jak inhibitor compound derived from imidazopiperidine fusion

专利摘要:
the invention provides a compound of formula 1 or a pharmaceutically acceptable salt, useful as a jak inhibitor. the invention also provides crystalline forms of the compound, pharmaceutical compositions comprising the compound, methods of using the compound to treat diseases that can be treated with a jak inhibitor and the processes and intermediates useful for preparing the compound.
公开号:BR112019022648A2
申请号:R112019022648
申请日:2018-04-30
公开日:2020-05-19
发明作者:D Crater Glenn；Zhang Hao；Nzerem Jerry；Jiang Lan；Dabros Marta；A Kleinschek Melanie；Benjamin Noah；R Fatheree Paul；Murray Mckinnell Robert；R Thalladi Venkat
申请人:Theravance Biopharma R&D Ip Llc；
IPC主号:

专利说明:

JAK INHIBITOR COMPOUND DERIVED FROM THE FUSION OF
IMIDAZOPIPERIDINE
HISTORY OF THE INVENTION
Field of. invention
[001] The invention is directed to a JAK kinase inhibitory compound useful for the treatment of multiple diseases, in particular, eye, skin and respiratory diseases. The invention is also directed to pharmaceutical compositions that include such compounds, methods of using such compounds to treat diseases that can be treated with a JAK inhibitor, and processes and intermediates useful for preparing such compounds.
state of art
[002] Cytokines are intracellular signaling molecules that include chemokines, interferons, interleukins, lymphokines and tumor necrosis factor. Cytokines are critical for normal cell growth and immunoregulation, but they also trigger diseases mediated by the immune system and contribute to the growth of malignant cells. Elevated levels of various cytokines are involved in the pathology of a large number of diseases or conditions, in particular, those diseases characterized by inflammation. Many of the cytokines involved in the disease act via signaling pathways dependent on the Janus family of tyrosine kinases (JAKs), which signal via the Transcription Activator and Signal Transducer (STAT) family of transcription factors.
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[003] The JAK family is composed of four members, JAK1, ϋΑ, Κζ, JAK3 and tyrosine kinase 2 (lYKz). Binding of the cytokine to a JAK-dependent cytokine receptor induces dimerization of the receptor which results in the phosphorylation of tyrosine residues in the JAK kinase, activating JAK. Phosphorylated JAKs, in turn, bind and phosphorylate several STAT proteins that dimerize, internalize in the cell nucleus and directly modulate gene transcription, leading, among other effects, to downstream effects associated with inflammatory disease. JAKs generally associate with paired cytokine receptors such as homodimers and heterodimers. Specific cytokines are associated with specific JAK pairings. Each of the four members of the JAK family is involved in signaling at least one of the cytokines associated with the inflammation.
[004] Inflammation plays a prominent role in many eye diseases, including uveitis, diabetic retinopathy, diabetic macular edema, dry eye disease, age-related macular degeneration, retinal vein occlusion and atopic keratoconjunctivitis. Uveitis encompasses multiple inflammatory conditions and is often autoimmune, appearing without a known infectious trigger.
It is estimated that coi patients in the USA. associated with the main uveitis cause of ailment i. and t and c and r c a. α e In some patients, it leads to the destruction of blindness in the USA. The millions of chronic tissue inflammation and is the fifth
C _L L O C ± ΙΊ cL S β J_ Θ V <3. Cl 3 S in the eyes of patients with uveitis that signal via the JAK-STAT pathway include IL-2, IL-4, IL-5, IL-6, IL-10, IL-2.3 and IFN-γ. (Horai and Caspi, J Interferon Cytokine Res, 2011, 31, 733-744; Ooi et al, Clinicai Medicine and Research, 2006, 4,
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294-309). Existing therapies for uveitis are often suboptimal and many patients are poorly controlled. Steroids, although often effective, are associated with cataracts and increased blood pressure / g1aucoma.
[005] Diabetic retinopathy (RD) is caused by damage to blood vessels in the retina. It is the most common cause of vision loss among people with diabetes. Angrogenesis, like inflammatory pathways, plays an important role in the disease. Often, RD progresses to diabetic macular edema (EMD), the most frequent cause of vision loss in patients with diabetes. The condition is estimated to affect about 1.5 million patients in the US alone, of which about 20% have diseases that affect both eyes. It is believed that the cytokines that signal, via the JAK-STAT pathway, such as IL-6, as well as other cytokines, such as IP-10 and MCP-1 (alternatively known as CCL2), whose production is partially driven via the JAK-STAT signaling pathway play a role in inflammation associated with DR / EMD (Abcouwer, J Clin Cell Immunol, 2013, Suppl 1, 1-12; Sohn et al., American Journal of Optnalmiology, 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, 1734-1746; Funatsu et al, Ophthalmology, 2009, 116, 73-79). Existing therapies for EMD are suboptimal: intravitreal anti-VEGF treatments are only effective in a fraction of patients and steroids are associated with cataracts and increased intraocular pressure.
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[006Ί Dry eye disease (DED) is a multifactorial disease that affects approximately 5 million patients in the USA. 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 ΤΕΝ-γ have been observed in the eye fluids of patients with DED. (Stevenson et al, Arch Ophthalmol, 2012, 130, 90-100), and levels often correlate with disease severity. Age-related macular degeneration and atopic keratoconjunctivitis are also associated with JAK-dependent cytokines.
[007J Retinal vein occlusion (OVR) is a disease that compromises vision and has. high prevalence. Obstruction of blood flow to the retina can damage the retinal vasculature, cause hemorrhage and tissue ischemia. Although OVR cases are multifactorial, vascular and inflammatory mediators have been shown to be important (Deobhakta et al, International Journal of Inflammation, 2013, article ID 438412). Cytokines that signal via the JAK-STAT pathway, such as IL-6 and IL-13, as well as other cytokines, such as MCP-1, whose production is boosted in. partly by the JAK-STAT signaling pathway, were detected at high levels in the eye tissues of patients with OVR (Shchuko et al, Indian Journal of Ophthalmology, 2015, 63 (12), 905-911). Many patients with OVR are treated with photocoagulation, an inherently destructive therapy. Anti-VEGF agents are also used, but they are effective only in a fraction of patients. It has been shown that steroid drugs that reduce the level of inflammation in the
Petition 870190110020, of 10/29/2019, p. 31/160 eye (acetonide triamcinolone and dexamethasone implants) provide beneficial results for patients with certain forms of OVR, but they have also been shown to cause cataracts and increased intraocular pressure / glaucoma.
[008] Atopic dermatitis (AD) is a chronic inflammatory skin disease that affects about 14 million people in the United States alone. It is estimated that AD affects 10-20% of children and 1-3% of adults in developed countries (Bao et al., JAK-STAT, 2013, 2, e24137) and the prevalence is increasing. The elevation of the proinflammatory cytokines that depend, on the JAK-STAT pathway, in. in particular, IL-4, IL-5, IL-10, IL-13, IFNy is associated with AD (Bao et al., Leung et al., The Journal of Clinical Investigation, 2004, 113, 651-657). In addition, it has been shown that the overregulation of IL-31, another cytokine that signals through a JAK pairing, participates in the pruritus process associated with the chronic state of AD. (Sunkoly et al., Journal of Allergy and Clinical Immunology, 2006, 117, 411-417)
[009] Asthma is a chronic disease of the airways for which there is no prevention or cure. The disease is characterized by inflammation, fibrosis, hyperresponsiveness and airway remodeling, which contribute to the limitation of airflow. An estimated 300 million people worldwide suffer from asthma and it is estimated that the number of people with asthma will grow by more than 100 million by 2025. Although most patients can get control of asthma symptoms with. the use of inhaled corticosteroids that can be combined with. a leukotriene modifier and / or a long-acting beta-agonist, there is still a subset of patients with severe asthma whose disease is not
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6/117 controlled with conventional therapies. The cytokines involved in asthma inflammation that signal via the JAKSTAT pathway include IL-2, IL-3, IL-4, TL-5, IL-6, IL-9, IL-11, IL13, IL-23, IL-31 , IL-27, thymic stromal lymphopoietin (TSLP), gamma interferon (ΙΕΝγ) and granulocyte-macrophage colony stimulating factor (GM-CSF). Inflammation of the airways is characteristic of respiratory diseases other than asthma. Chronic obstructive pulmonary disease (COPD), cystic fibrosis (CF), pneumonitis, pulmonary interstitial diseases (including idiopathic pulmonary fibrosis), acute lung injury, acute respiratory distress syndrome, bronchitis, emphysema, obliterating bronchiolitis and sarcoidosis are also diseases of the tract in which the pathophysiology is believed to be related to JAK signaling cytokines.
[0010] Considering the number of cytokines elevated in inflammatory diseases and that each cytokine is associated with a particular JAK pairing, a chemical inhibitor with. Comprehensive activity against all members of the JAK family could have wide use for the treatment of eye, skin and respiratory diseases. There remains a need for a potent broad-spectrum JAK inhibitor.
SUMMARY OF THE INVENTION
[0011] In one aspect, the invention provides a JAK inhibitor compound useful for the treatment of inflammatory disease.
[0012] In particular, in one aspect, the invention provides 1- (2- (6- (2-ethyl-5-fluorO-4-hydrOxyphenyl) -4-fluoroPetition 870190110020, from 10/29/2019, pg. 33 / 160 lif-indazol-3-yl) -1,4,6,7-tetrahydro-5.H-imidazo [4, o-c] pyridin5-yl) -2-morpholinoetan-l-one of the formula
hereinafter compound 1, or a pharmaceutically acceptable salt.
[0013] The invention also provides crystalline forms of the compound, form 1 and form 2.
[0014] The invention also provides a pharmaceutical composition comprising compound 1 or a pharmaceutically acceptable salt and a pharmaceutically acceptable carrier.
[0015] In one aspect, the invention provides one. a method of treating an eye disease in a mammal, the method comprising administering compound 1, or a pharmaceutical composition of the invention to the mammal. In one aspect, eye disease is uveitis, diabetic retinopathy, diabetic macular edema, dry eye disease, age-related macular degeneration, retinal vein occlusion and atopic keratoconjunctivitis. In particular, eye disease is diabetic macular edema or uveitis.
[0016] In yet another aspect of the method, the invention provides a method of treating an inflammatory skin disease, in particular atopic dermatitis, the method comprising applying compound 1 or a pharmaceutical composition of the invention to the skin of a mammal.
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[0017] In another aspect, the invention provides a method of treating a respiratory disease in a mammal, the method comprising administering compound 1 or a pharmaceutically acceptable salt of the invention to the mammal.
[0018] In separate and distinct aspects, the invention also provides synthetic and intermediate processes described herein, useful for preparing compound 1.
[0019] The invention also provides compound 1 as described herein for use in medical therapy, as well as the use of the compound of the invention in the manufacture of a formulation or medicament to treat eye, skin or respiratory disease in a mammal.
QUICK DESCRIPTION OF THE DRAWINGS
[0020] Various aspects of the present invention are illustrated by references accompanying the drawings.
[0021] The Figure. 1 shows a powder X-ray diffraction pattern (PXRD) of crystalline Form 2 of compound 1 (hereinafter Form 2).
[0022] Figure 2 shows a differential scanning calorimetry (DSC) thermogram of Form 2 c r i s t a 1.1. at .
[0023] Figure 3 shows a graph of thermal gravimetric analysis (TGA) of crystalline Form 2.
[0024] Figure 4 shows an isotherm for dynamic moisture adsorption (DMS) of crystalline Form 2 observed at a temperature of about 25 ° C
[0025] Figure 5 shows a polarized light microscope image of Form 2.
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[0026] Figure 6 shows one. X-ray powder diffraction (PXRD) pattern of crystalline Form 1 of compound 1 (hereinafter referred to as Form 1).
[0027] Figure 7 shows a crystalline Form 1 differential scanning calorimetry (DSC) thermogram.
[0028] Figure 8 shows a graph of thermal gravimetric analysis (TGA) of crystalline Form 1.
[0029] Figure 9 shows one. isotherm of dynamic moisture adsorption (DMS) of crystalline Form 1 observed at a temperature of about 25 ° C ^{, J} C
[0030] Figure 10 shows a polarized light microscope image of Form 1.
DETAILED DESCRIPTION OF THE INVENTION
[0031] Chemical structures are named in this report in accordance with IUPAC conventions as implemented in the ChemDraw program (PerkinElmer, Inc., Cambridge, MA).
[0032] Furthermore, the imidazo portion of the tetrahydroimidazopyridine portion in the structure of the present compound exists in tautomeric forms. The compound could be represented in an equivalent way as
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10/117 of the tetrahydroimidazopyridine portion. Therefore, this structure is called 1- (2- (6- (2-ethyl-5-fluoro-4-hydroxyphenyl) -4fluoro-1 H-indazol-3-yl) -3,4,6,7-tetrahydro- 5H-imidazo [4,5c] pyridin-5-yl) -2-morpholinoetan-1-one. It can also be designated 1- (2- (6- (2-ethyl-5-fluoro-4-hydroxyphenyl) -4-fluorol.H-indazol-3-yl) -1,4,6,7-tetrahydro-5H -imidazo [4,5-c] pyridin5-yl) -2-morpholinoetan-1-one. It must be understood that, although the structures are presented or named, it was a particular form, the invention also includes its tautomer.
[0034] The compounds of the invention have several basic groups and, therefore, the compounds can exist as the free base or various forms of salt, as a form of monoprotonated, diprotonated, triprotonated salt or mixtures thereof. All of these forms are included in the scope of application of the present invention, unless otherwise stated.
[0035] The present invention also includes isotope-labeled compounds of formula 1, that is, compounds of formula 1 where an atom has been replaced or enriched with an atom with. the same atomic number, but different atomic mass from that which predominates in nature. Examples of isotopes that can be incorporated into a compound of formula 1 include, but are not limited to, ² u, ⁸ H, ^1J -C, ² “0, ~ ⁸ N, ^1J O, ¹⁷ O, ¹⁸ O and ¹⁸ F. Of particular interest are compounds of formula 1, enriched with tritium, or carbon-14, which can be used, for example, in tissue distribution studies. Of particular interest are also compounds of formula 1, enriched with deuterium, especially at a site of metabolism, whose compounds have greater metabolic stability. In addition, of particular interest are compounds of formula 1, enriched with. an isotope emitting
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11/117 positrons, such as ^U C, ¹⁸ F, ¹⁵ 0 and ¹³ N, whose compounds can be used, for example, in Positron Emission Tomography (PET) studies.
Definitions
[0036] In describing the present invention, including its various aspects and modalities, the following
[0037] The term therapeutically effective amount means an amount sufficient to treat when administered to a patient in need of treatment,
[0038] The term to treat or treat means to alleviate or suppress the medical condition, disease or medical disorder to be treated (eg, a respiratory illness) a patient (in particular, a human); or relieve symptoms of the disease or medical disorder.
[0039] The term pharmaceutically acceptable salt means a salt that is acceptable for administration to a patient or mammal, such as a human (e.g., salts with acceptable safety in mammals for a given dosage schedule ). Pharmaceutically acceptable salts include acetic, ascorbic, benzenesulfonic, benzoic, camphorsulfonic, citric, ethanesulfonic, edisyl, fumaric, gentisic, glyconic, glycuronic, glutamic, hypuric, hydrobromic, hydrochloric, isatoonic, lactic, lactic, lactic, lactic, lactic, lactic, lacto, lacto, salts. maleic, malic, mandelic, methanesulfonic, musclic, naphthalenesulfonic, naphthalene-l, 5 ~ disulfonic, naphthalene2,6-disulfonic, nicotinic, nitric, orotic, pamoic, pantothenic, phosphoric, succinic, sulfuric, tartaric, and p-toluene similar.
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[0040] The term its salt means a compound formed when the hydrogen of an acid is replaced by a cation, such as a metal cation or an organic cation and the like. For example, the cation can be a protonated form of a compound of formula 1, that is, a form where one or more amino groups have been protonated by an acid. Normally, salt is a pharmaceutically acceptable salt, although this is not necessary for salts of intermediate compounds that are not suitable for administration to one. patient.
[0041] The term amino protection group means an adequate protection group to prevent undesirable reactions in an amino nitrogen. Representative amino-protecting groups include, but are not limited to, formyl; acyl groups, for example, alkanoyl groups, such as acetyl and trifluoroacetyl groups; alkoxycarbonyl groups, such as tert-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), tert-butyldimethylsilyl (TBDMS), [2 (trimethylsilyl) -ethoxy] methyl (SEM); and the like.
[0042] The term hydroxy protecting group means a suitable protecting group to prevent undesirable reactions in a hydroxy group. Representative 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), ρ-methoxybenzyl (PMB),
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9-fluorenylmethyl (Fm) and diphenylmethyl (benzhydryl, DPM); silyl groups, such as trimethylsilyl (TMS) and tertbutyldimethylsilyl (TBS); and the like.
[0043] Numerous protecting groups and 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
[0044] Compound 1 and its intermediates can be prepared according to the following general methods and procedures using commercially available or routinely available starting materials and reagents. In addition, compounds with an acid or basic atom or functional group can be used or produced acids as one. salt, unless otherwise stated (in. some cases, the use of a salt in a particular reaction will require conversion of the salt to the non-salt form, for example, a free base, using routine procedures before performing the reaction).
[0045] Although a particular embodiment of the present invention can be shown or described in the following procedures, those skilled in the art will recognize that other embodiments or aspects of the present invention can also be prepared with such procedures, or using other methods, reagents and materials known to those skilled in the art. In particular, it will be appreciated that compound 1 can be prepared by various process routes in which the reagents are combined in different orders to provide different intermediates in the route and to produce the final products.
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[0046] The preparation of 1- (2- (6- (2-ethyl-5-fluoro-4hydroxyphenyl) -4-fluoro ~ 1H-indazol-3 "yl) -1,4,6,7-tetrahydro577-imidazo [4,5-c] pyridin-5-yl) -2-morpholinoethane-1-one (compound 1) is described in detail in the attached examples. The key steps are summarized in Scheme 1
Layout 1
where reagent 7 is 2,5-dioxopyrrolidin-1-ii 2morpholinoacetate, that is, the variable R ^A represents the activating agent 2,5 ~ dioxopyrrolidinyl, as described in Example 1. On the other hand, morpholin-4-yl acid acetic, that is, R ^A represents hydrogen, is used as reagent 7 under
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15/117 typical amide bond formation conditions, as described in Example 4.
[0047] Intermediate 3 can be prepared as described in Preparations 1 and 3 below. An alternative method of preparing the protected key intermediate 5 is illustrated in Scheme 2.
Layout 2
[0048] The aldehyde bromoindazole 8 can react with the imine 2 compound with protected benzyl to produce the intermediate 9. The reaction is conducted in the presence of sodium disulfide, at a temperature between 130 ^U C and 140 ° C for about 1 to about 6 hours or until the reaction is considerably complete. Compound 9 is reduced using a reducing agent such as sodium borohydride to provide compound 10, which is combined with protected phenyltrifluoroborate 11, under typical SuzukiMiyaura coupling conditions, to provide intermediate 5. The reaction is normally conducted at an elevated temperature in the presence of the palladium catalyst. The Suzuki 11 partner, shown in
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Scheme 2, how trifluoroborate potassium salt can be prepared by reacting the corresponding boronate (intermediate 1-5 in Preparation 1 below) with potassium hydrogen difluoride to provide intermediate 11. On the other hand, the boronate intermediate can be used in place of the rooo ra to 11.
[0049] Thus, in one aspect of the method, the invention provides a process for preparing a compound of formula 1 or a pharmaceutically acceptable salt, the process comprising reacting a compound of formula 6 with a compound of formula 7, as illustrated in Scheme 1 to provide a compound of formula 1 or a pharmaceutically acceptable salt.
[0050] In a further aspect of the method, the invention provides one. compound of formula 5 and a compound of formula 6, useful in the preparation of a compound of formula 1. Crystalline forms 100.i. ] In. another aspect, the invention provides r— (2 - I.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) -2morpholinoetan-1-one (1) in crystalline form.
rma
[0052] The crystalline Form 1 of the invention is a free crystalline form of compound 1. In one aspect, Form 1 is characterized by a powder X-ray diffraction pattern (PXRD), with significant diffraction peaks, among other peaks , at 2Θ values of 8.16: 10.20, 8.9710.20, 15.2910.20, 16.7010.20, 18.0010.20 and 20.1810.20. Form 1 can be characterized by a PXRD pattern with two or more additional diffraction peaks, including three or more and four or
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7/117 plus additional diffraction peaks at 2Θ values selected from 7, 69 + 0.20, 10, 66 + 0.20, 11, 4 6: 10.20, 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 a PXRD pattern with three, four, five or six diffraction peaks at 2Θ values selected from 8.1610.20, 8.9710.20, 15.29 + 0.20 , 16.7010.20, 18.0010.20 and 20.18 + 0.20.
[0053] As is well known in the powder X-ray diffraction field, the PXRD standard peak positions are relatively less sensitive to experimental details, such as sample preparation details and instrument geometry, than are the relative heights of peak. Thus, in one aspect, crystalline Form 1 is characterized by an X-ray powder diffraction pattern in which the peak positions are substantially in line with the positions shown in figure 6.
[0054] In. another aspect, crystalline Form 1 is characterized by its behavior when exposed to high temperature. As shown in Figure 7, the differential scanning calorimetry (DSC) trace recorded at one. heating rate of 10 ° C per minute displays one. peak in endothermic heat flow, identified as melting transition, in the transition range of about 210 ° C to about 234 ° C, or in the range of about 215 ° C to about 229 ° C, or in the range between about 220 ° C to about 224 ° C. Crystalline Form 1 is characterized by a trace of exploratory differential calorimetry recorded at a heating rate of 10 ° C per minute, which shows a maximum endothermic heat flow at a peak temperature of about
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18/117 of 221.7 ° C. The thermal gravimetric analysis (TGA) of Figure 8 does not show significant weight loss at temperatures below the start of decomposition at about 293 ° C.
[0055] Figure 9 shows a dynamic moisture sorption (DMS) trace for the free crystalline Form 1 of the invention. Crystalline Form 1 demonstrated signal hysteresis between two sorption and desorption cycles. Form 1 showed a weight gain of about 0.99% in the range of 5% to 70% relative humidity and a weight gain of about 1.32% in the range of 5% to 90% relative humidity environment, as shown in Figure 9. Form 1 is considered to be slightly hygroscopic.
[0056] It has been shown that crystalline form 1 is stable after exposure to high temperature and humidity. After 36 weeks in. accelerated conditions of 40 ° C and 75% relative humidity, no statistically relevant changes in chemical purity were observed.
[0057] Form 1 can be prepared by dissolving compound 1 in ethanol under heating, followed by the addition of acetonitrile, where the ratio of acetonitrile to ethanol is about 1: 1 or from about 1: 3 to 3: 1. The resulting mixture is then heated, followed by the addition while stirring at a temperature, between about 20 ° C and about 25 ° C for about 4 hours to 30 hours or. for about 16 hours. The solid is then filtered and dried to provide Form 1.
[0058] Form 1 can also be prepared by mixing compound 1 with ethanol and stirring the mixture at a temperature between about 50 and about 80 ° C for about 2 to 30 minutes or about 10 minutes, followed by the addition slow acetonitrile at a temperature between about
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19/117 of 50 and about 80 ° C, where the volume ratio of acetonitrile to ethanol is from 3: 1 to 1: 1 or about 1.5: 1. Grains of Form 1 can be added and the resulting mixture is then stirred at a temperature between about 20 ° C and about 25 ° C for about 4 hours and about 30 hours or for about 18 hours. The solid is then filtered and dried to provide Form 1.
Form 2
[0059] The crystalline Form 2 of the invention is a free crystalline form of compound 1. In one aspect, Form 2 is characterized by one. powder X-ray diffraction pattern (PXRD) having significant diffraction peaks, among other peaks, at 2Θ values of 10.6110.20, 11.8410.20, 14.94 1 0.20, 18.26 1 0 , 20 and 19, 06 1 0.20. The shape. 2 can also be characterized by a PXRD pattern with additional diffraction peaks at 2Θ values of 13.3210.20, 17.6910.20 and 21.1010.20. Form 2 can be characterized by a PXRD standard with two or more additional diffraction peaks, including three or more and four or more additional diffraction peaks at 2Θ values selected from 10, 8510.20, 16, 1410.20, 16, 3510.20, 18.43 1 0.20, 19, 20 1 0.20, 19, 49 1 0.20, 20, 72 1 0.20, 21.94 1 0.20, 22, 64 1 0.20, 2 3.6410.20, 25.1910.20 and 28.08 1 0.20.
[0060] In another aspect, Form 2 is characterized by a PXRD pattern with three, four, five or six diffraction peaks at 2Θ values selected from 10.6110.20, 11.8410.20, 13, 3210, 20, 14.9410,20, 1.7, 6910.20, 1.8.2 610.20, 19.0610.20 and 21.1010.20.
[0061] As is well known in the powder X-ray diffraction field, the peak positions of the PXRD pattern are
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20/117 relatively less sensitive to experimental details, such as sample preparation details and instrument geometry, than are relative peak heights. Thus, in one aspect, crystalline Form 2 is characterized by an X-ray powder diffraction pattern in which the peak positions are substantially in line with the positions indicated in figure 1.
[0062] The structure of crystalline form 2 was further characterized by the analysis of single crystal x-ray diffraction. The crystals belong to an orthorhombic crystalline system and space group Pbca. The cell dimensions of the unit are: a ~ 9, 7245 (11) Â, b ~ 16.8197 (14) Â, c 32, 604 (4) À, a- 90 °, 3 == 90 °, y = == ^: 90 °, volume ^ 5332.8 (10) Â ³ . The calculated density is 1.302 g / cm ³ . The crystals contain eight molecules per cell in the unit. The structure confirms that the crystals have no water or other solvent molecules and that the molecular structure is consistent with the structure of the compound of Example 1 as described here. The powder X-ray diffraction peaks predicted from the derived atomic positions are in good harmony with the observed results.
[0063] In another aspect, the crystalline Form 2, is characterized by its behavior when exposed to high temperature. As shown in Figure 2, the differential scanning calorimetry (DSC) trace recorded at a heating rate of 10 ° C per minute exhibits a peak in endothermic heat flow, identified as a melting transition, in the transition range of about 268 ° C to about 277 ° C, or in the range between about 270 ° C to about 275 ° C, or in the range between about 271 ° C to about 274 ° C. The shape
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21/117 crystalline is characterized by a trace of differential exploratory calorimetry recorded at a heating rate of 10 ° C per minute, which shows a maximum in the endothermic heat flow with a peak of about
272.6 ± 2 ° C.
[0064] Gravimetric analysis. (TGA) of Figure 3 does not show significant weight loss at temperatures below the onset of decomposition at about 269 ° C.
[0065] Figure 4 shows a trace of dynamic moisture sorption (DMS) for the free crystalline Form 2 of the invention. Crystalline Form 2 showed no hysteresis between two sorption and desorption cycles and showed an exceptionally small propensity for hygroscopicity. Form 2 demonstrated a weight gain of about 0.18% in the range of 5% to 90% relative humidity and a weight gain of about 0.12% in the range of 5% to 70% relative humidity environment, as shown in Figure 4. Form 2 is considered to be non-hygroscopic.
[0066] Form 2 can be prepared by dissolving the compound 1 of example 1 in. DMSO (dimethylsulfoxide, in a ratio of 1 g of compound 1 to 1 to 3 ml or 2 ml of DMSO) at a temperature between about 45 and 75 ° C or about 60 ° C, followed by the addition of methanol, where the proportion volume of methanol to. DMSO is about 1: 4 to about 1: 1 or about 1: 2. A. homogeneous mixture is then added dropwise to a premixed solution of methanol and water (where the ratio of methanol to DMSO is between 1.5 and 3 to 1) at a temperature between about 60 and about 90 ° C or about 75 ° C, where the volume ratio of the premixed methanol solution to water is between about 0.5: 1 at
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22/117 about 1: 2 or about 1: 0.9. The mixture remains stirred at a temperature between about 60 ° C or about 90 ° C or about 75 ° C for about 30 minutes to hours or for about 1 hour. The water can then be added slowly at a temperature between about 60 and about 90 ° C or about 75 ° C, where the volume ratio of water to methanol is between 2 and 4. The resulting slurry is then slowly cooled to room temperature (generally between about 20 and about 25 ° C), typically over about 2 to about 12 or about 6 hours. The slurry is kept at room temperature and then filtered and washed with a mixture of water to about 50 to 90% methanol or about 70% water, to provide the Form. 2.
[00 67] In. In one aspect, the invention provides a method of preparing crystalline form 2 comprising:
(a) form a homogeneous mixture of 1- (2- (6- (2 ~ ethyl-5 ~ fluoro-4-hydroxyphenyl j -4-fluoro-lif-indazol-3-yl) 1,4,6, 7 "tetrahydro ~ 5ff" imidazo [4,5-c] pyridin ~ 5 ~ yl) -2morpholinoetan-l-one in a polar aprotic solvent or a water miscible polar solvent, or in a mixture of polar aprotic solvent and a solvent polar miscible in water at a temperature between 45 and Γ-O. -s / 5 u;
(b) adding the homogeneous mixture to a mixture of a water and water miscible solvent at a temperature between 60 and 90 ° C to produce a second mixture;
(c) slowly adding water to the second mixture at a temperature between 60 and 90 ° C to form a slurry; and
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23/117 (d) isolating the crystalline form from the slurry.
[00 68] In. In some respects, the polar aprotic solvent from step (a) is selected from the group consisting of DMSO, DMF, NMP, DMAc and nitromethane, and the water-miscible polar solvent from step (a) is selected from the group consisting of acetonitrile, acetone, methanol, ethanol and THF and the water miscible solvent from 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. in some respects, the polar aprotic solvent from step (a) is DMSO, the water miscible polar solvent from step (a) is methanol and the water miscible solvent from step (b) is methanol.
