CYCLLOBUTAN AND METHYLCYCLOBUTAN DERIVATIVES AS JANUS KINASE INHIBITORS AND COMPOSITION THAT UNDERST

专利摘要:
cyclobutane and methylcyclobutane derivatives as janus kinase inhibitors. the present invention relates to cyclobutane and methylcyclobutane derivatives, as well as their salts, compositions, and methods of use, which are inhibitors of janus kinase (jak) useful in the treatment of diseases associated with jak including, for example, inflammatory and autoimmune disorders, as well as cancer and myeloproliferative disorders.
公开号:BR112012020693B1
申请号:R112012020693-1
申请日:2011-02-18
公开日:2020-05-12
发明作者:Yun-Long Li；James D. Rodgers
申请人:Incyte Holdings Corporation；
IPC主号:

专利说明:

Invention Patent Descriptive Report for CYCLOBUTAN AND METHYLCYCLOBUTAN DERIVATIVES AS INHIBITORS OF JANUS KINASE AND COMPOSITION THAT UNDERSTANDS THEM.
RELATED ORDER [0001] This order claims priority for the Interim Order
No. 61 / 305,630, filed February 18, 2010, entitled CYCLOBUTANE AND METHYLCYCLOBUTANE DERIVATIVES AS JANUS KINASE INHIBITORS. The contents of all patents, patent applications and references cited throughout this specification are hereby incorporated by reference in their entirety.
FIELD OF THE INVENTION [0002] The present invention relates to cyclobutane and methylcyclobutane derivatives, as well as their salts, compositions, and methods of use. These compounds are Janus kinase (JAK) inhibitors useful in the treatment of diseases associated with JAK including, for example, inflammatory and autoimmune disorders, as well as cancer and myeloproliferative disorders.
BACKGROUND OF THE INVENTION [0003] Protein kinases (PKs) are a group of enzymes that regulate several important biological processes including cell development, survival and differentiation, organ formation and morphogenesis, neovascularization, tissue repair and regeneration, among others. Protein kinases exert their physiological functions by catalyzing the phosphorylation of proteins (or substrates) and thereby modulating the cellular activities of the substrates in various biological contexts. In addition to normal tissue / organ functions, many protein kinases also play more specialized roles in a host of
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2/50 human diseases, including cancer. A subset of protein kinases (also referred to as oncogenic protein kinases), when deregulated, can cause tumor formation and growth, and also contribute to tumor maintenance and progression. So far, oncogenic protein kinases represent one of the largest and most attractive groups of protein targets for cancer intervention and drug development.
[0004] The Janus Kinase (JAK) family plays a role in cytokine-dependent regulation of proliferation and function of cells involved in the immune response. Currently, there are four known members of the JAK mammal family: JAK1 (also known as Janus kinase-1), JAK2 (also known as Janus kinase-2), JAK3 (also known as Janus kinase, leukocytes; JAKL; L-JAK and Janus kinase-3) and TYK2 (also known as protein-tyrosine kinase 2). JAK proteins range in size from 120 to 140 kDa and comprise seven conserved domains of JAK homology (JH); one is a functional catalytic kinase domain, and the other is a pseudokinase domain that potentially serves as a regulatory function and / or serves as a coupling site for STATs.
[0005] Blocking signal transduction at the level of kinases
JAK keeps the promise of developing treatments for inflammatory diseases, autoimmune diseases, myeloproliferative diseases, and human cancers, to name a few. Inhibition of JAK kinases is also expected to have therapeutic benefits in patients suffering from immune skin disorders such as psoriasis, and skin sensitization.
[0006] Thus, new or improved agents that inhibit kinases, such as Janus kinases, are continually needed for the development of new and more effective products
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3/50 pharmacists to treat cancer and other diseases. The compounds, salts and compositions described herein are directed to these needs and other purposes.
SUMMARY OF THE INVENTION [0007] The present invention provides a compound that is 3cyclobutyl-3- [4- (7H-pyrrolo [2,3-d] pyrimidin-4-yl) -1H-pyrazol-1yl] propanonitrile, or a salt pharmaceutically acceptable thereto. In some embodiments, the aforementioned compound is the R or S enantiomer.
[0008] The present invention further provides a compound that is 3 (4- (7H-pyrrolo [2,3-d] pyrimidin-4-yl) -1H-pyrazol-1-yl) -3- (3-methylcyclobutyl) propanonitrile, or a pharmaceutically acceptable salt thereof. The present invention further includes the various stereoisomers of the aforementioned compound, including the R and S enantiomers and cis and trans geometric isomers.
[0009] The present invention further provides a phosphoric acid salt of any of the cyclobutyl or methylcyclobutyl compounds described herein.
[00010] The present invention further provides a composition comprising a compound as described herein, or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable carrier.
[00011] The present invention further provides methods of treating a disease or disorder associated with JAK in a patient comprising administering to the patient a therapeutically effective amount of a compound as described herein, or a pharmaceutically acceptable salt thereof.
[00012] The present invention further provides the compounds described herein, or their pharmaceutically acceptable salts, for use in therapy.
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[00013] The present invention further provides the use of the compounds described herein, or their pharmaceutically acceptable salts, for the preparation of a medicament for use in therapy.
[00014] A method of treating an autoimmune disease in a patient is also provided here comprising administering to said patient a therapeutically effective amount of a compound of the invention, or a pharmaceutically acceptable salt thereof. In one embodiment, autoimmune disease is a skin disease, multiple sclerosis, rheumatoid arthritis, psoriatic arthritis, juvenile arthritis, type I diabetes, lupus, inflammatory bowel disease, Crohn's disease, myasthenia gravis, immunoglobulin nephropathies, myocarditis, or autoimmune thyroid disorders. In another embodiment, the autoimmune disease is rheumatoid arthritis. In yet another modality, autoimmune disease is a skin disorder, such as atopic dermatitis, psoriasis, skin sensitization, skin irritation, rash, contact dermatitis or allergic contact sensitization.
[00015] In another aspect, a method of treating cancer in a patient comprising administering to said patient a therapeutically effective amount of a compound of the invention, or a pharmaceutically acceptable salt thereof, is provided herein. In one embodiment, cancer is a solid tumor. In another embodiment, the cancer is prostate cancer, kidney cancer, liver cancer, breast cancer, lung cancer, thyroid cancer, Kaposi's sarcoma, Castleman's disease or cancer of the pancreas. In yet another modality, cancer is lymphoma, leukemia, or multiple myelomas.
[00016] In yet another aspect, a method of treating a myeloproliferative disorder in a patient is provided herein comprising administering to said patient a therapeutically effective amount of a compound of the invention, or a pharmaceutically acceptable salt thereof. In one embodiment, the disorder
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5/50 myeloproliferative (MPD) is polycythemia vera (PV), essential thrombocythemia (ET), primary myelofibrosis (PMF), myelofibrosis with myeloid metaplasia (MMM), chronic myeloid leukemia (CML), chronic myelomonocytic leukemia (CMML), hypereosinophilic syndrome (HES), idiopathic myelofibrosis (IMF), systemic mast cell disease (SMCD), or myelofibrosis after polycythemia vera / essential thrombocythemia (PostPV / ET MF).
[00017] In another aspect, a method of treating an inflammatory disease in a patient is provided herein comprising administering to said patient a therapeutically effective amount of a compound of the invention, or a pharmaceutically acceptable salt thereof.
[00018] In yet another aspect, a method of treating organ transplant rejection in a patient is provided herein, comprising administering to said patient a therapeutically effective amount of a compound of the invention, or a pharmaceutically acceptable salt thereof.
[00019] In yet another aspect, a method of treating dry eye in a patient is provided herein, comprising administering to said patient a therapeutically effective amount of a compound of the invention, or a pharmaceutically acceptable salt thereof.
DETAILED DESCRIPTION [00020] The present invention provides, inter alia, the JAK inhibiting compound:
3-cyclobutyl-3- [4- (7H-pyrrolo [2,3-d] pyrimidin-4-yl) -1H-pyrazol-1-yl] propanonitrile (Formula I), and its pharmaceutically acceptable salts.
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6/50
[00021] The present invention further provides the compounds (R) -3cyclobutyl-3- [4- (7H-pyrrolo [2,3-d] pyrimidin-4-yl) -1H-pyrazol-1-yl] propanonitrile (Formula IR) and (S) -3-cyclobutyl-3- [4- (7H-pyrrole [2,3-
d] pyrimidin-4-yl) -1H-pyrazol-1-yl] propanonitrile (Formula I-S), and its pharmaceutically acceptable salts.

[00022] The present invention further provides the JAK inhibiting compound 3- (4- (7H-pyrrolo [2,3-d] pyrimidin-4-yl) -1H-pyrazol-1-yl) -3- (3methylcyclobutyl ) propanonitrile (Formula II), and its pharmaceutically acceptable salts.
[00023] The present invention further provides the cis and trans isomers of the compound of Formula II. These cis and trans isomers are:
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7/50 [00024] 3- (4- (7H-pyrrolo [2,3-d] pyrimidin-4-yl) -1H-pyrazol-1-yl) -3 ((trans) -3-methylcyclobutyl) propanonitrile ( Formula II-trans); and [00025] 3- (4- (7H-pyrrolo [2,3-d] pyrimidin-4-yl) -1H-pyrazol-1-yl) -3- (cis) 3-methylcyclobutyl) propanonitrile (Formula II- cis).

