.29±0.20, 16.70±0.20, 18.00±0.20 and 20.18±0.20. Form 1 may also be characterized by an X-ray powder diffraction pattern having two or more additional diffraction peaks, including three or more and four or more additional diffraction peaks at 2θ values selected from 7.69±0.20, 10.66±0, 20, 11.46±0.20, 11.91±0.20, 15.80±0.20, 17.02±0.20, 18.83±0.20, 22.39±0.20, 22.98±0.20, 24.89±0.20 and 26.54±0.20. In another aspect, Form 1 is characterized by an X-ray powder diffraction pattern having three, four, five, or six diffraction peaks at 2θ values selected from 8.16±0.20, 8.97±0.20, 15.29±0.20, 16.70±0.20, 18.00±0.20 and 20.18±0.20.

As is well known in the field of X-ray powder diffraction, the positions of peaks in an X-ray powder diffraction pattern are relatively less sensitive to experimental conditions such as sample preparation conditions and instrumental geometry than relative peak heights. Thus, in one aspect, crystalline Form 1 is characterized by an X-ray powder diffraction pattern in which the peak positions substantially correspond to those shown in FIG. 6.

In another aspect, the crystalline form 1 is characterized by behavior when exposed to high temperature. As shown in FIG. 7, a differential scanning calorimetry (DSC) thermogram taken at a heating rate of 10° C./min shows a peak in endothermic heat flux, identified as a melt transition, in the range of about 210 to about 234° C., or in the range of about 215 up to about 229°C, or in the range from about 220 to about 224°C. Crystal form 1 is characterized by a differential scanning calorimetry thermogram obtained at a heating rate of 10°C/min, which represents a maximum in the endothermic heat flux with a peak at about 221.7°C. Thermogram of thermogravimetric analysis (TGA) in FIG. 8 shows no significant weight loss at temperatures below the decomposition start temperature at about 293°C.

A representative dynamic moisture sorption (DMS) curve for Form 1 of the crystalline free form of the invention is shown in FIG. 9. Crystal form 1 showed a small hysteresis between two cycles of sorption and desorption. Form 1 showed a weight increase of about 0.99% in the humidity range of 5 to 70% RH and a weight increase of about 1.32% in the humidity range of 5 to 90% RH at room temperature, as shown in FIG. 9. Form 1 is considered slightly hygroscopic.

Crystal Form 1 has been shown to be stable under conditions of elevated temperature and humidity. After 36 weeks under accelerated conditions of 40° C. and 75% relative humidity, no statistically significant change in chemical purity was observed.

Form 1 can be prepared by dissolving compound 1 in ethanol with heating followed by the addition of acetonitrile, where the ratio of acetonitrile to ethanol is about 1:1, or about 1:3 to 3:1. The resulting mixture is then heated followed by stirring at about 20 to about 25°C for about 4 to about 30 hours, or about 16 hours. The solid is then filtered and dried to give Form 1.

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Form 1 can also be prepared by mixing Compound 1 with ethanol and stirring the mixture at about 50 to about 80°C for about 2-30 minutes or about 10 minutes, followed by slow addition of acetonitrile at about 50 to about 80°C. C, wherein the volume ratio of acetonitrile to ethanol is from about 3:1 to 1:1, or about 1.5:1. Form 1 seeds may be added and the reaction mixture stirred at about 20 to about 25°C for about 4 hours to about 30 hours, or for about 18 hours. The solid is then filtered and dried to give Form 1.

Form 2.

Crystal Form 2 of the invention is the crystalline free form of Compound 1. In one aspect, Form 2 has a powder X-ray diffraction (PXRD) pattern having characteristic diffraction peaks, among other peaks at 2θ values of 10.61±0.20, 11.84± 0.20, 14.94±0.20, 18.26±0.20 and 19.06±0.20. Form 2 can also be characterized by an X-ray powder diffraction pattern having additional diffraction peaks at 2θ values of 13.32±0.20, 17.69±0.20 and 21.10±0.20. Form 2 may also be characterized by an X-ray powder diffraction pattern having two or more additional diffraction peaks, including three or more and four or more additional diffraction peaks at 2θ values selected from 10.85±0.20, 16.14±0, 20, 16.35±0.20, 18.43±0.20, 19.20±0.20, 19.49±0.20, 20.72±0.20, 21.94±0.20, 22.64±0.20, 23.64±0.20, 25.19±0.20 and 28.08±0.20.

In another aspect Form 2 is characterized by an X-ray powder diffraction pattern having three, four, five or six diffraction peaks at 2θ values selected from 10.61±0.20, 11.84±0.20, 13.32±0.20, 14.94±0.20, 17.69±0.20, 18.26±0.20, 19.06±0.20 and 21.10±0.20.

As is well known in the field of X-ray powder diffraction, the positions of peaks in an X-ray powder diffraction pattern are relatively less sensitive to experimental conditions such as sample preparation conditions and instrumental geometry than relative peak heights. Thus, in one aspect, crystalline Form 2 is characterized by an X-ray powder diffraction pattern in which the peak positions substantially correspond to those shown in FIG. 1.

The structure of crystalline form 2 was also characterized by single crystal X-ray diffraction. The crystals belong to the orthorhombic crystal system and the Pbca space group. Unit cell dimensions are as follows: a=9.7245(11) A, b=16.8197(14) A, c=32.604(4) A, α=90°, β=90°, γ=90°, volume= 5332.8(10) ^A3 . The calculated density is 1.302 g/cm ³ . Crystals contain eight molecules per unit cell. The structure confirms that the crystals do not contain a water molecule or other solvent and the molecular structure corresponds to the structure of the compound of example 1 shown in this application. The X-ray diffraction peaks predicted from the derived atomic positions are in good agreement with the observed results.

In another aspect, the crystalline form 2 is characterized by behavior when exposed to high temperature. As shown in FIG. 2, a differential scanning calorimetry (DSC) thermogram taken at a heating rate of 10° C./min shows a peak in endothermic heat flux, identified as a melt transition, in the range of about 268 to about 277° C., or in the range of about 270 up to about 275°C, or in the range from about 271 to about 274°C. Crystal form 2 is characterized by a differential scanning calorimetry thermogram obtained at a heating rate of 10°C/min, which represents a maximum in the endothermic heat flux with a peak at about 272.6±2°C.

Thermogram of thermogravimetric analysis (TGA) in FIG. 3 shows no significant weight loss at temperatures below the decomposition start temperature at about 269°C.

A representative dynamic moisture sorption (DMS) isotherm for the crystalline free form Form 2 of the invention is shown in FIG. 4. Crystal Form 2 showed no hysteresis between the two sorption and desorption cycles and showed an exceptionally low tendency towards hygroscopicity. Form 2 showed a weight gain of about 0.18% in the humidity range of 5 to 90% RH and a weight gain of about 0.12% in the humidity range of 5 to 70% RH at room temperature, as shown in FIG. 4. Form 2 is considered non-hygroscopic.

Form 2 can be prepared by dissolving compound 1 of example 1 in DMSO (for example, at a ratio of 1 g of compound 1 to 1-3 ml or about 2 ml of DMSO) at a temperature of about 45-75°C or at about 60°C, followed by addition methanol, where the volume ratio of methanol to DMSO is from about 1:4 to about 1:1 or about 1:2. The homogeneous mixture is then added dropwise to a premixed solution of methanol and water (wherein the ratio of methanol to DMSO is in the range of 1.5 to 3 to 1), at a temperature between about 60 and about 90°C, or about 75°C, where in the premixed solution, the volume ratio of methanol to water is from about 0.5:1 to about 1:2, or about 1:0.9. The mixture is then allowed to stir at a temperature between about 60 and about 90°C, or about 75°C for about 30 minutes to about 2 hours or about 1 hour.

- 7 040670 water at a temperature between about 60 and about 90°C, or about 75°C, where the volume ratio of water to methanol is in the range from 2 and 4. The resulting suspension is then slowly cooled to room temperature (typically to a temperature of from about 20 to about 25° C.), typically from about 2 to about 12 hours or about 6 hours. with form 2.

In one aspect, the invention provides a process for preparing crystalline Form 2, comprising:

(a) formation of a homogeneous mixture of 1-(2-(6-(2-ethyl-5-fluoro-4-hydroxyphenyl)-4-fluoro-1H-indazol-3yl)-1,4,6,7-tetrahydro-5H -imidazo[4,5-c]pyridin-5-yl)-2-morpholinoethan-1-one in a polar aprotic solvent, or in a polar water-miscible solvent, or in a mixture of a polar aprotic solvent and a polar water-miscible solvent at a temperature between 45 and 75°C;

(b) adding a homogeneous mixture to a mixture of water-miscible solvent and water at a temperature between 60 and 90° C. to obtain a second mixture;

(c) slowly adding water to the second mixture at a temperature between 60 and 90° C. to form a slurry; and (d) separating the crystalline form from the suspension.

In some aspects, the polar aprotic solvent of step (a) is selected from the group consisting of DMSO, DMF, NMP, DMAc and nitromethane, the polar water-miscible solvent of step (a) is selected from the group consisting of acetonitrile, acetone, methanol, ethanol and THF and the water-miscible solvent of step (b) is selected from the group consisting of acetonitrile, acetone, methanol, ethanol, n-propanol, isopropanol, n-butanol, THF, DMSO, DMF, NMP, DMAc, and nitromethane. In some aspects, the polar aprotic solvent of step (a) is DMSO, the polar water-miscible solvent of step (a) is methanol, and the water-miscible solvent of step (b) is methanol.

In some aspects, the suspension obtained in stage (C), cooled to a temperature between about 20 and 25°C before stage (d).

Alternatively, form 2 can be prepared by mixing compound 1, obtained in example 1, in a mixture of polar water-miscible solvent and water at a temperature between 60 and 90°C. In some aspects, the ratio of solvent to water is about 1:1, or 2:1 to 0.5:1. In some aspects, the polar water-miscible solvent is selected from the group consisting of acetonitrile, acetone, methanol, ethanol, n-propanol, isopropanol, n-butanol, THF, DMSO, DMF, NMP, DMAc, and nitromethane.

pharmaceutical compositions.

Compound 1 and its pharmaceutically acceptable salts are typically used in the form of a pharmaceutical composition or preparation. Such pharmaceutical compositions may be useful for administration to a patient by any suitable route of administration, including, but not limited to, oral, inhalation, ophthalmic injection, topical (including transdermal), rectal, nasal, and parenteral routes of administration.

Accordingly, in one of its compositional aspects, the invention is directed to a pharmaceutical composition comprising a pharmaceutically acceptable carrier or excipient and Compound 1, wherein, as defined above, Compound 1 means Compound 1 or a pharmaceutically acceptable salt thereof. Optionally, such pharmaceutical compositions may contain other therapeutic agents and/or formulation agents, as appropriate. When discussing compositions and their uses, Compound 1 may also be referred to as the active substance.

In some aspects, the invention provides a pharmaceutical composition comprising Compound 1, or a pharmaceutically acceptable salt or Form 1 or Form 2 thereof, and a pharmaceutically acceptable carrier. In some aspects, the pharmaceutical composition is suitable for ophthalmic use. In some aspects, the composition is suitable for injection into the eye. In some aspects, the composition is suitable for intravitreal injection. In some aspects, the composition is a suspension. In some aspects, the composition is a crystalline suspension. In some aspects, the composition is a Form 1 or Form 2 suspension.

Pharmaceutical compositions of the present invention generally contain a therapeutically effective amount of Compound 1. Those skilled in the art will appreciate, however, that a pharmaceutical composition may contain more than a therapeutically effective amount, i.e. bulk compositions, or less than a therapeutically effective amount, i. separate unit doses intended for repeated administration to achieve a therapeutically effective amount.

Typically, such pharmaceutical compositions will contain from about 0.01% to about 95% by weight of the active substance; including, for example, from about 0.05 to about 30 wt.%; and from about 0.1 to about

- 8 040670 wt.% active substance.

Any conventional carrier or excipient may be used in the pharmaceutical compositions of the present invention. The choice of a particular carrier or excipient, or combinations of carriers or excipients, will depend on the route of administration used to treat a particular patient, or on the type of medical condition or disease state. In this regard, obtaining a suitable pharmaceutical composition for a particular route of administration is within the competence of experts in the field of pharmaceuticals. In addition, carriers or excipients used in the pharmaceutical compositions of the present invention are commercially available. As a further illustration, conventional formulation methods are described in Remington: The Science and Practice of Pharmacy, ^20th Edition, Lippincott Williams & White, Baltimore, Maryland (2000); and H. C. Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, 7th Edition, Lippincott Williams & White, Baltimore, Maryland (1999).

Representative examples of substances that can serve as pharmaceutically acceptable carriers include, but are not limited to, the following: sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose such as microcrystalline cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols such as propylene glycol; polyols such as glycerol, sorbitol, mannitol and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethanol; phosphate buffer solutions; and other non-toxic compatible substances used in pharmaceutical compositions.

Pharmaceutical compositions are generally prepared by intimately mixing or admixing the active substance with a pharmaceutically acceptable carrier and one or more optional ingredients. The resulting uniformly mixed mixture can then be formed or loaded into tablets, capsules, pills, and the like using conventional procedures and equipment.

The pharmaceutical compositions of the invention are preferably packaged in unit dosage form. The term unit dosage form refers to a physically discrete unit suitable for administration to a patient, ie. each unit contains a predetermined amount of the active substance, calculated to provide the desired therapeutic effect, either alone or in combination with one or more additional units. For example, such unit dosage forms may be capsules, tablets, pills, and the like, or individual packs suitable for ophthalmic or parenteral administration.

In one embodiment, the pharmaceutical compositions of the invention are suitable for oral administration. Suitable pharmaceutical compositions for oral administration may be in the form of capsules, tablets, pills, lozenges, sachets, dragees, powders, granules; or as a solution or suspension in an aqueous or non-aqueous liquid; or as an oil-in-water or water-in-oil liquid emulsion; or as an elixir or syrup; and so on.; wherein each of these forms contains a predetermined amount of Compound 1 as the active ingredient.

If they are intended for oral administration in solid dosage form (ie, in the form of capsules, tablets, pills, etc.), the pharmaceutical compositions of the present invention will generally include the active substance and one or more pharmaceutically acceptable carriers. Optionally, such solid dosage forms may also include bulking agents or additives such as starches, microcrystalline cellulose, lactose, dicalcium phosphate, sucrose, glucose, mannitol and/or silicic acid; binders such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and/or gum arabic; humectants such as glycerin; disintegrants such as croscarmellose sodium, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates and/or sodium carbonate; retarding dissolution agents such as paraffin; absorption accelerators such as quaternary ammonium compounds; wetting agents such as cetyl alcohol and/or glycerol monostearate; absorbents such as kaolin and/or bentonite clay; lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate and/or mixtures thereof; dyes; and buffer agents.

Release agents, wetting agents, coating agents, sweeteners, flavoring agents, preservatives and antioxidants may also be present in the pharmaceutical compositions of the invention. Examples of pharmaceutically acceptable antioxidants include water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfate, sodium sulfite, and the like; oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, lecithin, propyl gallate, tttocopherol, and the like; and metal chelating agents such as citric acid, EDTA

- 9 040670 tartaric acid, sorbitol, tartaric acid, phosphoric acid, and the like. Coating agents for tablets, capsules, pills and the like. include agents used for enteric coatings such as cellulose acetate phthalate, polyvinyl acetate phthalate, hydroxypropyl methyl cellulose phthalate, hydroxypropyl methyl cellulose, methacrylic acid, methacrylic acid ester copolymers, cellulose acetate trimethylate, carboxymethyl ethyl cellulose, hydroxypropyl methyl cellulose acetate succinate, and the like.

The pharmaceutical compositions of the invention can also be formulated to provide slow or controlled release of the active substance using, for example, hydroxypropyl methylcellulose in varying proportions; or other polymeric matrices, liposomes and/or microspheres. In addition, the pharmaceutical compositions of the invention may optionally contain opacifying agents and may be formulated to release the active ingredient only or preferentially in a certain part of the gastrointestinal tract, optionally in a delayed manner. Examples of potting compositions that can be used include polymeric materials and waxes. The active substance may also be in microencapsulated form, if desired, with one or more of the excipients described above.

Suitable liquid dosage forms for oral administration include, by way of illustration, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. Liquid dosage forms usually include the active substance and an inert diluent such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol , oils (particularly cottonseed, peanut, corn, germ, olive, castor and sesame oils), oleic acid, glycerin, tetrahydrofuryl alcohol, polyethylene glycols and sorbitan fatty acid esters, and mixtures thereof. Alternatively, some liquid compositions may be converted, for example by spray drying, to a powder which is used to prepare solid dosage forms by conventional methods.

Suspensions may contain, in addition to the active ingredient, suspending agents such as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.

Compound 1 can also be administered parenterally (eg, by intravenous, subcutaneous, intramuscular or intraperitoneal injection). For parenteral administration, the active substance is usually mixed with a suitable parenteral carrier, including, by way of example, sterile aqueous solutions, saline, low molecular weight alcohols such as propylene glycol, polyethylene glycol, vegetable oils, gelatin, fatty acid esters such as ethyl oleate, etc. .P. Parenteral compositions may also contain one or more antioxidants, solubilizers, stabilizers, preservatives, wetting agents, emulsifiers, buffering agents or dispersing agents. These compositions can be rendered sterile by the use of a sterile injectable medium, a sterilizing agent, filtration, irradiation, or heating.

Compound 1 can also be formulated as a sterile aqueous suspension or solution for ophthalmic injection. Useful excipients that may be included in such an aqueous composition include polysorbate 80, cellulose polymers such as carboxymethylcellulose, hydroxypropylmethylcellulose, methylcellulose, potassium chloride, calcium chloride, sodium chloride, magnesium chloride, sodium acetate, sodium citrate, histidine, α-dihydrate trehalose, sucrose, polysorbate 20, hydroxypropyl-β-cyclodextrin, benzalkonium chloride, amberlite IRP-69, polyoxyethylene glycol esters (lauryl, stearyl and oleyl), ethylenediaminetetraacetic acid sodium, sodium taurocholate, saponins and Cremophor EL, polycarbophil cysteine, xanthan gum, gellanic gum , hyaluronic acid, liposomes and sodium phosphate. Penetration enhancers, surfactants, bile acids, cyclodextrins such as 2-hydroxypropyl-v-cyclodextrin, and chelating agents may be included in the composition. Cylindrical oligonucleotides with a hydrophilic outer surface and a lipophilic inner surface, which are capable of forming complexes with the active substance, can also be included in the composition. Benzyl alcohol may serve as a preservative and sodium chloride may be included to control tonicity. Additionally, hydrochloric acid and/or sodium hydroxide can be added to the solution to adjust the pH. Can be obtained aqueous compositions for intraocular injection containing no preservatives.

The ophthalmic composition may allow for sustained release of the active ingredient into the eye. The ophthalmic composition may be formulated as an emulsion (oil-in-water or water-in-oil), suspension or ointment. The suspension composition may contain Compound 1, or a pharmaceutically acceptable salt thereof, in crystalline form, such as Form 1 or Form 2, or in an amorphous state.