[0069] In some respects, the slurry obtained in step (c) is cooled to one. between about 20 and 25 ° C before step (d).
[0070] On the other hand, Form 2 can be produced by stirring compound 1 obtained in example 1 in a mixture of a water-miscible polar solvent at a temperature between 60 and 90 ° C. In some respects, the solvent to water ratio is about 1: 1 or from 2: 1 to 0.5; 1. In some respects, the water-miscible polar 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
[0071] Compound 1 and its pharmaceutically acceptable salts are normally used in the form of a pharmaceutical composition or formulation. Such pharmaceutical compositions can be advantageously administered at
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24/117 a patient by any acceptable route of administration, including, but not limited to, oral, inhalation, topical, topical (including transdermal), rectal, nasal and parenteral injection.
[0072] Thus, in one aspect of the compositions, the invention is directed to a pharmaceutical composition comprising a pharmaceutically acceptable carrier or excipient 'and compound 1, where, as defined above, compound 1 means compound 1 or a pharmaceutically acceptable salt. Optionally, such pharmaceutical compositions can, have other formulation and / or therapeutic agents if desired. When it comes to compositions and their uses, compound 1 can also be referred to here as the active agent.
[0073] On. in some aspects, the disclosure provides a pharmaceutical composition comprising compound 1 or one. pharmaceutically acceptable salt or Form 1 or Form. 2 and a pharmaceutically acceptable carrier. In. In some respects, the pharmaceutical composition is suitable for application to the eye. In some ways, the composition is suitable for injection into the eye. In some ways, the composition is suitable for intravitreal injection. In. in some ways, the composition is a suspension. In some ways, the composition is a crystalline suspension. In some respects, the composition is a suspension of Form 1 or Form 2.
[0074] The pharmaceutical compositions of the invention generally contain a therapeutically effective amount of compound 1. Those skilled in the art will recognize, however, that a pharmaceutical composition may have
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25/117 more than one effective therapeutic amount, i.e., bulk compositions or less than one effective therapeutic amount, i.e., individual unit doses designed for the administration of multiple doses to achieve an effective therapeutic amount.
[0075] Typically, such pharmaceutical compositions will contain about 0.01 to 95%, by weight, of the active agent, including, for example, from about 0.05 to 30%, by weight, of the active agent, and from 0.1% to about 10% by weight of the active agent.
[0076] Any conventional carrier or excipient can be used in the pharmaceutical compositions of the invention. The choice of a particular carrier or excipient, or combinations of carriers or excipients, will depend on the mode of administration to be used to treat a particular patient or type of illness or medical condition. In this context, the preparation of a pharmaceutical composition suitable for a particular mode of administration is well within the scope of those skilled in the pharmaceutical art. In addition, carriers or excipients used in the pharmaceutical compositions of this invention are commercially available. Through further illustration, conventional formulation techniques are described in Remington: The Science and Practice of Pharmacy, 20th Edition, Lippincott Williams & White, Baltimore, Maryland (2000); and HC Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, 7 ^th Edition, Lippincott Williams & White, Baltimore, Maryland (1999).
[0077] Representative examples of materials that can serve as acceptable carriers from the point of view
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Pharmaceutical 26/117 include, but are not limited to, the following: sugars, such as lactose, glucose and sucrose; starches, such as corn and starch; cellulose, such as microcrystalline cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatine; baby powder; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cotton oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, 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 solution; Ringer's solution; ethyl alcohol; phosphate buffer solutions; and other compatible non-toxic substances used in pharmaceutical compositions.
[0078] Pharmaceutical compositions are generally prepared by stirring or mixing the active agent thoroughly and efficiently with a carrier and one or more optional pharmaceutically acceptable ingredients. The resulting uniformly stirred mixture can then be molded or loaded into. tablets, capsules, tablets and using conventional procedures and equipment.
[0079] The pharmaceutical compositions of the invention are preferably packaged in unit pharmaceutical form. The term unitary dosage form refers to a separate unit physically suitable for administration to a patient, that is, each unit containing a quantity
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27/117 predetermined active agent calculated to produce the desired therapeutic effect, alone or combined with. one o 'plus additional units. For example, such unit dosage forms can be capsules, tablets, pills, and the like or packaging suitable for ocular or parenteral administration.
[0080] In one embodiment, the pharmaceutical compositions of the invention are suitable for oral administration. Pharmaceutical compositions suitable for oral administration may be in the form of capsules, tablets, lozenges, pills, pills, powders, granules, or as a solution or suspension in an aqueous or non-aqueous liquid; or as a liquid oil-in-water or water-in-oil emulsion; or as an elixir or syrup and the like; each containing a predetermined amount of compound 1 of the present invention as an active ingredient.
[0081] When intended for oral administration in. in a solid dosage form (i.e., as capsules, tablets, pills and the like), the pharmaceutical compositions of the invention will include the active agent and one or more pharmaceutically acceptable carriers. Optionally, such solid dosage forms may include: fillers or extenders, such as starches, microcrystalline cellulose, lactose, dicalcium phosphate, sucrose, glucose, mannitol and / or silicic acid; binders, such as carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and / or acacia; humectants, such as glycerol; disintegrating agents, such as croscarm.elo.se sodium, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, some silicates and / or carbonate
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11/28 sodium; solution retarding agents, such as paraffin; absorption accelerators, such as quaternary ammonium-based 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, polyethylene glycols, sodium lauryl sulfate, and / or mixtures thereof; dyes and buffering agents.
[0082] Release agents, wetting agents, coating agents, sweeteners, sweeteners and flavorings, preservatives and antioxidants may also be present in the composition of pharmaceutical compositions of the invention. Examples of pharmaceutically acceptable antioxidants include: water-soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisol, butylated hydroxytoluene, lecithin, propyl gallate, alpha-tocopherol, and the like and metal chelating agents, such as citric acid, ethylenediamine tetra-acetic acid, sorbitol, tartaric acid, sorbitol, tartaric acid phosphoric and the like. Coating agents for tablets, capsules, pills and the like include those used for enteric coatings, such as cellulose acetate phthalate, polyvinyl acetate phthalate, hydroxypropylmethylcellulose phthalate, hydroxypropylmethylcellulose, methacrylic acid, methacrylic acid copolymers, trimellitate cellulose acetate, carboxymethyl ethyl cellulose, hydroxypropyl methyl cellulose succinate acetate and the like.
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11/29
[0083] The pharmaceutical compositions of the invention can also be formulated to provide slow or controlled release of the active agent used, for example, hydroxypropylmethylcellulose in different proportions; or other polymeric matrices, liposomes and / or microspheres. In addition, the pharmaceutical compositions of the invention can have opacifying agents and can be formulated so that they release only the active ingredient, or, preferably, in a certain portion of the gastrointestinal tract, in a delayed manner. Examples of incorporated compositions that can be used include polymeric substances and waxes. The active agent can also be in microencapsulated form, if appropriate, with one or more of the excipients described above.
[0084] Liquid dosage forms suitable for oral administration include, by way of illustration, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. Liquid dosage forms suitable for oral administration include., By way of illustration, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, sodium benzoate, propylene glycol, 1,3-butylene glycol, oils (esp. cottonseed oils, peanuts, corn, wheat germ, olive oil, castor and sesame), oleic acid, glycerol, tetrahydrofuryl alcohol, polyethylene glycols and sorbitan fatty acid esters and their mixtures. On the other hand, some liquid formulations can be converted, for example, by spray drying to a powder, which is
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11/30 used to prepare solid dosage forms using conventional procedures.
[0085] The suspensions, in addition to the active ingredient, may contain suspending agents, such as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitan, sorbitol and sorbitol esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar and tragacanth, and their mixtures.
[0086] Compound 1 can also be administered parenterally (eg, intravenous, subcutaneous, intramuscular or intraperitoneal injection). In the case of parenteral administration, the active agent is mixed with a vehicle suitable for parenteral administration including, for example, sterile aqueous solutions, saline solution, low molecular weight alcohols, such as propylene glycol, polyethylene glycol, vegetable oils, gelatin, fatty acid esters such as ethyl oleate and the like. Parenteral formulations can also contain one. or more antioxidants, solubilizers, stabilizers, preservatives, wetting agents, emulsifiers, buffering agents or dispersing agents. These formulations can be sterilized using a sterile injectable medium, a sterilizing agent, filtration, irradiation, or heat.
[0087] Compound 1 can also be formulated as a sterile aqueous solution or suspension for eye injection. Useful excipients that can be included in such an aqueous formulation include polysorbate 80, polymers of c e1u1o s and hydroxypropylmethylcellulose, methylcellulose, potassium chloride, sodium chloride, sodium chloride, sodium chloride
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31/117 magnesium, sodium acetate, sodium citrate, histidine, α-trehalose dihydrate, sucrose, polysorbate 20, hydroxypropyl-p-cyclodextrin, benzalkonium chloride, Amberlite IRP-69, polyoxyethylene glycol ethers (lauryl, stearyl and oleyl), sodium salt of ethylenediaminetetraacetic acid, sodium taurocholate, saponins and EL chromophore, polycarbophyl-cysteine, xanthan gum, gellan gum, hyaluronic acid, liposomes and sodium phosphate. Permeability enhancers, surfactants, bile acids, cyclodextrins such as 2-hydroxypropyl-p-cyclodextrin and chelating agents can be included in the formulation. Cylindrical oligonucleotides with a hydrophilic outer surface and a lipophilic inner surface with the ability to form complexes with an active agent can also be included in the formulation. Benzyl alcohol can serve as a preservative and sodium chloride can be included to adjust tonicity. In addition, hydrochloric acid and / or sodium hydroxide can be added to the solution to adjust the pH. Aqueous formulations for eye injection can be prepared without preservatives.
[0088] The ocular formulation can allow the sustained release of the active ingredient to the eye. The ocular formulation can be formulated as an emulsion (oil in water or water in oil), a suspension or an ointment. The suspension formulation can contain compound 1 or a pharmaceutically acceptable salt, as a crystalline form, for example, Form 1 or Form 2 or in one. amorphous state.
[0089] Compound 1 can also be formulated to be suitable for administration of eye drop or as an intravitreal implant. The implant can allow the release
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32/117 of constant therapeutic levels of the drug. Reservoir implants are usually made with. a pelleted drug core surrounded by non-reactive substances such as silicon, ethylene vinyl acetate (EVA) or polyvinyl alcohol (ÍPVA), these implants are not biodegradable and can release continuous amounts of a drug for months to years. Matrix implants can also be used. They are generally used to deliver a loading dose followed by reduced doses of the drug for one. 1 day to 6 months. They are most commonly produced from polylactic acid (PLA) and / or polylactic glycolic acid (PLGA) copolymers, which degrade in water and carbon dioxide. Iontophoresis can also be used. Iontophoresis is a non-invasive technique in which a small electrical current is applied to enhance the penetration of the ionized drug into the skin.
[0090] Encapsulated cell technology (TCE), which is a cell-based delivery system as well, can be used to deliver the therapeutic agent to the eyes. Typically, genetically modified cells are housed in a hollow tube of semipermeable membrane, which prevents the entry of immune cells and allows nutrients and therapeutic molecules to disperse freely across the membrane. The two ends of the polymer cut are sealed and a titanium loop is placed on the anchoring end, which is implanted in the flat part and anchored in the sclera.
[0091] Compound 1 can be formulated in. any form that allows release at the back of the eye. Examples of release modes are known in the literature
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33/117 (Kuno et al, Polymers, 2011, 3, 193-221, del Amo et al, Drug Discovery Today, 2008, 13, 135-143, Short, Toxicologic Pathology, 2008, 36, 49-62). Such release modes include, but are not limited to, supracoroid release, which allows for release in the choroid and retina through the supracoroid space, sub-Tenon release, periocular release, contact lenses, point plugs and sclera plugs. Compound 1 can also be released by periocular, suprascleral, retrobulbar, peribulbar or subconjunctival injection.
[0092] Compound 1 can be released as an emulsion, polymeric micro or nanospheres, liposomes, micro or nanoparticles, microspheres, micelles or dendrimers. Biodegradable and biocompatible polymers, such as polytactic acid and PLGA, can be used. Compound 1 can be encapsulated.
[0093] In addition, compound 1 can be formulated for topical administration, to the skin as an ointment or cream. Ointment formulations are semi-solid preparations with one. base of a greasy or oily material that, in general, is clear. Oily materials suitable for use in ointment formulations: petrolatum (petroleum jelly), beeswax, cocoa butter, shea butter, and cetyl alcohol. Ointments may additionally include emollients and penetration enhancers, if desired.
[0094] Cream formulations can be prepared as emulsions, comprising an oil phase and an aqueous phase, usually including purified water. Components of cream formulations may include: oil bases, such as petrolatum, mineral oils, vegetable and animal oils, and
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34/117 triglycerides; cream bases, such as lanolin alcohols, stearic acid and ceto-stearyl alcohol; gel-based, such as polyvinyl alcohol; solvents, such as propylene glycol and polyethylene glycol; emulsifiers, such as polysorbates, stearates, such as glyceryl stearate, octyhydroxistearate, polyoxyl stearate, PEG stearyl ethers, isopropyl palmitate and sorbitan monostearate; stabilizers, such as polysaccharides and sodium sulfite; emollients (that is, moisturizers), such as medium chain triglycerides, isopropyl myristate, and dimethicone; stiffening agents, such as cetyl alcohol and stearyl alcohol, antimicrobial agents, such as methylparaben, propylparaben, phenoxyethanol, sorbic acid, diazolidinyl urea and butylated hydroxyanisole;
penetration potenti ers, such as N-methylpyridine, propylene glycol, polyethylene glycol monolaurate and the like and chelating agents, such as disodium edda.
[0095] Alternatively, the pharmaceutical compositions of the invention are formulated for administration by inhalation. Pharmaceutical compositions suitable for administration by inhalation will be in the form of an aerosol or powder. Such compositions are generally administered by well-known devices, such as metered-dose inhaler, dry powder inhaler, nebulizer or similar device.
[0096] When administered by inhalation using a pressurized container, the pharmaceutical compositions of the invention will include the active ingredient and a suitable propellant, such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In addition, the composition
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Pharmaceutical 35/117 can be in the form of a capsule or cartridge (prepared, for example, from gelatin) comprising compound 1 and one. powder suitable for use in a powder inhaler. Suitable powder bases include, for example, lactose or starch.
[0097] The following non-limiting examples illustrate pharmaceutical compositions representative of the present invention.
Solid oral tablet pharmaceutical form
[0098] Compound 1, or a pharmaceutically acceptable salt, is mixed dry with microcrystalline cellulose, polyvinyl pyrrolidone and croscarmellose sodium in the ratio of 4: 5: 1: 1 and pressed in. tablets to provide a unit dose of, for example, 5 mg, 20 mg or 40 mg of active agent per tablet.
Solid oral capsule dosage form
[0099] Compound 1, or a pharmaceutically acceptable salt is mixed dry with microcrystalline cellulose, polyvinyl pyrrolidone and croscarmellose sodium in a 4: 5: 1: 1 ratio and loaded into gelatin or hydroxypropylmethylcellulose capsules to provide a unit dose of, for example, 5 mg, 20 mg or 40 mg of active agent qoi capsule *
Liquid formulation.
[00100] A liquid formulation comprising compound 1 (0.1%), water (98.9%) and ascorbic acid (1.0%) is formed by adding a compound of the invention to a mixture of water and ascorbic acid .
Pharmaceutical form, oral with enteric coating
[00101] Compound 1 is dissolved in an aqueous solution containing polyvinyl pyrrolidone and applied as a spray coating on microcrystalline cellulose beads
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36/117 or sugar in a ratio of 1: 5 w / w active ingredient / beads and then a gain of approximately 5% of an enteric coating comprising an acrylic copolymer is applied. Enteric coated beads are loaded into gelatin or hydroxypropyl methylcellulose capsules to provide a unit dose of, for example, 30 mg of active agent per capsule.
Oral dosage form with enteric coating [00102] An enteric coating comprising a combination of Eudragit-L® and Eudragit-S®, or hydroxypropylmethylcellulose or acetate succinate is applied to an oral, tablet or capsule dosage form, described above.
Aqueous formulation for, eye injection
[00103] Each ml of a sterile aqueous suspension includes 5 mg to 50 mg of compound 1, sodium chloride for tonicity, 0.99% (w / v) benzyl alcohol as a preservative, 0.75 sodium carboxymethylcellulose % and 0.04% polysorbate. Sodium hydroxide or hydrochloric acid can be included to adjust the pH to 5 to 7.5.
Aqueous formulation for eye injection
[00104] A sterile aqueous suspension without. preservatives includes 5 mg / ml to 50 mg / ml of compound 1 in 10 mM sodium phosphate, 40 mM sodium chloride, 0.03% polysorbate 20 and 5% sucrose.
Ointment formulation for topical administration [00105] Compound 1 is combined with petrolatum, Cs-Cio chain triglycerides, octyhydroxistearate, and nmethylpyrrolidone in a proportion to provide a composition containing 0.05% to 5% active agent by weight.
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Ointment formulation for topical administration
[00106] Compound 1 is combined with white petrolatum, propylene glycol, mono- and di-glycerides, paraffin, butylated hydroxytoluene and disodium calcium in a proportion to provide a composition containing 0.05% to 5% active agent by weight .
Ointment formulation for topical administration
[00107] Compound 1 is combined with mineral oil, paraffin, propylene carbonate, white petrolatum and white wax to provide a composition containing 0.05% to 5% active agent by weight.
Cream formulation for topical administration
[00108] Mineral oil is combined with compound 1, propylene glycol, isopropyl palmitate, polysorbate 60, cetyl alcohol, sorbitan monostearate, polyoxyl 40 stearate, sorbic acid, methylparaben and propylparaben to form an oil phase, which is combined with water purified by shear mixture to provide a composition containing 0.05% to 5% active agent by weight.
Cream formulation for topical administration
[00109] A cream formulation comprising compound 1, benzyl alcohol, cetyl alcohol, anhydrous citric acid, mono and diglycerides, oleyl alcohol, propylene glycol, sodium ketostearyl sulfate, sodium hydroxide, stearyl alcohol, triglycerides and water contains 0, 05% to 5% of active agent by weight.
Cream formulation for topical administration
[00110] A cream formulation comprising compound 1, keto stearyl alcohol, isopropyl myristate, propylene glycol, ketomacrogol 1000, dimethicone 360, acid
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38/117 citrus, sodium citrate and purified water, with imidurea, methylparaben, propylparaben, as preservatives, contains from 0.05% to 5% of active agent by weight.
Dry powder composition
[00111] The micronized compound 1 (1 g) is mixed with crushed lactose (25 g). This combined mixture is then loaded into individual blisters from an easily opened blister pack in an amount sufficient to provide between about 0.1 mg to 4 mg of compound 1 per dose. The contents of the blisters are administered using an inhaler
Q Θ S6CO powder *
Composition of the metered-dose inhaler
[00112] The micronized compound 1 (10 g) is dispersed in a solution prepared by dissolving lecithin (0.2 g) in demineralized water (200 ml). The resulting suspension is dried with. spray and micronized to form a micronized composition comprising particles with an average diameter of less than 1.5 µm. The micronized composition is then loaded into metered-dose inhaler cartridges containing 1,1,1,2 tetrafluoroethane pressurized in an amount sufficient to provide about 0.1 to about 4 mg of compound 1 per dose when administered by a metered-dose inhaler.
Nebulizer composition
[00113] Compound 1 (25 mg) is dissolved in a solution containing 1.5-2.5 equivalents of hydrochloric acid, followed by the addition of sodium hydroxide to adjust the pH to 3.5 to 5.5 and 3% by weight of glycerol. The solution is stirred well until all components are dissolved. The solution is administered with a nebulizer device that provides about 0.1 mg to about 4 mg of compound 1 per dose.
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11/39
U t .1.11 d a α θ
[00114] Compound 1 has been shown to be a potent inhibitor of the JAK family of enzymes: JAK1, JAK2, JAK3 and TYK2. Eye diseases
[00115] It has been shown that many eye diseases are associated with. elevation of proinflammatory cytokines that depend on the JAK-STAT pathway. As compound 1 has a potent inhibition for all four JAK enzymes, it is expected that the signaling and pathogenic effects of various cytokines (such as IL-6, IL-2 and IFN-γ), which signal through JAK are potentially inhibited, as well as the increase in other cytokines (such as MCP-1 and ΤΡΙΟ), whose production is triggered by signaling via JAK-STAT, is avoided.
[00116] On. In particular, compound 1 exhibited pICso values of 6.4 or greater (IC 50 values of 400 nM or less) for inhibiting IL-6, IL-2, and IFNy signaling in cell assays described in Assays 3 to 6 , including assays that record inhibition of downstream effects of cytokine elevation.
[00117] The pharmacokinetic study of Trial 7 demonstrated sustained exposure in the eyes of rabbits after a single intravitreal injection and a plasma concentration of at least three orders of magnitude lower than the concentration observed in vitreous tissue.
In addition, intravitreal administration of compound 1 demonstrated significant inhibition of IL-6-induced pSTAT3 in rat retina / choroidal tissue as well as significant and sustained inhibition of IFN-γ-induced IP-10 in rabbit vitreous as well as in the retina / tissue
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40/117 choroid. Intravitreal administration of compound 1 demonstrated significant and sustained inhibition of pSTAT1 induced by IFN-γ in the rabbit.
[00119] It is expected that sustained ocular JAK inhibition in the absence of significant systemic levels will result in. potent local anti-inflammatory activity in the eye without targeted systemic adverse effects. Thus, compound 1 is expected to be beneficial in several eye diseases that include, but are not limited to, uveitis, diabetic retinopathy, diabetic macular edema, dry eye disease, age-related macular degeneration, retinal vein occlusion and atopic keratoconjunctivitis.
[00120] In particular, uveitis (Horai and Caspi, J Interferon Cytokine Res, 2011, 31, 733-744), diabetic retinopathy (Abcouwer, J Clin Cell Immunol, 2013, Suppl 1, 112), diabetic macular edema (Sohn et al., American Journal of Opthalmology, 2011, 152, 686-694), dry eye disease (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 the elevation of some pro-inflammatory cytokines that signal through the JAK-STAT route. In this way, compound 1 may be able to alleviate eye inflammation, associate and reverse disease progression or provide symptom relief in these diseases.
[00121] In one aspect, therefore, the invention provides a method of treating an eye disease in a mammal, the method comprising administering a pharmaceutical composition comprising 1- (2- (6- (2-ethyl-5-fluorO-4 -hydroxyphenyl) -4
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41/117 fluoro-lif-indazol-3-yl) -1,4,6,7-tetrahydro-5H-imidazo [4,5c] pyridin-5-yl) -2-morpholinoethane-l-one or an acceptable salt from a pharmaceutical point of view and a pharmaceutical carrier for the mammalian eye. In one aspect, eye disease is uveitis, diabetic retinopathy, diabetic macular edema, dry eye disease, age-related macular degeneration, retinal vein occlusion or atopic keratoconjunctivitis. In one aspect, the method comprises administering compound 1 by intravitreal injection.
Inflammatory skin disease
[00122] A. atopic dermatitis, for example, is associated with elevation of pro-inflammatory cytokines that depend on the JAK-STAT pathway, in particular, IL-4, IL-5, IL-10, IL-13 and IFNv . In addition to cytokine inhibition in the cell assays cited above, compound 1 exhibited an IC 50 value of 13 nM for inhibition of IL-13, as described in Assay 2. In addition, cream ointment formulations and formulations of compound 1 demonstrated levels continuous dermal for at least 2 days in mice and at least 7 days in piglets with no detectable plasma exposure.
[00123] It is expected that the continuous cutaneous levels of compound 1 in the absence of significant systemic levels will result in potent local anti-inflammatory and anti-itchy activity on the skin without targeted systemic adverse effects. Compound 1 is expected to be beneficial in a variety of inflammatory or itchy dermal conditions, which include, but are not limited to, alopecia area, vitiligo, cutaneous T-cell lymphoma, nodular prurigo, lichen planus, localized primary cutaneous amyloidosis, bullous pemphigoid , cutaneous manifestations of
Petition 870190110020, of 10/29/2019, p. 68/160 graft versus host disease, pemphigoid, discoid lupus, annular granuloma, chronic simple lichen, vulvar / scrotal / perianal pruritus, sclerous lichen, post-herpes neural pruritus, planopilar lichen and decalvating folliculitis. It was particular, alopecia areada (Xing et al., Nat Med. 2014 Sep; 20 (9): 1043-9), vitiligo (Craiglow et al, JAMA Dermatol. 2015 Oct; 151 (10): 1110-2), lymphoma cutaneous T cells (Netchiporouk et al., Cell Cycle.
2014; 13 (21): 3331-5), nodular prurigo (Sonkoly et al., J Allergy Clin Immunol. 2006 Feb; 117 (2): 411-7), lichen piano (Welz-Kubiak et al., J Immunol Res. 2015; 2015: 854747), localized primary cutaneous amyloidosis (Tanaka et al., Br J Dermatol. 2009 Dec; 161 (6): 1217-24), bullous pemphigoid (Feliciani et al., Int J Immunopathol Pharmacol. 1999 MayAgo; 12 (2): 55-61) and dermal manifestations of graft versus host disease (Okiyama et al., J Invest Dermatol. 2014 fibr; 134 (4): 992-1000) are characterized by the elevation of some cytokines that signal via activation JAK. In this way, compound 1 may be able to relieve the itching associated with skin inflammation or triggered by these cytokines. In particular, compound 1 is expected to be useful for the treatment of atopic dennatitis and other inflammatory skin diseases.
[001241 In one aspect, therefore, the invention provides a method of treating an inflammatory skin disease in a mammal (e.g., a human), the method comprising applying a pharmaceutical composition comprising 1 (2- (6- (2 -ethyl-5-fluoro-4-hydroxyphenyl) -4-fluoro-1-indazol-3yl) -1,4,6,7-tetrahydro-5JF-imidazo [4,5-c] pyridin-5-yl) - 2morpholinoetan-l-one or a salt acceptable from the point of view
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43/117 pharmaceutical and a pharmaceutical carrier for mammalian skin. In one aspect, inflammatory skin disease is atopic dermatitis.
[00125] Compound 1 can also be used in combination with gram positive antibiotics, such as mupirocin and fusidic acid, for the treatment of inflammatory skin diseases. In one aspect, therefore, the invention provides a method of treating an inflammatory skin disease in a mammal, the method comprising applying compound 1 and a gram-positive antibiotic to the skin of mammals. In. another aspect, a. invention provides a pharmaceutical composition comprising 1- (2- (6- (2-ethyl “5-fluoro-4-hydroxyphenyl) -4fluoro-1A-indazol-3-yl) -1,4,6,7-tetrahydro-5 / i-imidazo [4,5c] pyridin-5-yl) -2-morpholinoetan-1-one or a pharmaceutically acceptable salt, a gram positive antibiotic and a pharmaceutically acceptable carrier.
Respiratory diseases
[00126] Cytokines that signal via the JAK-STAT pathway, in particular IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-11, IL13, IL-23, IL -31, IL-27, thymic stromal lymphopoietin (TSLP), gamma interferon (IFNy) and granulocyte-macrophage colony stimulating factor (GM-CSF) are also involved in the inflammation of asthma and other inflammatory respiratory diseases. As described above, compound 1 has been shown to be a potent inhibitor of the enzymes JAK1, JAK2, JAK3 and TYK2 and a potent inhibition of pro-inflammatory cytokines in cell assays.
[00127] The anti-inflammatory activity of JAK inhibitors has been demonstrated in preclinical models of asthma (Malaviya et al., Int Immunopharmacol, 2010, 10, 829, -836;
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Matsunaga et al., Biochem Sind Biophys B.es Comuiun, 2011, 404, 261-267; Kudlacz et al., Eur J Pharmacol, 2008, 582, 154161.) Thus, compound 1 is expected to be useful for the treatment of inflammatory respiratory disorders, in particular, a. asthma. Inflammation and fibrosis of the lung is characteristic of other respiratory diseases in addition to asthma such as chronic obstructive pulmonary disease (COPD), cystic fibrosis (CF), pneumonitis, interstitial lung diseases (including idiopathic pulmonary fibrosis), acute lung injury, distress syndrome acute respiratory disease, bronchitis, emphysema and bronchiolitis obliterans. Therefore, compound 1 is expected to be useful for 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.
[0012 8] On. one aspect, therefore, the invention provides a method of treating a respiratory disease in a mammal (e.g., a human), the method comprising administering 1- (2- (6- (2-ethyl-5-fluoro-) 4-hydroxyphenyl) -4fluoro-1H-indazol-3-yl) -1,4,6,7-tetrahydro-5H-imidazo [4,5c] pyridin-5-yl) -2-morpholinoethane-l-one or a pharmaceutically acceptable salt to one. mammal.
[00129] In one aspect, respiratory disease is 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. a gold
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45/117 aspect, respiratory disease is asthma or chronic obstructive pulmonary disease.