[00026] The present invention further provides the R and S enantiomers of the compound of Formula II. These R and S isomers are:
[00027] (3R) -3- (4- (7H-pyrrolo [2,3-d] pyrimidin-4-yl) -1H-pyrazol-1-yl) -3 (((3-methylcyclobutyl) propanonitrile (Formula II -R); and [00028] (3S) -3- (4- (7H-pyrrolo [2,3-d] pyrimidin-4-yl) -1H-pyrazol-1-yl) -3 (3-methylcyclobutyl) propanonitrile (Formula II-S).

[00029] The present invention also provides the R / trans isomers,
R / cis, S / trans, and S / cis of the compound of Formula II. These isomers are: [00030] (3R) -3- (4- (7H-pyrrolo [2,3-d] pyrimidin-4-yl) -1H-pyrazol-1-yl) -3 ((trans) -3 -methylcyclobutyl) propanonitrile (Formula II-R / trans), [00031] (3S) -3- (4- (7H-pyrrolo [2,3-d] pyrimidin-4-yl) -1H-pyrazol-1-yl ) -3 ((trans) -3-methylcyclobutyl) propanonitrile (Formula II-S / trans), [00032] (3R) -3- (4- (7H-pyrrolo [2,3-d] pyrimidin-4-yl ) -1H-pyrazol-1-yl) -3Petition 870190113460, of 11/06/2019, p. 13/63
8/50 ((cis) -3-methylcyclobutyl) propanonitrile (Formula II-R / cis), and [00033] (3S) -3- (4- (7H-pyrrolo [2,3-d] pyrimidin-4- yl) -1H-pyrazol-1-yl) -3 ((cis) -3-methylcyclobutyl) propanonitrile (Formula II-S / cis).

II-S / trans
II-S / cis [00034] The compounds described above are referred to herein as the compounds of the invention. Here and elsewhere, where there are discrepancies between a compound name and a compound structure, the chemical structure will control.
[00035] The present invention further provides pharmaceutically acceptable salts of any of the aforementioned compounds. In some embodiments, the pharmaceutically acceptable salt is a phosphoric acid salt.
[00036] The compounds described herein are asymmetric (for example, having one or more stereocenters). All stereoisomers, such as enantiomers and diastereomers, are designed, unless otherwise indicated. The compounds of the present invention that contain asymmetrically carbon atoms
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Substituted 9/50 can be isolated in optically active or racemic forms. Methods on how to prepare optically active forms are known in the art, such as by resolving racemic mixtures or by stereoselective synthesis. Geometric isomers can also be present in the compounds described herein, and all such stable isomers are contemplated in the present invention. The cis and trans geometric isomers of the compounds of the present invention are described and can be isolated as a mixture of isomers or as substantially separate isomeric forms. Where a compound capable of stereoisomerism (for example, optical and / or geometric isomerism) is designated in its structure or name without reference to specific R / S or cis / trans configurations, it is intended that all such isomers be contemplated. For example, Formulas I and II as represented above should be understood as inclusive of both R and S isomers and cis and trans isomers insofar as the molecules take into account such isomerism.
[00037] The resolution of racemic mixtures, or the separation of a mixture of optical and / or geometric isomers, can be carried out by any of the numerous methods known in the art including chromatographic methods, (for example, chiral column chromatography) or fractional recrystallization.
[00038] The compounds of the invention can also include tautomeric forms. Tautomeric forms result from the exchange of a single bond with an adjacent double bond together with the concomitant migration of a proton. Tautomeric forms include prototropic tautomers that are states of isomeric protonation having the same empirical formula and full charge. Exemplary prototropic tautomers include ketone - enol pairs, amide - imidic acid pairs, lactam - lactime pairs, amide - imidic acid pairs,
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10/50 pairs of enamine - imine, and the annular forms where a proton can occupy two or more positions in a heterocyclic system, for example, 1H- and 3H-imidazole, 1H-, 2H- and 4H- 1,2,4 -triazole, 1H- and 2H-isoindole, and 1H- and 2H-pyrazole.
[00039] The compounds and salts of the present invention can be found together with other molecules, such as solvent and water molecules, to form hydrates and solvates.
[00040] The compounds and salts of the invention can also include all isotopes of atoms present internally. Isotopes include those atoms having the same atomic number, but different mass numbers. For example, hydrogen isotopes include tritium and deuterium.
[00041] In some embodiments, the compounds of the invention, and their salts, are substantially isolated. By substantially isolated means that the compound is at least partially or substantially separated from the environment in which it was formed or detected. Partial separation can include, for example, a composition enriched in the compound or salt of the invention. Substantial separation can include compositions containing at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least at least about 97%, or at least about 99% by weight of the compound of the invention, or its salt.
[00042] The phrase pharmaceutically acceptable is used here to refer to those compounds, salts, materials, compositions, and / or dosage forms that are, within safe medical judgment, suitable for use in contact with human tissues and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.
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11/50 [00043] The present invention also includes the pharmaceutically acceptable salts of the compounds described herein. As used herein, the phrase pharmaceutically acceptable salts refers to derivatives of the disclosed compounds in which the parent compound is modified by converting an existing acid or base component to its salt form. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkaline or organic salts of acid residues such as carboxylic acids; and more. The pharmaceutically acceptable salts of the present invention include conventional non-toxic salts of the parent compound formed, for example, of non-toxic inorganic or organic acids. The pharmaceutically acceptable salts of the present invention can be synthesized from the parent compound which contains a basic or acidic component by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or an organic solvent, or a mixture of the two. Lists of suitable salts are found in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418 and Journal of Pharmaceutical Science, 66, 2 (1977), each of which is incorporated herein by reference in its entirety.
Methods [00044] The compounds and salts of the invention can inhibit the activity of one or more Janus kinases (JAKs). The JAKs in which the present compounds bind and / or inhibit include any member of the JAK family. The present compounds inhibit the activities of both JAK1 and JAK2.
[00045] Another aspect of the present invention pertains to methods
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12/50 treatment of a disease or disorder associated with JAK in an individual (e.g., patient) by administering to the individual in need of such treatment a therapeutically effective amount or dose of a compound or salt of the present invention or a pharmaceutical composition of this. A disease associated with JAK may include any disease, disorder or condition that is directly or indirectly linked to JAK expression or activity, including abnormal overexpression and / or levels of activity. A disease associated with JAK can also include any disease, disorder or condition that can be prevented, ameliorated, or cured by modulating JAK activity.
[00046] Examples of diseases associated with JAK include diseases involving the immune system including, for example, organ transplant rejection (for example, allograft rejection and graft versus host disease).
[00047] Other examples of diseases associated with JAK include autoimmune diseases such as multiple sclerosis, rheumatoid arthritis, juvenile arthritis, psoriatic arthritis, type I diabetes, lupus, psoriasis, inflammatory bowel disease, ulcerative colitis, Crohn's disease, myasthenia gravis, immunoglobulin nephropathies, autoimmune thyroid disorders, and more. In some embodiments, autoimmune disease is an autoimmune bullous skin disorder such as pemphigus vulgaris (PV) or bullous pemphigoid (BP).
[00048] Other examples of diseases associated with JAK include allergic conditions such as asthma, food allergies, atopic dermatitis and rhinitis. Other examples of diseases associated with JAK include viral diseases, such as the Epstein Barr virus (EBV), hepatitis B, hepatitis C, HIV, HTLV 1, varicella-zoster virus (VZV) and human papilloma virus (HPV) .
[00049] Other examples of diseases or conditions associated with
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13/50 to JAK include skin disorders such as psoriasis (for example psoriasis vulgaris), atopic dermatitis, rash, skin irritation, skin sensitization (for example, contact dermatitis or allergic contact dermatitis). For example, certain substances including some pharmaceuticals when applied topically can cause skin sensitization. In some embodiments, co-administration or sequential administration of at least one JAK inhibitor of the invention together with the agent causing the unwanted sensitization may be useful in the treatment of such unwanted sensitization or dermatitis. In some embodiments, the skin disorder is treated by the topical administration of at least one JAK inhibitor of the invention.
[00050] In other modalities, the disease associated with JAK is cancer, including one that is characterized by solid tumors (eg, prostate cancer, kidney cancer, liver cancer, pancreatic cancer, gastric cancer, breast cancer, cancer of lung, head and neck cancer, thyroid cancer, glioblastoma, Kaposi's sarcoma, Castleman's disease, melanoma, etc.), hematological cancers (for example, lymphoma, leukemia, such as acute lymphoblastic leukemia, acute myeloid leukemia (AML) or multiple myeloma), and skin cancer such as cutaneous T-cell lymphoma (CTCL) and cutaneous B-cell lymphoma. Example of cutaneous T-cell lymphoma includes Sezary's syndrome and fungal mycosis.
[00051] Diseases associated with JAK may further include those characterized by the expression of a mutant JAK2, such as those having at least one mutation in the pseudo-kinase domain (for example, JAK2V617F).