Compound 1 can also be formulated to be suitable for administration as

- 10 040670 eye drops or intravitreal implant. The implant can deliver consistent therapeutic levels of the drug. Reservoir implants are usually prepared using a granular drug core surrounded by non-reactive substances such as silicon, ethylene vinyl acetate (EVA) or polyvinyl alcohol (PVA); these implants are not biodegradable and can deliver an amount of drug continuously over a period of several months to a year. Matrix implants can also be used. Typically they are used to deliver a loading dose followed by a dose reduction over a period of 1 day to 6 months. They are most commonly made from polylactic acid (PLA) and/or polylactic-glycolic acid (PLGA) copolymers that degrade to water and carbon dioxide. You can also use iontophoresis. This is a non-invasive technique in which a small electrical current is applied to enhance the penetration of an ionized drug into tissues.

Cell encapsulation technology (ECT), which is a cell-based delivery system, can also be used to deliver a therapeutic agent to the eye. Typically, genetically modified cells are packaged in a hollow tube with a semi-permeable membrane that prevents immune cells from entering and allows nutrients and therapeutic molecules to freely diffuse through the membrane. The two ends of the polymer section are hermetically sealed, and the titanium loop is located on the anchor end, which is implanted in the flat part of the ciliary body and attached to the sclera.

Compound 1 can be formulated into any form that allows delivery to the back of the eye. Examples of delivery methods are known from the literature (Kuno et al., Polymers, 2011, 3, 193-221, del Amo et al., Drug Discovery Today, 2008, 13, 135-143, Short, Toxicologic Pathology, 2008, 36 4962) . Such delivery methods include, but are not limited to, suprachoroidal delivery, which provides delivery to the choroid and retina through the suprachoroidal space, delivery to the sub-Tenon space, periocular delivery, contact lenses, punctal obturators, and scleral plugs. Compound 1 can also be delivered by periocular, suprascleral, retrobulbar, peribulbar, or subconjunctival injection.

Compound 1 can be delivered as an emulsion, polymeric micro- or nanospheres, liposomes, micro- or nanoparticles, microspheres, micelles, or dendrimers. Biodegradable and biocompatible polymers such as polyactide and PLGA can be used. Compound 1 may be encapsulated.

In addition, Compound 1 can be formulated for topical application to the skin as an ointment or cream. Ointment compositions are semi-solid preparations having an oily or fatty substance base which is usually clear. Suitable oily substances for use in ointment compositions include petrolatum (petroleum jelly), beeswax, cocoa butter, shea butter and cetyl alcohol. Ointments may optionally further include emollients and penetration enhancers, if desired.

Cream compositions can be prepared as emulsions containing an oil phase and an aqueous phase, usually including purified water. Components of cream compositions may include: oil bases such as petroleum jelly, mineral oils, vegetable and animal oils, and triglycerides; cream bases such as lanolin alcohols, stearic acid and cetostearyl alcohol; a gel base such as polyvinyl alcohol; solvents such as propylene glycol and polyethylene glycol; emulsifiers such as polysorbates, stearates such as glyceryl stearate, octylhydroxystearate, polyoxyl stearate, PEG stearyl esters, isopropyl palmitate and sorbitan monostearate; stabilizers such as polysaccharides and sodium sulfite; emollients (ie, humectants) such as medium chain triglycerides, isopropyl myristate, and dimethicone; firming agents such as cetyl alcohol and stearyl alcohol; antimicrobial agents such as methylparaben, propylparaben, phenoxyethanol, sorbic acid, diazolidinyl urea and butylated hydroxyanisole; penetration enhancers such as N-methylpyrrolidone, propylene glycol, polyethylene glycol monolaurate and the like; and chelating agents such as disodium edetate.

Alternatively, the pharmaceutical compositions of the invention are formulated for administration by inhalation. Suitable pharmaceutical compositions for administration by inhalation are usually in the form of an aerosol or powder. Such compositions are typically administered using well known delivery devices such as a metered dose inhaler, dry powder inhaler, nebulizer or similar delivery device.

When administered by inhalation using a pressurized container, the pharmaceutical compositions of the invention generally comprise the active ingredient and a suitable propellant such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gas. In addition, the pharmaceutical composition may be in the form of a capsule or cartridge (made, for example, from gelatin) containing Compound 1 and a powder suitable for use in a dry powder inhaler. Suitable powder bases include, by way of example, lactose or starch.

The following non-limiting examples illustrate representative pharmaceutical companies.

- 11 040670 positions according to the present invention.

Oral solid dosage form in the form of tablets.

Compound 1 or its pharmaceutically acceptable salt is dry mixed with microcrystalline cellulose, polyvinylpyrrolidone and croscarmellose sodium in a ratio of 4:5:1:1 and compressed into tablets to obtain a standard dose, for example, 5 mg, 20 mg or 40 mg of active substance per tablet .

Oral solid dosage form in the form of capsules.

Compound 1 or a pharmaceutically acceptable salt thereof is combined with microcrystalline cellulose, polyvinylpyrrolidone and croscarmellose sodium in a ratio of 4:5:1:1 by wet granulation and loaded into gelatin or hydroxypropyl methylcellulose capsules to obtain a standard dose, for example, 5 mg, 20 mg or 40 mg active ingredient per capsule.

liquid composition.

A liquid composition containing compound 1 (0.1%), water (98.9%) and ascorbic acid (1.0%) is obtained by adding a compound of the invention to a mixture of water and ascorbic acid.

Enteric-coated oral dosage form.

Compound 1 is dissolved in an aqueous solution containing polyvinylpyrrolidone and sprayed onto microcrystalline cellulose or sugar granules in a ratio of 1:5 w/w. active: granules and then coated with an approximately 5% weight gain enteric coating comprising an acrylic copolymer. The enteric-coated granules are loaded into gelatin or hydroxypropyl methylcellulose capsules to give a unit dose, eg 30 mg active per capsule.

Enteric-coated oral dosage form.

An enteric coating comprising a combination of Eudragit-L® and Eudragit-S® or hydroxypropyl methylcellulose acetate succinate is applied to the oral dosage form as a tablet or oral dosage form as a capsule as described above.

Aqueous composition for ophthalmic injection.

Each ml of the sterile aqueous suspension contains 5 to 50 mg of compound 1, sodium chloride for tonicity, 0.99% (w/v) benzyl alcohol as a preservative, 0.75% sodium carboxymethyl cellulose, and 0.04% polysorbate. You can include sodium hydroxide or hydrochloric acid to bring the pH to 5-7.5.

Aqueous composition for ophthalmic injection.

A sterile, preservative-free aqueous suspension comprises 5 to 50 mg/ml Compound 1 in 10 mM sodium phosphate, 40 mM sodium chloride, 0.03% polysorbate 20, and 5% sucrose.

Composition of ointment for topical administration.

Compound 1 is combined with petrolatum, C8-C10 triglyceride, octylhydroxystearate and N-methylpyrrolidone in such a ratio to obtain a composition containing from 0.05 to 5 wt.% of the active substance.

Composition of ointment for topical administration.

Compound 1 is combined with medical petrolatum, propylene glycol, mono- and diglycerides, paraffin, butylated hydroxytoluene and calcium disodium edetate in such a ratio to obtain a composition containing from 0.05 to 5 wt.% of the active substance.

Composition of ointment for topical administration.

Compound 1 is combined with mineral oil, paraffin, propylene carbonate, medical petrolatum and white wax to obtain a composition containing 0.05-5 wt.% of the active substance.

Cream composition for topical administration.

The mineral oil is combined with compound 1, propylene glycol, isopropyl palmitate, polysorbate 60, cetyl alcohol, sorbitan monostearate, polyoxyl 40 stearate, sorbic acid, methyl paraben and propyl paraben to form an oil phase which is combined with purified water by mixing under high shear to form a composition containing from 0.05 to 5% by weight of the active substance.

Cream composition for topical administration.

Cream composition containing compound 1, benzyl alcohol, cetyl alcohol, anhydrous citric acid, mono- and diglycerides, oleyl alcohol, propylene glycol, sodium cetostearyl sulfate, sodium hydroxide, steayl alcohol, triglycerides and water, contains from 0.05 to 5 wt.% active substance.

Cream composition for topical administration.

Cream composition containing compound 1, cetosteayl alcohol, isopropyl myristate, propylene glycol, cetomacrogol 1000, dimethicone 360, citric acid, sodium citrate and purified water, with imidurea, methyl paraben and propyl paraben as preservatives, contains from 0.05 to 5 wt.% active substances.

Dry powder composition.

Finely divided compound 1 (1 g) is mixed with milled lactose (25 g).

- 12 040670

The resulting mixture is then loaded into individual blisters of a peelable blister pack in an amount sufficient to provide about 0.1 to about 4 mg of Compound 1 per dose. The contents of the blisters are administered using a dry powder inhaler.

Composition for metered dose inhaler.

Finely divided compound 1 (10 g) is dispersed in a solution obtained by dissolving lecithin (0.2 g) in demineralized water (200 ml). The resulting slurry is spray dried and then finely ground to obtain a finely divided composition containing particles having an average diameter of less than about 1.5 microns. The finely divided composition is then loaded into metered dose inhaler cartridges containing pressurized 1,1,1,2-tetrafluoroethane in an amount sufficient to provide about 0.1 to about 4 mg of Compound 1 per dose when administered via a metered dose inhaler.

Composition for a nebulizer.

Compound 1 (25 mg) is dissolved in a solution containing 1.5-2.5 eq. hydrochloric acid, followed by the addition of sodium hydroxide to bring the pH to 3.5-5.5 and 3 wt.% glycerol. The solution is thoroughly mixed until all components are completely dissolved. The solution is administered using a nebulizer that provides about 0.1 to about 4 mg of Compound 1 per dose.

Utility.

Compound 1 has been shown to be a strong inhibitor of the JAK family of enzymes: JAK1, JAK2, JAK3 and TYK2.

Eye diseases.

Many eye diseases have been shown to be associated with increased levels of pro-inflammatory cytokines that are dependent on the JAK-STAT pathway. Since Compound 1 exhibits strong inhibition of all four JAK enzymes, it is expected to strongly inhibit the signaling and pathogenic effects of numerous cytokines (such as IL-6, IL-2, and IFN-γ) that signal through JAK, as well as interfere with increased levels of other cytokines (such as MCP-1 and IP-10) whose production is dependent on the JAK-STAT signaling pathway.

In particular, compound 1 showed pIC ₅₀ values of 6.4 or greater (IC ₅₀ values of 400 nM or less) for inhibition of IL-6, IL-2 and IFNy signaling in the cellular assays described in assays 3-6, including assays registering inhibition of the downstream effects of increased levels of cytokines.

A pharmacokinetic study in Analysis 7 demonstrated long-term exposure in rabbit ocular tissues after a single intravitreal injection and a plasma concentration at least three orders of magnitude lower than that observed in vitreous tissue.

In addition, intravitreal administration of Compound 1 demonstrated significant inhibition of IL-6-induced pSTAT3 in rat retinal/choroid tissue, as well as significant and sustained inhibition of IFN-γ-induced IP-10 in rabbit vitreous as well as retinal tissues. choroid of the eyes. Intravitreal administration of Compound 1 demonstrated significant and sustained inhibition of IFN-γ-induced pSTAT1 in the rabbit.

Sustained inhibition of JAK in ocular tissues in the absence of significant systemic levels is expected to result in strong local anti-inflammatory activity in the eye without systemically induced side effects. Thus, compound 1 is expected to be useful for various ocular diseases, which include, but are not limited to, uveitis, diabetic retinopathy, diabetic macular edema, dry eye syndrome, age-related macular degeneration, retinal vein occlusion, and atopic keratoconjunctivitis.

In particular, uveitis (Horai and Caspi, J. Interferon Cytokine Res, 2011, 31, 733-744), diabetic retinopathy (Abcouwer, J. Clin. Cell. Immunol., 2013, Suppl 1, 1-12), diabetic macular edema (Sohn et al., American Journal of Opthalmology, 2011, 152, 686-694), dry eye syndrome (Stevenson et al., Arch Ophthalmol, 2012, 130, 90-100), retinal vein occlusion (Shchuko et al. , Indian Journal of Ophthalmology, 2015, 63(12), 905-911) and age-related macular degeneration (Knickelbein et al., Int. Ophthalmol Clin, 2015, 55(3), 63-78) are characterized by increased levels of certain pro-inflammatory cytokines , which signal through the JAK-STAT path. Accordingly, Compound 1 can alleviate associated ocular inflammation and induce regression of progressive disease or provide relief from the symptoms of these diseases.

Therefore, in one aspect, the invention provides a method for treating an ocular disease in a mammal, comprising administering a pharmaceutical composition comprising 1-(2-(6-(2-ethyl-5fluoro-4-hydroxyphenyl)-4-fluoro-1H-indazol-3-yl )-1,4,6,7-tetrahydro-5H-imidazo[4,5-c]pyridin-5-yl)-2morpholinoethan-1-one or a pharmaceutically acceptable salt thereof and a pharmaceutical carrier, into the eye of a mammal. In one aspect, the ocular disease is uveitis, diabetic retinopathy, diabetic macular edema, dry eye syndrome, age-related macular degeneration, retinal vein occlusion, or atopic keratoconjunctivitis. In one aspect, the method comprises administering Compound 1 by intravitreal injection.

- 13 040670

Inflammatory skin disease.

For example, atopic dermatitis is associated with increased levels of pro-inflammatory cytokines that are dependent on the JAK-STAT pathway, specifically IL-4, IL-5, IL-10, IL-13, and IFNy. In addition to cytokine inhibition in the cellular assays above, Compound 1 showed an IC ₅₀ value of 13 nM for IL-13 inhibition as described in Assay 2. In addition, cream and ointment model formulations of Compound 1 showed sustained skin levels in for at least 2 days in mice and at least 7 days in mini-gilts without detectable plasma exposure.

Sustained skin levels of Compound 1 in the absence of significant systemic levels are expected to result in potent local anti-inflammatory and antipruritic activity in the skin without side effects due to systemic exposure. Therefore, Compound 1 is expected to be useful in a variety of inflammatory or pruritic skin conditions, which include, but are not limited to, alopecia areata, vitiligo, cutaneous T-cell lymphoma, pruritus nodosum, lichen planus, primary localized cutaneous amyloidosis, bullous pemphigoid , cutaneous manifestations of graft-versus-host disease, pemphigoid, discoid lupus, granuloma annulare, lichen simplex chronicus, vaginal/scrotal/perianal pruritus, lichen sclerosus, pruritus after herpetic neuralgia, lichen planus follicularis, and folliculitis decalvans. In particular, alopecia areata (Xing et al., Nat. Med. 2014, 20, 1043-9), vitiligo (Craiglow et al., JAMA Dermatol. 2015, 151, 1110-2), nodular pruritus (Sonkoly et al. , J. Allergy Clin Immunol. 2006, 117, 411-7), lichen planus (Welz-Kubiak et al., J. Immunol Res. 2015; 2015:854747), primary localized skin amyloidosis (Tanaka et al., Br J. Dermatol. 2009, Dec; 161(6): 1217-24), bullous pemphigoid (Feliciani et al., Int. J. Immunopathol Pharmacol. 1999, May-Aug; 12(2); 55-61) and dermal manifestations of graft-versus-host disease (Okiyama et al., J. Invest Dermatol. 2014, Apr; 134(4):992-1000) are characterized by increased levels of certain cytokines that signal through JAK activation. Accordingly, Compound 1 can alleviate skin inflammation or pruritus associated with these cytokines. In particular, Compound 1 would be expected to be useful in the treatment of atopic dermatitis and other inflammatory skin diseases.

Therefore, in one aspect, the invention provides a method for treating an inflammatory skin disease in a mammal (e.g., a human) comprising the application of a pharmaceutical composition comprising 1-(2-(6-(2-ethyl-5-fluoro-4-hydroxyphenyl)-4-fluoro -1H-indazol-3-yl)-1,4,6,7tetrahydro-5H-imidazo[4,5-c]pyridin-5-yl)-2-morpholinoethan-1-one or a pharmaceutically acceptable salt thereof and a pharmaceutical carrier , on the skin of a mammal. In one aspect, the inflammatory skin disease is atopic dermatitis.

Compound 1 can also be used in combination with gram positive antibiotics such as mupirocin and fusidic acid to treat inflammatory skin conditions. Therefore, in one aspect, the invention provides a method for treating an inflammatory skin disease in a mammal, comprising applying Compound 1 and a Gram-positive antibiotic to the skin of the mammal. In another aspect, the invention provides a pharmaceutical composition comprising 1-(2-(6-(2-ethyl-5-fluoro-4-hydroxyphenyl)-4-fluoro-1H-indazol-3-yl)-1,4,6, 7-tetrahydro-5H-imidazo[4,5c]pyridin-5-yl)-2-morpholinoethan-1-one or a pharmaceutically acceptable salt thereof, a Gram-positive antibiotic, and a pharmaceutically acceptable carrier.

Respiratory diseases.

Cytokines that signal through the JAK-STAT pathway, specifically IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-11, IL-13, IL-23, IL -31, IL-27, thymic stromal lymphopoietin (TSLP), interferon-γ (IFNy), and granulocyte-macrophage colony-stimulating factor (GM-CSF) are also implicated in asthma inflammation and other inflammatory respiratory diseases. As described above, Compound 1 was shown to be a strong inhibitor of JAK1, JAK2, JAK3 and TYK2 enzymes and also showed strong inhibition of pro-inflammatory cytokines in cellular assays.

The anti-inflammatory activity of JAK inhibitors has been clearly demonstrated in preclinical models of asthma (Malaviya et al., Int. Immunopharmacol, 2010, 10, 829,-836; Matsunaga et al., Biochem and Biophys Res Commun, 2011, 404, 261-267; Kudlacz et al., Eur. J. Pharmacol, 2008, 582, 154-161). Accordingly, Compound 1 is expected to be useful in the treatment of inflammatory respiratory disorders, in particular asthma. Inflammation and fibrosis of the lung are characteristic of respiratory diseases other than asthma, such as chronic obstructive pulmonary disease (COPD), cystic fibrosis (CF), pneumonitis, interstitial lung disease (including idiopathic pulmonary fibrosis), acute lung injury, acute respiratory distress syndrome , bronchitis, emphysema, and bronchiolitis obliterans. Therefore, Compound 1 is also expected to be useful in the treatment of chronic obstructive pulmonary disease, cystic fibrosis, pneumonitis, interstitial lung diseases (including idiopathic pulmonary fibrosis), acute lung injury, acute respiratory distress syndrome, bronchitis, emphysema, bronchiolitis obliterans and sarcoidosis.