[00130] In another aspect, the respiratory disease is a pulmonary infection, a helminth infection, pulmonary arterial hypertension, sarcoidosis, lymphangioleiomyomatosis, bronchiectasis or an infiltrative lung disease. In another aspect, the respiratory disease is drug-induced pneumonitis, fungus-induced pneumonitis, allergic bronchopulmonary aspergillosis, hypersensitivity pneumonitis, eosinophilic granulomatosis with. polyangiitis, acute idiopathic eosinophilic pneumonia, chronic idiopathic eosinophilic pneumonia, hypereosinophilic syndrome, Loffler syndrome, bronchiolitis obliterans with organizing pneumonia or pneumonia.
[001311 The invention provides a method for treating asthma in a mammal, the method comprising administering a pharmaceutical compound comprising 1- (2- (6- (2-ethyl-5-fluoro ~ 4hydroxyphenyl) -4-fluoro-1H-indazole- 3-yl) -1,4,6,7-tetrahydro-5Himidazo [4,5-c] pyridin-5-yl) -2-morpholinoetan-1-one or a pharmaceutically acceptable salt and an acceptable carrier from a pharmaceutical point of view to a mammal.
[00132] Compound 1 or a pharmaceutically acceptable salt can also be useful for treating eosinophilic lung diseases. Eosinophilic inflammation of the airways is a characteristic of diseases collectively called pulmonary 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 helminth infections), drug-induced pneumonitis
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46/117 (induced, for example, by drugs such as antibiotics, phenytoin or 1-tryptophan), fungal-induced pneumonitis (eg, allergic bronchopulmonary aspergillosis), hypersensitivity pneumonitis and eosinophilic granulomatosis with polyangiitis (formerly known as ChurgStrauss syndrome) . Eosinophilic pulmonary diseases of unknown etiology include acute idiopathic eosinophilic pneumonia, chronic idiopathic eosinophilic pneumonia, hypereosinophilic syndrome and Löffler's syndrome.
[00133] Compound 1 or a pharmaceutically acceptable salt can also be useful for treating PAH. A polymorphism in the IL-6 gene has been associated with high 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). Corroborating the role of IL-6 in PAH, inhibition of the IL-6 gp! 30 receptor chain improved the disease in a mouse model of PAH (Huang et al., Can J Cardiol., 2016, 32 (11 ), 13 5 6.e1-13 5 6.e10).
[00134] Compound 1 or a pharmaceutically acceptable salt may also be useful for treating non-allergic lung diseases, such as sarcoidosis and lymphangioleiomyomatosis. Cytokines like ΙΡΝγ, IL-12 and IL-6 are involved in a variety of non-allergic lung diseases such as sarcoidosis and lymphangioleiomyomatosis (ElHashemite et al., Am. J. Respir. Cell Mol. Biol., 2005, 33, 227- 230, and El-Hashemite et al., Cancer Res. _F 2004, 64, 34363443).
[00135] Compound 1 or a pharmaceutically acceptable salt can also be useful in the treatment of
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47/117 bronchiectasis and infiltrative lung diseases that are diseases associated with chronic neutrophilic inflammation.
Some cytokines are associated with neutrophilic inflammation (eg, IL ~ 6, IFNy).
[00136] The pathological activation of T cells is fundamental in the etiology of several respiratory diseases. Autoreactive T cells play a role in bronchiolitis obliterans with organizing pneumonia (also called BOOP). Similar to BOOP, the etiology of lung transplant rejections is associated with an aberrant activation of receptor T cell activation by the donor transplanted lung. Lung transplant rejections can occur early as primary graft dysfunction (PGD), organizing pneumonia (PO), acute rejection (RA) or lymphocytic bronchiolitis (BL) or can occur years after lung transplantation as chronic lung graft dysfunction (CLAD). CLAD was previously known as bronchiolitis obliterans (BO), but is now considered a syndrome that can have different pathological manifestations including BO, restrictive CLAD (rCLAD or RAS) and graft neutrophilic dysfunction. Chronic pulmonary graft dysfunction (CLAD) is a major challenge for management in. long-term lung transplant recipients, as it causes the transplanted lung to gradually lose its functionality (Gauthier et al., Curr Transplant Rep., 2016, 3 (3), 185-191). CLAD is not very sensitive to treatment and, therefore, there remains a need for effective compounds capable of preventing or treating this condition. Several JAK-dependent cytokines, such as IFNy and IL-5, are over-regulated in CLAD and in transplant rejection.
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48/117 lung (Berastegui et al, Clin Transplant. 2017, 31, el2898). In addition, high levels of lung CXCR3 chemokines such as CXCL9 and CXCL10 that are downstream (downstream) to JAK-dependent IFN signaling are related to worse outcomes in patients undergoing lung transplantation (Shino et al, PLOS One, 2017, 12 (7), e0180281). 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 has the potential to be effective in the treatment or prevention of lung transplantation and CLAD. Events similar to T cell activation as described as the basis for lung transplant rejection are also considered to be the main factor in graft versus lung host disease (GVHD) that can occur after hematopoietic stem cell transplantation. Similar to CLAD, pulmonary GVHD is a progressive condition with extremely poor outcomes and there is no currently approved treatment. One, retrospective multicenter research study of 95 patients with acute or chronic steroid-refractory GVHD who received the systemic JAK inhibitor ruxolitini.be as rescue therapy demonstrated a complete or partial response to ruxolitinib in. majority of patients, including those with. Pulmonary GVHD (Zeiser et al, Leukemia, 2015, 29, 10, 2062-68). More recently, the immune checkpoint inhibitor induced pneumonitis, another T cell-mediated lung disease that came up with. the increasing use of immunological checkpoint inhibitors. Cancer patients treated with these T cell stimulating agents can
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49/117 develop fatal pneumonitis. Compound 1 or a pharmaceutically acceptable salt has the potential to present a new treatment for these unassisted severe respiratory diseases.
Gastrointestinal diseases
[00137] As a JAK inhibitor, compound 1 or a pharmaceutically acceptable salt can also be useful for a variety of other diseases. Compound 1 or a pharmaceutically acceptable salt can be useful for various gastrointestinal inflammatory indications that include, but are not limited to, inflammatory bowel disease, ulcerative colitis (proctosigmoiditis, pancolitis, ulcerative proctitis and left colitis), Disease Crohn's disease, collagenous colitis, lymphocytic colitis, Behcet's disease, celiac disease, immunological checkpoint inhibitor-induced colitis, ileitis, eosinophilic esophagitis, graft versus host disease 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., Rmmunol Res, 2013, 56, 249-260), colitis related to graft versus host disease (Coghill et al., Blood, 2001, 117, 32683276), infectious colitis (Stallmach et al., Int J Colorectal Dis, 2004, 19, 308-315), Behcet disease (Zhou et al., Autoímmun Rev, 2012, 11, 699-704), celiac disease (de Nitto et al .. World J Gastroenterol, 2009, 15, 4609-4614), inhibitor-induced colitis (checkpoint) checkpoint
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Immunological 50/117 (eg, CTLA-4 inhibitor-induced colitis; (Yano et al., J Translation Med, 2014, 12, 191), or PD-1- or PD-L1 inhibitor-induced colitis ) and ileitis (Yamamoto et al., Dig Liver Dis, 2008, 40, 253-259) are characterized by increased levels of some pro-inflammatory cytokines. As many pro-inflammatory cytokines signal via JAK activation, compound 1 or one. pharmaceutically acceptable salt may be able to relieve inflammation and provide relief for your symptoms. In particular, compound 1 or a pharmaceutically acceptable salt may be useful for inducing and maintaining remission of nonspecific ulcerative colitis, and for the treatment of Crohn's disease, checkpoint inhibitor-induced colitis (checkpoint) immune system, and the gastrointestinal adverse effects of graft versus host disease. In one aspect, therefore, the invention provides a method for treating an inflammatory gastrointestinal disease in. a mammal (e.g., human), the method comprising administering to the mammal compound 1 or a pharmaceutically acceptable salt or a pharmaceutical composition comprising a pharmaceutically acceptable carrier and compound 1 or an acceptable salt of the point of v .1 stafa rma c ê u 11. co.
Other diseases
[00138] 0 compound 1 or one. salt. pharmaceutically acceptable can also be useful for the treatment of other diseases such as inflammatory diseases, autoimmune diseases or cancers.
[00139] Compound 1 or a pharmaceutically acceptable salt can be useful for the treatment of a
Petition 870190110020, of 10/29/2019, p. 77/160 or more of arthritis, rheumatoid arthritis, juvenile rheumatoid arthritis, transplant rejection, xerophthalmia, psoriatic arthritis, diabetes, insulin dependent diabetes, motor neuron disease, myelodysplastic syndrome, pain, sarcopenia, cachexia, septic shock, lupus erythematosus , leukemia, chronic lymphocytic leukemia, chronic myelocytic leukemia, acute lymphoblastic leukemia, acute myelogenic leukemia, ankylosing spondylitis, myelofibrosis, B-cell lymphoma, hepatocellular carcinoma, Hodgkin's disease, breast cancer, multiple myeloma, melanoma, melanoma, melanoma non-small cell lung, clear cell ovarian cancer, ovarian tumor, pancreatic tumor, polycythemia vera, Sjoegrens syndrome, soft tissue sarcoma, sarcoma, splenomegaly, T-cell lymphoma and major thalassemia.
Combination therapy
[00140] The compounds of the disclosure or a pharmaceutically acceptable salt can also be used in combination with. one or more agents that act, by the same or different mechanisms to treat a disease. The different agents can be administered in sequence or simultaneously, in separate compositions or in the same composition. Useful classes of agents for combination therapy include, but are not limited to, antiangiogenic, steroid, anti-inflammatory, plasma kallikrein inhibitor, placental growth factor inhibitor, VEGF-A ligand inhibitor, angiopoietin ligand inhibitor ~ 2, protein tyrosine phosphatase beta inhibitor, Tek tyrosine kinase receptor stimulator, calcineurin inhibitor, VEGF ligand inhibitor, inhibitor
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52/117 of the mTOR 1 complex, mTOR inhibitor, IL-17 antagonist, calmodulin modulator, FGF receptor antagonist, PDGF receptor antagonist, VEGF receptor antagonist, alpha-TNF ligand inhibitor, TNF binding agent, stimulator of proteoglycan 4, VEGF-C ligand inhibitor, VEGF-D ligand inhibitor, CD126 antagonist, complement cascade inhibitor, glucocorticoid agonist, complement C5 factor inhibitor, cannabinoid receptor antagonist, 1-receptor modulator sphingosine-l-phosphate, sphingosine-l-phosphate-3 receptor modulator, sphingosine-l-phosphate-4 receptor modulator, sphingosine-l-phosphate 5-receptor modulator, acetaldehyde dehydrogenase inhibitor, tyrosine kinase inhibitor Flt3, tyrosine kinase inhibitor Kit, protein kinase C inhibitor, adrenocorticotrophic hormone ligand, factor 1 ligand inhibitor derived from stromal cells, immunoglobulin Gl agonist; interleukin-1 beta ligand inhibitor, mucin stimulator; nuclear factor kappa B modulator, protein-4 stimulator of cytotoxic T lymphocytes, inhibitor of CD28 glycoprotein on the surface of the T cell, stimulator of lipoprotein lipase; PPAR-alpha agonist, adenosine A3 receptor agonist, angiotensin II receptor antagonist, VEGF receptor antagonist, beta-interferon ligand, SMAD-2 modulator; TGF beta 1 ligand inhibitor, somatostatin receptor agonist, IL-2 receptor alpha subunit inhibitor, VEGF-B ligand inhibitor, thymosin beta 4 receptor antagonist, angiotensin II AT-1 receptor antagonist, antagonist chemokine CCR2, amino copper oxidase inhibitor with membrane copper, CD11a antagonist, ICAM-1 inhibitor,
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53/117 type 1 insulin-like growth factor antagonist, kallikrein inhibitor, fucosyltransferase 6 stimulator, GDP-fucose synthase 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, PPAR-alpha agonist, Rho GTPase inhibitor, protein-associated Rho kinase inhibitor, C3 complement inhibitor, EGR-1 transcription factor inhibitor, modulator factor-related erythroid nuclear factor 2, kappa B nuclear factor inhibitor, alpha-V / beta-3 integrin antagonist, erythropoietin receptor agonist, glucagon-like peptide agonist, TNFRSF1A gene stimulator, ligand 2 inhibitor angiopoietin, alpha 2-antiplasmin inhibitor, collagen antagonist, fibronectin inhibitor, laminin antagonist, plasmin stimulator, nerve growth factor ligand, FGF1 receptor antagonist, FGF3 receptor antagonist, itk tyrosine kinase inhibitor, inhibitor tyrosine kinase Lck, Ltk tyrosine kinase inhibitor, PDGF alpha-receptor antagonist, PDGF beta-receptor antagonist, protein tyrosine kinase inhibitor, receptor antagonist ptor VEGF-3, amino oxidase inhibitor with. membrane copper, subtype 2 somatostatin receptor agonist, subtype 4 somatostatin receptor agonist, subtype 5 somatostatin receptor agonist, protein kinase C alpha inhibitor, protein kinase C beta inhibitor, delta protein kinase inhibitor, inhibitor protein kinase C epsilon protein kinase C eta inhibitor, protein kinase C theta inhibitor, ankylrin modulator,
Petition 870190110020, of 10/29/2019, p. 80/160 mucin stimulator, P2Y2 purinoceptor agonist, alpha-Γι protein inhibitor. gap junction, CCR3 chemokine antagonist; eotaxin ligand inhibitor, amiloride sensitive sodium channel inhibitor, PDGF receptor antagonist, protein tyrosine kinase inhibitor, retinal pigment epithelium protein inhibitor, matrix metalloprotease inhibitor, PDGF receptor antagonist, beta- antagonist PDGF receptor, PDGFB ligand inhibitor, growth hormone receptor antagonist, cell adhesion molecule inhibitor, integrin modulator, chemokine antagonist. CX.CR4, coiled coil domain containing protein inhibitor, Hsp 90 modulator, protein-associated Rho kinase inhibitor, VEGF gene inhibitor, endoglobin inhibitor, CCR3 chemokine antagonist, maxi K potassium channel modulator, stimulator of the maxi K potassium channel, PGF2 alpha agonist, prostanoid receptor agonist, voltage-dependent chloride channel 2 modulator, complement C5a receptor antagonist, inosine monophosphate dehydrogenase inhibitor, interleukin 18 ligand inhibitor, channel stimulator cationic TRP M8, agonist of the CNTF receptor, inhibitor of the TRPV1 gene, stimulator of deoxyribonuclease I, inhibitor of the gene IRS1, inhibitor of Rho kinase associated with protein, inhibitor of poly ADP ribose polymerase 1, inhibitor of poly ADP ribose polymerase 2, poly ADP ribose polymerase 3 inhibitor, VR1 vanyloid agonist, NFAT5 gene stimulator, Mucin stimulator, Syk tyrosine kinase inhibitor, adrenaline alpha 2 agonist ceptor, cyclooxygenase inhibitor, inhibitor of amyloid protein deposition, glycogen synthase kinase-3 inhibitor, PARP stimulator, inhibitor
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55/117 of tau deposition, DDIT4 gene inhibitor, hemoglobin synthesis modulator, interleukin-1 beta ligand inhibitor, TNF antagonist, voltage-dependent KCNQ potassium channel stimulator, NMDA receptor antagonist, cycle inhibitor -oxygenase 1, cyclooxygenase inhibitor, 5-HT 1α agonist, calcium channel inhibitor, FGF-2 ligand modulator, phosphoinositol 3 kinase inhibitor, CD44 antagonist, hyaluronidase modulator, hyaluronic acid agonist, antagonist of IL-1, type I IL-1 receptor antagonist, complement factor P inhibitor, tubulin antagonist, beta amyloid antagonist, IL2 gene stimulant, kinase I-kappa B inhibitor, kappa nuclear factor modulator B, plasminogen activator inhibitor 1, FGF-2 ligand, protease modulator and corticotropin modulator.
[00141] Specific agents that can be used in combination with the current JAK inhibitor compound include, but are not limited to, lanadelumab, aflibercept, RG-7716, AKB-9778, cyclosporine, bevacizumab, everolimus, secukinumab, acetonide fluocinolone, RP -101, squalamine lactate, recombinant human lubricin, OPT-302, sarilumab, dexamethasone, eculizumab, fingolimod, adalimumab, reproxalape, midostaurine, corticotropin, olaptesed pegol, canakinumab, recoflavone, abatacepte, golatenate, pencenidate, fenofenate, penhofen pegaptanib, betainterferone, disitertide, octreotate acetate, anecortave, basiliximab, supracoroidal acetonide triamcinolone, RGN-259, di fluprednate, HL-036, avacincaptad pegol sodium, irbesartan, propagermanium, triamcinegone azonide, 14-azonetron, acetonide, BI-5
Petition 870190110020, of 10/29/2019, p. 82/160 of loteprednol, teprotumumab, KVD-001, TZ-101, atesidorsen, Nov-03, bevacizuma.be, AVA-101, RU-101, voclosporine, vorolanib, sirolimus, choline fenofibrate, VX-210, APL- 2, CPC-551, elamipretide, SF-0166, cybinetide, elamipretide, 1iragluti da, EYS- 6 0 6, ne svacumab, flibercept, ocriplasmin, filgotinib, cenegermine, adipocell, brolucizumab, ranibizumab, afliberinipine, afliberinipine, therapy with afliberinipid, therapy with afliberin, lipid therapy pazopanib, ASP-8232, veldoreotide, sotrastaurine, abicipar pegol, diquafosol tetrasodium, HCB-1019, conbercept, bertilimumab, SHP-659, THR-317, ALK-001, PAN-90806, alpha-2b-interferone, fluocinolone malate, malate sunitinib, emixustate, hi-conl, TB403, minocycline, MA09-hRPE cells, pegpleranib sodium, pegvisomant, luminate, burixafor, H-1129, carotuxima.be, AXP 1275, ranibizumab, isopropyl unoprostone, tesidolumab, tesidolumab, sodium mycophenolate , tadekinig alfa, triamcinolone acetonide, cyclosporine, ST-266, 7WX-012 NT-501-ECT, tivanisiran, ve rteporfin, alpha-dornase, aganirsen, ripasudil, rucaparib phosphate, zucapsaicin, tetrathiomolybdate, diclofenac, LHA-510, AGN-195263, tacrolimus, rebamipide, R-348, brimonidine tartrate, vizomitine, T -36, 89 BI-1026706, rimexolone, tobramycin, TOP-1630, talaporfin, sodium bronfenac, triamcinolone acetonide, davunetide, loteprednol etabonate, XED-60, EG-Mirotin, APD-209, adenovir, PF04523655, hydroxycarpamide, retinal, hydroxycarbamide, hydroxycarbamide ranibizumab, flupirtin, B27PD, S-646240, GLY-230, hydralazine, nepafenac, DexNP, Trealose, hyaluronic acid, sustained release deposition of dexamethasone-Ca, naluzotane, hyaluronidase, sodium hyaluronate, isunakinra,
Petition 870190110020, of 10/29/2019, p. 83/160 somatostatins, CLG-561, OC-IOX, UCA-002, recombinant human epidermal growth factor, pemirolaste, VM-100, MB11316, monosodium al-luminol, ranibizumab, IMD-1041, LMG324, HE-10, sodium kinialuronate, BDM-E, precursor cells that are sensitive, dissolve, CT C-9 6, PG-101, Beifushu, chymotrypsin.
[001421 Here also provided is a pharmaceutical composition comprising compound 1 or a pharmaceutically acceptable salt and one or more therapeutic agents. The therapeutic agent can be selected from the class of agents specified above and from the list of specific agent described above. In some embodiments, the pharmaceutical composition of the invention is suitable for delivery to the eyes. In some embodiments, the pharmaceutical composition is a liquid or a liquid composition. suspension.
[00143] Furthermore, in one aspect of the method, the invention provides a method for treating a disease or disorder in a mammal comprising administering to the mammal compound 1 or a pharmaceutically acceptable salt and one or more therapeutic agents.
[00144] When used in therapeutic combination, the agents can be formulated in a single pharmaceutical composition, or the agents can be supplied in different compositions that are administered simultaneously or at different times, by the same route or by different routes of administration. Such compositions can be packaged separately or can be packaged as a kit. The two or more therapeutic agents in the kit can be administered by the same route of administration or by different routes of administration.
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EXAMPLES
[00145] The following synthetic and biological examples are provided to illustrate the invention, and are not to be construed in any way, as a limitation on the scope of the invention. In the examples below, the abbreviations have. the following meanings, unless otherwise indicated. Abbreviations not defined below have their accepted meanings.
ACN = acetonitrile
DCC = dicyclohexylcarbodiimide
DIPEA = N, AFdi-isopropiethylamine
DMAc = dimethylacetamide
DMF = N, AZ-dimethyltormamide
DMSO = dimethyl sulfoxide
EtOAc == ethyl acetate
HATU = Ν, Ν, Ν ', N'-tetramethyl-O (7-azabenzotriazol-l-yl) uranium hexafluorophosphate
LDA = lithium diisopropylamide min. === minute (s)
MTBE = tert-butyl methyl ether
NBS = N-bromosuccinimide
NMP = N-methyl-2-pyrrolidone
TA = room temperature
THF === tetrahydrofuran bis (pinacolate) diboron = 4.4,5, 5, 4 ', 4', 5 ', 5'-octamethyl [2,2'] bi [[1,3,2] dioxaborolanil]
Pd (dppf) C2 "CH ₂ C12 = dichloro (1,1 '~ bis (diphenylphosphine) -ferrocene) dipaladium (II) complex with dichloromethane
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[001461 Q reagents and solvents were purchased from commercial suppliers (Aldrich, Fluka, Sigma etc.), and used without purification. The progress of the reaction mixture was monitored by thin layer chromatography (TLC), high performance liquid chromatography (CLA.E / analytical HPLC) and mass spectrometry. The reaction mixtures were worked out as described specifically in each reaction; were purified by extraction and other purification methods, such as temperature and solvent-dependent crystallization and precipitation. In addition, the reaction mixtures were purified by column chromatography or preparative HPLC, generally using C18 or column BDS packaging and conventional eluents. Typical conditions for preparative HPLC are described below.
[00147] The characterization of the reaction products was made using mass spectrometry and ¹ H-NMR. For PMN analysis, the samples were dissolved; in deuterated solvents (such as CD3OD, CDCI3, or dg-DMSO), and the ^ H-NMR spectra were acquired with the Varian Gemini 2000 (400 MHz) instrument under standard observation conditions. The identification by mass spectrometry of the compounds was done by one. electrospray ionization method (ESMS) with. one. Applied Biosystems instrument, (Foster City CA) model A.PI 150 EX or a Waters instrument (Milford, MA) 3100, coupled with self-purification systems.
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HPLC conditions ρ r and p a. r a t i v a
Column:
C18, 5 pm .. 21.2 x 15 0 mm or C18, pm 21 x 250 or C14, 5 pm 21x150 mm
Column temperature:
Flow rate:
Mobile phases:
Injection volume:
Wavelength d
Ambient temperature 20.0 ml / min.
A = Water + 0.05% TFA B = ACN + 0.05% TFA, (100-1500 pl)
214 nm.
detector:
[001481 The crude compounds were dissolved in. 1: 1 water: acetic acid at about 50 mg / ml. A 4 minute run of the toi test analytical scale performed using a 2.1 x 50 mm C18 column, followed by a 15 or 20 minute preparative scale run using 100 pL injection with 0 'gradient based on% B retention of the test analytical scale run. The precise gradients depended on the sample. . Samples with impurities with close retention times were checked with a C18 21 x 250 mm column and / or C14 21 x 150 mm column for better separation. Fractions with the desired product were identified by mass spectrometry analysis.
Preparation 1: 2- (4- (benzyloxy) -2-ethyl-S-fluorophenyl) 4,4,5,5-tetramethyl-1,2,2-dioxaborolane (1-5)
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[0014 9] Two reactions were carried out on. parallel and combined for work. A mixture of 5-bromo-2fluorophenol (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 h. The reactions were combined and concentrated, diluted with. water (8 1) and extracted with EtCAc (3 x 3 1). The organic layer was separated, washed with brine (3 L), dried over sodium sulfate and concentrated. The crude product was purified through a block of silica gel (elution with. 3: 1 petroleum ether: EtCAc) to yield the intermediate title (1.83 kg, 73% yield) as a white solid. NMR Hi (400 MHz, CDC1 ₃ ) δ 7.38-7.4 6 (m, 5H), 7.15 (dd, J ------ 7.6, 2.0 H z, 1H) , 6.98-7.15 1, m, 1H), 5, 12 (s, 2 H, 1.
(b) 2- (Benzyloxy) -4-ethyl-1-fluorobenzene (1-3)
[00150Ί Six reactions were performed in parallel and combined for the job. To a solution of the product from the previous step (200 g, 711 mmol) in THE (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) CI2-CH2Cl2 (11.6 g, 14.2 mmol). The reaction mixture was cooled to 0 ° C, diethylzinc (1 M, 1.07 1) was added dropwise and the reaction mixture was stirred at 70 ° C for 1 h. The reactions were combined, cooled to 20 ° C and poured into water (7 1) slowly. To the mixture was added aq. 4 M HCl to pH 6. The organic layer was separated and the aqueous phase was extracted with EtCAc (3 x 2 1). The combined organic layer was washed with brine (5 l), dried over sodium sulfate, concentrated and purified through a block of silica gel (eluted with 50: 1
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62/117 oil: EtOAc) to produce the intermediate title (900 g,
92% yield) as a light yellow oil. ¹ H 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), 2.522.58 (m, 2H), 1.17 (t, J = 7.6 Hz, 3H ).
(c) 1- (Benzyloxy) -4-bromo-5 ~ ethyl-2-fluorobenzene (1-4)
[00151] Four reactions were carried out in parallel and combined. To a solution of 2- (benzyloxy) -4-ethyl-1-fluorobenzene (1-3) (293 g, 1.3 mol) in AON (11) was added NBS (249 g, 1.4 mol) in. portions at 20 ° C. The reaction mixture was stirred at 20 ^U C for 2 h. The reaction mixtures were combined and concentrated. The residue was diluted with water (5 1) and extracted with EtOAc (2 x 5 1). The organic layer was washed with brine (41), dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. The crude product was purified by chromatography on silica gel (eluting with 100: 1 - 10: 1 petroleum ether: EtOAc) to produce the intermediate title (1.4 kg, 89% yield) as light yellow oil. NMR ^{: 1} H (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), 5.0 6 (s, 2H), 2.6 (q, J ------- 7.6 Hz, 2 H), 1.1 (t , J = 7.6 Hz, 3H).
(d) 2- (4- (benzyloxy) -2-ethyl-5-fluorophenyl) -4,4,5,5
t.etramethyl-1,3,2-dioxaborolane (1-5)
[00152] Seven reactions were carried out in parallel and combined for work. To a solution of the product from the previous step (200 g, 647 mmol) in dioxane (21) was added potassium acetate (190 g, 1.9 mol), bis (pinacolate) diboron (181 g, 712 mmol) and Pd (dppf) CI2-CH2CI2 (10.6 g, 12.9 mmol) under nitrogen at 20 ° C. The mixture was
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63/117 stirred at 120 ° C for 2 h. The reaction mixtures were combined, concentrated, diluted with. water (5 1) and extracted with EtOAc (3 x 4 1). The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. The crude product was purified by chromatography on. silica gel (elution 1: 0 - 5: 1 petroleum ether: EtOAc) to yield the compound title (1.35 kg, 84% yield) as a white solid. PÍMN HI (400 MHz, CDC1 ₃ ) δ 7.33-7.51 (m,
6H), 6.82 (d, J = 1.6 Hz, 1H), 5.17 (s, 2H), 2.85 (q, J =
7.6 H z, 2 H), 1.33 (s, 12 H), 1.15 (t, J = /, 6 H z, 3 H).
Preparation 2: 1-Benzyl-4-imino-1,4-di-idropyridin-3amine (2)
[00153] 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 insoluble material was washed with ACN (500 ml) and dried to produce the HBr salt of the compound title (600 g, 2.14 mol, 58% yield) as a white powder. ³ H NMR (400 MHz, MeOD) δ 7.83 (d, J = 5.6 Hz, 1H), 7.64 (s, 1H), 7.32-7.4 0 (m, 5H), 6 , 76 (d, J = 6.8 Hz, 1H), 5.28 (s, 2H).
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Preparation 3: 6 "(4- (Benzyloxy) -2-ethyl" 5-fluorophenyl) -4fluoro-1Jf-indazol-3-carbaldehyde (3)
(3-1)
[00154] Nine reactions were carried out in parallel and combined for work. A solution of l-bromo-3,5difluorobenzene (100 g, 518 mmol) in THF (700 ml) was degassed and purged with nitrogen three times. Then 2 M LDA (311 ml) was added at -70 ° C and the reaction mixture was stirred at -70 ° C for 0.5 h under nitrogen. A solution of ethyl 2,2-diethoxyacetate (96 g, 544 mmol) in THF (200 ml) was added dropwise at -70 ° C under nitrogen and the reaction mixture was stirred for 1 h. The reactions were combined and poured into ice saturated ammonium chloride (10 1) in portions and extracted with. EtOAc (3 x 3 1). The organic layer was separated, washed, with brine (5 l), dried over sodium sulfate, concentrated and purified
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65/117 with chromatography on silica gel (eluted with 1: 0 - 100: 1 petroleum ether: EtOAc) to produce the intermediate title (1.26 kg, 84% yield) as a yellow oil. NMR ² Η (400 MHz, CDCI3) δ 7.12 (d, J == 7.2 Hz, 2H), 5.15 (s, 1H), 3, 61 - 3, / (, m, 4H) , 1.2 (t, J = 7.2 Hz, OH).
(b) 1- (4 '- (benzyloxy) -2'-ethyl-3, 5,5'-trifluoro- [1,1'biphenyl] -4-yl) -2,2-dietoxietan-l-one ( 3-2)
[00155] Five reactions were carried out in parallel and combined for work. A. a mixture of 1- (4-bromo-2,6difluorophenyl) -2,2-dietoxietan-l-one (3-1) (189 g, 586 mmol) in ethanol (150 ml) and toluene (1.5 L) water (150 ml), sodium carbonate (84.8 g, 800 mmol) and 2- (4 (benzyloxy) -2-ethyl-5-fluorophenyl) -4,4,5,5-tetramethyl- 1,2,2dioxaborolane (1-5) (190 g, 533 mmol) at 20 ° C. The suspension was degassed under vacuum and purged with. nitrogen several times. Pd (dppf) CI2 - CH2CI2 (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 (3 x 4 L) .. The organic layers were washed with brine (5 L), dried over sodium sulfate, filtered, concentrated and purified by chromatography on silica gel (eluted with 100: 1 - 5: 1 petroleum ether : EtOAc) to produce the intermediate title (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, 2 H), 3.6 / -3, / 7 (m, 4 H), 2.52 (, q, J = 7.6 H z, 2 H), 1.25 (t, J = 6.8 Hz, 6H), 1.07 (t, J = 7.2 Hz, 3H).
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66/117 (c) 6- (4- (benzyloxy) -2-ethyl-5-fluorophenyl) -3 (dietoxymethyl) -4-fluoro-1H-indazole (3—3)
[00156] Four reactions were carried out in parallel and combined for work. To a solution of the product from the previous step (220 g, 466 mmol) in THF (21) was added hydrazine monohydrate (47.6 g, 931 mmol) at 20 ° C. The reaction mixture was stirred at 100 ° C for 12 h. Four reactions were combined and cooled to 20 ° C and concentrated. The residue was dissolved with EtOAc (5 ml) and washed with. 0.1 HCl (2 x 1.5 1). The combined organic layers were washed with brine (1.5 l), dried over sodium sulfate, filtered and concentrated to give the intermediate title (900 g, crude) as yellow gum, which was used directly in the next step. NMR (400 MHz, CDCI3) 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 (q, 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-1Hindazole-3-carbaldehyde (3)
[001571 Three reactions were carried out in parallel and combined for work. To a solution of the product from the previous step (300 g, 643 mmol) in acetone (1.5 L) was added 4 M HCl (16 ml) dropwise at. 20 ° C and the reaction mixture was. stirred at 20 ° C for 0.17 h. The reactions were combined, concentrated, diluted with MTBE (11) and filtered. The insoluble material was washed with MTBE (2 x 300 ml) and dried under reduced pressure to produce the intermediate title (705 g, crude) as one. yellow solid, which was used directly in the next step, (m / z): [M + HJ ⁺ calculated for 393, 1 of C23H18F2N2O2 was found 393, 13. NMR -Ή (4 00 MHz, DMSO-dg) δ 14, 51 (s, 1H),
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10.17 (d, J = 3.6 Hz, 1H), 7.50 (d, J = 7.2 Hz, 2H), 7.407.42 (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: S-Benzyl-2- (6- (4- (benzyloxy) -2-ethyl-Sfluorophenyl) -4-fluoro-1H-indazol-3-yl) -SH-imidazo [4,5 cjpiridine (4)
combined for work. A. a solution of 6- (4 (benzyloxy) -2-ethyl 1 - 5 - f 1 uoropheni 1) -4-fluoro ro-1H-indazo 1-3 carbaldehyde (3), the product of Preparation 3 (172 g , 440 mmol) in DMF (1.11), sodium bisulfite (68.6 g, 659 mmol) and 1-benzyl-4-imino-1,4-dihydropyridine-3-amine (2) ( 136 g, 484 mmol) at 20 ° C and the reaction mixture was stirred at 150 ° C for 2 h. The four reactions were combined and the reaction mixture was concentrated under reduced pressure. The residue was poured into water (10 1) and filtered. The insoluble material was dried under reduced pressure to produce the intermediate title (990 g, crude) as a yellow solid, which was used directly without purification, (m / z): [M + H] ⁺ calculated for 572.2 C35H27F2N5O 572.3 was found.
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Preparation 5: 5-Benzyl-2- (6- (4- (benzyloxy) -2-ethyl-5fluorophenyl) -4-fluoro-1H-indazol-3-yl) -4,5,6,7-tetrahydro-1Himidazo [4,5-c] pyridine (5)