[00052] Diseases associated with JAK may further include myeloproliferative disorders (SMP) such as polycythemia vera (PV), essential thrombocythemia (ET), myelofibrosis with myeloid metaplasia
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14/50 (MMM), chronic myeloid leukemia (CML), chronic myelomonocytic leukemia (CMML), hypereosinophilic syndrome (HES), systemic mast cell disease (SMCD), and more. In some modalities, the myeloproliferative disorder is primary myelofibrosis (PMF) or post-polycythemia vera / myelofibrosis essential thrombocythemia (Post-PV / ET MF). [00053] Other diseases associated with JAK include inflammation and inflammatory diseases. Exemplary inflammatory diseases include inflammatory diseases of the eye (for example, iritis, uveitis, scleritis, conjunctivitis, or related disease), inflammatory diseases of the respiratory tract (for example, the upper respiratory tract including the nose and sinuses such as rhinitis or sinusitis or the lower respiratory tract including bronchitis, chronic obstructive pulmonary disease, and more), inflammatory myopathy such as myocarditis, and other inflammatory diseases.
[00054] The JAK inhibitors described herein can further be used to treat ischemia reperfusion injury or a disease or condition related to an inflammatory ischemic event such as stroke or cardiac arrest. The JAK inhibitors described herein can further be used to treat anorexia, cachexia, or fatigue, such as those that result from or are associated with cancer. The JAK inhibitors described herein can also be used to treat restenosis, sclerodermatitis, or fibrosis. The JAK inhibitors described herein can further be used to treat conditions associated with hypoxia or astrogliosis such as, for example, diabetic retinopathy, cancer, or neurodegeneration. See, for example, Dudley, A.C. et al. Biochem. J. 2005, 390 (Pt 2): 427-36 and Sriram, K. et al. J. Biol. Chem. 2004, 279 (19): 19936-47. Epub 2004 Mar 2. The JAK inhibitors described here can be used to treat Alzheimer's disease.
[00055] The JAK inhibitors described here can still be
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15/50 used to treat other inflammatory diseases such as systemic inflammatory response syndrome (SIRS) and septic shock.
[00056] The JAK inhibitors described herein can further be used to treat gout and enlarged prostate size due to, for example, benign prostatic hypertrophy or benign prostatic hyperplasia.
[00057] The JAK inhibitors described herein, as well as other JAK inhibitors capable of influencing IL-6 / STAT3 signaling, can further be used to treat the proliferative diseases associated with inflammation. Inflammation has been shown to be linked to the development of certain types of cancers. For example, patients suffering from inflammatory bowel disease such as ulcerative colitis have been shown to be at a much higher risk of developing colorectal cancer. These types of cancers linked to inflammation have been termed cancer associated with colitis (CAC). Several studies have shown that IL-6 / STAT3 signaling is involved in the promotion of CAC. For example, mice deficient in STAT3 intestinal epithelial cells had reduced tumor size and incidence in an animal model of CAC. Bromberg, et al., Inflammation and cancer: IL-6 and STAT3 complete the link, Cancer Cell, 15: 79-80 (2009). Similar results were obtained with IL-6 deficient mice, which developed fewer and smaller adenomas than wild-type mice. Grivennikov, et al., IL-6 and STAT3 are required for survival of intestinal epithelial cells and the development of colitis-associated cancer, Cancer Cell, 15: 103-111 (2009). See also, Bollrath, et al., Gp130Mediated STAT3 activation in enterocytes regulatres cell survival and cell-cycle progression during colitis-associated tumorigenesis, Cancer Cell, 15: 91-102 (2009); and Kortylewski, et al., Regulation of the IL-23 and IL-12 balance by Stat3 signaling in the tumor microenvironment, Cancer
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16/50
Cell, 15: 114-123 (2009).
[00058] Consequently, in some embodiments, the JAK inhibitors of the invention and those that influence IL-6 / STAT3 signaling, can be used to treat cancers associated with inflammation. In some modalities, cancer is associated with inflammatory bowel disease. In some embodiments, inflammatory bowel disease is ulcerative colitis. In some embodiments, inflammatory bowel disease is Crohn's disease. In some embodiments, the cancer associated with inflammation is the cancer associated with colitis. In some modalities, the cancer associated with the inflammation is colon cancer or colorectal cancer. In some modalities, the cancer is gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), adenocarcinoma, small intestine cancer, or rectal cancer. In addition to the compounds provided herein, exemplary JAK inhibitors that can be used to treat cancers associated with inflammation include those described in US 2006/0106020; US 2006/0183906; US 2007/0149506; US 2007/0135461; US 2008/0188500; US 2008/0312258; US 2008/0312259; and U.S. Ser. No. 12 / 270,135.
[00059] JAK inhibitors can be tested in animal models for potential effectiveness in treating cancers associated with inflammation. For example, CAC can be induced in mice treated (for example, with JAK inhibitors) or untreated by the method summarized in Grivennikov, et al., IL-6 and STAT3 are required for survival of intestinal epithelial cells and the development of colitis-associated cancer, Cancer Cell, 15: 103-111 (2009). The progression of the disease can be followed by measuring body weight and monitoring signs of rectal hemorrhage and diarrhea. After sacrificing the animals, parts of the distal colon are removed to
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17/50 analysis.
[00060] In some embodiments, the JAK inhibitors described herein can still be used to treat a dry eye disorder. As used here, dry eye disorder is intended to encompass the sick states summarized in a recent official report by the Dry Eye Workshop (DEWs), which defined dry eye as a multifactorial disease of tears and ocular surface that results in symptoms of discomfort. , visual disturbance and instability of the tear film with potential damage to the ocular surface. It is accompanied by increased osmolarity of the tear film and inflammation of the ocular surface, Lemp, The Definition and Classification of Dry Eye Disease: Report of the Definition and Classification Subcommittee of the International Dry Eye WorkShop, The Ocular Surface, 5 (2), 75- 92 April 2007, which is incorporated herein by reference in its entirety. The dry eye is also sometimes referred to as dry keratoconjunctivitis. In some embodiments, treatment of dry eye disorder involves ameliorating a particular symptom of dry eye disorder, such as eye discomfort, visual disturbance, tear film instability, tear hyperosmolarity, and inflammation of the eye surface. The use of JAK inhibitors for the treatment of dry eye is provided in U.S. Ser. No. 12 / 571,834, filed October 1, 2009, which is incorporated herein by reference.
[00061] In another aspect, the present invention provides a method of treating conjunctivitis, uveitis, (including chronic uveitis), chorioditis, retinitis, cyclitis, scleritis, episcleritis or iritis; treatment of inflammation or pain related to corneal transplantation, LASIK (laser-assisted keratomileusis), photorefractive keratectomy, or LASEK (laser-assisted subepithelial keratomileusis); loss of visual acuity inhibition related to corneal transplantation, LASIK, photorefractive keratectomy, or LASEK; or
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18/50 inhibition of transplant rejection in a patient in need thereof, which comprises administering to the patient a therapeutically effective amount of a compound of Formula I, or a pharmaceutically acceptable salt or N-oxide thereof. In some embodiments, the compound, or its pharmaceutically acceptable salt or N-oxide, is administered prior to surgery to a patient about to undergo a selected corneal transplant procedure, LASIK, photorefractive keratectomy, and LASEK. In some embodiments, the compound, or its pharmaceutically acceptable salt or N-oxide, suppresses or decreases inflammation or pain during and after the procedure. In some embodiments, the compound, or its pharmaceutically acceptable salt or N-oxide, is administered about 1 day to about 2 days before the procedure. In some embodiments, the compound, or its pharmaceutically acceptable salt or N-oxide, is administered postoperatively to a patient who has undergone a selected corneal transplant procedure, LASIK, photorefractive keratectomy, and LASEK. In some embodiments, inhibiting loss of visual acuity means decreasing loss of visual acuity. In some modalities, postoperative or preoperative treatment decreases the amount of scarring and fibrous deposits after the procedure. In some embodiments, inhibiting loss of visual acuity means that the patient retains visual acuity. In some embodiments, the inhibition of transplant rejection means that the compound, or its pharmaceutically acceptable salt or N-oxide, is immunosuppressive, thus preventing complete rejection of the corneal transplant.
[00062] In one embodiment, a method of treating cancer in an individual is provided herein, comprising administering to the individual 3-cyclobutyl-3- [4- (7H-pyrrole [2,3d] pyrimidin-4-yl) -1H-pyrazol-1-yl] propanonitrile, an isomer thereof, or a pharmaceutically acceptable salt thereof. In another modality, it is here
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19/50 provided a method of treating myelofibrosis in an individual, comprising administering to the individual 3-cyclobutyl-3- [4- (7Hpyrrolo [2,3-d] pyrimidin-4-yl) -1H-pyrazole-1 -il] propanonitrile, an isomer thereof, or a pharmaceutically acceptable salt thereof. In another embodiment, a method of treating rheumatoid arthritis (RA) in an individual is provided herein, which comprises administering to the individual 3-cyclobutyl-3- [4- (7H-pyrrole [2,3-d] pyrimidine- 4-yl) -1H-pyrazol1-yl] propanonitrile, an isomer thereof, or a pharmaceutically acceptable salt thereof. In another embodiment, a method of treating polycythemia vera (PV) in an individual is provided herein, comprising administering to the individual 3-cyclobutyl-3- [4- (7H-pyrrole [2,3d] pyrimidin-4-yl ) -1H-pyrazol-1-yl] propanonitrile, an isomer thereof, or a pharmaceutically acceptable salt thereof. In another embodiment, a method of treating essential thrombocythemia (ET) in an individual is provided herein, comprising administering to the individual 3cyclobutyl-3- [4- (7H-pyrrole [2,3-d] pyrimidin-4- il) -1H-pyrazol-1yl] propanonitrile, an isomer thereof, or a pharmaceutically acceptable salt thereof. In another embodiment, a method of treating a solid tumor in an individual is provided herein, comprising administering to the individual 3-cyclobutyl-3- [4- (7H-pyrrole [2,3d] pyrimidin-4-yl) -1H-pyrazol-1-yl] propanonitrile, an isomer thereof, or a pharmaceutically acceptable salt thereof. In another embodiment, a method of treating psoriasis in an individual is provided herein, comprising administering to the individual 3-cyclobutyl-3- [4- (7Hpirrolo [2,3-d] pyrimidin-4-yl) -1H- pyrazol-1-yl] propanonitrile, an isomer thereof, or a pharmaceutically acceptable salt thereof.