Therefore, in one aspect, the invention provides a method for treating a respiratory disease in a mammal (e.g., a human) comprising administering to the mammal 1-(2-(6-(2-ethyl-5-14 040670 fluoro-4-hydroxyphenyl)-4-fluoro- N-indazol-3-yl)-1,4,6,7-tetrahydro-5H-imidazo[4,5-c]pyridin-5-yl)-2morpholinoethan-1-one or a pharmaceutically acceptable salt thereof.

In one aspect, the respiratory disease is asthma, chronic obstructive pulmonary disease, cystic fibrosis, pneumonitis, chronic obstructive pulmonary disease (COPD), cystic fibrosis (CF), pneumonitis, interstitial lung diseases (including idiopathic pulmonary fibrosis), acute lung injury, acute respiratory distress syndrome, bronchitis, emphysema, bronchiolitis obliterans or sarcoidosis. In another aspect, the respiratory disease is asthma or chronic obstructive pulmonary disease.

In another aspect, the respiratory disease is a pulmonary infection, helminthiasis, pulmonary arterial hypertension, sarcoidosis, lymphangioleiomyomatosis, bronchiectasis, infiltrative lung disease. In yet another aspect, the respiratory disease is drug-induced pneumonitis, fungal pneumonitis, allergic bronchopulmonary aspergillosis, hypersensitivity pneumonitis, eosinophilic granulomatosis with polyangiitis, idiopathic acute eosinophilic pneumonia, idiopathic chronic eosinophilic pneumonia, hypereosinophilic syndrome, Loeffler's syndrome, bronchiolitis obliterans with organizing pneumonia, or inhibitor-induced immune checkpoint pneumonitis.

The invention also provides a method for treating asthma in a mammal, comprising administering to the mammal a pharmaceutical composition containing 1-(2-(6-(2-ethyl-5-fluoro-4-hydroxyphenyl)4-fluoro-1H-indazol-3-yl)- 1,4,6,7-tetrahydro-5H-imidazo[4,5-c]pyridin-5-yl)-2-morpholinoethan-1-one or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

Compound 1 or a pharmaceutically acceptable salt thereof may also be useful in the treatment of eosinophilic lung diseases. Eosinophilic airway inflammation is a prominent feature of diseases collectively referred to as eosinophilic lung diseases (Cottin et al., Clin. Chest. Med., 2016, 37(3), 535-56). Eosinophilic diseases are associated with IL-4, IL-13, and IL 5 signaling. Eosinophilic lung diseases include infections (especially helminthiasis), drug-induced pneumonitis (induced, for example, by therapeutic drugs such as antibiotics, phenytoin or tryptophan or 1-tryptophan) , fungal pneumonitis (eg, allergic bronchopulmonary aspergillosis), hypersensitivity pneumonitis, and eosinophilic granulomatosis with polyangiitis (formerly known as Churg-Strauss syndrome). Eosinophilic lung diseases of unknown etiology include idiopathic acute eosinophilic pneumonia, idiopathic chronic eosinophilic pneumonia, hypereosinophilic syndrome, and Loeffler's syndrome.

Compound 1 or a pharmaceutically acceptable salt thereof may also be useful in the treatment of RAS. Polymorphisms in the IL-6 gene are associated with increased levels of IL-6 and an increased risk of developing pulmonary arterial hypertension (PAH) (Fang et al., J. Am. Soc. Hypertens., 2017, 11(3), 171-177). Confirming the role of IL-6 in RAS, inhibition of the gp130 chain of the IL-6 receptor alleviates disease in a rat model of RAS (Huang et al., Can. J. Cardiol., 2016, 32(11), 1356.e1-1356.e10).

Compound 1 or a pharmaceutically acceptable salt thereof may also be useful in the treatment of non-allergic lung diseases such as sarcoidosis and lymphangioleiomyomatosis. Cytokines such as IFNy, IL-12 and IL-6 are implicated in a number of non-allergic lung diseases such as sarcoidosis and lymphangioleiomyomatosis (El-Hashemite et al., Am. J. Respir. Cell. Mol. Biol., 2005, 33 , 227-230, and ElHashemite et al., Cancer Res., 2004, 64, 3436-3443).

Compound 1 or a pharmaceutically acceptable salt thereof may also be useful in the treatment of bronchiectasis and infiltrative lung diseases, which are diseases associated with chronic neutrophilic inflammation. Several cytokines are associated with neutrophilic inflammation (eg IL-6, IFNy).

Pathological T cell activation is critical in the etiology of multiple respiratory diseases. Autoreactive T cells play a role in bronchiolitis obliterans with organizing pneumonia (also called COS). Like COS, the etiology of lung transplant rejection is associated with aberrant T cell activation of recipient T cells by the transplanted donor lung. Lung transplant rejections may occur earlier as primary graft dysfunction (PGD), organizing pneumonia (OR), acute rejection (AR), or lymphocytic bronchiolitis (LB), or they may occur years after lung transplantation as chronic lung allograft dysfunction (CLAD). ). CLAD was formerly known as bronchiolitis obliterans (BO), but is now considered a syndrome that can have various pathological manifestations, including BO, restrictive CLAD (rCLAD or RAS), and neutrophilic allograft dysfunction. Chronic lung allograft dysfunction (CLAD) is a major problem in the long-term management of lung transplant recipients as it causes the transplanted lung to progressively lose functionality (Gauthier et al., Curr. Transplant Rep., 2016, 3(3), 185-191). CLAD does not respond well to treatment and therefore there remains a need for effective compounds capable of preventing or treating this condition. Some JAK- 15 040670 dependent cytokines, such as IFNy and IL-5, are upregulated in CLAD and lung transplant rejection (Berastegui et al., Clin Transplant. 2017, 31, e12898). In addition, high levels of CXCR3 chemokines in the lung, such as CXCL9 and CXCL10, which are on a downstream JAK-dependent IFN signaling pathway, are associated with worse outcomes in lung transplant patients (Shino et al., PLOS One, 2017, 12 (7 ), e0180281). Systemic JAK inhibition has been shown to be effective in kidney transplant rejection (Vicenti et al., American Journal of Transplantation, 2012, 12, 2446-56). Therefore, compound 1 may be effective in the treatment or prevention of lung transplant rejection and CLAD. Similar T cell activation events, described as the basis for lung transplant rejection, are also believed to be a major factor in causing graft-versus-host disease (GVHD) lung disease that can occur after hematopoietic stem cell transplantation. As with CLAD, GVHD of the lung is a chronic, progressive disease with extremely poor outcomes, and there is currently no approved treatment. A retrospective multicentre review study of 95 patients with steroid-resistant acute or chronic GVHD treated with the systemic JAK inhibitor ruxolitinib as conservative therapy demonstrated a complete or partial response to ruxolitinib in the majority of patients, including patients with pulmonary GVHD (Zeiser et al., Leukemia, 2015, 29, 10, 2062-68). More recently, with increased use of immune checkpoint inhibitors, immune checkpoint inhibitor-induced pneumonitis, another T-cell mediated lung disease, has emerged. Cancer patients treated with these T-cell stimulants may develop fatal pneumonitis. Compound 1, or a pharmaceutically acceptable salt thereof, is a potential new treatment for these underappreciated serious respiratory diseases.

Gastrointestinal diseases.

As JAK inhibitors, Compound 1, or a pharmaceutically acceptable salt thereof, may also be useful in a variety of other diseases. Compound 1, or a pharmaceutically acceptable salt thereof, may be useful in a variety of gastrointestinal inflammatory symptoms, which include, but are not limited to, inflammatory bowel disease, ulcerative colitis (proctosigmoiditis, pancolitis, ulcerative proctitis, and left-sided colitis), Crohn's disease, collagenous colitis, lymphocytic colitis, Behçet's disease, celiac disease, immune checkpoint inhibitor-induced colitis, ileitis, eosinophilic esophagitis, graft-versus-host-associated colitis, and infectious colitis. Ulcerative colitis (Reimund et al., J. Clin. Immunology, 1996, 16, 144-150), Crohn's disease (Woywodt et al., Eur. J. Gastroenterology Hepatology, 1999, 11, 267-276), collagenous colitis ( Kumawat et al., Mol. Immunology, 2013, 55, 355-364), lymphocytic colitis (Kumawat et al., 2013), eosinophilic esophagitis (Weinbrand-Goichberg et al., Immunol Res, 2013, 56, 249-260) graft-versus-host-associated colitis (Coghill et al., Blood, 2001, 117, 3268-3276), infectious colitis (Stallmach et al., Int. J. Colorectal Dis, 2004, 19, 308-315) , Behçet's disease (Zhou et al., Autoimmun Rev, 2012, 11, 699-704), celiac disease (de Nitto et al., World J. Gastroenterol, 2009, 15, 4609-4614), immune checkpoint inhibitor-induced colitis ( for example, CTLA-4 inhibitor-induced colitis (Yano et al., J. Translation Med., 2014, 12, 191), PD-1 or PD-L1 inhibitor-induced colitis) and ileitis (Yamamoto et al., Dig Liver Dis , 2008, 40, 253-259) are characterized by an increase in the level of some x pro-inflammatory cytokines. Because many pro-inflammatory cytokines signal through JAK activation, Compound 1 or a pharmaceutically acceptable salt thereof may alleviate inflammation and provide symptomatic relief. In particular, Compound 1, or a pharmaceutically acceptable salt thereof, may be useful in inducing and maintaining remission of ulcerative colitis, as well as in the treatment of Crohn's disease, immune checkpoint inhibitor-induced colitis, and gastrointestinal side effects of graft versus host reaction. Therefore, in one aspect, the invention provides a method of treating a gastrointestinal inflammatory disease in a mammal (e.g., a human) comprising administering Compound 1, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising a pharmaceutically acceptable carrier and Compound 1, or a pharmaceutically acceptable salt thereof, to the mammal.

Other diseases.

Compound 1 or a pharmaceutically acceptable salt thereof may also be useful in the treatment of other diseases such as other inflammatory diseases, autoimmune diseases, or cancers.

Compound 1, or a pharmaceutically acceptable salt thereof, may be useful in the treatment of one or more diseases selected from arthritis, rheumatoid arthritis, juvenile rheumatoid arthritis, transplant rejection, xerophthalmia, psoriatic arthritis, diabetes, insulin dependent diabetes, motor neurone disease, myelodysplastic syndrome, pain, sarcopenia, cachexia, septic shock, systemic lupus erythematosus, leukemia, chronic lymphocytic leukemia, chronic myelocytic leukemia, acute lymphoblastic leukemia, acute myelogenous leukemia, ankylosing spondylitis, myelofibrosis, B-cell lymphoma, hepatocellular carcinoma, Hodgkin's disease, breast cancer, multiple myeloma, melanoma, non-Hodgkin's lymphoma, non-small cell lung cancer, clear cell ovarian cancer, tumors

- 16 040670 ovary, pancreatic tumor, polycythemia vera, Sjogren's syndrome, soft tissue sarcoma, sarcoma, splenomegaly, T-cell lymphoma and thalassemia major.

Combined therapy.

The compounds of the present invention, or a pharmaceutically acceptable salt thereof, may be used in combination with one or more agents that act by the same or different mechanisms to treat a disease. The different agents may be administered sequentially or simultaneously, in separate compositions, or in the same composition. Suitable classes of agents for combination therapy include, but are not limited to, anti-angiogenic agents, steroids, anti-inflammatory agents, plasma kallikrein inhibitor, placental growth factor ligand inhibitor, VEGF-A ligand inhibitor, angiopoietin ligand-2 inhibitor, protein tyrosine phosphatase β inhibitor, tyrosine kinase stimulator Tek receptor, calcineurin inhibitor, VEGF ligand inhibitor, mTOR complex 1 inhibitor, mTOR inhibitor, IL-17 antagonist, calmodulin modulator, FGF receptor antagonist, PDGF receptor antagonist, VEGF receptor antagonist, TNF-α ligand inhibitor, TNF-binding agent, proteoglycan stimulator 4, VEGF-C ligand inhibitor, VEGF-D ligand inhibitor, CD126 antagonist, complement cascade inhibitor, glucocorticoid agonist, complement factor C5 inhibitor, cannabinoid receptor antagonist, sphingosine-1-phosphate receptor-1 modulator, sphingosine-1 receptor-3 modulator -phosphate, sphingosine-1-phosphate receptor-4 modulator, m sphingosine-1-phosphate receptor-5 modulator, acetaldehyde dehydrogenase inhibitor, Flt3 tyrosine kinase inhibitor, Kit tyrosine kinase inhibitor, protein kinase C inhibitor, adrenocorticotropic hormone ligand, stromal cell factor 1 ligand inhibitor, immunoglobulin G1 agonist; interleukin-1b ligand inhibitor, mucin stimulant; nuclear factor kappa B modulator, cytotoxic T-lymphocyte protein-4 stimulator, T-cell surface glycoprotein CD28 inhibitor, lipoprotein lipase stimulator; PPARa agonist, adenosine A3 receptor agonist, angiotensin II receptor antagonist, VEGF receptor antagonist, interferon-β ligand, SMAD-2 modulator; TGF β 1 ligand inhibitor, somatostatin receptor agonist, IL-2 receptor α-subunit inhibitor, VEGF-B ligand inhibitor, thymosin β4 ligand, angiotensin II AT-1 receptor antagonist, CCR2 chemokine antagonist, membrane copper amine oxidase inhibitor, CD11a antagonist, inhibitor ICAM-1, insulin-like growth factor 1 antagonist, kallikrein inhibitor, fucosyl transferase 6 stimulator, GDP fucose synthetase modulator, GHR gene inhibitor, IGF1 gene inhibitor, VEGF-1 receptor antagonist, albumin agonist, IL-2 antagonist, CSF-1 antagonist; PDGF receptor antagonist, VEGF-2 receptor antagonist, mTOR inhibitor, PPARa agonist, Rho GTPase inhibitor, Rho-associated protein kinase inhibitor, complement C3 inhibitor, EGR-1 transcription factor inhibitor, nuclear erythroid-related factor-2 modulator, nuclear factor kappa inhibitor B, α-Υ/β-3 integrin antagonist, erythropoietin receptor agonist, glucagon-like peptide 1 agonist, TNFRSF1A gene stimulator, angiopoietin ligand2 inhibitor, alpha-2 antiplasmin inhibitor, collagen antagonist, fibronectin inhibitor, laminin antagonist, plasmin stimulator, growth factor ligand nerves, FGF1 receptor antagonist, FGF3 receptor antagonist, Itk tyrosine kinase inhibitor, Lck tyrosine kinase inhibitor, Ltk tyrosine kinase receptor inhibitor, PDGF receptor-a antagonist, PDGF receptor-β antagonist, protein tyrosine kinase inhibitor, VEGF-3 receptor antagonist, membrane copper amine oxidase inhibitor somatostatin 2 receptor, somatostatin 4 agonist -receptor, somatostatin5 receptor agonist, protein kinase C α inhibitor, protein kinase C β inhibitor, protein kinase C δ, protein kinase C ε inhibitor, protein kinase C eta inhibitor, protein kinase C theta inhibitor, ankyrin modulator, mucin stimulator, purinergic receptor P2Y2 agonist, intercellular protein inhibitor gap junctions a-1, CCR3 chemokine antagonist; eotaxin ligand inhibitor, amiloride-responsive sodium channel inhibitor, PDGF receptor antagonist, protein tyrosine kinase inhibitor, retinal pigment epithelial protein inhibitor, matrix metalloproteinase inhibitor, PDGF receptor antagonist, PD receptor-β antagonist, PDGF-B GF ligand inhibitor, growth hormone receptor antagonist, inhibitor cell adhesion molecule, integrin modulator, CXCR4 chemokine antagonist, supercoiled domain-containing protein inhibitor, Hsp 90 modulator, Rho-associated protein kinase inhibitor, VEGF gene inhibitor, endoglin inhibitor, CCR3 chemokine antagonist, maxiK potassium channel modulator, maxiK potassium channel stimulator, PGF2a agonist, prostanoid receptor agonist, voltage-gated chloride channel 2 modulator, complement C5a receptor antagonist, inosine monophosphate dehydrogenase inhibitor, interleukin 18 ligand inhibitor, TRP cation channel M8 stimulator, CNTF receptor agonist, TRPV1 gene inhibitor, deoxyri stimulator bonuclease I, IRS1 gene inhibitor, Rho-associated protein kinase inhibitor, poly(ADP-ribose) polymerase 1 inhibitor, poly(ADP-ribose) polymerase 2 inhibitor, poly(ADP-ribose) polymerase 3 inhibitor, vanilloid VR1 agonist, NFAT5 gene stimulator , mucin stimulator, Syk tyrosine kinase inhibitor, a2-adrenergic receptor agonist, cyclooxygenase inhibitor, amyloid protein deposition inhibitor, glycogen synthase kinase-3 inhibitor, PARP stimulator, tau deposition inhibitor, DDIT4 gene inhibitor, hemoglobin synthesis modulator, interleukin-17 ligand inhibitor 040670 β, TNF antagonist, KCNQ voltage-gated potassium channel stimulator, NMDA receptor antagonist, cyclooxygenase 1 inhibitor, cyclooxygenase inhibitor, 5-HT 1a receptor agonist, calcium channel inhibitor, FGF-2 ligand modulator, phosphoinositide 3-kinase inhibitor, CD44 antagonist, hyaluronidase modulator , hyaluronic acid agonist, IL-1 antagonist, IL-1 type I receptor antagonist, complement factor P inhibitor, antagonist tubulin ist, amyloid β antagonist, IL2 gene stimulator, kappa B kinase β inhibitor, nuclear factor kappa B modulator, plasminogen activator inhibitor 1 inhibitor, FGF-2 ligand, protease modulator, and corticotropin modulator.