5
[00159] Three reactions were carried out in parallel and combined for work. To a mixture of 5-benzyl-2- (6 (4- (benzyloxy) -2-ethyl-5-fluorophenyl) -4-fluoro-1H-indazol-3yl) -Sn-imidazo [4,5-c] pyridine (4), the product of Preparation 4 (330 g, 577 mmol) in methanol (1.5 1) and THF (1 L) in sodium borohydride (267 g, 6.9 mol) in. portions at 20 ° C and the reaction mixture was stirred at 20 ° C for 24 h. The three reactions were combined and the reaction mixture was added to the water (10 1), stirred for 10 min. and filtered. The filtrate was extracted with. EtOAc (2 x 5 L) and the combined organic layer was dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. The crude product was diluted with EtOAc (21), stirred for 30 min. and filtered. The insoluble material was washed with MTBE (3 x 200 ml) to produce the intermediate title (275 g, 28% yield) as a light yellow solid, (m / z): [M + H] + calculated for 57 6, 25 of C35H31F2N5O was found 57 6.3. NMR JH (400 MHz, DMSO-dg) 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 Hz, 1H), 6.81 (d, J ------- 12.0 Hz, 1H), 5.25 (s, 2H), 3.72 (s, 2H), 3.43 (brs, s,
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2H), 2, / 8 (br, s, 2H), 2.66 (br, s, 2H), 2.5b (q, 2H), 1.04 (t, J = 7.6 Hz, 3H) .
Preparation 6: 5 ~ Ethyl ~ 2 ~ fluoro ~ 4 ~ (4 "fluoro ~ 3 ~ (4,5,6,7 tetrahydro-1H-imidazo [4,5- ~ dpiridin-2-yl) - 1H-m. nda.zo 1 --- 6-yl) phenol (6)