[00063] In one embodiment, a method of treating cancer in an individual is provided, comprising administering to the individual 3- (4- (7H-pyrrolo [2,3-d] pyrimidin-4-yl) -1Hpyrazole -1-yl) -3- (3-methylcyclobutyl) propanonitrile, an isomer thereof, or a
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20/50 pharmaceutically acceptable salt thereof. In another embodiment, a method of treating myelofibrosis in an individual is provided herein, comprising administering to the individual 3- (4- (7H-pyrrolo [2,3d] pyrimidin-4-yl) -1H-pyrazole-1 -il) -3- (3-methylcyclobutyl) propanonitrile, an isomer thereof, or a pharmaceutically acceptable salt thereof. In another embodiment, a method of treating rheumatoid arthritis (RA) in an individual is provided herein, comprising administering to the individual 3- (4- (7H-pyrrolo [2,3-d] pyrimidin-4-yl) - 1H-pyrazol-1-yl) -3- (3methylcyclobutyl) propanonitrile, an isomer thereof, or a pharmaceutically acceptable salt thereof. In another embodiment, a method of treating polycythemia vera (PV) in an individual is provided herein, comprising administering to the individual 3- (4- (7Hpirrolo [2,3-d] pyrimidin-4-yl) -1H -pyrazol-1-yl) -3- (3-methylcyclobutyl) propanonitrile, an isomer thereof, or a pharmaceutically acceptable salt thereof. In another embodiment, a method of treating essential thrombocythemia (ET) in an individual is provided here, comprising administration to the individual of 3- (4- (7H-pyrrolo [2,3-d] pyrimidin-4-yl) - 1Hpirazol-1-yl) -3- (3-methylcyclobutyl) propanonitrile, an isomer thereof, or a pharmaceutically acceptable salt thereof. In another embodiment, a method of treating a solid tumor in an individual is provided herein, comprising administering to the individual 3- (4- (7Hpirrolo [2,3-d] pyrimidin-4-yl) -1H-pyrazole- 1-yl) -3- (3-methylcyclobutyl) propanonitrile, an isomer thereof, or a pharmaceutically acceptable salt thereof. In another embodiment, a method of treating psoriasis in an individual is provided herein, comprising administering to the individual 3- (4- (7H-pyrrolo [2,3-d] pyrimidin-4-yl) -1H-pyrazole -1-yl) -3- (3methylcyclobutyl) propanonitrile, an isomer thereof, or a pharmaceutically acceptable salt thereof.
[00064] As used herein, the term contact refers to the combination of components indicated in an in vitro system or an in vivo system.
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21/50
For example, contacting a JAK with a compound of the invention includes administering a compound of the present invention to an individual or patient, such as a human, having a JAK, as well as, for example, introducing a compound of the invention in a sample containing a cell or purified preparation that contains JAK.
[00065] As used herein, the term individual or patient used interchangeably refers to any animal, including mammals, preferably mice, rats, other rodents, rabbits, dogs, cats, pigs, cattle, sheep, horses , or primates, and most preferable humans.
[00066] As used herein, the phrase therapeutically effective amount refers to the amount of active compound or pharmaceutical agent that elicits the biological or medicinal response that is being sought in a tissue, system, animal, individual or human by a researcher, veterinarian, doctor or other clinician.
[00067] As used herein, the term treat or treatment refers to one or more of (1) disease prevention; for example, prevention of a disease, condition or disorder in an individual who may be predisposed to the disease, condition or disorder, but who does not yet experience or present the disease pathology or symptoms; (2) inhibition of the disease; for example, inhibition of a disease, condition or disorder in an individual who experiences or exhibits the pathology or symptoms of the disease, condition or disorder; and (3) improvement of the disease; for example, improvement of a disease, condition or disorder in an individual who experiences or exhibits the disease pathology or symptomatology, condition or disorder (i.e., reversing the pathology and / or symptomatology) such as decreasing the severity of the disease.
[00068] The term use includes any one or more of the following
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22/50 embodiments of the invention, respectively: use in the treatment of a disorder; the use for the manufacture of pharmaceutical compositions for use in the treatment of a disorder, for example, in the manufacture of a medicine; methods of using the compounds of the invention in the treatment of these diseases; pharmaceutical preparations having the compounds of the invention for the treatment of these diseases; and compounds of the invention for use in the treatment of such diseases; when appropriate and convenient, if not mentioned otherwise. In particular, the diseases to be treated which are, therefore, preferred for use of a compound of the present invention are selected from diseases associated with JAK kinase activity.
Combination Therapies [00069] One or more additional pharmaceutical agents such as, for example, chemotherapeutics, anti-inflammatory agents, steroids, immunosuppressants, as well as Bcr-Abl, Flt-3, RAF and FAK kinase inhibitors such as, for example , those described in WO 2006/056399, or other therapeutic agents can be used in combination with the compounds or salts of the present invention for the treatment of diseases, disorders or conditions associated with JAK. The one or more additional pharmaceutical agents can be administered to a patient simultaneously or sequentially.
[00070] Exemplary chemotherapy includes proteasome inhibitors (eg, bortezomib), thalidomide, revlimid, and DNA damaging agents such as melphalan, doxorubicin, cyclophosphamide, vincristine, etoposide, carmustine, and more.
[00071] Exemplary steroids include coriticosteroids such as dexamethasone or prednisone.
[00072] The exemplary Bcr-Abl inhibitors include the compounds, and pharmaceutically acceptable salts thereof, of the genera and species disclosed in U.S. Patent No. 5,521,184, WO 04/005281 and WO
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23/50
2005/123719.
[00073] Exemplary suitable Flt-3 inhibitors include compounds, and their pharmaceutically acceptable salts, as disclosed in WO 03/037347, WO 03/099771 and WO 04/046120.
[00074] Exemplary suitable RAF inhibitors include compounds, and their pharmaceutically acceptable salts, as disclosed in WO 00/09495 and WO 05/028444.
[00075] Exemplary suitable FAK inhibitors include compounds, and their pharmaceutically acceptable salts, as disclosed in WO 04/080980, WO 04/056786, WO 03/024967, WO 01/064655, WO 00/053595, and WO 01 / 014402.
[00076] In some embodiments, one or more of the compounds of the invention can be used in combination with one or more other kinase inhibitors including imatinib, particularly for the treatment of patients resistant to imatinib or other kinase inhibitors.
[00077] In some embodiments, one or more JAK inhibitors of the invention can be used in combination with a chemotherapeutic in the treatment of cancer and can potentially improve the response to treatment compared to the response to the chemotherapeutic agent alone, without exacerbating its effects toxic. Examples of additional pharmaceutical agents used in the treatment of multiple myeloma, for example, may include, without limitation, melphalan, melphalan plus prednisone [MP], doxorubicin, dexamethasone, and Velcade (bortezomib). Other additional agents used to treat multiple myeloma include Bcr-Abl, Flt-3, RAF and FAK kinase inhibitors. Additive or synergistic effects are desirable results of combining a JAK inhibitor of the present invention with an additional agent. In addition, the resistance of multiple myeloma cells to agents such as
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24/50 dexamethasone, can be reversible after treatment with a JAK inhibitor of the present invention. The agents can be combined with the present compounds in a single or continuous dosage form, or the agents can be administered simultaneously or sequentially as separate dosage forms.
[00078] In some embodiments, a corticosteroid such as dexamethasone is administered to a patient in combination with at least one JAK inhibitor where dexamethasone is administered intermittently as opposed to continuously.
[00079] In some additional embodiments, combinations of one or more JAK inhibitors of the invention with other therapeutic agents can be administered to a patient before, during and / or after a bone marrow transplant or stem cell transplant.
[00080] In some embodiments, at least one additional therapeutic agent can be used in connection with the treatment of dry eye disorders and other disorders of the eye. In some embodiments, the additional therapeutic agent is fluocinolone acetonide (Retisert®), or rimexolone (AL-2178, Vexol, Alcon). In some embodiments, the additional therapeutic agent is cyclosporine (Restasis®). In some embodiments, the additional therapeutic agent is a corticosteroid. In some modalities, the corticosteroid is triaminolone, dexamethasone, fluocinolone, cortisone, prednisolone or flumetolone.