Specific agents that can be used in combination with the JAK inhibitor compounds of the present invention include, but are not limited to, lanadelumab, aflibercept, RG-7716, AKB-9778, cyclosporine, bevacizumab, everolimus, secukinumab, fluocinolone acetonide, RP101, squalamine lactate, recombinant human lubricin, ORT-302, sarilumab, dexamethasone, eculizumab, fingolimod, adalimumab, reproxalap, midostaurin, corticotropin, oleptesed pegol, canakinumab, recoflavon, abatacept, fenofibrate, piclidenoson, OpRegen, candesartan, pegapap-golimumab , disitertide, octreotide acetate, anekortav, basiliximab, suprachoroidal triamcinolone acetonide, RGN-259, difluprednate, HL-036, avacincaptad pegol sodium, irbesartan, propgermanium, triamcinolone acetonide, azithromycin, BI-1467335, lilifetrast K-etamubon, tenrednol 001, TZ-101, atezidorsen, Nov-03, bevacizumab, AVA-101, RU-101, voclosporin, vorolanib, sirolimus, choline fenofibrate, VX-210, APL-2, CPC-551, Elamipretide, SF-0166, Cibinetide, Elamipretide, Liraglutide, EYS-606, Nesvacumab, Aflibercept, Ocriplasmin, Filgotinib, Cenegermine, Adiposel, Brolicizumab, Ranibizumab, Aflibercept, Padeliporfin PDT, Pazopanib, ASP-8232, ASP-8232 sotrastaurin, abicipar pegol, diquafozol tetrasodium, NSV-1019, conbercept, bertilimumab, SHP-659, THR-317, ALK-001, PAN-90806, interferon a-2b, fluocinolone, sunitinib malate, emixustat, hI-conl, TB- 403, minocycline, MA09-hRPE cells, pegpleranib sodium, pegvisomant, luminate, burixafor, H-1129, carotuximab, AChR-1275, ranibizumab, isopropylunoprostone, tesidolumab, enteric-coated mycophenolate sodium, tadekinig a, triamcinolone acetonide, cyclosporine, ST- 266, AVX-012, NT-501-ECT, tivaniziran, verteporfin, dornase α, aganirsen, ripasudil, rucaparib phosphate, zucapsaicin, tetrathiomolybdate, diclofenac, LHA-510, AGN-195263, tacrolimus, rebamipide, R-348, brimonitine tartrate , visomitin, T-89, LME-636, BI-1026706, rimexolone, then bramycin, TOP-1630, thalaporphin, bromfenac sodium, triamcinolone acetonide, davunetide, loteprednol etabonate, XED-60, EG-myrotin, APD-209, adenovir, PF-04523655, hydroxycarbamide, napyrvamepent, retinalamine, CNTO-2476, ranibizumab, flup , B27PD, S-646240, GLY-230, hydralazine, nepafenac, DexNP, trehalose, hyaluronic acid, sustained release dexamethasone-Ca depot, naluzotane, hyaluronidase, sodium hyaluronate, isunakinra, somatostatin, CLG-561, OC-10X , UCA002, recombinant human epidermal growth factor, pemirolast, VM-100, MB-11316, monosodium α luminol, ranibizumab, IMD-1041, LMG-324, HE-10, sodium cyngialuronate, BDM-E, mesenchymal progenitor cells, disulfiram , CTC-96, PG-101, BeifUshu, chymotrypsin.

The invention also provides a pharmaceutical composition containing Compound 1 or a pharmaceutically acceptable salt thereof and one or more other therapeutic agents. The therapeutic agent may be selected from the class of agents defined above and from the list of specific agents described above. In some embodiments, the pharmaceutical composition is suitable for delivery to the eye. In some embodiments, the implementation of the pharmaceutical composition is a liquid or suspension composition.

In addition, in a method aspect, the present invention provides a method of treating a disease or disorder in a mammal, comprising administering Compound 1, or a pharmaceutically acceptable salt thereof, and one or more other therapeutic agents to the mammal.

When used in combination therapy, the agents may be formulated into a single pharmaceutical composition, or the agents may be presented in separate compositions that are administered simultaneously or at different times by the same or different routes of administration. Such compositions may be packaged separately or may be packaged together as a kit. The two or more therapeutic agents in a kit may be administered by the same route of administration or by different routes of administration.

Examples

The following synthetic examples and biological examples are provided to illustrate the invention and should in no way be construed as limiting the scope of the invention. In the examples below, the following abbreviations have the following meanings unless otherwise noted. Abbreviations not defined below have their conventional meanings.

- 18 040670

ACN= acetonitrile DCC= dicyclohexylcarbodiimide DIPEA= Ν,N-diisopropylethylamine DMAc= dimethylacetamide DMF= Ν,N-dimethylformamide DMSO= dimethisulfoxide EtOAc= ethyl acetate

HATU= Ν, Ν, BR, BR-tetramethyl-O-(7- azabenzotriazol-1-yl)uronium hexafluorophosphate

LDA= lithium diisopropylamide min = minute(minutes) MTWE = A1ethyl-shrccsh butyl ether NBS= N-bromosuccinimide ΝΜΡ = Lg-methyl-2-pyrrolidone R.T.= room temperature THF= tetrahydrofuran bis(pinacolato)diboron = 4,4,5,5,4',4',5',5'-octamethyl- [2,2']bi[[1,3,2]dioxaborolanyl] Pd(dppf)Cl ₂ -CH ₂ Cl ₂ = dichloro(1,G- bis(diphenylphosphino)-ferrocene)dipalladium(II) with dichloromethane

Reagents and solvents were purchased from commercial suppliers (Aldrich, Fluka, Sigma, etc.) and used without further purification. Reactions were monitored by thin layer chromatography (TLC), analytical high performance liquid chromatography (anal HPLC) and mass spectrometry. The reaction mixtures were worked up as described specifically for each reaction; they have usually been purified by extraction and other purification methods such as temperature- and solvent-dependent crystallization, as well as precipitation. In addition, the reaction mixtures were usually purified by column chromatography or preparative HPLC, usually using C18 or BDS packed columns and conventional eluents. Typical preparative HPLC conditions are described below.

The reaction products were usually characterized by mass and ¹ H-NMR spectrometry. For NMR analysis, samples were dissolved in a deuterated solvent (such as CD ₃ OD, CDCl ₃ or d ₆ -DMSO) and ¹ H-NMR spectra were obtained using a Varian Gemini 2000 (400 MHz) device under standard observation conditions. Mass spectrometric identification of compounds was performed by electrospray ionization (ESMS) using an Applied Biosystems (Foster City, CA) model API 150 EX or Waters (Milford, MA) 3100 device connected to auto-cleaning systems.

Preparative HPLC conditions. Column: С18 5 µm 21.2+150 mm, or С18 5 µm 21+250, or С14 5 µm 21*150 mm Column temperature: Room temperature Flow rate: 20.0 ml/min Mobile phases: A=Water+0.05% TFA B=ACN+0.05% TFA Volume of injected sample: (100-1500 µl) Detector wavelength: 214 nm

The crude compounds were dissolved in 1:1 water:acetic acid at about 50 mg/mL. A 4 minute cycle on the analytical scale was performed using a C18 2.1x50 mm column followed by a 15- or 20-minute cycle on the preparative scale using an injection volume of 100

- 19 040670 µl, with a gradient based on the % B retention of the experiment on the analytical scale. The exact gradients depended on the sample. Samples with closely eluted impurities were tested on a C18 column

21x250mm and/or C14 21x150mm column for better separation. Fractions containing the desired product were identified by mass spectrometric analysis.

Preparation 1. 2-(4-(Benzyloxy)-2-ethyl-5-fluorophenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (1-5)

(a) 2-(Benzyloxy)-4-bromo-1-fluorobenzene (1-2).

The two reactions were run in parallel and combined for further processing. A mixture of 5-bromo2-fluorophenol (1-1) (850 g, 4.5 mol), benzyl bromide (837 g, 4.9 mol) and potassium carbonate (923 g, 6.7 mol) in ACN (5 L) was stirred at 20° C. for 12 hours. The reactions were combined and concentrated, diluted with water (8 L) and extracted with EtOAc (3x3 L). The organic layer was separated, washed with brine (3 L), dried over sodium sulfate and concentrated. The crude product was purified through a pad of silica gel (eluted with 3:1 petroleum ether:EtOAc) to give the title intermediate (1.83 kg, 73% yield) as a white solid.

1H NMR (400 MHz, CDCl ₃ ) δ 7.38-7.46 (m, 5H), 7.15 (dd, J=7.6, 2.0 Hz, 1H), 6.98-7.15 (m, 1H), 5.12 (s, 2H).

(b) 2-(Benzyloxy)-4-ethyl-1-fluorobenzene (1-3).

Six reactions were carried out in parallel and combined for further processing. To a solution of the product of the previous step (200 g, 711 mmol) in THF (100 ml) was added potassium carbonate (197 g, 1.4 mol). The reaction mixture was purged with nitrogen 3 times followed by the addition of Pd(dppf)Cl ₂ -CH ₂ Cl ₂ (11.6 g, 14.2 mmol). The reaction mixture was cooled to 0°C, diethylzinc (1M, 1.07 L) was added dropwise and the reaction mixture was stirred at 70°C for 1 h. The reactions were combined, cooled to 20°C and slowly poured into water (7 L) . An aqueous solution of 4M HCl was added to the mixture until pH 6. The organic layer was separated and the aqueous phase was extracted with EtOAc (3x2 L). The combined organic layer was washed with brine (5 L), dried over sodium sulfate, concentrated and purified through a pad of silica gel (eluted with 50:1 petroleum ether:EtOAc)) to give the title intermediate (900 g, 92% yield) as a light yellow oil.

1H NMR (400 MHz, CDCl ₃ ) δ 7.29-7.43 (m, 5H), 6.94-6.97 (m, 1H), 6.82 (d, J=8.0 Hz, 1H ), 6.70 (m, 1H), 5.09 (s, 2H), 2.52-2.58 (m, 2H), 1.17 (t, J=7.6 Hz, 3H).

(c) 1-(Benzyloxy)-4-bromo-5-ethyl-2-fluorobenzene (1-4).

Four reactions were carried out in parallel and combined. To a solution of 2-(benzyloxy)-4-ethyl-1fluorobenzene (1-3) (293 g, 1.3 mol) in ACN (1 L) was added NBS (249 g, 1.4 mol) in portions at 20°C . The reaction mixture was stirred at 20° C. for 2 hours. The reaction mixtures were combined and concentrated. The residue was diluted with water (5 L) and extracted with EtOAc (2x5 L). The organic phase was washed with brine (4 L), dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. The crude product was purified by silica gel chromatography (eluting with petroleum ether:EtOAc 100:1-10:1) to give the title intermediate (1.4 kg, 89% yield) as a light yellow oil.

1H NMR (400 MHz, CDCl ₃ ) δ 7.29-7.38 (m, 5H), 7.2 (d, J=10.4 Hz, 1H), 6.8 (d, J=8.8 Hz, 1H), 5.06(s, 2H), 2.6(q, J=7.6Hz, 2H), 1.1(t, J=7.6Hz, 3H).

(d) 2-(4-(Benzyloxy)-2-ethyl-5-fluorophenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (1-5).

Seven reactions were carried out in parallel and combined for further processing. To a solution of the product of the previous step (200 g, 647 mmol) in dioxane (2 L) were added potassium acetate (190 g, 1.9 mol), bis(pinacolato)diboron (181 g, 712 mmol) and Pd(dppf)Cl ₂ -CH ₂ Cl ₂ (10.6 g, 12.9 mmol) under nitrogen at 20°C. The mixture was stirred at 120° C. for 2 hours. The reactions were combined, concentrated, diluted with water (5 L) and extracted with EtOAc (3x4 L). The combined organic phase was dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. The crude product was purified by silica gel chromatography (eluting with petroleum ether:EtOAc 1:0-5:1) to give the title compound (1.35 kg, 84% yield) as a white solid.

1H NMR (400 MHz, CDCl ₃ ) δ 7.33-7.51 (m, 6H), 6.82 (d, J=7.6 Hz, 1H), 5.17 (s, 2H), 2, 85 (sq., J=7.6 Hz, 2H), 1.33 (s, 12H), 1.15 (t, J=7.6 Hz, 3H).

Preparation 2. 1-Benzyl-4-imino-1,4-dihydropyridine-3-amine (2)

- 20 040670

To a solution of pyridine-3,4-diamine (400 g, 3.67 mol) in ACN (3 L) was added benzyl bromide (596 g, 3.49 mol) in portions at 0°C and the reaction mixture was stirred for 30 min and then at 20°C for 12 h and filtered. The filter cake was washed with ACN (500 mL) and dried to give the HBr salt of the title compound (600 g, 2.14 mol, 58% yield) as a white powder.

¹ H NMR (400 MHz, MeOD) δ 7.83 (d, J=5.6 Hz, 1H), 7.64 (s, 1H), 7.32-7.40 (m, 5H), 6, 76 (d, J=6.8 Hz, 1H), 5.28 (s, 2H).

Preparation 3. 6-(4-(Benzyloxy)-2-ethyl-5-fluorophenyl)-4-fluoro-1H-indazole-3-carbaldehyde (3)

(a) 1-(4-Bromo-2,6-difluorophenyl)-2,2-diethoxyethan-1-one (3-1).

Nine reactions were carried out in parallel and combined for further processing. A solution of 1-bromo-3,5-difluorobenzene (100 g, 518 mmol) in THF (700 ml) was degassed and purged with nitrogen three times. Then 2M LDA (311 ml) was added at -70°C and the reaction mixture was stirred at -70°C for 0.5 h under nitrogen atmosphere. A solution of ethyl 2,2-diethoxyacetate (96 g, 544 mmol) in THF (200 ml) was added dropwise at -70°C under nitrogen atmosphere and the reaction mixture was stirred for 1 h. The reactions were combined and poured into ice-cold saturated chloride solution ammonium (10 L) in portions and extracted with EtOAc (3x3 L). The organic layer was separated, washed with brine (5 L), dried over sodium sulfate, concentrated and purified by silica gel chromatography (eluted with petroleum ether EtOAc 1:0-100:1) to give the title compound (1.26 kg, 84% yield) as a yellow oil.

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.12 (d, J=7.2 Hz, 2H), 5.15 (s, 1H), 3.61-3.7 (m, 4H), 1 .2 (t, J=7.2 Hz, 6H).

(b) 1-(4'-(Benzyloxy)-2'-ethyl-3,5,5'-trifluoro-[1,1'-biphenyl]-4-yl)-2,2-diethoxyethane-1-one (3-2).

Five reactions were carried out in parallel and combined for further processing. To a mixture of 1-(4bromo-2,6-difluorophenyl)-2,2-diethoxyethan-1-one (3-1) (189 g, 586 mmol) in ethanol (150 ml) and toluene (1.5 L) was added water (150 ml), sodium carbonate (84.8 g, 800 mmol) and 2-(4-(benzyloxy)-2-ethyl-5fluorophenyl)-4,4,5,5-tetramethyl-1,3,2- dioxaborolane (1-5) (190 g, 533 mmol) at 20°C. The suspension was degassed under vacuum and purged with nitrogen several times. Pd(dppf)Cl ₂ -CH ₂ Cl ₂ (13 g, 16 mmol) was added and the reaction mixture was purged with nitrogen several times and stirred at 120°C for 2 h. The reactions were combined, cooled to 20°C, poured into water ( 5 L) and extracted with EtOAc (3x4 L). The combined organic layers were washed with brine (5 L), dried over sodium sulfate, filtered, concentrated and purified by silica gel chromatography (eluted with petroleum ether:EtOAc 100:1-5:1) to give the title intermediate (880 g , 70% yield) as a yellow oil.

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.36-7.48 (m, 5H), 6.94-6.96 (m, 2H), 6.86-6.92 (m, 2H), 5.29 (s, 1H), 5.19 (s, 2H), 3.67-3.77 (m, 4H), 2.52 (sq. J=7.6 Hz, 2H), 1, 25 (t, J=6.8 Hz, 6H), 1.07 (t, J=7.2 Hz, 3H).

(c) 6-(4-(Benzyloxy)-2-ethyl-5-fluorophenyl)-3-(diethoxymethyl)-4-fluoro-1H-indazole (3-3).

Four reactions were carried out in parallel and combined for further processing. To a solution of the product of the previous step (220 g, 466 mmol) in THF (2 L) was added hydrazine monohydrate (47.6 g, 931 mmol) at 20°C. The reaction mixture was stirred at 100°C for 12 hours. The four reactions were combined and cooled to 20°C and concentrated. The residue was dissolved in EtOAc (5 L) and washed with a solution

0.1M HCl (2x1.5 L). The combined and organic layers were washed with brine (1.5

l), dried over sodium sulfate, filtered and concentrated to give the title intermediate (900 g, crude) as a yellow gum, which was used directly in the next step.

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.36-7.48 (m, 5H), 6.94-6.96 (m, 2H), 6.86-6.92 (m, 2H), 5.29 (s, 1H), 5.19 (s, 2H), 3.67-3.77 (m, 4H), 2.52 (sq. J=7.6 Hz, 2H), 1, 25 (t, J=6.8 Hz, 6H), 1.07 (t, J=7.2 Hz, 3H).

(d) 6-(4-(Benzyloxy)-2-ethyl-5-fluorophenyl)-4-fluoro-III-indazole-3-carbaldehyde (3).

Three reactions were carried out in parallel and combined for further processing. To a solution of the product of the previous step (300 g, 643 mmol) in acetone (1.5 L) was added 4M HCl (16 ml) dropwise at 20°C and the reaction mixture was stirred at 20°C for 0.17 h. , concentrated, diluted with MTBE (1 L) and filtered. The filter cake was washed with MTBE (2x300 mL) and dried under reduced pressure to give the title intermediate (705 g, crude) as a yellow solid which was used directly in the next step, (m/z): [M+H] + calculated for C ₂₃ H ₁₈ F ₂ N ₂ O ₂ 393.13 found 393.1.

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 14.51 (s, 1H), 10.17 (d, J=3.6 Hz, 1H), 7.50 (d, J=7.2 Hz , 2H), 7.40-7.42 (m, 4H), 7.24 (d, J=8.4 Hz, 1H), 7.15 (d, J=12.4 Hz, 1H), 7 .06 (d, J=8.4 Hz, 1H), 5.25 (s, 2H), 2.52-2.53 (m, 2H), 1.03 (t, J=7.6 Hz, 3H).

Preparation 4. 5-Benzyl-2-(6-(4-(benzyloxy)-2-ethyl-5-fluorophenyl)-4-fluoro-1H-indazol-3-yl)-5Nimidazo[4,5-c]pyridine (4)

4

Four reactions were carried out in parallel and combined for further processing. To a solution of 6-(4-(benzyloxy)-2-ethyl-5-fluorophenyl)-4-fluoro-1H-indazole-3-carbaldehyde (3), product of preparation 3 (172 g, 440 mmol), in DMF (1 1 L) sodium bisulfite (68.6 g, 659 mmol) and 1-benzyl-4-imino1,4-dihydropyridine-3-amine (2) (136 g, 484 mmol) were added at 20°C and the reaction mixture was stirred at 150° C. for 2 hours. The four reactions were combined and the reaction mixture was concentrated under reduced pressure. The residue was poured into water (10 L) and filtered. The filter cake was dried under reduced pressure to give the title intermediate (990 g, crude) as a yellow solid which was used directly without purification, (m/z): [M+H]+ calculated for C ₃₅ H ₂₇ F ₂ N ₅ O 572.2 found 572.3.

Preparation 5 -imidazo[4,5-c]pyridine (5)

5

Three reactions were carried out in parallel and combined for further processing. To a mixture of 5benzyl-2-(6-(4-(benzyloxy)-2-ethyl-5-fluorophenyl)-4-fluoro-1H-indazol-3-yl)-5H-imidazo[4,5-c]pyridine ( 4), product 4 (330 g, 577 mmol), in methanol (1.5 L) and THF (1 L), sodium borohydride (267 g, 6.9 mol) was added in portions at 20° C. and the reaction mixture was stirred at 20°C for 24 hours Three reactions were combined and the reaction mixture was added to water (10 l), stirred for 10 min and filtered. The filtrate was extracted with EtOAc (2x5 L) and the combined organic phase was dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. The crude product was diluted with EtOAc (2 L), stirred for 30 min and filtered. The filter cake was washed with MTBE (3x200 mL) to give the title intermediate (275 g, 28% yield) as a light yellow solid, (m/z): [M+H]+ calculated for C35H31F2N5O 576.25 found 576.3.