[001601 Five reactions were carried out in parallel and combined for work. To a mixture of 5-benzyl-2- (6 (4- (benzyloxy) ~ 2 ~ ethyl ~ 5-fluorophenyl) ~ 4-fluoro ~ 1H - indazol ~ 3yl) ~ 4,5,6,7- ~ tetrahydro -lH- ~ imidazo [4,5- ~ c] pyridine (5), the product of Preparation 5 (55 g, 95.5 nunol) in THF (500 ml) and methanol (500 ml) was added palladium on carbon ( 15 g, 9.6 mmol) and aq. 12 M HCl (10 ml). The suspension was degassed under vacuum, purged with hydrogen several times and stirred under hydrogen (50 psi) at 50 ° C for 12 h. The reactions were combined and the reaction mixture was filtered. The filtrate was concentrated in vacuo to produce the intermediate title HCl salt (150 g, crude) as an off-white solid, (m / z): [M + H] ⁺ calculated for 396.16 of C21H19F2N5O found 396, 2 ¹ H NMR (400 MHz, MeOD) δ
7.43 (s, 1H), 7.07 (O, J --- 11.6 Ηz, 1H), 6.9 7 (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 (q, J = 7.6 Hz, 2H), 1.08 (t, J =
7.6 Hz, 3H).
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Preparation 7: 2,5-dioxopyrrolidin-1-i 2morpholinoacetate (7 ’)
(a) tert-Butyl 2-morpholinoacetate (7-1)
[00161] To a mixture of morpholine (160 g, 1.84 mol) and potassium carbonate (381 g, 2.76 mol) in THF (3 1) 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 (3 x 1 1). The organic layer was separated, washed with brine (500 ml), dried over sodium sulfate and concentrated to give the intermediate title (300 g, 81% yield) as a yellow oil. ¹ H NMR (400 MHz, CDCl 3) δ 3.74 (t, J 4.8 Hz, 4H), 3.10 (s, 2H), 2.57 (t, J ------ 4.8
H z, 4 H), 1.46 (s, 9H).
(b) 2-morpholinoacetic acid (7 ’’)
[00162] A mixture of the product from the previous step (7-1) (300 g, 1.49 mol) in 3 M HCl in dioxane (2.0 1) was stirred at 20 ° C for 12 h and concentrated to produce a salt HC1 of the compound title (270 g, 99% yield) as a clear solid, which was used directly in the next step NMR ^] -H (400 MHz, MeOD) δ 4.13 (s, 2H), 3.93 (br s, 4H), 3.64 (br. s, 4H).
(c) 2,5-dioxopyrrolidin-1-yl 2-morpholinoacetate (7 ') [00163] A mixture of the product from the previous step (150 g, 826 ml), l-hydroxypyrrolidine-2,5-dione (95 g, 826 mmol), DCC (256 g, 1.24 mol) and DIPEA (160 g, 1.24 mol) in
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DCM (21) 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 provide the compound title (150 g, 75% yield) as a white solid. NMR (400 MHz, DMSO-dg) 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-1J-indazol-3-yl) -1,4,6,7 ~ tet.hydro -5Jf-imidazo [4,5c] pyridin-5-yl) -2-morpholinoetan-l-one (1)