[00081] In some embodiments, the additional therapeutic agent is selected from Dehydrex ™ (Holies Labs), Civamide (Opko), sodium hyaluonate (Vismed, Lantibio / TRB Chemedia), cyclosporine (ST-603, Sirion Therapeutics), ARG101 ( T) (testosterone, Argentis), AGR1012 (P) (Argentis), sodium ecabet (Senju-Ista), gefarnate (Santen), 15-hydroxyieicosatetraenoic acid (s) (15 (S) -HETE), cevilemine, doxycline (ALTY
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25/50
0501, Alacrity), minocycline, iDestrin ™ (NP50301, Nascent Pharmaceuticals), cyclosporine A (Nova22007, Novagali), oxytetracycline (Duramycin, MOLI1901, Lantibio), CF101 (2S, 3S, 4R, 5R) -3,4-dihydrox 5 [6 - [(3-iodophenyl) methylamino] purin-9-yl] -N-methyl-oxolane-2-carbamyl, CanFite Biopharma), voclosporin (LX212 or LX214, Lux Biosciences), ARG103 (Agentis), RX- 10045 (synthetic resolvin analog, Resolvyx), DYN15 (Dyanmis Therapeutics), rivoglitazone (DE011, Daiichi Sanko), TB4 (RegeneRx), OPH-01 (Ophtalmis Monaco), PCS101 (Pericor Science), REV1-31 (Evolutec), Lacritin (Senju), rebamipide (Otsuka-Novartis), OT-551 (Othera), PAI-2 (University of Pennsylvania and Temple University), pilocarpine, tacrolimus, pimecrolimus (AMS981, Novartis), loteprednol etabonate, rituximab, diquafosoletrassassium INS365, Inspire), KLS-0611 (Kissei Pharmaceuticals), dehydroepiandrosterone, anaquinra, efalizumab, mycophenolate sodium, etanercept (Embrel®), hydroxychloroquine, NGX267 (TorreyPines Therapeutics), or t alidomide.
[00082] In some embodiments, the additional therapeutic agent is an antiangiogenic agent, cholinergic agonist, TRP-1 receptor modulator, a calcium channel blocker, a mucin secretagogue, MUC1 stimulant, a calcineurin inhibitor, a corticosteroid, a corticosteroid, a P2Y2 receptor agonist, a muscarinic receptor agonist, another JAK inhibitor, Bcr-Abl kinase inhibitor, Flt-3 kinase inhibitor, RAF kinase inhibitor, and FAK kinase inhibitor such as, for example, those described in WO 2006/056399. In some embodiments, the additional therapeutic agent is a tetracycline derivative (for example, minocycline or doxycline).
[00083] In some embodiments, additional therapeutic agents are demulcent eye drops (also known as artificial tears), which include, but are not limited to, compositions containing polyvinyl alcohol, hydroxypropyl methylcellulose,
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26/50 glycerin, polyethylene glycol (e.g., PEG400) and or carboxymethyl cellulose. Artificial tears can help treat dry eye by compensating for the reduced capacity for moistening and lubricating the tear film. In some embodiments, the additional therapeutic agent is a mucolytic drug, such as N-acetyl-cysteine, which can interact with mucoproteins and, therefore, decrease the viscosity of the tear film. [00084] In some embodiments, the additional therapeutic agent includes an antibiotic, antiviral, antifungal, anesthetic, anti-inflammatory agents including steroidal and non-steroidal anti-inflammatory drugs, and antiallergic agents. Examples of suitable medications include aminoglycosides such as amikacin, gentamycin, tobramycin, streptomycin, netylmycin, and kanamycin; fluoroquinolones such as ciprofloxacin, norfloxacin, ofloxacin, trovafloxacin, lomefloxacin, levofloxacin, and enoxacin; naphthyridine; sulfonamides; polymyxin; chloramphenicol; neomycin; paramomycin; colistimethate; bacitracin; vancomycin; tetracyclines; rifampicin and its derivatives (rifampins); cycloserine; beta-lactams; cephalosporins; amphotericin; fluconazole; flucytosine; natamycin; miconazole; ketoconazole; corticosteroids; diclofenac; flurbiprofen; ketorolac; suprofen; comoline; lodoxamide; levocabastine; naphazoling; antazoline; feniramimane; or azalide antibiotic.
Pharmaceutical Formulations and Dosage Forms [00085] When used as pharmaceutical products, the compounds and salts of the invention can be administered in the form of pharmaceutical compositions. These compositions can be prepared in a manner well known in the pharmaceutical art, and can be administered by a variety of routes, depending on whether local or systemic treatment is desirable and after the area is treated. Administration can be topical (including transdermal, ophthalmic, epidermal and in
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27/50 mucous membranes including intranasal, vaginal and rectal release), pulmonary (for example, by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal or intranasal), oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal, intramuscular injection or infusion; intracranial, for example, intrathecal or intraventricular administration. Parenteral administration can be in the form of a single bolus dose, or it can be, for example, by a continuous infusion pump. Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powdered or oily bases, thickeners and more may be necessary or desirable. Condoms, gloves and the like may also be useful. [00086] This invention also includes pharmaceutical compositions that contain, as the active ingredient, one or more of the compounds of the above invention in combination with one or more acceptable pharmaceutical carriers (excipients). In the manufacture of the compositions of the invention, the active ingredient is typically mixed with an excipient, diluted by an excipient or included within such a vehicle in the form of, for example, a capsule, sache, paper or other container. When the excipient serves as a diluent, it can be a solid, semi-solid or liquid material, which acts as a vehicle, vehicle or medium for the active ingredient. Thus, the compositions can be in the form of tablets, pills, powders, lozenges, sacks, seals, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium), ointments containing, for example, up to 10% by weight of the active compound, soft and solid gelatin capsules, suppositories, sterile injectable solutions, and powder packaged sterile.
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28/50 [00087] In preparing a formulation, the active compound can be ground to provide the appropriate particle size before combining with the other ingredients. If the active compound is substantially insoluble, it can be ground to a particle size smaller than the 200 mesh. If the active compound is substantially soluble in water, the particle size can be adjusted by grinding to provide a substantially uniform distribution. in the formulation, for example, around 40 mesh.
[00088] The compounds of the invention can be ground using known milling procedures such as wet milling to obtain a particle size suitable for tabletting and other types of formulation. The finely divided preparations (nanoparticles) of the compounds of the invention can be prepared by processes known in the art, for example, see International Patent Application No. WO 2002/000196.
[00089] Some examples of suitable excipients include lactose, dextrose, sucrose, sorbitol, mannitol, starches, acacia gum, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, and methyl cellulose. The formulations can additionally include: lubricating agents such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifiers and suspending agents; preservative agents such as methyl- and propylhydroxy benzoates; sweetening agents; and flavoring agents. The compositions of the invention can be formulated so as to provide a rapid, sustained or delayed release of the active ingredient after administration to the patient through the use of procedures known in the art. [00090] The compositions can be formulated in a unit dosage form, each dosage containing from about 5 to about
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29/50
1000 mg (1 g), more usually about 100 to about 500 mg, of the active ingredient. The term unit dosage forms refers to physically discrete units suitable as unit dosages for humans and other mammals, each unit containing a predetermined amount of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient.
[00091] The active compound can be effective over a wide dosage range and is generally administered in a pharmaceutically effective amount. It will be understood, however, that the amount of the compound actually administered will generally be determined by a physician, according to the relevant circumstances, including the condition to be treated, the chosen route of administration, the actual compound administered, the age, weight and individual patient's response, the severity of the patient's symptoms, and more.
[00092] For the preparation of solid compositions such as tablets, the main active ingredient is mixed with a pharmaceutical excipient to form a solid preformulation composition containing a homogeneous mixture of a compound of the present invention. When referring to these preformulation compositions as homogeneous, the active ingredient is typically dispersed evenly throughout the composition so that the composition can be easily subdivided into equally effective unit dosage forms such as tablets, pills and capsules. This solid preformulation is then subdivided into unit dosage forms of the type described above containing, for example, from about 0.1 to about 1000 mg of the active ingredient of the present invention.
[00093] The tablets or pills of the present invention can
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30/50 be coated or otherwise combined to provide a dosage form that provides the advantage of prolonged action. For example, the tablet or pill may comprise an internal dosage component and an external dosage component, the latter being in the form of an envelope over the former. The two components can be separated by an enteric layer that serves to resist disintegration in the stomach and allow the internal component to pass intact into the duodenum or to be delayed in release. A variety of materials can be used for such enteric layers or coatings, such materials including various polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol and cellulose acetate.
[00094] Liquid forms in which the compounds and compositions of the present invention can be incorporated for oral or injection administration include aqueous solutions, suitably flavored syrups, aqueous or oily suspensions and emulsions flavored with edible oils such as seed oil cotton, sesame oil, coconut oil or peanut oil, as well as elixirs and similar pharmaceutical vehicles.
[00095] Compositions for inhalation or insufflation include solutions and suspensions in pharmaceutically acceptable aqueous or organic solvents, or mixtures thereof, and powders. Liquid or solid compositions can contain suitable pharmaceutically acceptable excipients as described above. In some embodiments, the compositions are administered via the oral or nasal airway for local or systemic effect. The compositions can be nebulized using inert gases. The nebulized solutions can be breathed directly from the nebulizer device or the nebulizer device can be connected to a face hood tent, or positive pressure breathing machine
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Intermittent 31/50. Solution, suspension or powder compositions can be administered orally or nasally from devices that deliver the formulation in an appropriate manner.