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 7.50-7.52 (m, 2H), 7.35-7.43 (m, 7H), 7.23-7.25 (m, 3H ), 7.15 (d, J=12.0

- 22 040670

Hz, 1H), 6.81 (d, J=12.0 Hz, 1H), 5.25 (s, 2H), 3.72 (s, 2H), 3.43 (broad s, 2H), 2.78 (brs, 2H), 2.66 (brs, 2H), 2.55 (sq., 2H), 1.04 (t, J=7.6 Hz, 3H).

Preparation 6 6-yl)phenol (6)

6

Five reactions were carried out in parallel and combined for further processing. To a mixture of 5benzyl-2-(6-(4-(benzyloxy)-2-ethyl-5-fluorophenyl)-4-fluoro-1H-indazol-3-yl)-4,5,6,7-tetrahydro-1Himidazo[ 4,5-c]pyridine (5), product of preparation 5 (55 g, 95.5 mmol), in THF (500 ml) and methanol (500 ml) was added palladium on carbon (15 g, 9.6 mmol) and aqueous solution of 12M HCl (10 ml). The suspension was degassed in vacuo, purged with hydrogen several times, and stirred under a hydrogen atmosphere (50 psi (3.5 kg/cm ² )) at 50° C. for 12 hours. The reactions were combined and the reaction mixture was filtered. The filtrate was concentrated in vacuo to give the HCl salt of the title intermediate (150 g, crude) as an off-white solid, (m/z): [M+H]+ calculated for C21H19F2N5O 396.16, found 396.2 .

¹ H NMR (400 MHz, MeOD) δ 7.43 (s, 1H), 7.07 (d, J=11.6 Hz, 1H), 6.97 (d, J=11.6 Hz, 1H) , 6.91 (d, J=8.8 Hz, 1H), 4.57 (s, 2H), 3.74 (s, 2H), 3.24 (s, 2H), 2.55 (sq. , J=7.6 Hz, 2H), 1.08 (t, J=7.6 Hz, 3H).

Preparation 7 2,5-Dioxopyrrolidin-1-yl-2-morpholinoacetate (7')

7-1 7” 7’ (a) tert-Butyl-2-morpholinoacetate (7-1).

To a mixture of morpholine (160 g, 1.84 mol) and potassium carbonate (381 g, 2.76 mol) in THF (3 L) was added tert-butyl-2-bromoacetate (341 g, 1.75 mol) slowly at 0 °C. The reaction mixture was stirred for 30 min and then at 20° C. for 12 h and concentrated. Water (1.5 L) was added and the reaction mixture was extracted with EtOAc (3x1 L). The organic layer was separated, washed with brine (500 ml), dried over sodium sulfate and concentrated to give the title intermediate (300 g, 81% yield) as a yellow oil.

1H NMR (400 MHz, CDCl ₃ ) δ 3.74 (t, J=4.8 Hz, 4H), 3.10 (s, 2H), 2.57 (t, J=4.8 Hz, 4H ), 1.46 (s, 9H).

(b) 2-Morpholinoacetic acid (7).

A mixture of the product of the previous step (7-1) (300 g, 1.49 mol) in 3M HCl in dioxane (2.0 L) was stirred at 20° C. for 12 h and concentrated to give the HCl salt of the title compound (270 g, 99% yield) as a pale solid which was used directly in the next step 1 H NMR (400 MHz, MeOD) δ 4.13 (s, 2H), 3.93 (br s, 4H), 3.64 (broad s, 4H).

(c) 2,5-Dioxopyrrolidin-1-yl-2-morpholinoacetate (7').

Mixture of previous step product (150 g, 826 mmol), 1-hydroxypyrrolidine-2,5-dione (95 g, 826 mmol), DCC (256 g, 1.24 mol) and DIPEA (160 g, 1.24 mol) in DCM (2 L) was stirred at 15°C for 12 h and filtered. The filtrate was concentrated and washed with EtOAc (800 ml). The solid was collected by filtration and concentrated to give the title compound (150 g, 75% yield) as a white solid.

1H NMR (400 MHz, DMSO-d ₆ ) δ 3.68 (s, 2H), 3.58 (t, J=4.8 Hz, 4H), 2.82 (s, 4H), 2.57 ( t, J=5.2 Hz, 4H).

Example 1 1-(2-(6-(2-Ethyl-5-fluoro-4-hydroxyphenyl)-4-fluoro-1H-indazol-3-yl)-1,4,6,7-tetrahydro5H-imidazo[ 4,5-c]pyridin-5-yl)-2-morpholinoethan- 1-one (1)

A mixture of 5-ethyl-2-fluoro-4-(4-fluoro-3-(4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol6- yl)phenol (6), 2 HCl (100 g, 214 mmol), 2,5-dioxopyrrolidin-1-yl-2-morpholinoacetate (7') (67.2 g, 278 mmol) and DIPEA (69 g, 534 mmol) in DMF (600 ml) was stirred at 15°C for 12 h and filtered. The solution was purified by reverse phase chromatography (Agela FLEXATM FS-1L device; 2 kg

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Agela C18 DAC column; 200 g sample dissolved in DMF (900 ml); flow rate 300 ml/min; solvent A water, solvent B ACN; gradient (% B, time (min): 0/15, 0-40/45, 40/50) to give the title compound (50.0 g, 44.8% yield) as a light yellow solid, (m/z):

[M+H]+ calculated for C ₂₇ H ₂₈ F ₂ N ₆ O ₃ 523.22, found 523.0.

1H NMR (400 MHz, MeOD) δ 7.22 (s, 1H), 6.80-6.96 (m, 3H), 4.68-4.78 (m, 2H), 3.96 (s, 2H), 3.65-3.95 (m, 4H), 3.35-3.38 (m, 2H), 2.77-2.92 (m, 2H), 2.52-2.56 ( m, 6H), 1.06 (t, J=7.6 Hz, 3H).

Example 2 Crystalline 1-(2-(6-(2-ethyl-5-fluoro-4-hydroxyphenyl)-4-fluoro-1H-indazol-3-yl)1,4,6,7-tetrahydro-5H- imidazo[4,5-c]pyridin-5-yl)-2-morpholinoethan-1-one (1) form 1.

1-(2-(6-(2-ethyl-5-fluoro-4-hydroxyphenyl)-4-fluoro-1H-indazol-3-yl)1,4,6,7-tetrahydro- 5H-imidazo[4,5-c]pyridin-5-yl)-2-morpholinoethan-1-one (1), product of Example 1 (5 g), and ethanol (50 ml) and the reaction mixture was stirred at 50-80 °C over 10 min and then slowly add ACN (75 ml) at 50-80°C, followed by the addition of seed crystals from example 3. The reaction mixture was stirred at 20-25°C for 18 h. The resulting solid was collected by filtration and dried at 50° C. in vacuo for 18 h to give Form 1 of the title compound (3.6 g, 72% yield).

Example 3 Crystalline 1-(2-(6-(2-ethyl-5-fluoro-4-hydroxyphenyl)-4-fluoro-1H-indazol-3-yl)1,4,6,7-tetrahydro-5H- imidazo[4,5-c]pyridin-5-yl)-2-morpholinoethan-1-one (1) form 1.

Compound 1, product of Example 1 (1 g) was added to ethanol (10 ml) and heated to dissolve. Acetonitrile (10 ml) was added and the reaction mixture was stirred and heated and then stirred at room temperature for 16 h, filtered and dried at 50° C. under vacuum for 18 h to give Form 1 of the title compound (0.23 g) .

Example 4 1-(2-(6-(2-Ethyl-5-fluoro-4-hydroxyphenyl)-4-fluoro-1H-indazol-3-yl)-1,4,6,7-tetrahydro5H-imidazo[ 4,5-c]pyridin-5-yl)-2-morpholinoethan-1-one (1)

N,N-diisopropylethylamine (0.298 mL, 1.707 mmol) was added to a solution of 5-ethyl-2-fluoro-4-(4-fluoro3-(4,5,6,7-tetrahydro-1H-imidazo[4,5-c ]pyridin-2-yl)-1H-indazol-6-yl)phenol (135 mg, 0.341 mmol) (6), HATU (156 mg, 0.410 mmol) and 2-morpholinoacetic acid (7) (54.5 mg, 0.376 mmol) in DMF (0.5 ml) and the reaction mixture was stirred at room temperature for 24 h. Lithium hydroxide (49.1 mg, 2.049 mmol) was added and the reaction mixture was stirred at 65°C for 1 h and concentrated in vacuum to a clear yellow liquid. The crude liquid was purified by preparative HPLC to give the TFA salt of the title compound (142 mg, 0.223 mmol, 65.3% yield) as a beige solid.

Example 5 Crystalline 1-(2-(6-(2-ethyl-5-fluoro-4-hydroxyphenyl)-4-fluoro-1H-indazol-3-yl)1,4,6,7-tetrahydro-5H- imidazo[4,5-c]pyridin-5-yl)-2-morpholinoethan-1-one (1) form 2.

Compound 1 of Example 1 (2.5 g) was dissolved in DMSO (5 ml) at 60°C. Immediately after obtaining a homogeneous solution, MeOH (2.5 ml) was added to the solution. The homogeneous mixture was added dropwise over 30 min to a pre-mixed solution of MeOH (12.75 ml) and H2O (11.25 ml) at 75°C. After all of the mixture was added, the combined mixture was left to stir at 75° C. for 1 hour, whereupon a crystalline suspension formed. H2O (36 ml) was added dropwise over 2 h at 75°C. After completion of the H2O addition, the suspension was stirred at 75°C for 1 h, then slowly cooled to 20°C for 6 h. The suspension was kept at 20°C for another 10 h, then filtered, washed with 70% H ₂ O/MeOH (10 ml), dried at 50° C. in vacuo for 18 hours to give form 2 of the title compound (2.13 g).

Properties of solid forms according to the invention.

Samples of two anhydrous forms, form 1 and form 2, 1-(2-(6-(2-ethyl-5-fluoro-4-hydroxyphenyl)-4fluoro-1H-indazol-3-yl)-1,4,6, 7-tetrahydro-5H-imidazo[4,5-c]pyridin-5-yl)-2-morpholinoethan-1-one (1) of Examples 2 and 5, respectively, was analyzed using X-ray powder diffraction (PXRD), differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), dynamic moisture sorption (DMS), and polarized light microscopy. Form 2 was also analyzed by single crystal X-ray diffraction.

Example 6 X-ray powder diffraction.

The X-ray powder diffraction patterns shown in Figs. 1 and 6 were obtained using a Bruker D8-Advance X-ray diffractometer with Cu-Κα radiation (λ=1.54051 A), output voltage 45 kV and current 40 mA. The device operated in a Bragg-Bretano geometry with the entrance slit, divergence slit, and scattering slit set to maximize the intensity on the sample. For

- 24 040670 measurements A small amount of powder (5-25 mg) was gently pressed onto the sample holder to obtain a flat surface and subjected to X-ray irradiation. Samples were scanned in 2Θ-2Θ mode from 2 to 35° in 2Θ with a step size of 0.02° and a scan rate of 0.30° s per step. Data collection was monitored using the Bruker DiffracSuite measurement software and analyzed using the Jade software (version 7.5.1). The device was calibrated using a corundum standard, within ±0.02° of the 2Θ angle. The observed positions of the PXRD 2Θ peaks and interatomic distances are shown in Table 1. 1 and 2, respectively, for crystalline form 1 and crystalline form 2.

Table 1

PXRD data for crystalline form 1

2-Theta d(A) Square A% 7.69 11.49 5570 7.30 8.16 10.83 36847 48.60 8.97 9.85 75877 100.00 10.66 8.29 7323 9.70 11.46 7.72 5841 7.70 11.91 7.43 1496 2.00 15.29 5.79 7115 9.40 15.80 5.60 7841 10.30 16.70 5.31 14679 19.30 17.02 5.20 8024 10.60 18.00 4.92 17834 23.50 18.83 4.71 2658 3.50 20.18 4.40 18636 24.60 22.39 3.97 7067 9.30 22.98 3.87 9029 11.90 24.89 3.57 8561 11.30 26.54 3.36 7831 10.30

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table 2

PXRD data for crystalline form 2_____________

2-Theta d(A) Square A% 10.61 8.33 706299 100.00 10.85 8.15 192921 27.30 11.84 7.47 487816 69.10 13.32 6.64 97980 13.90 14.94 5.93 519386 73.50 16.14 5.49 110314 15.60 16.35 5.42 75483 10.70 17.69 5.01 197341 27.90 18.26 4.85 445270 63.00 18.43 4.81 152845 21.60 19.06 4.65 564088 79.90 19.20 4.62 427174 60.50 19.49 4.55 266328 37.70 20.72 4.28 72244 10.20 21.10 4.21 236517 33.50 21.94 4.05 287485 40.70 22.64 3.93 121406 17.20 23.64 3.76 152841 21.60 25.19 3.53 68220 9.70 28.08 3.17 139597 19.80

Example 7 Thermal Analysis

Differential Scanning Calorimetry (DSC) was performed using a TA Instruments Model Q-100 module with a Thermal Analyst controller. Data were collected and analyzed using TA Instruments Thermal Analysis software. A sample of each crystal form was accurately weighed into a lidded aluminum bowl. After a 5 minute isothermal equilibration period at 5°C, the sample was heated using a temperature ramp of 10°C/min from 0 to 300°C.

A representative DSC thermogram of Form 1 of the crystalline free form of the invention is shown in FIG. 7. A differential scanning calorimetry (DSC) thermogram taken at a heating rate of 10°C/min shows a peak in endothermic heat flux, identified as a melt transition, in the range of about 210 to about 234°C, or in the range of about 215 up to about 229°C, or in the range from about 220 to about 224°C. The crystalline form is characterized by a differential scanning calorimetry thermogram obtained at a heating rate of 10°C/min, which shows a maximum in endothermic heat flux with a peak at about 221.7°C or at 221.7±3°C.

A representative DSC thermogram of form 2 of the crystalline free form of the invention is shown in FIG. 2. A Differential Scanning Calorimetry (DSC) thermogram taken at a heating rate of 10°C/min shows a peak in endothermic heat flux, identified as a melt transition, in the range of about 268 to about 277°C, or in the range of about 270 up to about 275°C, or in the range from about 271 to about 274°C. The crystalline form is characterized by a differential scanning calorimetry thermogram obtained at a heating rate of 10°C/min, which shows a maximum in endothermic heat flux with a peak at about 272.6°C or at 272.6±2°C.

Thermogravimetric analysis (TGA) was performed using a high resolution TA Instruments Model Q-50 module. Data was collected using a TA Instruments Thermal Analyst controller and analyzed using TA Instruments Universal Analysis software. The weighed sample was placed on a platinum dish and scanned at a heating rate of 10°C from ambient temperature to 300-350°C. The weighing chambers and ovens were purged with a stream of nitrogen during use.

A representative TGA curve of Form 1 of the crystalline free form of the invention is shown in FIG. 8. Thermogravimetric analysis (TGA) curve in FIG. 8 shows no significant weight loss at temperatures below the decomposition start temperature at about 293°C.

A representative TGA curve of form 2 of the crystalline free form of the invention is shown in FIG. 3. Thermogravimetric analysis (TGA) curve in FIG. 3 shows no significant weight loss at temperatures below the decomposition start temperature at about 269°C.

Example 8. Evaluation by the method of dynamic moisture sorption.

Dynamic Moisture Sorption (DMS) determinations were made using a VTI atmospheric microbalance, SGA-100 system (VTI Corp., Hialeah, FL 33016). A weighed sample was used, and at the beginning of the analysis, the humidity was at the lowest possible value (close to 0% RH (relative humidity)). DMS analysis consisted of an initial drying step (0% RH) for 120 min, followed by two sorption and desorption cycles, at a scan rate of 5% RH/stage over a humidity range of 5 to 90% RH. The DMS experiment was carried out isothermally at 25°C.

A representative DMS curve for Form 1 of the crystalline free form of the invention is shown in FIG. 9.

Crystal form 1 showed a small hysteresis between two cycles of sorption and desorption. Form 1 showed a weight gain of about 0.99% in the humidity range of 5 to 70% RH and about a weight gain of 1.32% in the humidity range of 5 to 90% RH at room temperature, as shown in FIG. 9. Form 1 is considered slightly hygroscopic.

A representative DMS curve for Form 2 of the crystalline free form of the invention is shown in FIG. 4. Crystal form 2 showed no hysteresis between the two cycles of sorption and desorption and showed an exceptionally low tendency to hygroscopicity. Form 2 showed a weight increase of about 0.12% in the humidity range of 5 to 70% RH and a weight increase of about 0.18% in the humidity range of 5 to 90% RH at room temperature, as shown in FIG. 4. Form 2 is considered non-hygroscopic.

Example 9. X-ray diffraction of a single crystal of form 2.

Data were collected on a Rigaku Oxford Diffraction Supernova Dual Source, Cu at Zero, Atlas CCD diffractometer equipped with an Oxford Cryosystems Cobra cooler. Data was collected using Cu Ka radiation. The structure was determined and refined using the Bruker AXS SHELXTL crystallographic analysis software package. Full details can be found in CIF. Unless otherwise indicated, the hydrogen atoms attached to the carbons were arranged geometrically and allowed refinement with the isotropic displacement parameter. Hydrogen atoms attached to heteroatoms were located in the difference Fourier map, which provided the possibility of free refinement with the isotropic displacement parameter.

Table 3

Single crystal X-ray diffraction analysis data for form 2

Empirical formula C ₂₇ H ₂₈ F ₂ N ₆ O ₃ formula mass 522.55 Crystal size 0.14 x 0.10 x 0.02 mm ³ Data collection temperature 293(2)K Wavelength used for data collection 1.54178 A Crystal system orthorhombic space group Pbca Unit cell parameters 0=9.7245(11) A />=16.8197(14) A c=32.604(4) A a=90° /1=90° ^90°

- 27 040670

Unit cell volume 5332.8(10) A ³ Z (Number of molecules in a unit cell) 8 Density (calculated) 1.302 g/ ^cm3 Theta range for data collection 5.26-66.60° Index ranges -P<A<11 -12<K20 -38</<38 Collected Reflections 24516 Independent Reflections 4708 [7^=0.0927] End R indices [F2 > 2sigma(P2)] 7?1=0.0808, wR2=0.2159 R indices (all data) 7?1=0.1452, wR2=Q.2859

Example 10 Form 1 Solid State Stability Evaluation

Samples of Form 1 crystalline free form of the invention were stored at 25°C and 60% relative humidity (RH) and at 40°C and 75% RH in two configurations a) an open glass vial and b) a closed glass vial placed inside an HDPE bottle, containing a desiccant. The HDPE bottle was induction sealed. At certain intervals, the contents of a representative sample were removed and analyzed by HPLC for chemical purity (shown below as HPLC purity (% a/a)).