[00164] A mixture of 5-ethyl-2-fluoro-4- (4-fluoro-3 (4,5,6,7-tetrahydro-1H-imidazo [4,5-c] pyridin-2-yl) - 1Hindazol-6-yl) phenol (6) 2 HCl (100 g, 214 mmol), 2,5dioxopyrrolidin-1-yl 2-m.orf olinoacetate (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 instrument; 2 kg of DAC Cl8 Agela column, 200 g of sample dissolved in DMF (900 ml); flow rate of
300 ml / min .; solvent A: water, solvent B: ACN; gradient (% B, time (min.): 0/15, 0-40 / 45, 40/50) to obtain the compound title (50.0 g, 44.8% yield) as a light yellow solid, ( m / z): [M + H] ⁺ calculated for 523.0 C27H28F2N6O3 was found 523.22. ^I H NMR (400 MHz, MeOD) δ 7.22 (s, IH), 6, 80-6, 96 (m, 3H), 4. 68 to 4.78 (m, 2H), 3.96 (s ,
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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 Form 1 1- (2- (6- (2-ethyl ~ 5 ~ fluoro4-hydroxyphenyl) --4 - fluoro ~ 1Jf - indazole - 3-11) -1,4,6,7 -tetrahydro5H-imidazo [4,5-c] pyridin-5 "yl) -2-morpholinoetan-l-one (1) [001651 To a 250 ml flask was added 1- (2- (6 (2-ethyl) 5-fluoro-4-hydroxyphenyl) -4-fluoro-1H-indazo1-3-i1) 1,4,6,7-tetrahydro-5H-imidazo [4,5-c] pyridin-5-yl) -2morpholinoethane- 1-one (1), the product of Example 1 (5 g) and ethanol (50 ml) and the reaction mixture was stirred at 5080 ° C for 10 min. and then ACN (75 ml) was added slowly at 50-80 ° C followed by the seeds of 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 under vacuum for 18 h to provide the compound title of Form 1 (3.6 g, 72% yield).
Example 3: Crystalline Form 1 1- (2- (6- (2-ethyl-S-fluoro4-hydroxyphenyl) -4-fluoro-1 "indazol" 3-yl) -1,4,6,7-tetrahydroSH-imidazo [4,5-c] pyridin-5 "yl) -2-morpholinoethane-1-one (1) [00166] Compound 1, the 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 RT for 16 h, filtered and dried at 50 ° C under vacuum for 18 h to provide the compound title of Form 1 (0.23 g) .
Example 4: 1- (2- (6- (2-Ethyl "5-fluorO" 4-hydroxyphenyl) -4fluoro-1-indazol-3-yl) -1,4,6,7-tetrahydro-5J-imidazo [ 4.5c] pyridin-5-yl) -2-morpholinoethane-l-one (1)
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[00167] Ν, Λί-diisopropylethylamine (0.298 ml, 1.707 mmol) was added to a solution of 5-ethyl-2-fluoro4- (4-fluoro-3- (4,5,6,7-tetrahydro- 1H-imidazo [4,5-c] pyridin-2yl) -LA-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 RT 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 vacuo to produce a light yellow liquid. The crude liquid was purified by preparative HPLC to provide the TFA salt of the compound title (142 mg, 0.223 mmol, 65.3% yield) as a beige solid.
Example 5: Crystalline Form 2 1- (2- (6- (2-ethyl5-fluoro ··· 4-hydroxyphenyl) -4-fluoro-1Jf-indazol-3 "yl) -1,4,6,7-tetrahydro5H -imidazo [4,5-c] pyridin-5-yl) -2-morpholinoetan-1-one (1) [001681 Compound 1 of example 1 (2.5 g) was dissolved in DMSO (5 ml) at 60 ° C. As soon as the homogeneous solution was obtained, MeOH (2.5 ml) was added to the solution. The homogeneous mixture was added dropwise over 30 min. to a premixed solution of MeOH (12.75 ml) and H ₂ O (11.25 ml) at 75 ° C. After the mixture was completely added, the combined mixture was stirred at 75 ° C for 1 h, while a crystalline paste was formed. H ₂ O (36 ml) was added dropwise over 2 h at 75 ° C. After
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74/117 the H ₂ 0 charge is complete, the slurry was stirred at 75 ° C for 1 h and then slowly cooled to 20 ° C for 6 h. The slurry was kept at 20 ° C for an additional 10 h before it was filtered, washed with 70% H ₂ O / MeOH (10 ml), dried at 50 ° C under vacuum for 18 h to provide the compound title of Form 2 (2.13 g).
Properties of the solid forms of the invention
[00169] Samples of the two anhydrous forms, Form 1 and Form 2 of 1- (2- (6- (2-Ethyl-5-fluoro-4-hydroxyphenyl) -4fluoro-1H-indazol-3-yl) -1, 4, 6, 7-tetrahydro-5.fi-imidazo [4,5c] pyridin-5-yl) -2-morpholinoethane-1-one (1) of Examples 2 and 5, respectively, were analyzed by X-ray diffraction dust (PXRD), differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), dynamic moisture sorption (DMS) and microscope image. Form 2 was also analyzed by single crystal X-ray diffraction.
Example 6: X-ray powder diffraction 001/0 j The X-ray diffraction units of Figures 1 and 6 were obtained with a Bruker D8-Advance X-ray diffractometer using Cu-Koí radiation (λ = 1, 54051 Â) with 45 kV output voltage and 40 mA current. The instrument was operated in Bragg-Brentano geometry with incident, divergence and scattering slits adjusted to maximize the intensity in the sample. For measurement, a small amount of powder (5-25 mg) was gently pressed onto a sample holder to form a smooth surface and subjected to exposure to X-rays. The samples were examined in 2Θ-2Θ mode from 2 ° to 35 ° ° in. 2Θ with; a step size of 0.02 ° and a reading speed of 0.30 ° second per step. Data acquisition was
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75/117 controlled by the DiffracSuite Bruker measurement program and analyzed by the Jade program (version 7.5.1). The instrument was calibrated with a corundum standard, within the angle of two teats ± 0.02 °. The positions of the PXRD 2-theta peak and the observed d-spacings are shown in Tables 1 and
2, respectively for crystalline Forms 1 and 2.
Table 1: PXRD data for Crystalline Form 1
2-Theta gives) Area THE% 7, 6 9 11.49 5 5 7 0 7.3 0 8.16 10.83 3 68 4 7 4 8, 60 8.97 9, 8 5 7 5 8 7 7 100.00 10, 6 6 8.2 9 7 32 3 9.70 11.46 7.7 2 5 8 41 7.7 0 11.91 7.43 1496 2.00 15.29 5, 7 9 7115 9, 4 0 15, 80 5.60 7841 10.30 1 6, 7 0 5.31 14 679 19.30 17, 02 5.20 8024 10.60 18.00 4.92 17834 23.50 18.83 4.71 2 658 3.50 2 0.18 4.4 0 18 636 24.60 22.39 3.97 7067 9.30 22, 98 3.8 7 9 u 2 9 11.90 2 4.89 3.57 8561 11.30 2 6, 5 4 3.36 7 8 31 10.30
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Table 2: PXRD data for Crystalline Form 2
2 ~ Theta gives) Area THE% 10, 61 8.33 706299 100.00 10.85 8.15 192921 27.30 11.84 7.4 7 487816 6 9, 1 0 13.32 6.64 97 98 0 13, 90 14.94 5, 93 519 3 8 6 73.50 16, 14 5.49 110314 15, 60 16, 35 5.42 7 5483 10.70 17, 69 5, 01 197341 27, 90 18.26 4.85 445270 63.00 18.43 4.81 152845 21, 60 19, 0 6 4.65 564088 7 9.90 19.2 0 4.62 427174 60.50 19, 49 4.55 266328 37.7 0 2 0, / 2 4.2 8 72244 10.20 21.10 4.21 236517 33.50 21.94 4, 05 2 8 7 4 8 5 40.70 22, 64 3.93 121406 17.2 0 23, 64 .3.7 6 152841 21, 6 0 25.19 3 3 3 6822 0 9.70 28.08 3.17 139597 19.80
Example 7: Thermal analysis
[00171] Exploratory differential calorimetry (DSC) was performed using a TA Instruments Model Q-100 module with a Thermal Analyst controller. Data were collected and analyzed using the TA Instruments thermal analysis program. A sample of each crystalline form was
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77/117 weighed accurately in an aluminum crucible. After a period of 5 minutes of isothermal equilibration at 5 ° C, the sample was heated through a linear heating ramp of 10 ° C / min. from 0 ° C to 300 ° C.
[00172] Figure 7 shows a representative DSC thermogram for the free crystalline Form 1 of the invention. The differential scanning calorimetry (DSC) trace recorded at a heating rate of 10 ° C per minute shows a peak in the endothermic heat flow, identified as a melting transition, in the transition range from about 210 ° C to about 234 ° C, or in the range between about 215 ° C to about 229 ° C, or in the range between about 220 ° C to about 224 ° C. The crystalline form is characterized by a trace of exploratory differential calorimetry recorded at a heating rate of 10 ° C per minute, which shows a maximum in the endothermic heat flow at a temperature of about 221.7 ° C or 221.713 ° C .
[00173] Figure 2 shows a representative DSC thermogram for the free crystalline Form 2 of the invention. The differential scanning calorimetry (DSC) trace recorded at a heating rate of 10 ° C per minute shows a peak in the endothermic heat flow, identified as a melting transition, in the transition range from about 268 ° C to about 277 ° C, or in the range of about 270 ° C to about 275 ° C, or in the range of about 271 ° C to about 274 ° C. The crystalline form is characterized by a trace of differential exploratory calorimetry recorded at a heating rate of 10 ° C per minute, which shows a maximum in the endothermic heat flow at a temperature of about 272.6 ° C or 272.612 ° C .
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[00174] Thermogravimetric analysis (TGA) measurements were performed using a TA Instruments Model Q-50 module, equipped with a high resolution capacity. Data were collected using the TA Instruments Thermal Analyst controller and analyzed using the TA Instruments Universal Analysis program. A heavy sample was placed in a platinum crucible and examined at a heating rate of 10 ° C from room temperature to 300-350 ° C. The balance and oven chambers were purged with nitrogen flow during use.
[001751 Figure 8 shows one. significant trace of TGA for the free crystalline Form 1 of the invention. The thermal gravimetric analysis (TGA) of Figure 8 does not show significant weight loss at temperatures below the start of decomposition at about 293 ° C.
[001761 Figure 3 shows a significant trace of TGA for the free crystalline Form 2 of the invention. The thermal gravimetric analysis (TGA) of Figure 3 does not show significant weight loss at temperatures below the start of decomposition at about 269 ° C.
Example 8: Evaluation of dynamic moisture sorption [001771 A. Measurement of dynamic moisture sorption (DMS) was performed using a VTI atmospheric microbalance, SGA-100 system (VTI Corp., Hialeah, FL 33016). A heavy sample was used and the humidity was the lowest possible value (close to 0% RH), at the beginning of the analysis. The DMS analysis consisted of. a first drying step (0% RH) for 120 minutes, followed by two sorption and desorption cycles with a reading rate of 5% RH / step in the humidity range of 5% to 90% RH. DMS was performed with isotherm at 25 ° C.
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[001781 Figure 9 shows a significant trace of DMS for the free crystalline Form 1 of the invention.
[00179] Crystalline Form 1 demonstrated signal hysteresis between two sorption and desorption cycles. Form 1 showed a weight gain of about 0.99% in the range of 5% to 70% relative humidity and a weight gain of about 1.32% in the range of 5% to 90% relative humidity environment, as shown in Figure 9. Form 1 is considered to be slightly hygroscopic.
[00180] Figure 4 shows a significant trace of DMS for the free crystalline Form 2 of the invention. Crystalline Form 2 showed no hysteresis between two sorption and desorption cycles and showed an exceptionally small propensity for hygroscopicity. Form 2 demonstrated a weight gain of about 0.12% in the range of 5% to 70% relative humidity and weight gain of about 0.18% in the range of 5% to 90% relative humidity environment, as shown in Figure 4. Form 2 is considered to be non-hygroscopic.
Example 9: Form 2 single crystal x-ray diffraction [00181] Data were collected on a Rigaku Oxford Diffraction Supernova Dual Source diffractometer, Cu at Zero, Atlas CCD, equipped with a. Oxford Cryosystems Cobra cooling device. The data were collected using Cu Κα radiation. The structure was resolved and refined using the Bruker AXS SHELXTL suite crystallography program. The CIF can provide complete details. Unless otherwise indicated. On the contrary, hydrogen atoms bonded to carbon were geometrically placed and refined with an isotropic displacement parameter. The hydrogen atoms attached to the
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80/117 heteroatoms were located on a different Fourier map and were freely refined with. an isotropic displacement parameter.
Table 3: X-ray diffraction analysis data for single crystal for Form 2
Empirical formula C27H28F2F6O3 Formula weight (molecular) 522.55 Crystal size 0.14 x 0.10 x 0.02 mm ³ Data collection temperature 2 93 (2) K Wavelength used for data collection 1.54178 Â Crystallographic system Orthorhombic G r up o e s p a. c i a 1 Pb ca Unit cell dimensions a == 9, 7245 (11) Âb = 16.8197 (14) Âc = 32, 604 (4) Âu = 90 °fi == 90 °γ = 90 ° Unit cell volume 5332.8 (10) A ³ Z (number of molecules in the unit cell) 8 D e n s i d e (c a 1 c u .1 a d a) 1.3 0 2 g / cm ³ Theta interval for collecting d 3 d O S 5, 2 6 - 6 6, 6 0 ° Index ranges -11 <h <11-12 <k <2 0- 3 8 <1 <3 8
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Reflections collected 24516 Reflections i n d and p and n d and n t. and s 47 08 [Rint = 0, 0 927] Final indexes [F2> IR == 0, 0808, wR2 == 2 s i gma (F2)] 0.2159 R indexes (all data) IR = 0, 1452, wR2 =0.2859
Example 10: Evaluation of Form 1 solid state stability
[00182] Samples of free crystalline form 1 of the invention were stored at 25 ° C and 60% relative humidity (RH) and at 40 ° C and 75% RH under two configurations a) open glass bottle and b) glass bottle closed in a HOPE bottle containing desiccant. The HOPE bottle was sealed by induction. At specific intervals, the content of a representative sample was removed and analyzed by HPLC to obtain chemical purity (shown below as HPLC purity (% a / a)).
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Table 4: Crystalline Form 1 stability study
Times (weeks) Condition 40 ° C / 75% RH Closed With desiccant 40 ° C / 75%UROpen 25 ° C / 60% RH Closed With desiccant 25 ° C / 60%UROpen r. 'Ώ CO CO 2 NT 98.72 NT NT 4 98.75 9 8 7 0 9 8.7 5 98, '5 8 9 8.80 98.97 98.87 98.75 2 4 99, 06 98.89 99, 02 98.7 8 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
[00183] Samples of Form 1 and Form 2 were examined under an optical microscope (Olympus BX.51) with a cross-polarized light filter. The images were collected with a PaxCam camera controlled by the Paxlt Imaging program (version 6.4). The samples were prepared on glass slides with light mineral oil as an immersion medium. Depending on the size of the particles, 4x, 10x or 20x objective lenses were used for magnification.
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Preparation 8: tert-butyl 2-iodo-1- ((2 (trimethylsilyl) ethoxy) methyl) -1,4,6,7-tetrahydro-5Himidazo [4,5-c] pyridine-5-carboxylate (C-5 )
2HC!
NH ₂
C-22 „BQC
M
IMAGES
C-Í8
O ^ .o.ü / mho 100 C.18 h ₄ ..Soc N
C-19
Q., .Q, O
C-21
H
X.HCÍ
1M NaOH,
Methanol,
RT, 18 h i DiPEA, ΐ Methanol,
RT.18h * O
NT
C-2.0
SEM-CI, NaH, THF, 0 Ό RT.6 h. , B®s
Ν '
Si
C-5
[00184] To a portion of the C-22 asosto (25 mmol) in HCI dimethoxymethane (21.64 ml, 244.54 mmol). The resulting solution was stirred at 100 ° C for 18 h. The solvent was removed in vacuo and the residue was triturated twice with ethyl ether and ethanol, filtered and dried to provide compound C-21 (25.2 g, 94.6% yield).
[00185] 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 the addition of (Βοο) ₂ 0
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84/117 (68.23 ml, 318.87 mol) and the reaction was stirred at RT for 18 h. The resulting reaction was diluted with water (150 ml) and extracted using ethyl acetate (3 x 200 ml). The combined organic layer was dried over anhydrous sodium sulfate, decanted and concentrated under reduced pressure to obtain the crude product C-20, which was taken to the next step without. additional purification.
[00186] To a solution of this crude compound C-20 (40.0 g, 123.8 mmol) in methanol (500 ml) was added 1 M NaOH solution (200 ml) and the resulting solution was stirred at RT for 18 hours. H. The solvent was distilled in vacuo and the resulting crude product was diluted with water (200 ml) and extracted using ethyl acetate (3 x 300 ml). The combined organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain the crude product which was purified by column chromatography (100-200 silica gel), eluted with 5-10% MeOH: DCM to obtain the desired product C-19 (20 g, 72.4% yield).
[00187] - 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 RT and the resulting solution was stirred for 2 hours at the same temperature. The reaction mixture was diluted with water (200 ml.) And extracted with ethyl acetate (2 x 300 ml), the organic layer was washed with. 10% aqueous sodium thiosulfate solution (3x100 ml) followed by brine, dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain the crude product C-18 which was used in the next step without further purification.
[00188] A. A stirred solution of compound C-18 (15.0 g, 42.97 mmol) in THF (150 ml) was added NaH
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85/117 (1.8 g, 45.11 mmol) at 0 ° C in portions and the reaction mixture was stirred for 1 hour at RT. Then SEM chloride (8.18 ml, 45.11 mmol) was added dropwise at 0 ° C. The reaction was stirred for 6 hours at RT. The progress of the reaction was monitored by chromatography on. thin layer (CCF), the reaction was quenched with. ice water (200 ml) at 0 ° C and extracted with ethyl acetate (2 x 200 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 obtain the crude product, which was purified by silica column chromatography (100-200) (eluted with 10-15% EtOAc: Hexane) to obtain the desired C-5 product as a viscous liquid (11 g, 55%).
Preparation 9: 2- (2-ethyl-5-fluoro-4-methoxyphenyl) 4,4,5,5-tetramethi1-1,3,2-dioxaborolane (C-12)