[00096] The amount of the compound or composition administered to a patient will vary depending on what is being administered, the purpose of the administration, such as prophylaxis or therapy, the condition of the patient, the manner of administration, and more. In therapeutic applications, the compositions can be administered to a patient who is already suffering from a disease in an amount sufficient to cure or at least partially interrupt the symptoms of the disease and its complications. Effective doses will depend on the sick condition being treated, as well as on the judgment of the attending physician depending on factors such as the severity of the disease, the age, weight and general condition of the patient, and more.
[00097] The compositions administered to a patient can be in the form of pharmaceutical compositions described above. These compositions can be sterilized by conventional sterilization techniques, or they can be sterilized by filtration. The aqueous solutions can be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile aqueous vehicle prior to administration. The pH of the compound preparations will typically be between 3 and 11, more preferably 5 to 9 and most preferably 7 to 8. It will be understood that the use of some of the above excipients, vehicles, or stabilizers will result in the formation of pharmaceutical salts.
[00098] The therapeutic dosage of the compounds of the present invention may vary according to, for example, the particular use for which the treatment is performed, the mode of administration of the compound, the health and condition of the patient, and the judgment of the patient. prescribing physician. The proportion or concentration of a compound of the invention in an
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32/50 pharmaceutical composition may vary depending on several factors including dosage, chemical characteristics (for example, hydrophobicity), and the route of administration. For example, the compounds of the invention can be supplied in an aqueous solution of physiological buffer containing about 0.1 to about 10% w / v of the compound for parenteral administration. Some typical dose ranges are from about 1 pg / kg to about 1 g / kg of body weight per day. In some embodiments, the dose range is about 0.01 mg / kg to about 100 mg / kg of body weight per day. The dosage probably depends on such variables as the type and extent of the progression of the disease or disorder, the general health status of the particular patient, the relative biological efficacy of the selected compound, formulation of the excipient, and its route of administration. Effective doses can be extrapolated from dose-response curves derived from in vitro test systems or in an animal model.
[00099] In some embodiments, the compound of the invention, or its pharmaceutically acceptable salt, is administered as an ophthalmic composition. Consequently, in some embodiments, the methods comprise administering the compound, or its pharmaceutically acceptable salt, and an ophthalmically acceptable carrier. In some embodiments, the ophthalmic composition is a liquid composition, semi-solid composition, insertion, film, microparticles or nanoparticles. Ophthalmic compositions are described in detail in U.S. Ser. No. 12 / 571,834, filed October 1, 2009, which is incorporated herein by reference.
[000100] It is further noted that certain aspects of the invention, which are, for reasons of clarity, described in the context of the specific modalities, can also be provided in combination with a single modality. Conversely, various aspects of the invention that are, for brevity's sake, described in the context of a modality
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33/50 only, can also be supplied separately or in any suitable sub-combination.
[000101] Several modifications of the invention, in addition to those described here, will be evident to those skilled in the art from the description. Each reference cited in this application is hereby incorporated by reference in its entirety.
[000102] The invention will be described in more detail by means of specific examples. The following examples are offered for illustrative purposes, and are not intended to limit the invention in any way. Those of skill in the art will easily recognize a variety of non-critical parameters that can be altered or modified to produce essentially the same results.
EXAMPLES
Example 1 (R or S) -3-Cyclobutyl-3- [4- (7H-pyrrolo [2,3-d] pyrimidin-4-yl) -1H-pyrazol-1yl] propanonitrile
Step 1. Cyclobutanecarboxaldehyde [000103] A solution of dimethyl sulfoxide (34.6 ml, 0.488 mol) in methylene chloride (100 ml) was added to oxalyl chloride (20.6 ml, 0.244 mol) in methylene chloride ( 700 ml, 10 mol) at -78 ° C. After 10 min, cyclobutylmethanol (Aldrich, 17.5 g, 0.203 mol) in methylene chloride (100 ml) was added and the resulting mixture was stirred at -78 ° C for 30 min. A solution of triethylamine (140 ml, 1.0 mol) in methylene chloride (100 ml) was then added and the mixture was stirred
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34/50 for 5 h with the temperature gradually allowed to warm to room temperature (rt). After quenching with water, the mixture was separated. The organic layer was washed with water (x2), brine, dried over sodium sulfate, and filtered. The filtrate was distilled, collecting the fraction at 86 to 92 ° C to provide the aldehyde (18.6 g, 54.4%).
Step 2. 3-Cyclobutylacrylonitrile [000104] To a 1.00 M solution of potassium tert-butoxide in tetrahydrofuran (116 ml, 0.116 mol) at 0 ° C was added a drip solution of diethyl cyanomethylphosphonate (Aldrich , 19.7 ml, 0.122 mol) in tetrahydrofuran (200 ml). The reaction was heated to the cup and then cooled to 0 ° C again. The reaction mixture was a solution of cyclobutanecarboxaldehyde (see Step 1, 18.6 g, 0.110 mol) in tetrahydrofuran (100 ml). The reaction was allowed to warm to room temperature (rt) and stirred at rt overnight. After quenching with water, the mixture was extracted with ether. The combined organic layers were washed with water, brine, dried and evaporated to dryness. The crude mixture was purified on silica gel, eluting with 0 to 40% EtOAc in hexane, to provide the desired product (5.30 g, 44.7%). LCMS calculated for C HwN (M + H) +: m / z = 108.1; Observed: 108.1.
Step 3. (R) -3-Cyclobutyl-3- [4- (7 - {[2- (trimethylsilyl) ethoxy] methyl} -7Hpyrrolo [2,3-d] pyrimidin-4-yl) -1H-pyrazole- 1-yl] propanonitrile and (S) -3Cyclobutyl-3- [4- (7 - {[2- (trimethylsilyl) ethoxy] -methyl} -7H-pyrrolo [2,3-d] pyrimidin-
4-yl) -1H-pyrazol-1-yl] propanonitrile [000105] To a solution of 4- (1H-pyrazol-4-yl) -7 - {[2- (trimethylsilyl) ethoxy] methyl} -7H-pyrrole [2,3-d] pyrimidine (see US Pub. No. US
2007/0135461, 15.6 g, 0.050 mol) in acetonitrile (124 ml, 2.37 mol) 3-cyclobutylacrylonitrile (5.30 g, 0.050 mol) was added, followed by 1.8 diazabicyclo [5.4.0] undec-7-ene (3.70 ml, 0.025 mol). The resulting mixture was stirred at rt overnight, then evaporated to
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35/50 dryness. The mixture was purified on silica gel, eluting with 0 to 60% EtOAc in hexane, to provide the desired product as a racemic mixture (16 g, 76%). LCMS calculated for C22H31N6OSKM + H) +: m / z = 423.2; Observed: 423.0. The racemic mixture was separated with chiral HPLC (Column: ChiralCel OJ-H, 30 x 250 mm, 5 pm; Mobile Phase: 30% Ethanol / 70% Hexanes; Flow Rate: 24 ml / min) to provide two enantiomers. On chiral analytical HPLC (Column: ChiralCel OJ-H, 4.6 x 250 mm, 5 pm; Mobile Phase: 30% ethanol / 70% hexanes; Flow Rate: 0.8 ml / min): First retention time maximum: 6.6 min; Second maximum retention time: 8.1 min.
Step 4. (R or S) -3-Cyclobutyl-3- [4- (7H-pyrrolo [2,3-d] pyrimidin-4-yl) -1Hpyrazol-1-yl] propanonitrile [000106] In a bottle of 500 ml round bottom adjusted with a stir bar, condenser, and nitrogen inlet acetonitrile (55 ml), water (4.8 ml) and (R or S) -3-cyclobutyl-3- [4 (7 - {[2- (trimethylsilyl) ethoxy] methyl} -7H-pyrrolo [2,3-d] pyrimidin-4-yl) -1H-pyrazol1-yl] propanonitrile (second chiral separation peak in Step 3, 2, 8 g, 6.6 mmol). Lithium tetrafluoroborate (7.50 g, 0.078 mol) was added. The resulting mixture was heated to reflux overnight, cooled to room temperature and charged with 3.00 M ammonium hydroxide in water (9.78 ml) in portions over a period of 5 minutes, adjusting the pH to 9 to 10 After 30 min, the resulting mixture was purified by RP-HPLC (XBridge C18 column 30 x 100 mm, with injection volume 5 ml (~ 50 mg / injection)), eluting with an acetonitrile / water gradient containing 0.15 % NH4OH, at a flow rate of 60 ml / min) to provide the desired product as a free base (1.51 g, 77.96%). LCMS calculated for CwH1 - N6 (M + H) +: m / z = 293.2; Observed: 293.1. ¹ H NMR (300 MHz, CD3OD) δ 8.65 (1H, s), 8.59 (1H, s), 8.34 (1H, s), 7.50 (1H, d, J = 3.6 Hz), 6.94 (1H, d, J = 3.6 Hz), 4.69 (1H, m), 3.07 ~ 2.97 (3H, m), 2.21 (1H, m), 1.97 ~ 1.84 (5H,
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36/50
m) ppm. ee 98.8%.
[000107] The other enantiomer can be prepared in the same way starting with the compound corresponding to the first peak obtained from the chiral separation in Step 3.