Table 4

Crystal form 1, stability test

Time point (weeks) Conditions 40°C/75%RH Closed with dehumidifier 40°C/75%RH Open 25°C/60%RH Closed with dehumidifier 25°C/60%R Η Open 0 98.73 2 NT 98.72 NT NT 4 98.75 98.70 98.75 98.75 8 98.80 98.97 98.87 98.75 24 99.06 98.89 99.02 98.78 36 98.86 98.71 98.80 98.75

NT: not tested

Example 11 Polarized Light Microscopy (PLM) image of Form 1 and Form 2.

Samples of form 1 and form 2 were examined under an optical microscope (Olympus BX51) with a cross-polarized light filter. Images were obtained using a PaxCam camera controlled by Paxlt Imaging Software (version 6.4). Samples were prepared on glass slides with light mineral oil as immersion medium. Depending on the particle size, a 4x, 10x, or 20x objective lens was used for magnification.

Preparation 8. tert-Butyl-2-iodo-1-((2-(trimethylsilyl)ethoxy)methyl)-1,4,6,7-tetrahydro-5Nimidazo[4,5-c]pyridine-5-carboxylate (C -5)

- 28 040670

SEM-CI, NaH, THF, 0°C to r.m. pace. , 6 h

Dimethoxymethane (21.64 ml, 244.54 mmol) was added to a solution of compound C-22 (25 g, 135.86 mmol) in 0.01 M HCl (500 ml). The resulting solution was stirred at 100°C for 18 hours. The solvent was removed in vacuo and the residue was triturated twice with diethyl ether and ethanol, filtered and dried to give compound C-21 (25.2 g, 94.6% yield) .

To a solution of compound C-21 (25.0 g, 127.55 mmol) in methanol (250 ml) was added DIPEA (57.23 ml, 318.87 mmol) followed by (Boc) ₂ O (68.23 ml, 318.87 mol) and the reaction mixture was stirred at room temperature for 18 hours. The resulting reaction mixture was diluted with water (150 ml) and extracted with ethyl acetate (3x200 ml). The combined organic layer was dried over anhydrous sodium sulfate, decanted and concentrated under reduced pressure to give the crude C-20 product, which was used in the next step without further purification.

To a solution of this crude C-20 compound (40.0 g, 123.8 mmol) in methanol (500 ml) was added 1M NaOH solution (200 ml) and the resulting solution was stirred at room temperature for 18 h. The solvent was distilled off in vacuo and the resulting crude material was diluted with water (200 ml) and extracted using ethyl acetate (3x300 ml). The combined organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure to give a crude product which was purified by column chromatography (100-200 silica gel), eluted with 5-10% MeOH:DCM to give the desired product C-19 (20 g, 72 .4% yield).

To a solution of compound C-19 (20 g, 89.68 mmol) in THF (400 ml) was added NIS (30.26 g, 134.52 mmol) at room temperature and the resulting solution was stirred for 2 h at the same temperature. The reaction mixture was diluted with water (200 ml) and extracted with ethyl acetate (2x300 ml), the organic layer was washed with 10% aqueous sodium thiosulfate solution (3x100 ml), then brine, dried over anhydrous sodium sulfate and concentrated under reduced pressure to give crude product C -18, which was used in the next step without further purification.

To a stirred solution of compound C-18 (15.0 g, 42.97 mmol) in THF (150 ml) was added NaH (1.8 g, 45.11 mmol) at 0°C in portions and the reaction mixture was stirred for 1 h at room temperature. SEM chloride (8.18 ml, 45.11 mmol) was then added dropwise at 0°C. The reaction mixture was stirred for 6 hours at room temperature. The progress of the reaction was monitored by TLC, the reaction was quenched with ice water (200 ml) at 0°C and was extracted with ethyl acetate (2x200 ml). The combined organic layer was washed with brine and dried over anhydrous sodium sulfate. The organic layer was filtered and concentrated under reduced pressure to give a crude product which was purified by column chromatography on (100-200) silica gel (eluted with 10-15% BULetexan) to give the desired C-5 product as a viscous liquid (11 g, 55% ).

Preparation 9

- 29 040670

To a stirred suspension of compound C-17 (347.6 g, 973.14 mmol) in anhydrous THF (1000 mL) cooled to -40°C was added n-butyllithium (2.5M in hexane, 362.6 mL, 905 .02 mmol) for 50 min, while retaining the characteristic yellow color of the phosphorus ylide. The reaction mixture was heated to -10°C and stirred for 1 h, then the mixture was cooled to -30°C and a solution of compound R-1 (50 g, 324.38 mmol) in anhydrous THF (200 ml) was added over 30 min . The resulting mixture was warmed to ambient temperature and stirred overnight. The progress of the reaction was monitored by TLC. After completion, the reaction was quenched by the gradual addition of water (500 ml) and was extracted with diethyl ether (3x500 ml). The combined organic layer was washed with water (2x500 ml), brine (250 ml), dried over anhydrous Na ₂ SO ₄ and concentrated under reduced pressure to give the crude compound. The resulting crude product C16 was used in the next step without purification.

To a solution of crude C-16 (110 g, 723.39 mmol) in ethanol (1000 ml) was added 10% Pd/C (50 g). A balloon of hydrogen gas was installed and the reaction mixture was evacuated and refilled with hydrogen three times. The reaction mixture was stirred under hydrogen atmosphere overnight at room temperature. After stirring at room temperature overnight, the reaction was complete. The mixture was filtered through a Celite plug and concentrated in vacuo to give the crude compound, which was purified by silica gel column chromatography (100-200), eluted with 3-5% ethyl acetate/hexane to give the desired C-15 product as a colorless liquid (24 d, 48% over 2 stages).

To a solution of compound C-15 (24.0 g, 155.84 mmol) in MeCN (200 ml) was added a solution of NBS (28.0 g, 157.40 mmol) in MeCN (100 ml). The resulting solution was stirred at room temperature for 18 hours. The solvent was removed in vacuo and the residue was diluted with diethyl ether (100 ml). A precipitate was observed which was removed by filtration, and the filtrate was washed with aqueous sodium sulfite (200 ml) and brine (100 ml), dried over anhydrous sodium sulfate and concentrated in vacuo to give the desired C-14 product as a yellow oil (35 .0 g, 97% yield).

To a solution of compound C-14 (20 g, 85.836 mmol) in dioxane (400 ml) was added compound R-2 (32.69 g, 128.755 mmol) and KOAc (25.27 g, 257.508 mmol). The reaction mixture was degassed with nitrogen for 15 min, then palladium catalyst (3.5 g, 4.29 mmol) was added. The reaction mixture was stirred and heated at 110° C. for 3 hours. The reaction mixture was filtered through a pad of celite and washed with ethyl acetate. The filtrate was diluted with ethyl acetate (200 ml) and washed with water (2x200 ml) and brine (100 ml), dried over sodium sulfate and concentrated in vacuo. The resulting crude product was purified by column chromatography (100-200 silica gel), eluted with 3-5% EtOAc:hexan to give the desired C-12 product (20 g, 83% yield).

Preparation 10

- 30 040670

Mixture of compound C-13 (25 g, 126.84 mmol), 3,4-dihydro-2H-pyran (134.5 ml, 1471.5 ml) and p-TSA (5.57 g, 29.18 mmol) taken up in THF (700 ml) and heated at 60° C. overnight. The reaction mixture was poured into ice water and the aqueous phase was extracted with ethyl acetate. The organic layer was dried over sodium sulfate and filtered. The filtrate was evaporated under reduced pressure and the residue was purified on a silica gel column (230-400) (eluting with 1-2% ethyl acetate in hexane) to give the desired compound C-11 (23.5 g, 67% yield).

Solution of compound C-11 (13.3 g, 47.5 mmol), compound C-12 (15.96 g, 57.0 mmol) and K ₃ PO ₄ (30.21 g, 142.5 mmol) in DMF :H ₂ O (396:99 ml) was degassed with nitrogen for 15 min, then palladium catalyst (1.6 g, 2.37 mmol) was added and the reaction mixture was purged with nitrogen for 5 min. The resulting reaction mixture was heated at 100°C for 12 h with constant stirring. The reaction mixture was filtered through a pad of celite and washed with ethyl acetate. The filtrate was diluted with ethyl acetate (200 ml), extracted with EtOAc (2x100 ml) and washed with cold water (100 ml) and brine (50 ml), dried over sodium sulfate and concentrated in vacuo to give the crude product, which was purified by flash -chromatography (100-200 silica gel), eluted with 10% EtOAc:hexane to give C-10 (14 g, 91.4% yield).

To a solution of compound C-10 (52 g, 146.89 mmol) in methanol (600 ml) was added concentrated HCl (50 ml) and the resulting solution was heated at 60° C. overnight. The reaction mixture was cooled to room temperature and concentrated in vacuo. The residue was diluted with EtOAc (200 ml) and washed with saturated aqueous NaHCO ₃ (2x150 ml). The organic layer was dried over Na ₂ SO ₄ and concentrated to give the desired product C-9 (35 g, 88.9% yield).

To a solution of compound C-9 (17.5 g, 64.81 mmol) in DMF (100 ml) was added KOH (14.5 g, 259.54 mmol) and the mixture was stirred for 15 minutes. A solution of iodine (32.7 g, 129.62 mmol) in DMF (50 ml) was added slowly at 0° C. and the reaction mixture was stirred at room temperature for 30 minutes. The progress of the reaction was monitored by TLC, then the reaction mixture was diluted with water (200 ml) and extracted with ethyl acetate (2x200 ml). The organic layer was washed with saturated aqueous sodium metabisulphite (2x150 ml) and water (3x100 ml), dried over Na ₂ SO ₄ and concentrated in vacuo to give the crude product C-8 (21 g).

To a solution of C-8 (21.0 g, 53.02 mmol) in DCM (230 ml) was added p-TsOH (1.8 g, 10.60 mmol) and the mixture was cooled to 0°C. Compound R-3 (7.04 ml, 79.54 mmol) was added dropwise to this solution, and the reaction mixture was stirred at room temperature overnight. TLC analysis showed completion of the reaction. The reaction mixture was diluted with DCM (2x150 ml) and washed with saturated aqueous NaHCO ₃ (200 ml) and brine (200 ml). The organic layer was dried over anhydrous Na ₂ SO ₄ and concentrated in vacuo to give a crude product which was purified

- 31 040670 flash chromatography to obtain C-7.

A solution of C-7 (10.0 g, 20.83 mmol) in toluene (200 ml) was degassed with nitrogen for 20 min followed by the addition of R-4 (4.89 ml, 22.91 mmol) and Pd(PPh ₃ ) ₄ (1.2 g, 1.04 mmol). The reaction mixture was purged with nitrogen for another 5 min and then stirred at 100°C. After 2 hours, TLC analysis indicated the reaction was complete. The reaction mixture was cooled to room temperature, filtered through a pad of Celite and the residue was washed with ethyl acetate. The concentrated filtrate was purified by column chromatography (neutral alumina), eluted with 2-5% EtOAc:hexane to give product C-6 (6.4 g, 57.6% yield).

Preparation 11 (S-3)

To a solution of compound C-6 (6.4 g, 12.37 mmol) in toluene (100 ml) was added compound C-5 (5.9 g, 12.37 mmol). The reaction mixture was degassed with nitrogen for 20 min followed by the addition of copper(I) iodide (470 mg, 2.47 mmol) and Pd(PPh ₃ ) ₄ (714 mg, 1.237 mmol), then stirred at 100°C for 12 h The progress of the reaction was monitored by TLC. The reaction mixture was cooled to room temperature and filtered through a pad of Celite, the residue was washed with ethyl acetate. The organic layer was diluted with water, separated and the organic washed with brine, dried over Na ₂ SO ₄ and filtered. The filtrate was concentrated under reduced pressure to give a crude product which was purified by column chromatography (silica gel, 100-200 mesh), eluted with 20% EtOAc:hexane to give C-4 (4 g, 45.9%).

To a solution of compound C-4 (4.0 g, 5.6 mmol) in dioxane (30 ml) was added concentrated HCl (30 ml). The reaction mixture was stirred at 70° C. for 16 hours. The progress of the reaction was followed by LCMS. The reaction mixture was cooled to room temperature, concentrated in vacuo, triturated with diethyl ether and purified by preparative HPLC to give the desired compound C-3 (0.65 g, 29.5%).

Example 12 1-(2-(6-(2-Ethyl-5-fluoro-4-hydroxyphenyl)-1H-indazol-3-yl)-1,4,6,7-tetrahydro-5-imidazo[4,5- c]pyridin-5-yl)-2-morpholinoethan-1-one (C-1)

To a mixture of C-3 (180 mg, 0.460 mmol) in DCM (0.5 ml) at room temperature was added a solution of 1M boron tribromide in DCM [100 ml) (2.299 ml, 2.299 mmol). The resulting mixture was stirred for 30 minutes, then concentrated. The resulting residue was co-evaporated with MeOH (3x3.0 ml), again

- 32 040670 was dissolved in 1:1 AcOH:H2O (3.0 ml), filtered and purified by reverse phase preparative

HPLC. The desired fractions were combined and freeze dried to give C-2 ((5-ethyl-2-fluoro-4(3-(4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-2- yl)-1H-indazol-6-yl)phenol) as the TFA salt.

HATU (25.5 mg, 0.067 mmol) and DIEA (0.043 ml , 0.244 mmol). The resulting mixture was stirred at room temperature overnight. The reaction mixture was diluted with MeOH (0.5000 ml) and water (0.500 ml). LiOH (2.193 mg, 0.092 mmol) was added. The resulting mixture was heated at 65°C for 1 h. The reaction mixture was then concentrated, the resulting residue was treated with a mixture of DCM (0.500 ml) and TFA (0.500 ml) at room temperature for 30 min, concentrated, redissolved in 1:1 AcOH :H ₂ O (1.5 ml), filtered and purified by reverse phase preparative HPLC to give compound C-1 as TFA salt, (m/z): [M+H]+ calculated for C27H29FN6O3 504.23, found 505.2.

biological analyses.

Compound 1 has been characterized in one or more of the following biological assays.

Analysis 1. Biochemical analyzes of JAK kinases.

A panel of four LanthaScreen JAK chemistry assays (JAK1, 2, 3, and Tyk2) were run in conventional kinase reaction buffer (50 mM HEPES, pH 7.5, 0.01% Brij-35, 10 mM MgCl ₂ and 1 mM EGTA) . Recombinant GST-labeled JAK enzymes and GFP-labeled STAT1 peptide substrate were obtained from Life Technologies.

Serially diluted compound was pre-incubated with each of the four JAK enzymes and substrate in white 384-well microplates (Corning) at ambient temperature for 1 h. ATP was then added to initiate kinase reactions in a total volume of 10 ml, with 1% DMSO. Final enzyme concentrations for JAK1, 2, 3, and Tyk2 were 4.2 nM, 0.1 nM, 1 nM, and 0.25 nM, respectively; the respective Km ATP concentrations used were 25 mM, 3 mM, 1.6 mM and 10 mM; the substrate concentration was 200 nM for all four assays. Kinase reactions were allowed to proceed for 1 h at ambient temperature, then 10 µl of EDTA preparation (final concentration 10 mM) and Tb-anti-pSTAT1 (pTyr701) antibody (Life Technologies, 2 nM final concentration) in TR-FRET buffer were added for breeding (Life Technologies). The plates were left to incubate at ambient temperature for 1 h, then read on an EnVision plate reader (Perkin Elmer). Emission signals (520 nm/495 nm) were recorded and used to calculate percent inhibition based on DMSO and background controls.

For dose-response analysis, percent inhibition was plotted against compound concentrations and IC ₅₀ values were determined from a 4-parameter robust fitting model using Prism (GraphPad Software). The results were expressed as pIC ₅₀ (negative logarithm of IC ₅₀ ) and then converted to pKi (negative logarithm of the dissociation constant, Ki) using the Cheng-Prusoff equation.

Compound 1 showed the following enzyme activity.

Table 5

JAK1 pK JAK 2 RK JAK3 pK Tyk2pK 10 10.6 9.7 8.7

Assay 2 Cellular assay for JAK activity.

Inhibition of IL-13.

AlphaScreen cellular assay for JAKI activity was performed by measuring interleukin-13 (IL-13, R&D Systems)-induced STAT6 phosphorylation in BEAS-2B human lung epithelial cells (ATCC). Ahtu-STAT6 antibody (Cell Signaling Technologies) was conjugated to AlphaScreen acceptor beads (Perkin Elmer), while anti-pSTAT6 (pTyr641) antibody (Cell Signaling Technologies) was biotinylated using EZ-Link sulfo-NHS-biotin (Thermo Scientific).

BEAS-2B cells were grown at 37°C in a 5% CO2 humidified incubator in 50% DMEM/50% F-12 medium (Life Technologies) supplemented with 10% FBS (Hyclone), 100 U/ml penicillin, 100 μg/ml streptomycin (Life Technologies); and 2 mM GlutaMAX (Life Technologies). On day 1 of analysis, cells were seeded at a density of 7500 cells/well in white poly-O-lysine coated 384-well plates (Corning) with 25 μl of medium and left in the incubator overnight for adhesion. On day 2 of the assay, the medium was removed and replaced with 12 ml of assay buffer (Hank's balanced salt solution/HBSS, 25 mM HEPES and 1 mg/ml bovine serum albumin/BSA) containing the dose responses of the test compounds. The compound was serially diluted in DMSO and then diluted 1000-fold again in medium to bring the final concentration of DMSO to 0.1%. Cells were incubated with test compounds at 37° C. for 1 hour and then 12 μl of prewarmed IL-13 (80 ng/ml in assay buffer) was added for stimulation. After incubation at 37°C for 30 min, assay buffer

- 33 040670 (containing compound and IL-13) was removed and 10 μl of cell lysis buffer (25 mM HEPES, 0.1% SDS, 1% NP-40, 5 mM MgCl ₂ , 1.3 mM EDTA, 1 mM EGTA supplemented with protease inhibitors Complete Ultra mini and PhosSTOP from Roche Diagnostics). The plates were shaken at ambient temperature for 30 minutes before adding detection reagents. First, a mixture of biotin-anti-pSTAT6 and anti-STAT6-conjugated acceptor beads was added and incubated at ambient temperature for 2 h, followed by the addition of streptavidin-conjugated donor beads (Perkin Elmer). After a minimum of 2 hours of incubation, the assay plates were read on an EnVision plate reader. AlphaScreen luminescence signals were recorded and used to calculate percent inhibition based on DMSO and background controls.

For dose-response analysis, percent inhibition was plotted against compound concentrations and IC ₅₀ values were determined from a 4-parameter robust fitting model using the Prism software. The results can also be expressed as the negative logarithm of the IC ₅₀ value, pIC ₅₀ . In this assay, Compound 1 showed a pIC ₅₀ value of 7.9.

Assay 3 Cellular JAK activity assay.

Inhibition of IL-2/anti-CD3-stimulated IFNy in human PBMCs.