Pd / C / H _a
Ethanol
C-15 nbs
MeCN
DCM PDA _{s (} dppf)
KOAc
...;,. O O ../
[BB i j-θ 'b-r
R-2
C-12
[00189]
To a stirred suspension of the C-17 component (347.6 g, 973.14 mmol) in anhydrous THF (1000 ml) cooled, n-Butyl lithium (2.5 M in hexane,
362.6 ml, 905.02 mmol) for 50 min., At the point where the color
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86/117 yellow characteristic of phosphorus ylide persisted. The reaction mixture was heated to -10 ^U C and stirred for 1 h and then the mixture was cooled to -30 ° C and a solution of compound R1 (50 g, 324.38 mmol) in anhydrous THF (200 ml) was added over 30 min. The resulting mixture was heated to temperature and stirred overnight. The progress of the reaction was monitored with CCF. Upon completion, the reaction was quenched by the gradual addition of water (500 ml) and extracted with ethyl ether (3 x 500 ml). The combined organic layer was washed with water (2 x 500 1), brine (250 ml), dried (anhydrous NaHSC) and concentrated under reduced pressure to produce the crude compound. The obtained crude product C16 was used in the next step without purification.
[00190] To a crude solution of C-16 (110 g, 723.39 mmol) in ethanol (1000 ml) was added 10% Pd / C (50 g). A balloon of hydrogen gas was assembled and the reaction was emptied and filled with hydrogen three times. The reaction was stirred under a hydrogen atmosphere overnight at room temperature. After stirring at RT overnight, the reaction was complete. It was filtered through a pad of Celite and concentrated in vacuo to provide the crude compound, which was purified by chromatography with silica gel (100-200), eluted with 3-5% ethyl acetate / hexane to obtain the desired product. C-15 as a colorless liquid (24 g, 48% in both stages).
[00191] 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 h. The solvent was removed in vacuo and the residue was diluted with
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87/117 ethyl ether (100 ml). The observed precipitation was removed by filtration and the filtrate washed with aqueous sodium sulfite solution (200 ml) and brine (100 ml), dried over anhydrous sodium sulfate and concentrated in vacuo to produce the desired product C-14 as yellow oil (35.0 g, 97% yield).
[00192] 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 minutes and then the palladium catalyst (3.5 g, 4.29 mmol) was added. The reaction mixture was stirred and heated to 110 ° C for 3 hours. The reaction was filtered through a biocide and washed with ethyl acetate. The filtrate was diluted with. ethyl acetate (200 ml) and washed with water (2 x 200 ml) and brine (100 ml), dried over sodium sulfate and concentrated in vacuo. The obtained crude product was purified by chromatography on. column (100-200 silica gel) and eluted with. 3-5% EtOAc: Hexane to obtain the desired product C-12 (20 g, 83% yield).
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Preparation 10: 3- (dimethyl stanyl) ~ 6 ~ (2 ~ ethyl "5 ~ fluoro"
4-methoxyphenyl) -1- (tetrahydro- ~ 2H ~ pyran - 2- ~ yl) -IH-indazole (C-6)
[00193] A 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) was taken up in THF (700 ml) and heated to 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 pressure
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89/117 reduced and the residue purified on the silica gel column (230-400) (eluting with 1-2% ethyl acetate in hexane) to yield the desired compound C-ll (23.5 g, 67% yield ).
[00194] A solution of compound C-ll (13.3 g, 47.5 mmol), compound C-12 (15.96 g, 57.0 mmol) and K3PO4 (30.21 g, 142.5 mmol) in DMFiPQO (396: 99 ml) it was degassed with nitrogen for 15 minutes and the palladium catalyst (1.6 g, 2.37 mmol) was added and the reaction mixture was purged with nitrogen for 5 minutes. The reaction mixture was heated at 100 ° C for 12 h with continuous 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 (2 x 100 ml) and washed with cold water (100 ml) and brine (50 ml), dried over sodium sulfate and concentrated in vacuo to obtain the crude product which was purified by flash chromatography (100-200 silica gel), eluted with. 10% EtOAc: Hexane to produce C-10 (14 g, 91.4% yield).
[00195] A. a solution of compound C-10 (52 g, 146, 89 mmol) in methanol (600 ml) was. Concentrated HCl (50 ml) is added and the resulting solution was heated to 60 ° C overnight. A. The reaction mixture was cooled to RT. and concentrated in vacuo. The residue was dissolved with EtOAc (200 ml) and washed with saturated NaHCOs solution (2 x 150 ml). The organic layer was dried over Na2SÜ4 and concentrated to obtain the desired product C-9 (35 g, 88.9% yield).
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[00196] To a solution of compound C-9 (17.5 g, 64.81 mmol) in. DMF (100 ml) KOH (14.5 g, 259.54 mmol) was added 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 RT for 30 min. The progress of the reaction was monitored with TLC so the reaction mixture was diluted with water (200 ml) and extracted with ethyl acetate (2 x 200 ml). The organic layer was washed with. saturated aqueous solution of sodium metabisulfite (2 x 150 ml) and water (3 x 100 ml), dried over Na ₂ SO4 and concentrated in vacuo to obtain the crude product C ~ 8 (21 g).
[00197] 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 the solution described above and the reaction mixture was stirred at RT overnight. CCF monitoring showed the completion of the reaction. The reaction mixture was dissolved with DCM (2 x 150 mil) and washed with saturated aqueous NaHCCg solution (200 ml) and brine (200 ml). The organic layer was dried over anhydrous Na ₂ SO4 and concentrated in vacuo to obtain the crude product which was purified by flash chromatography to obtain compound C-7.
[00198] A solution of C-7 (10.0 g, 20, 83 mmol) in. Toluene (200 ml) was degassed with nitrogen for 20 minutes followed by the addition of R-4 (4.89 ml, 22.91 mmol) and Pd (PPh3) 4 (1.2 g, 1.04 mmol). The reaction mixture was purged with nitrogen for an additional 5 minutes and then stirred at 100 ° C. After 2 h, CCF showed the completion of the reaction. THE
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91/117 reaction mixture was cooled to room temperature, filtered through a pad of Celite and the residue washed with. ethyl acetate. The concentrated filtrate was purified by column chromatography (neutral alumina), eluted with 2-5% EtOAc: Hexane to obtain product C-6 (6.4 g, 57.6% yield).
Preparation 11: 2 ~ (6 ~ (2 ~ ethyl. 5 ~ fluoro - 4 ~ methoxyphenyl) - 1H ~ indazol-3-yl) -4,5,6,7-tetrahydro-1H-imidazo [4 , 5-c] pyridine (C-3)
[00199] - A solution of compound C-6 (6.4 g, 12.37 mm.ol) in toluene (100 ml) was added compound C5 (5.9 g, 12.37 ml). A. The reaction mixture was degassed with nitrogen for 20 minutes, followed by the addition of copper (I) iodide (470 mg, 2.47 mmol) and Pd (PPh ₃ ) 4 (714 mg, 1.237 mmol) and then stirred at 100 ° C for 12 h. The progress of the reaction was monitored with CCF. The reaction was cooled to RT and filtered through a pad of Celite, the residue washed with ethyl acetate. The organic layer was diluted with water, separated and the organic part was washed with.
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92/117 brine, dried over Na ₂ SO4 and filtered. The filtrate was concentrated under reduced pressure to obtain the crude product which was purified by column chromatography (100-200 mesh size silica), eluted with 20% EtOAc: Hexane to obtain C-4 (4 g, 45.9% ).
[00200] 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 h. The progress of the reaction was monitored with LCMS. The reaction was cooled to RT, concentrated in vacuo, triturated with ethyl ether and purified by preparative HPLC to obtain the desired C-3 compound (0.65 g, 29.5%).
Example 12: 1- (2 ~ (6 "(2 ~ Ethyl" 5 ~ fluoro-4 - hydroxyphenyl) 1 / f-indazol-3-yl) -1,4,6,7-tetrahydro-S-i-imidazo [4,5-a] pyridin5-yl) -2-morpholinoethane-l-one (Cl)
F
C-l
[00201] To the mixture of C-3 (180 mg, 0.460 mmol) in DCM (0.5 ml) at RT was added boron tribromide, 1 ml in DCM (100 ml) (2.299 ml, 2.299 mmol). The resulting mixture was stirred for 30 minutes before being concentrated. The resulting residue was coevaporated with MeOH (3 x 3.0 ml),
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93/117 redissolved in the 1: 1 mixture of AcOH: H ₂ O (3.0 ml), filtered and purified by reverse phase HPLC. The desired fractions were combined and freeze-dried to produce. C-2 (((5-ethyl-2-fluoro-4- (3- (4,5,6,7-tetrahydro-1H-imidazo [4,5— c] pyridin-2-yl) -1H-indazole- 6-yl) phenol) as a TFA salt.
[002021 To the mixture of C-2, TFA (15 mg, 0.031 mmol) and 7 '' (2 equivalents, 0.061 mmol) in DMF (0.5 ml) at RT was added HATU (25.5 mg, 0.067 mmol) and DIEA (0.043 ml, 0.244 mmol). The reaction mixture was stirred at RT overnight. The reaction 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 to 65 ° C for 1 h. The reaction was then concentrated, the resulting residue was treated with a mixture of DCM (0.500 ml) and TFA (0.500 ml) at RT for 30 minutes, concentrated, redissolved in the 1: 1 mixture of AcOH: H ₂ O (1, 5 ml), filtered and purified by preparative reverse phase HPLC to obtain compound Cl as TFA salt. ι, γώ / ζ): [M + u] ^T calculated for 504.23 of C27H29FN6U3 505.2 was found.
Biological assays
Compound 1 was characterized in one or more of the following biological assays.
Assay 1: JAK kinase biochemical assays
[00203] A panel of four LanthaScreen JAK biochemical assays (JAK1, 2, 3 and Tyk2) was performed in a common reaction buffer for kinase (50 mM HEPES, pH 7.5, 0.01% Brij-35, 10 mM MgCl.2 and 1 mM EGTA). Recombinant GST-labeled JAK enzymes and the GFP-labeled STAT1 peptide substrate were purchased from Life Technologies.
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[00204] The compound with serial dilution was preincubated with each of the four JAK enzymes and the substrate in 384 white well microplates (Corning) at room temperature for 1 h. ATP was added later to start the kinase reactions at 10 μL of total volume, with. 1% DMSO. The final enzyme concentrations for JAK1, 2, 3 and Tyk2 are 4.2 nM, 0.1 nM, 1 nM, and 0.25 nM, respectively; the corresponding concentrations of Km ATP used are 25 μΜ, 3 μΜ, 1.6 μΜ, and 10 μΜ; while the substrate concentration is 200 nM for all four assays. The kinase reactions continued for 1 hour at room temperature before a preparation of 10 μΐ EDTA (10 mM final concentration) and Tb-anti antibody, -pSTATl (pTyr701) (Life Technologies, 2 nM final concentration) was added in . TR-FRET dilution buffer (Life Technologies). The plates were incubated at room temperature for 1 hour before being read in the EnVision reader (Perkin Elmer). The emission rate signals (520 nm / 495 nm) were recorded and used to calculate the percentage of inhibition values based on DMSO and background controls.
[0020] For the dose-response analysis, the percentage inhibition was applied in. a graph versus the concentrations of the compounds, and the IC 50 values were determined from a robust 4-parameter fit model with the Prism software (GraphPad Software). The results were expressed as pICso (negative IC 50 logarithm) and subsequently converted to pKi (negative dissociation constant logar, Kl) using the Cheng-Prusoff equation.
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[00206] Compound 1 exhibited the following enzymatic potency.
Table 5
JAtC 1 pK _x pK _x JAtC 3 pK _x Tyk2 pK ± 10 10.6 9, 7 θ / 7
Assay 2: JAK cell potency assay: IL-13 inhibition
[00207] The AlphaScreen JAKI cell potency assay was done by measuring interleukin-13 induced STAT6 phosphorylation (IL-13, R&D Systems) in human lung epithelial cells BEAS-2B (ATCC). The anti-STAT6 antibody (Cell Signaling Technologies) was conjugated to AlphaScreen acceptor beads (Perkin Elmer), while the anti-pSTAT6 antibody (pTyr641) (Cell Signaling Technologies) was biotinylated using the sulfo-NHS-biotin EZ-Link (Thermo Scientific).
[00208] BEAS-2B cells were cultured at 37 ° C in a humidified incubator at 5% CO ₂ in 50% DMEM / 50% F-12 medium (Life Technologies), supplemented with 10% FBS (Hyclone), 100 U / ml penicillin, 100 ug / ml streptomycin (Life Technologies) and 2 mM GlutaMAX (Life Technologies). On day 1 of the assay, the cells were seeded; at a density of 7,500 cells / well in. white plates with. 384 wells coated with poly-D-lysine (Corning) with 25 μΐ of medium, and stayed overnight in the incubator for adherence. On day 2 of the assay, the medium was removed and replaced with 12 pL of assay buffer (Hank's Balanced
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Salt Solution, 25 mM HEPES, and 1 mg / ml of bovine serum albumin / BSA.) Containing dose responses of the test compounds. The compound was serially diluted in DMSO and then diluted
1000 times in the media to reach the final concentration of 0.1% DMSO. The cells were incubated with test compounds a. 37 ^{, J} C for 1 h, followed by the addition of 12 μΐ of pre-heated IL-13 (80 ng / ml in assay buffer) for stimulation. After incubation at 37 ° C for 30 min., The assay buffer (containing compound and IL-13) was removed, and 10 μΐ of Use cell buffer (25 mM HEPES, 0.1% SDS, 1%
NP-40, 5 mM MgC.l _2, 1.3 mM EDTA , 1 mM EGTA, and additional protease inhibitors with Mini Ultra and PhosSTOP Roche Diagnostics). The plates were shaken at room temperature for 30 minutes before adding the detection reagents. A mixture of anti-biotin-pSTA_T6 and anti-STAT6 conjugated acceptor beads was added first and incubated at room temperature for 2 h, followed by the addition of conjugated donor beads to. streptavidin (Perkin Elmer). After a minimum of 2 h of incubation, the assay plates were read on the EnVision plate reader. AlphaScreen luminescence signals were recorded and used to calculate percentage inhibition values based on DMSO and background controls.
[002091 For dose-response analysis, percentage inhibition data were plotted against compound concentrations, and IC50 values were determined from a robust 4-parameter fit model with. the program. Prism. The results can also be expressed as the negative log of the IC50 value,
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97/117 pIC ₅₀ . Compound 1 exhibited a pICso value of about 7.9 in that
Assay 3: JAK cellular potency assay: IFNy-stimulated IL-2 / anti-CD3 inhibition in human PBMCs [00210] The potency of the test compound for inhibiting interleukin-2 (IL-2) / stimulated anti-CD3 the interferon range (ΙΕΝγ) was measured in peripheral blood mononuclear cells (PBMCs) isolated from whole human blood (Stanford Blood Center). As IL-2 signals through JAK, this assay provides a measure of JAK cell potency.
[00211] (1) Peripheral blood mononuclear cells (PBMC) were isolated from whole human blood from healthy donors using a Ficoll gradient. The cells were cultured at 37 ° C in a humidified incubator at 5% CO ₂ in RPMI (Life Technologies), supplemented with 10% Heat Inactivated Bovine Fetal Serum (SFB, Life Technologies), 2 mM Glutamax (Life Technologies ), 25 mM HEPES (Life Technologies) and IX Pen / Strep (Life Technologies). The cells were seeded in 200,000 cells / well in the media (50 µl) and cultured for 1 h. The compound was serially diluted in DMSO and then diluted 500 times (to a final test concentration of 2x) in the media. Dilutions of the test compound (100 µl / well) were added to the cells and incubated at 37 ° C, 5% CO ₂ for 1 hour, followed by the addition of IL-2 (R&D Systems; final concentration of 100 ng / ml ) and anti-CD3 (BD Biosciences; final concentration of 1 pg / ml) in. preheated test media (50 pi) for 24 hours.
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[00212] (2) After cytokine stimulation, the cells were centrifuged at 500 g for 5 minutes and the supernatant removed and frozen at -80 ° C. To determine the inhibitory potency of the test compounds in response to IL-2 / anti-CD3, concentrations of IFNy supernatant were measured via ELISA (R&D Systems). IC50 values were determined from the analysis of the IFNv concentration curves versus the concentration of the compounds. Hi data was expressed in. pICso values (IC50 negative decadic logarithm). Compound 1 exhibited a pICso value of about 6.7 in that assay.
Assay 4: JAK cell potency assay: inhibition of IL-2-stimulated pSTAT5 in CD4 + T cells
[00213] The potency of the test compound for inhibiting STAT5 phosphorylation stimulated by interleukin-2 (IL · 2) / anti-CD3 was measured on CD4-positive T cells (CD4 +), isolated peripheral blood mononuclear cells (PBMCs) of total human blood (Stanford Blood Center) using flow cytometry. As IL-2 signals through JAK, this assay provides a measure of JAK cell potency.
[00214] CD4 + T cells were identified using an anti-CD4 antibody conjugated to phycoerythrobilin (PE) (Clone RPA-T4, BD Biosciences), while an Alexa Fluor 647 conjugated anti-pSTAT5 antibody was used (pY694, Clone 47, BD Biosciences ) to detect STAT5 phosphorylation.
[00215] (1) The protocol of paragraph 3 of the trial (1) fo.
followed with the exception that cytokine stimulation with. IL2 / anti-CD3 was performed for 30 minutes instead of 24 h.
[00216] (2) After cytokine stimulation, cells were fixed with preheated fixation solution
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99/117 (2UΟ μΐ; BD Biosciences) for 1U nun. The. 3 / ° C, 5% Cth, washed twice with DPBS buffer (1 ml, Life Technologies), and resuspended in cooled Perm III buffer (1000 μΐ, BD Biosciences) for 30 min. at 4 ° C. The cells were washed twice with 2% SFB DPBS (FACS buffer) and then resuspended in FACS buffer (100 μΐ) containing antiCD4 PE (1:50 dilution) and anti-CD3 anti ~ CD3Alexa Fluor 647 (dilution 1: 5) for 60 min. at room temperature in the dark. After incubation, cells were washed twice with FACS buffer before being analyzed using the LSRII flow cytometer (BD Biosciences). To determine the inhibitory potency of the test compound in response to IL-2 / anti-CD3, the median fluorescent intensity (IFM) of pSTA.T5 in CD4 + T cells was measured. IC50 values were determined from the analysis of the IMF inhibition curves versus compound concentration. The data were expressed as pICso values (IC50 negative decadic logarithm). Compound 1 exhibited a pICso value of about 7.7 in that assay.
Assay 5: JAK cell potency assay: inhibition of IL-6-stimulated CCL2 (MCP-1) in human PBMCs
[00217] The potency of the test compound to inhibit the production of CCL2 (MCP-1) stimulated by interleukin-6 (IL-6) was measured in. human peripheral blood mononuclear cells (PBMCs) isolated from human whole blood (Stanford Blood Center). As IL-6 signals via JAK, this assay provides a distal measure of JAK's c e1u1a power.
[00218] (1) The protocol of paragraph (1) of assay 3 was followed until incubation with the test compounds. At the
Petition 870190110020, of 10/29/2019, p. 126/160 this test, after the. addition of the test compounds to the wells and incubated, IL-6 (R&D Systems; final concentration 10 ng / ml) was added to the preheated test media (50 μΐ).
[00219] (2) After stimulation with. cytokine for h, cells were centrifuged at 500 g for 5 min. and the supernatant removed and frozen at -80 ° C. To determine the inhibitory potency of the test compound in response to IL-6, concentrations of CCL2 (MCP-1) supernatant were measured via ELISA (R&D Systems). IC50 values were determined from the analysis of the CCL2 / MCP-1 concentration curves versus the concentration of the compounds. The data were expressed as pICso values (IC50 negative decadic logarithm). Compound 1 exhibited a pICso value of about 6.4 in that assay.
Assay 6: JAK cell potency assay: pSTATl-induced INF-γ inhibition
[00220] The potency of the test compound for inhibiting STAT1 phosphorylation stimulated by gamma interferon (ΙΕΝγ) was measured in CD14-positive (CD14 +) monocytes derived from whole blood (Stanford Blood Center) using flow cytometry. As ΙΕΝγ signals through JAK, this assay provides a measure of JAK cell power.
[00221] Monocytes were identified using anti-CD14 antibody conjugated to fluorescein isothiocyanate (FITC) (Clone RM052, Beckman Coulter) and an Alexa Fluor 647 conjugated anti-pSTATl antibody (pY701, Clone 4a, BD Biosciences) was used to detect phosphorylation of STAT1.
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[00222] Peripheral blood mononuclear cells (PBMC) were isolated from whole human blood from healthy donors using a Ficoll gradient. The cells were cultured at 37 ° C, humidified incubator at 5% CO in RPMI (Life Technologies), supplemented with 10% Bovine Fetal Serum (FBS, Life Technologies), 2 mM Glutamax (Life Technologies), 25 mM HEPES (Life Technologies) and IX Pen / Strep (Life Technologies). The cells were seeded at a rate of 250,000 cells / well in. media (200 μΐ), grown for 2 h and resuspended in. test media (50 μΐ) (RPMI supplemented with 0.1% bovine serum albumin (Sigma), 2 mM Glutamax, 25 mM HEPES and IX Penstrep) containing different concentrations of the test compounds. The compounds were serially diluted in. DMSO and then diluted 1000 times in the media to reach the final concentration of 0.1% DMSO. Dilutions of the test compound were incubated with the cells at 37 ° C, 5% CO for 1 h, followed by the addition of preheated IFNy (R&D Systems) to the media (50 μΐ) at a final concentration of 0.6 ng / ml for 30 minutes. After stimulation with cytokines, the cells were fixed with preheated fixation solution (100 μΐ) (BD Biosciences) for 10 min. at 3 / ° C, 5% CO2, washed twice with. FACS buffer (1 ml) (1% BSA in. PBS), resuspended in 1:10 anti-CD14FITC: FACS buffer (100 μΐ), and incubated at 4 ° C for 15 min. The cells were washed once and then resuspended in ice-cold Perm III buffer, BD Biosciences) (100 μΐ, ι durance .30 minutes at 4 ° C. The cells were washed twice with FACS buffer and resuspended in Alexa Fluor 647 1:10 anti-pSTATl: FACS buffer (100 μΐ) for 30 min at RT in the dark
Petition 870190110020, of 10/29/2019, p. 128/160 washed twice in FACS buffer, and analyzed using an LSRII flow cytometer (BD Biosciences).
[00223] To determine the inhibitory potency of the test compound, the median fluorescent intensity (IFM) of pSTAT1 was measured on CD14 + monocytes. IC 50 values were determined from the analysis of the IMF inhibition curves versus concentration of the compounds. The data were expressed as pIC.50 values (IC50 negative decadic logarithm). Compound 1 exhibited a pIC ₅₀ value of about 7.1 in that assay.
Test 7: Ocular pharmacokinetics in rabbit eyes
[00224] The purpose of this test was to determine the pharmacokinetics of a test compound in o and c o and 1 η o s eye tissues.
Solution formulation
[00225] 1- (2- (6- (2-ethyl ~ 5-fluoro-4-hydroxyphenyl) -4 fluoro-1 ü-indazol-3-yl) -1,4,6,7-tetrahydro-5H- imidazo [4, δη Jpiridin-5-yl) -2-morpholinoetan-1-one (1), prepared in Example 2, was dissolved in 2% 2-hydroxypropyl-3cyclodextrin to obtain the target concentration of 1 mg / ml. Bilateral intravitreal injection (50 μΐ / eye) of the test compound solution was administered to New Zealand white rabbits (50 μΐ / eye). The concentration of the test compound was measured in the ocular tissues: aqueous vitreous, retina / choroid and body iris-ciliary at predetermined time points after injection (30 min., 4 h, 1 d, 3d, 7 d, 14 d). Two rabbits (four eyes) were dosed for each time point. In the vitreous tissue, compound 1 showed a two-phase decrease in concentration characterized by an initial decrease in concentration with a half-life of approximately 9 hours and
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103/117 finally a terminal half-life of approximately 2 days. It was found that the compound was rapidly distributed in the retina and choroid region as well and shows a similar pharmacokinetic profile as in vitreous tissue.
Suspension formulation
[00226] A suspension formulation was prepared by combining compound 1 of example 2 (Form 1) with 0.5% hydroxypropylmethylcellulose (HPMC E5) + 0.02% Tween 80 in normal saline to achieve a target concentration 5 mg / ml, 20 mg / ml and 80 mg / ml for the respective doses of 0.25 mg / eye, 1 mg / eye and 4 mg / eye. Bilateral intravitreal injection (50 μΐ / eye) of the test compound suspension was administered to New Zealand white rabbits. The concentration of the test compound was measured after injection into ocular tissues as in the solution formulation assay at 30 min, 4 h, 24 h, 72 h, 7 d, 14 d, 28 d, 56 d and 84 d. For the 4 mg / eye dose group, an additional 168 d time point was collected after injection. All dose groups demonstrated measurable drug concentration in the eye up to the last time point tested in this study. Robust and sustained exposure was observed for all doses at 12 weeks (84 d). Sustained exposure at 24 weeks (84 d) was observed for the 4 mg / eye dose group. The compound showed a linear decrease in drug concentration in the vitreous tissue for 30 min. at 24 weeks with a drug clearance rate of approximately 5 to 10 qg / ml / day. The clearance rate is consistent with. the solubility of compound 1 in the vehicle and the pharmacokinetic behavior of the eye in formulating the solution. All dose groups demonstrated measurable drug concentration in the eye up to the
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104/117 last time point tested in this study. Therefore, it is plausible that the exposure to the drug is longer than that observed in this study. The concentration of the drug in the plasma was measured and found to be at least 3 orders of magnitude lower than the concentration in the vitreous tissue at all three concentrations.
[00227] A suspension formulation was prepared by combining compound 1 of example 5 (Form 2) with 0.5% hydroxypropylmethylcellulose (HPMC E5) + 0.02% Tween 80 in normal saline to achieve a target concentration 0.4 mg / ml, 1 mg / ml, 2 mg / ml and 20 mg / ml for the respective doses of 0.02 mg / eye, 0.05 mg / eye, 0.1 mg / eye and 1 mg /eye. Bilateral intravitreal injection (50 μΐ / eye) of the test compound suspension was administered to white Dutch Belted rabbits. The concentration of the test compound was measured in the vitreous humor, aqueous humor, iris body, retina, retinal / choroidal pigment epithelium cells and plasma in 30 min., 7d, 14d, 28d, 42d and 56d post-injection. The compound showed a very gradual decrease in drug concentration in the vitreous tissue of 30 min. until the last time point tested (28d for 0.02 mg / eye dose, 42d for 0.05 mg / eye dose and 1 mg / eye and 56d for 0.1 mg / eye dose). All dose groups demonstrated measurable drug concentration in the eye up to the last time point tested in this study. Therefore, it is plausible that the exposure to the drug is longer than that observed in this study. The concentration of the drug in the plasma was measured and found to be at least 3 orders of magnitude lower than the concentration in the vitreous tissue at all three concentrations. The three orders of magnitude
Petition 870190110020, of 10/29/2019, p. 131/160 correspond to a logarithmic scale (that is, 1000 on a non-logarithmic scale).
Test 8: Pharmacodynamic test: inhibition of IL6-induced pSTAT3 in rats
[00228] The ability of a single intravitreal administration of the test compound to inhibit IL-6-induced pSTAT3 was measured in rat retina / choroid homogenates.
[00229] A suspension formulation was prepared by combining compound 1 of example 2 with 0.5% hydroxypropylmethylcellulose (HPMC E5 LV), 0.02% Tween 80 and 9 mg / ml sodium chloride in purified water for achieve a target concentration of 10 mg / ml.
[00230] Female Lewis rats received an intravitreal dose (IVT) (5 μΐ / eye) with the suspension formulation. Three days later, IL-6 (Peprotech;
0.1 mg / ml; 5 μΐ / eye) or vehicle intravitreally to induce pSTAT3. The ocular tissues were dissected one hour after the second IVT injection with IL-6. Retinal / choroidal tissues were homogenized and pSTAT3 levels were measured using an ELISA (Cell Signaling Technology). The percentage of IL-6-induced inhibition of pSTAT3 was calculated compared to. the vehicle / vehicle and vehicle / IL-6 groups. The inhibition of more than 100% reflects a reduction in pSTAT3 levels to below the values observed in the vehicle / vehicle group.
[00231] With a pretreatment of 3 days before the challenge with IL-6, a 50 pg dose of compound 1 administered by the suspension formulation inhibited IL-6-induced pSTAT3 by 116% in tissues.
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Trial 9: Pharmaoodinamic trial: IFNy-induced IP-10 inhibition in Rabbit Eyes
[00232] The ability of a single intravitreal administration of the test compound to inhibit levels of gamma interferon-induced IP-IO protein (IFNy) was measured in the vitreous and retina / choroid tissues.
[00233] A suspension formulation was prepared by combining compound 1 of example 2 (Form 1) with 0.5% hydroxypropylmethylcellulose (HPMC E5), 0.02% Tween 80 and 9 mg / ml sodium chloride in purified water to reach a target concentration of 20 mg / ml.
[00234] White male rabbits from New Zealand (Liveon Biolabs, India) were used for the studies. The animals were acclimated after arriving at the research facility (Jubilant Biosys Ltd., India). Each rabbit received a total of two intravitreal injections (IVT) with a total dose volume of 50 μΐ per eye. The first IVT injection (45 μΐ per eye) provided 0.9 mg of the test compound or vehicle. A week later, a second injection of TIV (5 μΐ per eye) provided IFNy (1 pg / eye; stock solution 1 mg / ml; Kingfisner Biotech) or vehicle for inducing the IB-iO pronein. On the day of the injections, the rabbits were anesthetized with an intramuscular injection of ketamine (35 mg / kg) and xylazine (5 mg / kg). As soon as they were deeply anesthetized, each eye was washed with sterile saline and IVT injections were made using a 0.5 ml insulin syringe (50 units = 0.5 ml) with a 31 gauge needle on the supranasal side of both. eyes when marking the position with a Braunstein caliper (2 3/4) 3.5 mm distant from the rectus muscle and 4 mm from the limbus.
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[00235] The tissues were collected 24 hours after the second IVT injection with IFNy. The vitreous humor (VH) and the retinal / choroidal tissues (R / C) were collected and homogenized and the levels of the IP-10 protein were measured using a rabbit ELISA kit CXCL10 (IP-10) (Kingfisher Biotech). The percentage of IPN-10 inhibition induced by IFNy was calculated in comparison with the vehicle / vehicle and vehicle / IFNy groups.
[00236] With a pretreatment of 1 week before the challenge with IFNy, the suspension formulation of compound 1 inhibited the IFNy-induced IP-10 in. 81% and 80% in vitreous humor and retinal / choroidal tissues, respectively. Similar efficacy was observed with a pre-treatment of 1 month before the challenge with. IFNy.
Test 10: Dermal pharmacokinetics on the skin of mice and mini pigs
[00237] The purpose of this trial is to determine the plasma pharmacokinetics of. epidermis, dermis and plasma of a compound after 24 hours exposure in intact skin of mice or mini pigs.
[00238] 1- (2- (6- (2-ethyl1-5-fluorO-4 ~ hydroxyphenyl) -4fluoro-1H-indazol-3-yl) -1,4,6,7-tetrahydro-5H- imidazo [4,5— c] pírídín-5-í1) -2-morpholinoetan-l-one (1) was formulated as a cream or ointment at 0.5% (ρ / ρ) as described, as Formulation A or Formulation B, respectively, in Table 6.
[00239] Twenty-four hours before administration, the hair was shaved off. part of the back of 25 g male Balb / c mice, exposing an area of at least 6 cm ² (about 10% of the body surface) and, in a separate experiment, 10 kg Gottingen mini-pigs, exposing an area
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108/117 of at least 450 cm ² (about 10% of the body surface). At time zero, after anesthesia with isoflurane, the test compound was applied to the back of mice or mini-pigs at a dose of 25 μΐ / cm ² . The skin was covered with an adhesive to prevent loss of the compound in the cage or pallet.
[002401 After 24 h of exposure, the backs were gently washed with soap and water to remove the unabsorbed drug and dried slightly. Immediately after this washing, blood was collected by cardiac puncture of the mice and by venipuncture of the mini-pigs. The outer skin (stratum corneum) was then removed with adhesive tape. After exposure of the epidermis, a biopsy was taken by perforation of 0.5 cm. A. epidermis and dermis were quickly separated, weighed and frozen. Similar samples were obtained after 48 h post-administration in mice and 48 h, 94 h and 168 h (7 days) post-administration in mini-pigs.
[00241] The epidermis and dermis samples were homogenized in 1:10 (w / v) of water using a Covaris ultrasonic homogenizer. The samples were extracted in 3 volumes of acetonitrile and quantified against a standard curve using LC-MS analysis. As evidenced by the pharmacokinetic parameter AUCo-t · for plasma, epidermis and dermis shown in Table 7 below, significant exposure of the compound in layers of the epidermis and dermis was observed, while plasma exposure was insignificant in mice in Formulation A and below the limit of quantification in Formulation B at. mice and in both Formulations in the mini-pig.
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Table 6
Formulation A Formulation B Compound 1 0.5% Compound 1 0.5% Stearic acid B% 0 ctylhydroxistearate b-è Alcoholc e t o e s t e a. r i 1 i c o 5% Triglyceride ofc to d. and 1 to C 8 — C10 Z) / P a. 1 m i t a t. the isopropyl 4% Vaseline.(Pet.ro lato) -.1<0·> ΣιO O Octyhydroxystearate 2 % Ai-Me t i ip i rro 1 i owner. 10% BRIJ S2 (PEG 2 etherstearyl) 1.08% BRIJ S20 (PEG 20stearyl ether) 6, 92% ϊ'Ρ-Methylpyrrolidine 10% PEG400 10% RO water 5 h 5%
Table 7
Fia smaAUCo-t (pg * h / ml) Epidermis AUCo-t (pg * h / g) DermisAUCo-t (pg * h / g) C ailin g a nd a til u 1 a ç a ο Ά 0.022 1370 99 MouseFo rmu1a tion B <0.001 107 0 0 1110 Mini pig fromr o r m u 1 a ç ο Ά <0.001 1220 44 Mini pig fromFo rmu1a tion B <0.001 2 4 60 8 8
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Trial 11: Pharmacoainetics of lung and plasma in mice
[00242j The plasma and lung concentrations of compound 1 and their proportions were determined as follows. BALB / c mice from Charles River Laboratories were used in the trial. Form 1 of compound 1 of example 2 was formulated in 0.01% Tween 80 in normal saline (0.9% sodium chloride in water) at a concentration of 0.1 mg / ml as a suspension. 50 µl of the suspension formulation was introduced into a mouse's trachea by oral aspiration. At various time points (0.083, 1, 4, 24, 48, 72 and 96 hr). After administration, blood samples were taken through cardiac puncture and intact lungs were taken from the rats. Blood samples were centrifuged (Eppendorf centrifuge, 5804R) for 4 minutes at approximately 12,000 rpm at 4 ° C to collect plasma. The lungs were dry padded, weighed and homogenized in a 1: 3 dilution in sterile water. The plasma and lung levels of compound 1 were determined by LC-MS analysis against analytical standards constructed on a standard curve in the test matrix. Good lung exposure was found with. an area under the curve (ASC) (0-96 h) of the lung of 360 pg h / g. The lung half-life was calculated at approximately 40 hours. A lung to plasma ratio was determined as the ratio of lung ASC in pg h / ml to plasma ASC in pg h / ml, where ASC is conventionally defined as the area under the test compound versus time curve. The lung to plasma ASC ratio was 1780, showing very low plasma exposure.
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Trial 12: Pharmacodynamic trial: IFNy-induced pSTAT1 inhibition in Rabbit Eyes
[00243] The ability of a single intravitreal administration of the test compound to inhibit gamma interferon (ΙΓΝγ) induced phosphorylation of the STATI protein (STγ) was measured in the vitreous and retina / choroid tissues.
[00244] A suspension formulation was prepared by combining compound 1 of example 2 (Form 1) with 0.5% hydroxypropylmethylcellulose (HPMC E5), 0.02% Tween 80 and 9 mg / ml sodium chloride in . purified water to reach a target concentration of 20 mg / ml.
[00245] White male rabbits from New Zealand (Liveon Biolabs, India) were used for the studies. The animals were acclimated after arriving at the research facility (Jubilant Biosys Ltd., India). Each rabbit received a total of two intravitreal injections (IVT) with a total dose volume of 50 μΐ per eye. The first IVT injection (45 μΐ per eye) provided 0.9 mg of the test compound or vehicle. A week later, a second injection of TIV (5 μΐ per eye) provided IFNy (1 pg / eye; stock solution 1 mg / ml; Kingfisner Biotech) or vehicle for the induction of the IP-i0 protein. On the day of the injections, the rabbits were anesthetized with an intramuscular injection of ketamine (35 mg / kg) and xylazine (5 mg / kg). As soon as they were deeply anesthetized, each eye was washed with sterile saline and IVT injections were made using a 0.5 ml insulin syringe (50 units = 0.5 ml) with a 31 gauge needle on the supranasal side of both. the eyes when marking the position with a Braunstein caliper (2 3/4) 3.5 mm distant from the rectus muscle and 4 mm from the limbus.
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[002461 The tissues were collected 2 hours after the second IVT injection with IFNy. Retinal / choroidal tissues (R / C) were collected and homogenized and pSTATl levels were measured by quantitative Western Blot in the ProteinSimple WES instrument. The percentage of pSTAT1 inhibition induced by IFNy was calculated in comparison with the vehicle / vehicle and vehicle / IFNy groups.
[002471 With a pretreatment of 1 week before the challenge with IFNy, the suspension formulation of compound 1 inhibited IFNy-induced pSTAT1 by 85%. After pretreatment of 3 months with a single dose of the suspension formulation before challenge with IFNy, the suspension formulation of compound 1 inhibited IFNy-induced pSTAT1 by 76%.
[00248] A suspension formulation was prepared by combining compound 1 of example 5 (Form 2) with. 0.5% hydroxypropylmethylcellulose (HPMC E5) + 0.02% Tween 80 in normal saline to achieve a target concentration of 11.1 3.3 and 1.1 mg / ml.
[00249] White male rabbits from New Zealand (Liveon Biolabs, India) were used for the studies. The animals were acclimated after arriving at the research facility (Jubilant Biosys Ltd., India). Each rabbit received a total of two intravitreal injections (IVT) with. a total dose volume of 50 μΐ per eye. The first IVT injection (45 μΐ per eye) provided 500 μμ, 150 μμ, or 50 μμ of the test compound or vehicle. Two weeks later, a second injection of TIV (5 μΐ per eye) provided IFNy (1 μμ / eye; stock solution 1 mq / ml; Kinqfisher Biotech) or vehicle for the induction of the IP-10 protein. On the day of the injections, the rabbits were
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113/117 anesthetized with an intramuscular injection of ketamine (35 mg / kg) and xylazine (5 mg / kg). Thus, when they were deeply anesthetized, each eye was washed with sterile saline and IVT injections were made using a 0.5 ml insulin syringe (50 units = 0.5 ml) with a 31 gauge needle on the supranasal side of both the eyes when marking the position with a Braunstein caliper (2 3/4) 3.5 mm distant from the rectus muscle and 4 mm from the limbus.
[00250] Qs tissues were collected 2 times after the second IVT injection with IFNy. Retinal / choroidal tissues (R / C) were collected and homogenized and pSTAT1 levels were measured by quantitative Western Blot in the ProteinSimple WES instrument. The percentage of pSTAT1 inhibition induced by IFNy was calculated in comparison with the vehicle / vehicle and vehicle / IFNy groups.
[00251] With a pre-treatment of 2 weeks before the challenge with IFNy, the suspension formulation of compound 1 inhibited IFNy-induced PSTAT1 by 79% for the 50 pg dose, 58% for a. 150 µg dose and 61% for the 50 pg dose.
Assay 13: Kinome screening and GINI coefficient [00252] Compounds 1 and C-1 were screened for other kinases to assess their s and 1 and t i v .1 d a profile. in .
[00253] Kinase-labeled T7 phage strains grew in parallel in 24-well blocks in an E. coli host derived from the BL21 strain. The E. coli bacterium was cultivated in a log phase and infected with. T7 phage from a frozen stock (multiplicity of infection = 0.4) and incubated with shaking at 32 ° C until cell rupture (90Petition 870190110020, 10/29/2019, page 140/160
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150 minutes). The used ones were centrifuged (6,000 x g) and filtered (0.2 μπι) to remove cell debris. The remaining kinases were produced in HEK-293 cells and later labeled with DNA for detection by qPCR.
[00254] Streptavidin-coated magnetic beads were treated with small biotinylated binding molecules for 30 minutes at room temperature to generate affinity resins for the kinase assays. The connected 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 of the phage. The binding reactions were set up by combining kinases, affinity linked beads and test compounds in. 1 x binding buffer (20% SeaBlock, 0.17x PBS, 0.05% Tween 20, 6 mM DTT). The test compounds were prepared as 40x stocks in 100% DMSO and diluted directly in the assay. All reactions were carried out in 384 wells in. polypropylene plates in. a final volume of 0.04 ml. The assay plates were incubated at temperature with shaking for 1 hour and the affinity beads were washed with wash buffer (Ix PBS, 0.05% Tween 20). The beads were then resuspended in elution buffer (Ix PBS, 0.05%; Tween 20, 0.5 μΜ of non-biotinylated affinity binder) and incubated at room temperature with shaking for 30 minutes. The kinase concentration in the animals was measured by pPCR.
[00255] The compounds were filtered at 1 μΜ and the results of the primary screening binding interactions in Tables 8 and 9 are reported as% inhibition (= 100Petition 870190110020, 29/10/2019, p. 141/160
115/117 ((test compound signal-positive control signal) / ((negative control signal) - (positive control signal)) x 100), where the negative control is DMSO and the positive control is a control compound .
Table 8
Compound / Kinase ALK AURKA CDK2 CDK7 CDK9 CSF1R EPHB61 / B 11 6 7 4 9 98 8 8 31 C-l 81 57 5 3 99 95 10 0 98 68
Table 9
Compo sto / Kinase KIT PAK4 PKAC-ALPHA PLK4 SLK SRC SYK VEGFR2 1 87 93 20 58 10 0 93 46 4 2 C-l 99 99 T Q 68 10 0 10 0 78 6 3
[00256] Compound 1 showed significantly less binding inhibition for CDK7 and CDK9 than compound C-1. Compound 1 also had less binding inhibition for several other kinases.
[00257] Both compounds 1 and C-1 were tested against 35 different kinases. The Gini coefficient was determined for both compounds. Compound 1 had a GINI coefficient of 0.62 and compound C-1 had a GINI coefficient of 0.46. The Gini coefficient is used to express the selectivity of a compound in. relation to a kinase panel (Graczyk, J. Med. Cheia., 2007, 50, 57735779). A higher number corresponds to one. more selective compound.
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[00258] The only structural difference between compound 1 and compound C-1 is the presence of a fluoro group in the nucleus. This structural difference has been shown to have an important effect on the selectivity of the compound's kinoma.
Test 14: Cytotoxicity test
[002591 A luminescent cell viability / Titer-Glo cytotoxicity assay was performed on human lung BEAS-2B epithelial cells (ATCC), under normal growth condition.
[002 60] The cells were grown at 37 ° C in. a humidified incubator at 5% CO ₂ in 50% DMEM / 50% F-12 medium (Life Technologies), supplemented with 10% FBS (Hyclone), 100 U / ml penicillin, 100 pg / 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 384 wells for tissue culture (Corning) with 25 μΐ of medium, and stayed overnight in the incubator for adherence. On day 2 of the assay, 5 μΐ of culture medium containing the dose-responses of test compounds were added, and incubated at 37 ° C for 48 hours. 30 pL of CellTiter-Gloe detection solution (Promega) was later added, mixed on an orbital shaker for 5 min. and incubated for another 10 min. before being read on the EnVision reader. The luminescence signals were recorded and the DMSO control values were calculated.
[00261] For dose-response analysis, DMSO percentage control data were plotted versus compound concentrations to obtain dose-response curves per line connecting each data point.
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The concentration at which each curve crosses the 15% inhibition threshold is defined as CC15.
[00262] Test compounds exhibiting a higher CC15 value in this assay are expected to be less likely to cause cytotoxicity.
[002631 Compound 1 exhibited a CC15 of 3.16 μΜ while compound C-1 exhibited a CC15 of 630 riM. Therefore, compound 1 is less likely to cause cytotoxicity than compound C-1 based on this assay.
[00264] The only structural difference between the compound and the C-1 compound is the presence of a fluoro group in the nucleus. This structural difference has been shown to have an important effect on the cytotoxicity of the compound.
[00265] Although the present invention has been described with reference to specific aspects or its modalities, those who are skilled in the art will understand that various changes can be made or equivalent can be replaced, without departing from the true spirit and scope of the invention . In addition, to the extent permitted by applicable statutes and applicable patent regulations, all publications, patents and applications cited here are incorporated by reference in. its entirety, in the same way as if each document had been incorporated individually by reference.