Example 2
(3R or 3S) -3- (4- (7H-Pyrrolo [2,3-d] pyrimidin-4-yl) -1H-pyrazol1 -yl) -3 - ((trans) -3-methylcyclobutyl) salt phosphoric propanonitrile
Step 1. 3-methylenocyclobutanecarboxylic acid [000108] In a round-bottomed flask equipped with a condenser, 3-methylenocyclobutanocarbonitrile (BePharma, 10.0 g, 0.107 mol) was added. To the flask, a solution of potassium hydroxide (24.1 g, 0.365 mol) in ethanol (112 ml) and water (88 ml) was added and the mixture was heated to 100 ° C. After about 2 hours, the evolution of ammonia ceased and the solvent was evaporated to dryness under reduced pressure. The solids were dissolved in water (75 ml), cooled in an ice bath, and acidified to a pH of about 1 with concentrated hydrochloric acid. The resulting upper layer was extracted with dichloromethane twice. The organic layers were combined and dried over anhydrous magnesium sulfate. Removal of organic solvents gave the desired crude product (11.8 g, 97.67%).
Step 2. N-methoxy-N-methyl-3-methylenocyclobutanecarboxamide [000109] To a mixture of 3-methylenocyclobutanecarboxylic acid (Step 1, 5.88 g, 52.4 mmol) in methylene chloride (100 ml) was
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37/50 oxalyl chloride (Aldrich, 5.33 ml, 62.9 mmol) is added, followed by a catalytic amount of dimethyl formamide (DMF). The reaction was stirred at rt for 2 h, then evaporated to dryness. The crude acid chloride was dissolved in methylene chloride (200 ml). To the resulting solution, N, O-dimethylhydroxylamine hydrochloride ((Aldrich, 6.14 g, 62.9 mmol) was added, followed by triethylamine (TEA) (21.9 ml, 0.157 mol), by dripping, at 0 ° C The reaction was stirred at rt overnight, and the TEA HCl salt was filtered The filtrate was washed with 1 N HCl, then aq. Sodium bicarbonate, brine, and dried over magnesium sulfate and evaporated to dryness. crude amide (7.30 g, 89.7%) was used directly in the next step LCMS calculated for CsHi4NO2 (M + H) +: m / z = 156.1; observed: 156.3.
Step 3. 3-Methylenocyclobutanecarbaldehyde [000110] To a suspension of lithium tetrahydroaluminate (2.18 g,
57.5 mmols) in ether (200 ml) a solution of N-methoxy-N-methyl-3-methylenocyclobutanecarboxamide (Step 2, 7.14 g, 46.0 mmol) in tetrahydrofuran (75 ml) was added dropwise. ) at -15 ° C. The reaction was stirred at 0 to -15 ° C for 30 min, then quenched with aq. The resulting mixture was extracted with ether. The combined organic layers were washed with brine, dried over magnesium sulfate, and evaporated. The crude product (6.70 g, 151.5%) was used directly in the next step.
Step 4. 3- (3-Methylenocyclobutyl) acrylonitrile [000111] To a 1.00 M solution of potassium tert-butoxide in tetrahydrofuran (48.3 ml, 48.3 mmol) at 0 ° C was added by drip a solution of diethyl cyanomethylphosphonate (Aldrich, 8.19 ml, 50.6 mmol) in tetrahydrofuran (80 ml). The reaction was heated to the cup and then cooled to 0 ° C. To the reaction mixture, a solution of 3-methylenocyclobutanecarbaldehyde (Step 4, 4.42 g, 46.0 mmol) in tetrahydrofuran (40 ml) was added. The reaction was allowed to warm up
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38/50 into the cup and then was stirred in the cup overnight. After quenching with water, the mixture was extracted with ether. The combined organic layers were washed with water, brine, dried and evaporated to dryness. The crude mixture (5.90 g, 107.7%) was used directly in the next step.
Step 5. 3- (3-Methylenocyclobutyl) -3- [4- (7 - {[2- (trimethylsilyl) ethoxy] methyl} -7Hpyrrolo [2,3-d] pyrimidin-4-yl) -1H-pyrazole- 1-yl] propanonitrile [000112] To a solution of 4- (1H-pyrazol-4-yl) -7 - {[2- (trimethylsilyl) ethoxy] methyl} -7H-pyrrolo [2,3-d] pyrimidine ( see the US Pub. No. US
2007/0135461, 7.25 g, 23.0 mmols) in acetonitrile (57.4 ml) was added 3- (3-methylenocyclobutyl) crude acrylonitrile (Step 4, 2.74 g, 23.0 mmols), followed by 1,8-diazabicyclo [5.4.0] undec-7-ene (3.44 ml, 23.0 mmol). The resulting mixture was stirred at rt over the weekend, then evaporated to dryness. The residue was purified on silica gel, eluting with 0 to 80% EtOAc in hexane, to provide the desired product (6.0 g, 60.1%). LCMS calculated for C23HsiN6OSi (M + H) +: m / z = 435.2; Observed: 435.4.
Step 6. (3R or 3S) -3 - ((trans) -3-Methylcyclobutyl) -3- (4- (7 - ((2- (trimethylsilyl) ethoxy) methyl) -7H-pyrrole [2,3-d ] pyrimidin-4-yl) -1H-pyrazol-1-yl) propanonitrile [000113] A mixture of 3- (3-methylenocyclobutyl) -3- [4- (7 - {[2 (trimethylsilyl) ethoxy] methyl} - 7H-pyrrolo [2,3-d] pyrimidin-4-yl) -1H-pyrazol-1-yl] propanonitrile (Step 5, 4.0 g, 9.2 mol) in 100 ml of methanol was hydrogenated in the presence of 0.6 g of 10% Pd / C, under hydrogen balloon pressure, for 1 h. After filtering the catalyst, the filtrate was evaporated to dryness and purified on silica gel, eluting with 0 to 100% EtOAc in hexane, to provide the desired product as a mixture of trans- and cis-isomers. LCMS calculated for C23H33N6OSKM + H) +: m / z = 437.3; Observed: 437.4. The product was subjected to chiral HPLC column purification twice. The first HPLC separation (Column: ChiralCel OD-H, 30 x 250 mm,
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39/50 pm; Mobile Phase: 15% ethanol / 85% hexanes; Flow Rate: 28 ml / min) provided two fractions, A and B. Fraction A was a cis / trans mixture of an enantiomer. Retention Time: 10.51 min. Fraction B was a cis / trans mixture of the other enantiomer, which showed two inseparable peaks with retention times 13.05 min and 13.92 min. The first fraction (A) was subjected to another chiral HPLC separation (Column: ChiralPak IA, 20 x 250 mm, 5 pm; Mobile Phase: 10% ethanol / 90% hexanes; Flow Rate: 15 ml / min) to provide two peaks, A1 and A2, one peak corresponding to cis and the other to trans. According to chiral analytical HPLC (Column: ChiralPak IA, 4.6 x 250 mm, 5 pm; Mobile Phase: 15% ethanol / 85% hexanes; Flow Rate: 1.0 ml / min): retention time from the first peak (A1): 11.79 min; second peak retention time (A2): 12.78 min. The second fraction (B) was subjected to chiral HPLC separation (Column: ChiralPak IA, 20 x 250 mm, 5 pm; Mobile Phase: 15% ethanol / 85% hexanes; Flow Rate: 15 ml / min) to provide two peaks, B1 and B2 (each peak 800 mg, 19.9%). B1 was later presented by nOe to be the cis-isomer and B2 was presented to be the trans-isomer of the other enantiomer. According to chiral analytical HPLC (Column: ChiralPak IA, 4.6 x 250 mm, 5 pm; Mobile Phase: 15% ethanol / 85% hexanes; Flow Rate: 1.0 ml / min): retention time first peak (B1): 12.48 min and retention time of the second peak (B2): 14.16 min.
Step 7. (3R or 3S) -3- (4- (7H-Pyrrolo [2,3-d] pyrimidin-4-yl) -1H-pyrazol-1-yl) 3 - ((trans) -3-methylcyclobutyl ) propanonitrile [000114] In a 500 ml round-bottom flask fitted with stir bar, condenser and nitrogen inlet, acetonitrile (9.69 ml), water (0.84 ml) and 3 - ((trans) - 3-methylcyclobutyl) -3- [4 (7 - {[2- (trimethylsilyl) ethoxy] methyl} -7H-pyrrolo [2,3-d] pyrimidin-4-yl) -1H-pyrazol1-yl] propanonitrile (0 , 60 g, 1.4 mol) (B2 from the chiral separation in the previous step (i.e., peak 2 of the second fraction)). Tetrafluoroborate
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40/50 lithium (1.31 g, 13.7 mmols) was added. The mixture was heated to reflux overnight, then charged with 7.2 M ammonium hydroxide in water (0.71 ml, 5.1 mmol) in portions over a period of 5 minutes at room temperature, adjusting the pH to 9 to 10. The reaction was stirred for 2 h at room temperature. The solid was removed by filtration and the filtrate was purified on RP-HPLC ((XBridge C18 column 30 x 100 mm, with 5 ml injection volume (~ 50 mg / injection), elution with an acetonitrile / water gradient containing 0, 15% NH4OH, flow rate 60 mL / min) to yield the desired product as the free base Ci7HioN6 LCMS calcd for (m + H) +: m / z = 307.2; Observed. 307.4 ¹ H NMR. (500 MHz, DMSO-d6) δ 12.08 (1H, s), 8.78 (1H, s), 8.68 (1H, s), 8.36 (1H, s), 7.59 (1H , d, J = 3.0 Hz), 6.99 (1H, d, J = 3.0 Hz), 4.78 (1H, m), 3.12 (2H, m), 2.88 (1H , m), 2.30 (1H, m), 2.06 (1H, m), 1.88 (1H, m), 1.74 (1H, m), 1.44 (1H, m), 1 .08 (3H, d, J = 7.0 Hz) ppm. Ee 93.3%.