The activity of the test compound to inhibit interleukin-2 (IL-2)/anti-CD3 stimulated interferon gamma (IFNy) was measured in human peripheral blood mononuclear cells (PBMC) isolated from human whole blood (Stanford Blood Center). Because IL-2 signals through JAK, this assay provides a measure of JAK activity in cells.

(1) Human peripheral blood mononuclear cells (PBMCs) were isolated from human whole blood from healthy donors using a ficoll gradient. Cells were cultured in a 37°C, 5% CO ₂ humidified incubator in RPMI (Life Technologies) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Life Technologies), 2 mM Glutamax (Life Technologies), 25 mM HEPES (Life Technologies) and IX penicillin/streptomycin (Life Technologies). Cells were seeded at a density of 200,000 cells/well in medium (50 μl) and cultured for 1 hour. The compound was serially diluted in DMSO and then diluted again 500-fold (to 2x final analyte concentration) in medium. Dilutions of test compound (100 µl/well) were added to cells and incubated at 37°C, 5% CO2 for 1 h followed by the addition of IL-2 (R&D Systems; final concentration 100 ng/ml) and anti-CD3 (BD Biosciences ; final concentration 1 µg/ml) in prewarmed quantitation medium (50 µl) for 24 h.

(2) After cytokine stimulation, the cells were centrifuged at 500 g for 5 min, and the supernatants were removed and frozen at -80°C. To determine the inhibitory activity of test compounds in response to IL-2/anti-CD3, IFNy concentrations in the supernatant were measured by ELISA (R&D Systems). IC ₅₀ values were determined from analysis of inhibition curves representing IFNy concentration versus compound concentration. Data are expressed as pIC ₅₀ (negative decimal logarithm of IC ₅₀ ) values. In this assay, compound 1 showed a pIC ₅₀ value of about 6.7.

Assay 4 Cellular assay for JAK activity.

Inhibition of IL-2-stimulated pSTAT5 in CD4+ T cells.

The activity of the test compound to inhibit interleukin-2 (IL-2)/anti-CD3-stimulated STAT5 phosphorylation was measured in CD4-positive (CD4+) T cells in human peripheral blood mononuclear cells (PBMC) isolated from human whole blood (Stanford Blood Center) , using flow cytometry. Because IL-2 signals through JAK, this assay provides a measure of JAK activity in cells.

CD4+ T cells were identified using a phycoerythrobilin (PE)-conjugated antu-CD4 antibody (clone RPA-T4, BD Biosciences), while an Alexa Fluor 647-conjugated anti-pSTATS antibody (pY694, clone 47, BD Biosciences) was used to detect STAT5 phosphorylation .

(1) Assay protocol 3, paragraph (1) was followed, except that cytokine stimulation using 1b-2/anti-CD3 was performed for 30 minutes instead of 24 hours.

(2) After cytokine stimulation, cells were fixed with prewarmed fixative solution (200 µl; BD Biosciences) for 10 min at 37°C, 5% CO2, washed twice with DPBS buffer (1 ml, Life Technologies) and resuspended in ice-cold Perm buffer III (1000 µl, BD Biosciences) for 30 min at 4°C. Cells were washed twice with 2% FBS in DPBS (FACS buffer) and then resuspended in FACS buffer (100 µl) containing anti-CD4 PE (1:50 dilution) and anti-CD3 anti-CD3Alexa Fluor 647 (1:5 dilution) , for 60 min at room temperature in the dark. After incubation, cells were washed twice in FACS buffer, then analyzed using an LSRII flow cytometer (BD Biosciences). To determine the inhibitory activity of a test compound in response to IL-2/anti-CD3, the mean fluorescence intensity (MFI) of pSTAT5 in CD4+ T cells was measured. IC ₅₀ values were determined from analysis of inhibition curves representing MFI versus compound concentration. Data are expressed as pIC ₅₀ (negative decimal logarithm of IC ₅₀ )

- 34 040670 values. In this assay, Compound 1 showed a pIC ₅₀ value of about 7.7.

Assay 5 Cellular assay for JAK activity.

Inhibition of IL-6-stimulated CCL2 (MCP-1) in human PBMCs.

The activity of the test compound to inhibit interleukin-6 (II-6)-stimulated production of CCL2 (MCP-1) was measured in human peripheral blood mononuclear cells (PBMCs) isolated from human whole blood (Stanford Blood Center). Because IL-6 signals through JAK, this assay provides a distal determination of JAK activity in cells.

(1) Followed assay protocol 3, paragraph (1), for incubation with test compounds. In this assay, after addition of test compounds to the wells and incubation, IL-6 (R&D Systems; final concentration 10 ng/mL) was added in prewarmed assay medium (50 µl).

(2) After 48 hours of cytokine stimulation, the cells were centrifuged at 500 g for 5 minutes, and the supernatants were removed and frozen at -80°C. To determine the inhibitory activity of the test compound in response to IL-6, the concentrations of CCL2 in the supernatant (MCP-1) were measured by ELISA (R&D Systems). IC ₅₀ values were determined from analysis of inhibition curves representing CCL2/MCP-1 concentration versus compound concentration. Data are expressed as pIC ₅₀ (negative decimal logarithm of IC ₅₀ ) values. In this assay, Compound 1 showed a pIC ₅₀ value of about 6.4.

Assay 6 Cellular assay for JAK activity.

Inhibition of IFNγ-induced pSTAT1.

The activity of the test compound for inhibiting interferon gamma (IFNy) stimulated STAT1 phosphorylation was measured in CD14-positive (CD14+) monocytes isolated from human whole blood (Stanford Blood Center) using flow cytometry. Because IFNy signals through JAK, this assay provides a measure of JAK activity in cells.

Monocytes were identified using a fluorescein isothiocyanate (FITC) conjugated anti-CD14 antibody (clone RM052, Beckman Coulter) and an Alexa Fluor 647 conjugated anti-pSTAT1 antibody (pY701, clone 4a, BD Biosciences) was used to detect STAT1 phosphorylation.

Human peripheral blood mononuclear cells (PBMCs) were isolated from human whole blood from healthy donors using a ficoll gradient. Cells were cultured in a 37°C, 5% CO ₂ humidified incubator in RPMI (Life Technologies) supplemented with 10% fetal bovine serum (FBS, Life Technologies), 2 mM Glutamax (Life Technologies), 25 mM HEPES (Life Technologies) and 1X penicillin/streptomycin (Life Technologies). Cells were seeded at a density of 250,000 cells/well in medium (200 µl), cultured for 2 h and resuspended in quantitation medium (50 µl) (RPMI supplemented with 0.1% bovine serum albumin (Sigma), 2 mM Glutamax, 25 mM HEPES and 1X penicillin/streptomycin) containing various concentrations of test compounds. Compounds were serially diluted in DMSO and then diluted 1000-fold again in medium to bring the final concentration of DMSO to 0.1%. Test compound dilutions were incubated with cells at 37°C, 5% CO2 for 1 hour followed by the addition of prewarmed IFNy (R&D Systems) in medium (50 µl) at a final concentration of 0.6 ng/ml for 30 minutes. After cytokine stimulation, cells were fixed with pre-warmed fixative solution (100 µl) (BD Biosciences) for 10 min at 37°C, 5% CO2, washed twice with FACS buffer (1 ml) (1% BSA in PBS), resuspended in a mixture 1:10 anti-CD14 FITC:FACS buffer (100 µl) and incubated at 4°C for 15 min. Cells were washed once and then resuspended in ice-cold Perm buffer III (BD Biosciences) (100 μl) for 30 min at 4°C. Cells were washed twice with FACS buffer and then resuspended in 1:10 anti-pSTAT1 Alexa Fluor 647:FACS buffer (100 µl) for 30 min at room temperature in the dark, washed twice with FACS buffer and analyzed using an LSRII flow cytometer. (BD Biosciences).

To determine the inhibitory activity of a test compound, the mean fluorescence intensity (MFI) of pSTAT1 was measured in CD14+ monocytes. IC ₅₀ values were determined from analysis of inhibition curves representing MFI versus compound concentration. Data are expressed as pIC50 (negative decimal logarithm of IC50) values. In this assay, Compound 1 showed a pIC50 value of about 7.1.

Analysis 7. Pharmacokinetics in rabbit ocular tissue.

The purpose of this assay was to determine the pharmacokinetics of the test compound in rabbit ocular tissues.

solution composition.

1-(2-(6-(2-ethyl-5-fluoro-4-hydroxyphenyl)-4-fluoro-1H-indazol-3-yl)-1,4,6,7-tetrahydro-5H-imidazo [4 ,5-c]pyridin-5-yl)-2-morpholinoethan-1-one (1), obtained in example 2, was dissolved in 2% 2

- 35 040670 hydroxypropyl-in-cyclodextrin until the target concentration of 1 mg/ml is reached. A bilateral intravitreal injection (50 μl/eye) of test compound solution was administered to New Zealand white rabbits (50 μg/eye). The concentration of the test compound was measured in the ocular tissues: vitreous, intraocular fluid, retina/choroid and irido-ciliary zone at predetermined time points after injection (30 min, 4 h, 1 d, 3 d, 7 d, 14 d). The introduction was carried out to two rabbits (four eyes) for each time point. In vitreous tissue, Compound 1 showed a biphasic decrease in concentration, characterized by an initial decrease in concentration with a half-life of about 9 hours, and finally with a final half-life of about 2 days. The compound was also found to distribute rapidly in the retina and choroid and exhibit the same pharmacokinetic profile as in vitreous tissue.

Suspension composition.

The suspension composition was obtained by combining compound 1 of example 2 (form 1) with 0.5% hydroxypropyl methylcellulose (HPMC E5) + 0.02% Tween 80 in saline to achieve a target concentration of 5 mg/ml, 20 mg/ml and 80 mg/ml ml for doses of 0.25 mg/eye, 1 mg/eye and 4 mg/eye, respectively. A bilateral intravitreal injection (50 μl/eye) of the test compound suspension was administered to New Zealand white rabbits. The concentration of the test compound was measured in ocular tissues, as in the analysis of the composition of the solution, at 30 min, 4 h, 24 h, 72 h, 7 d, 14 d, 28 d, 56 d and 84 d after injection. For the 4 mg/eye dosing group, data were also collected at an additional time point at 168 days. All dose groups showed measurable drug concentration in the eye up to the last time point analyzed in this study. Sustained long-term exposure was observed for all doses after 12 weeks (84 days). Long term exposure was observed at 24 weeks (84 days) for the 4 mg/eye dose group. The compound showed a linear decrease in drug concentration in vitreous tissue from 30 minutes to 24 weeks with a drug elimination rate of approximately 5-10 μg/mL/day. The rate of excretion corresponds to the solubility of compound 1 in the carrier and pharmacokinetics in the eye tissues when using the solution composition. All dose groups showed measurable drug concentration in the eye up to the last time point analyzed in this study. Therefore, it is likely that drug exposure is longer than observed in this study. The plasma drug concentration was measured and found to be at least 3 orders of magnitude lower than the vitreous tissue concentration at all three concentrations.

A suspension formulation was prepared by combining compound 1 of Example 5 (form 2) with 0.5% hydroxypropyl methylcellulose (HPMC E5) + 0.02% Tween 80 in saline to reach the target concentration of 0.4 mg/ml, 1 mg/ml, 2 mg/ml and 20 mg/ml for doses of 0.02 mg/eye, 0.05 mg/eye, 0.1 mg/eye and 1 mg/eye, respectively. A bilateral intravitreal injection (50 μl/eye) of a suspension of the test compound was administered to Dutch rabbits. The concentration of the test compound was measured in vitreous fluid, intraocular fluid, irido-ciliary zone, retina, retinal pigment epithelial cells/choroidal cells and plasma at 30 min, 7 d, 14 d, 28 d, 42 d and 56 d after injection. The compound showed a very gradual decrease in drug concentration in the vitreous tissue from 30 min to the last analyzed time point (28 d for the dose of 0.02 mg/eye, 42 d for the dose of 0.05 mg/eye and doses of 1 mg/eye and 56 e for a dose of 0.1 mg/eye). All dose groups showed measurable drug concentration in the eye up to the last time point analyzed in this study. Therefore, it is likely that drug exposure is longer than observed in this study. The plasma drug concentration was measured and found to be at least 3 orders of magnitude lower than the vitreous tissue concentration at all three concentrations. 3 orders of magnitude correspond to a logarithmic scale (ie, 1000 on a non-logarithmic scale).

Analysis 8. Pharmacodynamic analysis.

Inhibition of IL6-induced pSTAT3 in rats.

The ability of a test compound to inhibit IL-6-induced pSTAT3 as a single intravitreal injection was measured in rat retinal/choroid homogenates.

A suspension formulation was prepared by combining compound 1 of Example 2 with 0.5% hydroxypropyl methylcellulose (HPMC E5 LV), 0.02% Tween 80 and 9 mg/ml sodium chloride in purified water to reach the target concentration of 10 mg/ml.

Female Lewis rats were injected intravitreally (i/v) (5 μl/eye) with the suspension composition. Three days later, IL-6 (Peprotech; 0.1 mg/ml; 5 μl/eye) or vehicle to induce pSTAT3 was administered intravitreally. The ocular tissues were excised 1 hour after the second i/v injection of IL-6. Retinal/choroid tissues were homogenized and pSTAT3 levels were measured using ELISA (Cell Signaling Technology). Percent inhibition of IL-6-induced pSTAT3 was calculated compared to vehicle/vehicle and vehicle/III-6 groups. An inhibition greater than 100% reflects a reduction in pSTAT3 levels to below those seen in the vehicle/vehicle group.

In pre-treatment 3 days before IL-6 stimulation, a dose of 50 μg of Compound 1 administered with

- 36 040670 using a suspension formulation, inhibited IL-6-induced pSTAT3 by 116% in retinal/choroid tissue.

Analysis 9. Pharmacodynamic analysis.

Inhibition of IFNγ-induced IP-10 in rabbit eyes.

The ability of a test compound, when administered intravitreally, to inhibit interferon-gamma (IFNγ)-induced levels of IP-10 protein was measured in the vitreous and retinal/choroid tissues of the rabbit.

A suspension formulation was prepared by combining compound 1 of Example 2 (Form 1) with 0.5% hydroxypropyl methylcellulose (HPMC E5), 0.02% Tween 80 and 9 mg/ml sodium chloride in purified water to reach the target concentration of 20 mg/ml.

Male New Zealand white rabbits (Liveon Biolabs, India) were used for the studies. Animals were allowed to acclimate after being taken to the research laboratory (Liveon Biolabs, India). Each rabbit was given two intravitreal (i/v) injections with a total dose volume of 50 μl/eye. The first i/v injection (45 μl/eye) delivered 0.9 mg of test compound or vehicle. One week later, a second i/v injection (5 μl/eye) delivered IFNy (1 μg/eye; stock solution 1 mg/ml; Kingfisher Biotech) or IP-10 induction vehicle. On the day of injection, rabbits were anesthetized with an intramuscular injection of ketamine (35 mg/kg) and xylazine (5 mg/kg). When deep anesthesia was achieved, each eye was flushed with sterile saline and injected and/injected using a 0.5 ml insulin syringe (50 units=0.5 ml) with a 31 gauge needle on the suprasnasal side of both eyes, marking the position with a fixed Braunstein caliper (2 3/4) at a distance of 3.5 mm from the rectus muscle and 4 mm from the limbus.

Tissues were harvested 24 hours after the second and/or injection of IFNy. Vitreous (VH) and retinal/choroid (R/C) tissues were harvested and homogenized, and IP-10 protein levels were measured using the CXCL10 (IP-10) rabbit ELISA kit (Kingfisher Biotech). Percent inhibition of IFNy-induced IP-10 was calculated compared to vehicle/vehicle and vehicle/IFNy groups.

When pre-treated 1 week prior to IFNy stimulation, the suspension composition of Compound 1 inhibited IFNy-induced IP-10 by 81% and 80% in the vitreous and retinal/choroid tissues, respectively. Similar efficacy was also observed with pre-treatment 1 month prior to IFNy stimulation.

Analysis 10. Pharmacokinetics in skin tissues of mice and mini-pigs.

The aim of this assay was to determine the pharmacokinetics of the test compound in epidermis, dermis, and plasma following 24-hour exposure to the skin of an intact mouse or mini-mump.

-(2-(6-(2-Ethyl-5-fluoro-4-hydroxyphenyl)-4-fluoro-1 H-indazol-3-yl)-1,4,6,7-tetrahydro-5H-imidazo [4 ,5-c]pyridin-5-yl)-2-morpholinoethan-1-one (1) was formulated as a 0.5% (w/w) cream or ointment as described in Composition A or Composition B , respectively, shown in table. 6.

25 g male Balb/c mice were shaved off their backs 24 h prior to injection, exposing an area of at least 6 cm (about 10% of the body surface) and, in a separate experiment, an area of at least 450 cm was exposed in 10 kg Gottingen minipigs ( about 10% of the body surface). At time 0 after anesthesia with isoflurane, the test compound was applied to the back of mice or mini-pigs at a dose of 25 μl/cm ² . The skin was covered with an adhesive coating to prevent loss of connection when the animal was caged or bedded.

After 24 hours of exposure, the backs were gently washed with soap and water to remove unabsorbed drug and dried. Immediately after this washing, blood was taken by cardiac puncture in mice and venipuncture in mini-pigs. The outer layer (stratum corneum) was then removed using adhesive tape. After exposure to the epidermis, a 0.5 cm needle biopsy was made. The epidermis and dermis were quickly separated, weighed and quickly frozen. Similar samples were obtained 48 hours post-dose in mice and 48 hours, 94 hours and 168 hours (7 days) post-dose in mini-gilts.

Samples of the epidermis and dermis were homogenized in water 1:10 wt./about. using ultrasonic homogenizer Covaris. Samples were extracted with 3 volumes of acetonitrile and quantitated from a standard curve using LC-MS analysis. As evidenced by the pharmacokinetic parameters AUC ₀ _ _t for plasma, epidermis and dermis, shown in the table below. 7, significant compound exposure was in the epidermal and dermal layers, while plasma exposure was negligible in mice with Composition A and below the limit of quantitation in mice with Composition B and both formulations in minipigs.

- 37 040670

Table 6

Composition A Composition B Compound 1 0.5% Compound 1 0.5% Stearic acid 5% Octylhydroxystearate 5% Cetosteal alcohol 5% C8-C10 Triglyceride 5% Isopropyl palmitate 4% Vaseline (Vaseline oil) 79.5% Octylhydroxystearate 2% N-Methylpyrrolidone 10% BRU S2 (PEG 2 Steayl Ether) 1.08% BRU S20 (PEG 20 Steayl Ether) 6.92% N-methylpyrrolidine 10% PEG 400 10% Water purified by reverse osmosis 55.5%

Plasma AUCo_ _t (µg*h/ml) Epidermis AUCo_ _t (µg*h/g) Derma AUCo_ _t (µg*h/g) Mouse 0.022 1370 99 Composition A Mouse <0.001 10700 1110 Composition B mini pig <0.001 1220 44 Composition A mini pig <0.001 2460 88 Composition B

Table 7

Analysis 11 Pharmacokinetics in lung and plasma in mice.