权利要求:
Claims (43)
[1]
WHAT CLAIMS IS:
A compound of formula:

[2]
2. A compound of the formula:

[3]
The crystalline form of Claim 3, wherein the x-ray powder diffraction pattern is further characterized pc
Petition 870190110020, of 10/29/2019, p. 145/160 have additional dictation peaks in the 2Θ values of 13.3210.20, 17, 6910.20, and 21.1010.20.
[4]
The crystalline form of Claim 4, where the powder X-ray diffraction pattern is further characterized by having two or more diffraction peaks at the 2Θ values selected from 10.8510,20, 16,1410,20, 16 , 3510,20, 18,4310,20, 19,2010,20, 19,4910,20, 20,7210,20, 21,9410,20, 22,6410,20, 23,6410,20, 25,1910 , 20 and 28,0810,20.
[5]
6. The crystalline form of. Claim 3, wherein the crystalline form is characterized by a powder X-ray diffraction pattern, in which the peak positions are substantially in line with the peak positions of the pattern shown in Figure 1.
[6]
The crystalline form of Claim 3 where the crystalline form is c. a r a c t e r i z a d a by one. trace of differential exploratory calorimetry recorded at a heating rate of 10 ° C per minute, which shows a maximum in the endothermic heat flow at a temperature between 268 ° C and 277 ° C.
[7]
The crystalline form of Claim 3 where the crystalline form is characterized by a trace of exploratory differential calorimetry recorded at a heating rate of 10 ° C per minute, which shows one. maximum in the endothermic heat flow at a temperature between 272.6 ° C ± 2 ° C.
[8]
The crystalline form of Claim 3, wherein the crystalline form is characterized by a trace of exploratory differential calorimetry substantially as indicated in the Figure. 2.
[9]
10. A crystalline form of the compound of formula:
Petition 870190110020, of 10/29/2019, p. 146/160

[10]
The crystalline form of Claim 10 where the powder X-ray diffraction pattern is characterized with two or more additional diffraction peaks at 2Θ values selected from 7.69 1 0.20, 10.66 1 0.20 , 11.46 1 0, 20, 11, 91 1 0.20, 15, 80 1 0, 20, 17.02 1 0.20, 18, 83 1 0.20, 22.39 1 0.20, 22 , 98 1 0.20, 24.89 1 0, 20 and 2 6, 54 1 0.20.
[11]
The crystalline form of Claim 10, wherein the crystalline form is characterized by a powder X-ray diffraction pattern, in which the peak positions are substantially in accordance with the peak positions of the pattern shown in Figure 6.
[12]
The crystalline form of Claim 10 where the crystalline form is characterized by a trace of exploratory differential calorimetry recorded at a heating rate of 10 ° C per minute, which shows a maximum in the endothermic heat flow at a temperature between 215 ° C and 229 ° C.
[13]
The crystalline form of Claim 10 where the crystalline form is characterized by an exploratory differential calorimetry trace recorded at a heating rate of 10 ° C per minute, which shows a maximum in the endothermic heat flow at a temperature between 221.7 ° C ± 3 ° C.
Petition 870190110020, of 10/29/2019, p. 147/160
4/9
[14]
The crystalline form of Claim 10, wherein the crystalline form is corrected by an exploratory differential calorimetry trace substantially as indicated in Figure 7.
[15]
A pharmaceutical composition characterized by the compound of Claims 1 or 2 or the crystalline form of any one of Claims 3 to 15 and a pharmaceutically acceptable carrier.
[16]
The pharmaceutical composition of Claim 16, the composition of which is suitable for application or cure.
[17]
The pharmaceutical composition of Claim 17, wherein the composition is suitable for intravitreal injection.
[18]
The pharmaceutical composition of Claim 18, wherein the composition is one. suspension.

[19]
21. A compound of formula 6:

[20]
22. A method of preparing the crystalline form of. Claim 3 comprising:
(a) form a homogeneous mixture of 1- (2- (6- (2-ethyl-5fluoro-4-hydroxyphenyl ~ 4-fluoro-1H-indazol-S-yl) 1,4,6,7-tetrahydro-5H -imidazo [4,5-c] pyridin-5-yl) -2morpholinoetan-l-one in a polar aprotic solvent or a water miscible polar solvent, or in a mixture of polar aprotic solvent and a water miscible polar solvent at a temperature between 45 and 75 ° C;
(b) adding the homogeneous mixture to a mixture of a water and water miscible solvent, at a temperature between 60 and 90 ° C to produce a second mixture;
Petition 870190110020, of 10/29/2019, p. 149/160 (c) slowly add the water to the second mixture at a temperature between 60 and 90 ^J C to form a slurry; and (d) isolating the crystalline form from the slurry.
[21]
23. The method of Claim 22, wherein the polar aprotic solvent of step (a) is selected from the group consisting of DMSO, DMF, NMP, DM.AC and nitromethane, the water-miscible polar solvent of step (a) is selected from the group consisting of acetonitrile, acetone, methanol, ethanol and THF and the miscible solvent in. Water from step (b) is selected from the group consisting of acetonitrile, acetone, methanol, ethanol, n-propanol, isopropanol, nbutanol, THF, DMSO, DMF, NMP, DMAc and nitromethane.
[22]
The compound as claimed in Claim 1 or 2 or the crystalline form as claimed in any of Claims 3 to 15, for use in the treatment of an eye disease in a mammal.
[23]
The compound or crystalline form of Claim 24, wherein the eye disease is uveitis, diabetic retinopathy, diabetic macular edema, dry eye disease, age-related macular degeneration, retinal vein occlusion or atopic keratoconjunctivitis.
[24]
26. The compound or crystalline form of Claim 25, wherein the eye disease is diabetic macular edema or uveitis.
[25]
27. Use of a compound as claimed in Claims 1 or 2 or the crystalline form in any of Claims 3 to 15 for the production of a medicament for the treatment of an eye disease in. a mammal.
[26]
28. The use of Claim '27, where the eye disease is uveitis, diabetic retinopathy, diabetic macular edema,
Petition 870190110020, of 10/29/2019, p. 150/160 dry eye disease, age-related macular degeneration, retinal vein occlusion or atopic keratoconjunctivitis.
[27]
29. A method of treating an eye disease in a mammal, the method comprising administering a pharmaceutical composition comprising the compound of Claim 1 or 2 or the crystalline form of any one of Claims 3 to 15 and an acceptable carrier for the mammalian eye.
[28]
The method of Claim 29, wherein the eye disease is uveitis, diabetic retinopathy, diabetic macular edema, dry eye disease, age-related macular degeneration, retinal vein occlusion or atopic keratoconjunctivitis.
[29]
31. The method of Claim 30, wherein the eye disease is diabetic uveitis or macular edema.
[30]
The compound as claimed in Claim 1 or 2 or the crystalline form as claimed in any of Claims 3 to 15, for use in the treatment of a skin disease in a mammal.
[31]
33. The compound or crystalline form of Claim 32, wherein the inflammatory skin disease is atopic dermatitis.
[32]
34. Use of a compound as claimed in Claims 1 or 2 or the crystalline form in any of Claims 3 to 15 for the production of a medicament for the treatment of a skin disease in a mammal.
[33]
35. The use of Claim 34, wherein the inflammatory skin disease is atopic dermatitis.
[34]
36. A method of treating an inflammatory skin disease in. a mammal, the method comprising applying a pharmaceutical composition comprising the compound of Claim 1 or 2 or the crystalline form of any one
Petition 870190110020, of 10/29/2019, p. 151/160
8/9 of Claims 3 to 15 and a pharmaceutically acceptable carrier for mammalian skin.
[35]
37. The method of Claim 36, wherein the inflammatory skin disease is atopic dermatitis.
[36]
38. The compound of any one of Claims 1 or
2 or the crystalline form as claimed in. any of Claims 3 to 15 for use in the treatment of a respiratory disease in a mammal.
[37]
39. The compound or crystalline form of the Claim
38, where respiratory disease is asthma, chronic obstructive pulmonary disease, cystic fibrosis, pneumonitis, idiopathic pulmonary fibrosis, acute lung injury, acute respiratory distress syndrome, bronchitis, emphysema, lung transplant rejection, primary graft dysfunction, organizing pneumonia , acute lung transplant rejection, lymphocytic bronchiolitis, chronic restrictive pulmonary graft dysfunction, neutrophilic graft dysfunction or obliterating bronchiolitis.
[38]
40. The compound or crystalline form of Claim 39, wherein the respiratory disease is asthma, chronic pulmonary graft dysfunction or obstructive pulmonary disease.
[39]
41. Use of a compound as claimed in Claims 1 or 2 or the crystalline form in. any one of Claims 3 to 15 for the production of a medicament for the treatment of a respiratory disease in a mammal.
[40]
42. The use of Claim 41, where the respiratory disease is asthma, chronic obstructive pulmonary disease, cystic fibrosis, pneumonitis, idiopathic pulmonary fibrosis, acute lung injury, acute respiratory distress syndrome, bronchitis, emphysema, transplant rejection
Petition 870190110020, of 10/29/2019, p. 152/160 pulmonary, primary graft dysfunction, organizing pneumonia, acute lung transplant rejection, lymphocytic bronchiolitis, chronic restrictive pulmonary graft dysfunction, graft neutrophilic dysfunction or obliterating bronchiolitis.
[41]
43. A method of treating a respiratory disease in a mammal, the method comprising administering to the mammal a pharmaceutical composition comprising the compound of Claims 1 or 2, or the crystalline form of any one of Claims 3 to 15 and an acceptable point carrier. pharmaceutical point of view.
[42]
44. The method of Claim 43, wherein the respiratory disease is asthma, chronic obstructive pulmonary disease, cystic fibrosis, pneumonitis, idiopathic pulmonary fibrosis, acute lung injury, acute respiratory distress syndrome, bronchitis, emphysema, lung transplant rejection, dysfunction primary graft, organizing pneumonia, acute lung transplant rejection, lymphocytic bronchiolitis, chronic restrictive pulmonary graft dysfunction, graft neutrophilic dysfunction or obliterating bronchiolitis.
[43]
45. The method of Claim 44, wherein the respiratory disease is asthma, chronic pulmonary graft dysfunction or chronic obstructive pulmonary disease.

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PH12019502264A1|2020-09-21|

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EA201992601A1|2020-03-03|

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US11160810B2|2021-11-02|

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法律状态:
2021-10-19| B350| Update of information on the portal [chapter 15.35 patent gazette]|

优先权:

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申请号 | 申请日 | 专利标题

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US201762492574P| true| 2017-05-01|2017-05-01|

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