Plasma and lung concentrations of Compound 1 and their ratios were determined as follows.

BALB/c mice from Charles River Laboratories were used in the assay. Compound 1 of Form 1 of Example 2 was formulated as a suspension in 0.01% Tween 80 in saline (0.9% sodium chloride in water) at a concentration of 0.1 mg/ml. 50 μl of the suspension preparation was injected into the mouse trachea by oral aspiration. At various time points (0.083, 1, 4, 24, 48, 72 and 96 hours). After dosing, blood samples were taken by cardiac puncture and intact lungs were excised from mice. Blood samples were centrifuged (Eppendorf centrifuge, 5804R) for 4 min at approximately 12,000 rpm at 4° C. to collect plasma. The lungs were dried by blotting with a swab, weighed and homogenized at a 1:3 dilution in sterile water. Compound 1 plasma and lung concentrations were determined by LC-MS analysis against analytical standards plotted as a standard curve in the test matrix. Good exposure in the lungs was found with an AUC (0-96 h) of 360 µg-h/g. The lung half-life was calculated as being approximately 40 hours. The lung:plasma ratio was defined as the ratio of AUC in µg-h/g in lung to AUC µg-h/mL in plasma (where AUC is usually defined as the area under the curve for the concentration of test compound in depending on time). The AUC lung:plasma ratio was 1780, indicating a very low plasma exposure.

Analysis 12. Pharmacodynamic analysis.

Inhibition of IFNY-induced pSTAT1 in rabbit eyes.

The ability of a test compound to inhibit interferon-gamma (IFNγ)-induced phosphorylation of STAT1 protein (pSTAT1) as a single intravitreal injection was measured in rabbit retinal/choroid tissue.

A suspension formulation was prepared by combining compound 1 of Example 2 (Form 1) with 0.5% hydroxypropyl methylcellulose (HPMC E5), 0.02% Tween 80 and 9 mg/ml sodium chloride in purified water to reach the target concentration of 20 mg/ml.

Male New Zealand white rabbits (Liveon Biolabs, India) were used for the studies.

- 38 040670

Animals were allowed to acclimate after being taken to the research laboratory (Liveon Biolabs, India). Each rabbit was given two intravitreal (i/v) injections with a total dose volume of 50 μl/eye. The first i/v injection (45 μl/eye) delivered 0.9 mg of test compound or vehicle. One week later, a second i/v injection (5 μl/eye) delivered IFNy (1 μg/eye; stock solution 1 mg/ml; Kingfisher Biotech) or IP-10 induction vehicle. On the day of injection, rabbits were anesthetized with an intramuscular injection of ketamine (35 mg/kg) and xylazine (5 mg/kg). When deep anesthesia was achieved, each eye was flushed with sterile saline and injected and/injected using a 0.5 ml insulin syringe (50 units=0.5 ml) with a 31 gauge needle on the suprasnasal side of both eyes, marking the position with a fixed Braunstein caliper (2 3/4) at a distance of 3.5 mm from the rectus muscle and 4 mm from the limbus.

Tissues were harvested 2 hours after the second and/or injection of IFNy. Retinal/choroid (R/C) tissues were harvested and homogenized, and pSTAT1 protein levels were measured using quantitative Western blot analysis on a ProteinSimple WES device. Percent inhibition of IFNy-induced pSTAT1 was calculated compared to the vehicle/vehicle and vehicle/IFNy groups.

When pre-treated 1 week prior to IFNy stimulation, the suspension composition of Compound 1 inhibited IFNy-induced pSTAT1 by 85%. After pre-administration of a single dose of the suspension composition 3 months prior to IFNy stimulation, the suspension composition of Compound 1 inhibited IFNy-induced pSTAT1 by 76%.

A suspension formulation was prepared by combining Example 5 Compound 1 (Form 2), with 0.5% Hydroxypropyl Methylcellulose (HPMC E5) + 0.02% Tween 80 in saline to achieve a target concentration of 11.1, 3.3 and 1.1 mg /ml

Male New Zealand white rabbits (Liveon Biolabs, India) were used for the studies. Animals were allowed to acclimate after being taken to the research laboratory (Jubilant Biosys Ltd., India). Each rabbit was given two intravitreal (i/v) injections with a total dose volume of 50 μl/eye. The first i/v injection (45 μl/eye) delivered 500 μg, 150 μg or 50 μg of test compound or vehicle. Two weeks later, a second i/v injection (5 μl/eye) delivered IFNy (1 μg/eye; stock solution 1 mg/ml; Kingfisher Biotech) or IP-10 induction vehicle. On the day of injection, rabbits were anesthetized with an intramuscular injection of ketamine (35 mg/kg) and xylazine (5 mg/kg). When deep anesthesia was achieved, each eye was flushed with sterile saline and injected and/injected using a 0.5 ml insulin syringe (50 units=0.5 ml) with a 31 gauge needle on the suprasnasal side of both eyes, marking the position with a fixed Braunstein caliper (2 3/4) at a distance of 3.5 mm from the rectus muscle and 4 mm from the limbus.

Tissues were harvested 2 hours after the second and/or injection of IFNy. Retinal/choroid (R/C) tissues were harvested and homogenized, and pSTAT1 protein levels were measured using quantitative Western blot analysis on a ProteinSimple WES device. Percent inhibition of IFNy-induced pSTAT1 was calculated compared to the vehicle/vehicle and vehicle/IFNy groups.

When pre-treated 2 weeks prior to IFNy stimulation, the suspension formulation of Compound 1 inhibited IFNy-induced pSTAT1 by 79% for the 500 μg dose, 58% for the 150 μg dose, and 61% for the 50 μg dose.

Analysis 13. Screening of kinome and GINI coefficient.

Compounds 1 and C-1 were screened against other kinases to evaluate their selectivity profile.

Kinase-labeled T7 phage strains were grown in parallel in 24-well blocks in an E. coli host derived from strain BL21. E. coli were grown to log phase and infected with T7 phage from the frozen strain (multiplicity of infection=0.4) and incubated with shaking at 32°C until lysis (90-150 min). Lysates were centrifuged (6000xg) and filtered (0.2 μm) to remove cell debris. The remaining kinases were produced in HEK-293 cells and then labeled with DNA for qPCR detection.

Streptavidin-coated magnetic beads were treated with biotinylated low molecular weight ligands for 30 min at room temperature to prepare affinity resins for kinase assays. Ligand-bound beads were blocked with excess biotin and washed with blocking buffer (SeaBlock (Pierce), 1% BSA, 0.05% Tween 20, 1 mM DTT) to remove unbound ligand and reduce non-specific binding to phage. Binding reactions were pooled by combining kinases, ligan-bound affinity beads and test compounds in 1x binding buffer (20% SeaBlock, 0.17x PBS, 0.05% Tween 20, 6 mM DTT). Test compounds were prepared as 40x stock solutions in 100% DMSO and directly diluted in the assay. All reactions were performed in polypropylene 384-well plates in a final volume of 0.04 ml. The assay plates were incubated at room temperature with shaking for 1 hour and the affinity beads were washed with wash buffer (1 x PBS, 0.05% Tween 20). The beads were then resuspended in elution buffer (1x PBS, 0.05% Tween 20, 0.5 μM non-biotinylated affinity ligand) and

- 39 040670 were incubated at room temperature with shaking for 30 minutes. The kinase concentration in the eluates was measured by quantitative PCR.

Compounds were screened at 1 μM, and the results of binding interactions for the primary screen in table. 8 and 9 are shown as % inhibition (=100-((test compound signal-positive control signal)/((negative control signal)-(positive control signal))x100) where the negative control is DMSO and the positive control is control connection.

________________________________________________________________________ Table 8

Compound/Kinase ALK AURK A CDK2 CDK7 CDK9 CSF1 R ERNV6 GSK3B 1 75 eleven 6 74 9 98 88 31 C-1 81 57 53 99 95 100 98 68

Compound/Kinase KIT CANCER RKAS PLK4 SL SR SYK VEGFR2 4 ALF A TO WITH 1 87 93 20 58 100 93 46 42 C-1 99 99 70 68 100 100 78 63

Table 9

Compound 1 was found to show significantly lower binding inhibition for CDK7 and CDK9 than Compound C-1. Compound 1 also had lower binding inhibition for some other kinases.

Compounds 1 and C-1 were both screened against 35 different kinases. The GINI coefficient was determined for both compounds. Compound 1 had a GINI score of 0.62 and Compound C-1 had a GINI score of 0.46. The GINI coefficient is used to express the selectivity of a compound for a panel of kinases (Graczyk, J. Med. Chern., 2007, 50, 5773-5779). A higher value corresponds to a more selective compound.

The only structural difference between compound 1 and compound C-1 is the presence of a fluorine group in the core. This structural difference has been shown to significantly affect the selectivity of the compound for kinome.

Assay 14 Cytotoxicity assay.

The CellTiter-Glo luminescent cell viability/cytotoxicity assay was performed in BEAS-2B human lung epithelial cells (ATCC) under normal growth conditions.

Cells were grown at 37°C in a 5% CO ₂ humidified incubator in 50% DMEM/50% F-12 medium (Life Technologies) supplemented with 10% FBS (Hyclone), 100 U/ml penicillin, 100 μg/ml streptomycin ( Life Technologies) and 2 mM GlutaMAX (Life Technologies). On day 1 of the assay, cells were seeded at a density of 500 cells/well in white 384-well tissue culture plates (Coming) with 25 μl of medium and allowed to adhere overnight in the incubator. On day 2 of the assay, 5 µl of medium containing the dose-response test compounds was added and incubated at 37°C for 48 h. incubated for another 10 min, then read on an EnVision plate reader. Recorded luminescence signals and calculated the control values of the percentage of DMSO.

For dose-response analysis, percent DMSO control data was plotted against compound concentrations to obtain dose-response curves by drawing a line connecting each data point. The concentration at which each curve crosses the 15% inhibition threshold is defined as CC ₁₅ .

As expected, test compounds showing a higher CCi ₅ value in this assay are less likely to cause cytotoxicity.

Compound 1 showed a CCi ₅ value of 3.16 μM, while compound C-1 showed a CCi ₅ value of 630 nM. Therefore, based on this analysis, Compound 1 is significantly less likely to cause cytotoxicity than Compound C-1.

The only structural difference between compound 1 and compound C-1 is the presence of a fluorine group in the core. This structural difference has been shown to significantly affect the cytotoxicity of the compound.

Although the present invention has been described with reference to specific aspects or embodiments thereof, it will be appreciated by those skilled in the art that various

Claims (37)

changes, or substitute equivalents may be used without departing from the spirit and scope of the invention. In addition, to the extent permitted by applicable patent laws and regulations, all publications, patents and patent applications cited in this application are hereby incorporated by reference in their entirety to the same extent as if each document has been separately incorporated into this application by reference. CLAIM 1. Compound of formula 1 or a pharmaceutically acceptable salt thereof. 2. Compound of Formula 1 3. The crystalline form of the compound of formula 1 wherein the crystalline form is characterized by an X-ray powder diffraction pattern containing diffraction peaks at 2Θ values of 10.61±0.20, 11.84±0.20, 14.94±0.20, 18.26±0 .20 and 19.06±0.20. 4. The crystalline form of claim 3, wherein the X-ray powder diffraction pattern is further characterized by having additional diffraction peaks at 2θ values of 13.32±0.20, 17.69±0.20, and 21.10±0.20. 5. The crystalline form of claim 4, wherein the X-ray powder diffraction pattern is further characterized by having two or more additional diffraction peaks at 2θ values selected from 10.85±0.20, 16.14±0.20, 16.35 ±0.20, 18.43±0.20, 19.20±0.20, 19.49±0.20, 20.72±0.20, 21.94±0.20, 22.64±0 .20, 23.64±0.20, 25.19±0.20 and 28.08±0.20. 6. The crystalline form of claim 4, wherein the X-ray powder diffraction pattern is further characterized by having additional diffraction peaks at 2θ values selected from 10.85±0.20, 16.14±0.20, 16.35±0, 20, 18.43±0.20, 19.20±0.20, 19.49±0.20, 20.72±0.20, 21.94±0.20, 22.64±0.20, 23.64±0.20, 25.19±0.20 and 28.08±0.20. 7. A crystalline form according to claim 3, wherein the crystalline form is characterized by a differential scanning calorimetry curve obtained at a heating rate of 10°C/min, which represents a maximum in endothermic heat flux at a temperature between 268 and 277°C. 8. A crystalline form according to claim 3, wherein the crystalline form is characterized by a differential scanning calorimetry curve obtained at a heating rate of 10°C/min, which represents a maximum in endothermic heat flux with a peak at 272.6±2°C. 9. Crystalline form of the compound of formula 1 - 41 040670 where the crystalline form is characterized by an X-ray powder diffraction pattern containing diffraction peaks at 2Θ values of 8.16±0.20, 8.97±0.20, 15.29±0.20, 16.70±0.20, 18.00± 0.20 and 20.18±0.20. 10. The crystalline form of claim 9, wherein the X-ray powder diffraction pattern is further characterized by having two or more additional diffraction peaks at 2θ values selected from 7.69±0.20, 10.66±0.20, 11.46 ±0.20, 11.91±0.20, 15.80±0.20, 17.02±0.20, 18.83±0.20, 22.39±0.20, 22.98±0 .20, 24.89±0.20 and 26.54±0.20. 11. The crystalline form of claim 9, wherein the X-ray powder diffraction pattern is further characterized by having additional diffraction peaks at 2θ values selected from 7.69±0.20, 10.66±0.20, 11.46±0, 20, 11.91±0.20, 15.80±0.20, 17.02±0.20, 18.83±0.20, 22.39±0.20, 22.98±0.20, 24.89±0.20 and 26.54±0.20. 12. A crystalline form according to claim 9, wherein the crystalline form is characterized by a differential scanning calorimetry curve obtained at a heating rate of 10°C/min, which represents a maximum in endothermic heat flux at a temperature between 215 and 229°C. 13. A crystalline form according to claim 9, wherein the crystalline form is characterized by a differential scanning calorimetry curve obtained at a heating rate of 10°C/min, which represents a maximum in endothermic heat flux with a peak at 221.7±3°C. 14. A pharmaceutical composition containing a compound according to claim 1 or 2 or a crystalline form according to any one of claims 3 to 13 and a pharmaceutically acceptable carrier. 15. The pharmaceutical composition of claim 14, wherein the composition is suitable for ophthalmic use. 16. The pharmaceutical composition of claim 15, wherein the composition is suitable for intravitreal injection. 17. The pharmaceutical composition of claim 16, wherein the composition is a suspension. 18. Process for the preparation of a compound of formula 1 1, or a pharmaceutically acceptable salt thereof, comprising: (a) reacting a compound of formula 6 with compound of formula 7 O<O where R ^a is hydrogen or 2,5-dioxopyrrolidinyl, and (b) optionally obtaining a pharmaceutically acceptable salt, to obtain a compound of formula 1 or a pharmaceutically acceptable salt thereof. - 42 040670 19. Compound of Formula 6 F or its salt. 20. A method for obtaining a crystalline form according to claim 3, including: (a) formation of a homogeneous mixture of 1-(2-(6-(2-ethyl-5-fluoro-4-hydroxyphenyl)-4-fluoro-III-indazol-3yl)-1,4,6,7-tetrahydro-5H -imidazo[4,5-c]pyridin-5-yl)-2-morpholinoethan-1-one in a polar aprotic solvent, or in a polar water-miscible solvent, or in a mixture of a polar aprotic solvent and a polar water-miscible solvent at a temperature between 45 and 75°C; (b) adding a homogeneous mixture to a mixture of water-miscible solvent and water at a temperature between 60 and 90° C. to obtain a second mixture; (c) slowly adding water to the second mixture at a temperature between 60 and 90° C. to form a slurry; and (d) separating the crystalline form from the slurry. 21. The method according to claim 20, wherein the polar aprotic solvent of step (a) is selected from the group consisting of DMSO, DMF, NMP, DMAc and nitromethane, the polar water-miscible solvent of step (a) is selected from the group consisting of acetonitrile, acetone , methanol, ethanol and THF, and the water-miscible solvent of step (b) is selected from the group consisting of acetonitrile, acetone, methanol, ethanol, n-propanol, isopropanol, n-butanol, THF, DMSO, DMF, NMP, DMAc and nitromethane. 22. A compound according to claim 1 or 2 or a crystalline form according to any one of claims 3 to 13 for the treatment of an ocular disease in a mammal. 23. The compound or crystalline form of claim 22, wherein the ocular disease is uveitis, diabetic retinopathy, diabetic macular edema, dry eye syndrome, age-related macular degeneration, retinal vein occlusion, or atopic keratoconjunctivitis. 24. The compound or crystalline form of claim 23, wherein the ocular disease is diabetic macular edema or uveitis. 25. The use of a compound according to claim 1 or 2 or a crystalline form according to any one of claims 3 to 13 for the preparation of a medicament for the treatment of an ocular disease in a mammal. 26. Use according to claim 25, wherein the ocular disease is uveitis, diabetic retinopathy, diabetic macular edema, dry eye syndrome, age-related macular degeneration, retinal vein occlusion, or atopic keratoconjunctivitis. 27. A method of treating an ocular disease in a mammal, comprising administering a pharmaceutical composition containing a compound according to claim 1 or 2 or a crystalline form according to any one of claims 3 to 13 and a pharmaceutically acceptable carrier to the eye of a mammal. 28. The method of claim 27, wherein the ocular disease is uveitis, diabetic retinopathy, diabetic macular edema, dry eye syndrome, age-related macular degeneration, retinal vein occlusion, or atopic keratoconjunctivitis. 29. The method of claim 28 wherein the ocular disease is uveitis or diabetic macular edema. 30. A compound according to claim 1 or 2 or a crystalline form according to any one of claims 3 to 13 for the treatment of an inflammatory skin disease in a mammal. 31. The compound or crystalline form of claim 30 wherein the inflammatory skin disease is atopic dermatitis. 32. The use of a compound according to claim 1 or 2 or a crystalline form according to any one of claims 3 to 13 for the preparation of a medicament for the treatment of an inflammatory skin disease in a mammal. 33. Use according to claim 32, wherein the inflammatory skin disease is atopic dermatitis. 34. A method of treating an inflammatory skin disease in a mammal, comprising applying a pharmaceutical composition containing a compound according to claim 1 or 2 or a crystalline form according to any one of claims 3 to 13 and a pharmaceutically acceptable carrier to the skin of a mammal. 35. The method of claim 34 wherein the inflammatory skin disease is atopic dermatitis. 36. A compound according to claim 1 or 2, or a crystalline form according to any one of claims 3 to 13, for the treatment of a respiratory disease in a mammal. 37. The compound or crystalline form of claim 36, wherein the respiratory disease is asthma, chronic obstructive pulmonary disease, cystic fibrosis, pneumonitis, idiopathic -