[000115] The other enantiomer can be prepared by the same method starting with the compound corresponding to fraction A of Step
6.
Step 8. acid salt (3R or 3S) -3- (4- (7H-pyrrolo [2,3-d] pyrimidin-4-yl) 1H-pyrazol-1-yl) -3 - ((trans) - Phosphoric 3-methylcyclobutyl) propanonitrile [000116] To a solution of (3R or 3S) -3 - ((trans) -3-methylcyclobutyl) -3 [4- (7H-pyrrole [2,3-d] pyrimidin-4- il) -1H-pyrazol-1-yl] propanonitrile (Step 7, 0.275 g, 0.898 mmol) in isopropyl alcohol (5.83 ml) was added phosphoric acid (96.8 mg, 0.987 mmol) in 1.0 ml of isopropanol at 60 ° C. After stirring for 1 h, the mixture was allowed to cool to the bowl. The precipitate was filtered off and air dried, then rinsed with ethyl ether and air dried further to provide the desired phosphate product (330 mg, 90.9%).
Example 3
Acid salt (3R or 3S) -3- (4- (7H-pyrrolo [2,3-d] pyrimidin-4-yl) -1H-pyrazolePetition 870190113460, from 11/6/2019, page 46/63
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-il) -3 - ((cis) -3-methylcyclobutyl) -phosphoric propanonitrile I
I
ΗΟ'Ϊ ΟΗ N
Step 1. (3R or 3S) -3 - ((cis) -3-methylcyclobutyl) -3- (4- (7 - ((2 (trimethylsilyl) ethoxy) methyl) -7H-pyrrole [2,3-d] pyrimidin-4-yl) -1H-pyrazol-1il) propanonitrile [000117] In a 500 ml round-bottom flask fitted with a stir bar, condenser, and nitrogen inlet acetonitrile (8.1 ml), water was charged (0.70 ml) and (3R or 3S) -3 - ((cis) -3methylcyclobutyl) -3- [4- (7 - {[2- (trimethylsilyl) ethoxy] methyl} -7H-pyrrole [2,3d ] pyrimidin-4-yl) -1H-pyrazol-1-yl] propanonitrile (0.50 g, 1.1 mmol) (B1 from the chiral separation described in Example 2, Step 6 (ie peak 1 of second fraction)). Lithium tetrafluoroborate (1.10 g, 11.4 mmol) was added. The solution was heated to reflux overnight. Then a solution of ammonium hydroxide in water (7.2 M, 0.59 ml, 4.3 mmol) was loaded into the solution in portions over a period of 5 minutes at room temperature, adjusting the pH to 9 to 10. The reaction it was stirred for 2 h at room temperature. The solid was removed by filtration and the filtrate was purified on RP-HPLC (XBridge C18 column 30 x 100 mm, with injection volume 5 ml (~ 50 mg / injection), elution with an acetonitrile / water gradient containing 0.15 % NH4OH, at a flow rate of 60 ml / min) to provide the desired product. LCMS calculated for C17Hi9N6 (M + H) +: m / z = 307.2; Observed: 307.4. ¹ H NMR (500 MHz, DMSO-d6) δ 12.08 (1H, s), 8.75 (1H, s), 8.68 (1H, s), 8.36 (1H, s), 7, 59 (1H, d, J = 3.0 Hz), 6.99 (1H, d, J = 3.0 Hz), 4.66 (1H, m), 3.11 (2H, m), 2, 66 (1H, m), 2.20 (2H, m), 1.88 (1H, m), 1.42 (2H, m), 0.97 (3H, d, J =
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6.0 Hz) ppm. ee 99.8%.
[000118] The other enantiomer can be prepared by the same method starting with the corresponding compound for fraction A of Example 2, Step 6.
Step 2. Acid (3R or 3S) -3- (4- (7H-pyrrolo [2,3-d] pyrimidin-4-yl) 1H-pyrazol-1-yl) -3 - ((cis) - Phosphoric 3-methylcyclobutyl) propanonitrile [000119] To a solution of (3R or 3S) -3 - ((cis) -3-methylcyclobutyl) -3- [4 (7H-pyrrole [2,3-d] pyrimidin-4- il) -1H-pyrazol-1-yl] propanonitrile (Step 1, 0.23 g, 0.751 mmol) in isopropyl alcohol (4.87 ml) was added phosphoric acid (80.9 mg, 0.83 mmol) in 1 , 0 ml of isopropanol at 60 ° C. The mixture was stirred for 2 h, then allowed to cool to the bowl. The precipitate was filtered off and air dried, then rinsed with ethyl ether and air dried further to provide the desired phosphate product (300 mg, 98.8%). ¹ H NMR (400 MHz, DMSO-d6) δ 12.08 (1H, s), 8.75 (1H, s), 8.65 (1H, s), 8.34 (1H, s), 7, 58 (1H, d, J = 2.4 Hz), 6.97 (1H, d, J = 2.4 Hz), 4.63 (1H, m), 3.09 (2H, m), 2, 64 (1H, m), 2.18 (2H, m), 1.86 (1H, m), 1.40 (2H, m), 0.96 (3H, d, J = 6.4 Hz) ppm .
Example A: In vitro JAK Kinase assay [000120] The compounds here were tested for the inhibitory activity of JAK targets according to the following in vitro assay described in Park et al., Analytical Biochemistry 1999, 269, 94-104 . The catalytic domains of human JAK1 (a.a. 837-1142), JAK2 (a.a. 8281132) and JAK3 (a.a. 781-1124) with an N-terminal His label were expressed using baculovirus in insect cells and purified. The catalytic activity of JAK1, JAK2 or JAK3 was assessed by measuring the phosphorylation of a biotinylated peptide. The phosphorylated peptide was detected by the decided time homogeneous fluorescence (HTRF). The IC50s of the compounds were measured for each kinase in the reactions that contain the enzyme, ATP and 500 nM peptide in
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43/50 mM Tris buffer (pH 7.8) with 100 mM NaCI, 5 mM DTT, and 0.1 mg / ml (0.01%) BSA. The concentration of ATP in the reactions was 90 μΜ for JAK1,30 μΜ for JAK2 and 3 μΜ for JAK3. The reactions were carried out at room temperature for 1 h and then stopped with 20 μΙ 45 mM EDTA, 300 nM SA-APC, 6 nM Eu-Py20 in assay buffer (Perkin Elmer, Boston, MA). Binding to the europium-labeled antibody would occur for 40 minutes and the HTRF signal was measured on a Fusion plate reader (Perkin Elmer, Boston, MA). The compounds of Examples 1,2 and 3 were found to have ICso values less than 2 nM for JAK1 and less than 1 nM for JAK2. Example B: Cellular Assays [000121] One or more compounds here have been tested for inhibitory activity on JAK targets according to at least one of the following cell assays.
[000122] Cancer cell lines dependent on cytokines and, therefore, on JAK / STAT signal transduction, for growth, were plated at 6000 cells per well (96 well plate format) in RPMI 1640, FBS at 10 %, and 1 ng / ml of appropriate cytokine. The compounds were added to the cells in DMSO / medium (final concentration 0.2% DMSO) and incubated for 72 hours at 37 ° C, 5% CO2. The effect of the compound on cell viability was assessed using the CellTiter-Glo Luminescent Cell Viability Assay (Promega) followed by TopCount quantification (Perkin Elmer, Boston, MA). The potential off-target effects of the compounds were measured in parallel using a non-JAK-oriented cell line with the same assay reading. All experiments were performed in duplicate.
[000123] The cell lines above can also be used to examine the effects of the compounds on the phosphorylation of JAK kinase or potential downstream substrates such as STAT proteins,
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Akt, Shp2, or Erk. These experiments can be performed following an overnight cytokine starvation, followed by a brief preincubation with the compound (2 hours or less) and cytokine stimulation of approximately 1 hour or less. The proteins are then extracted from cells and analyzed by techniques familiar to those skilled in the art including Western blotting or ELISA that uses antibodies that can differentiate between phosphorylated and total protein. These experiments can use normal or cancer cells to investigate the activity of the compounds on the tumor cell's survival biology or on mediators of inflammatory disease. For example, in relation to the latter, cytokines such as IL-6, IL-12, IL-23, or IFN can be used to stimulate JAK activation resulting in phosphorylation of STAT proteins and potentially in transcription profiles (assessed by qPCR formation or technology) or protein production and / or secretion, such as IL-17. The ability of the compounds to inhibit these cytokine-mediated effects can be measured using techniques common to those skilled in the art.
[000124] The compounds of this invention can also be tested in cell models designed to assess their potency and activity against the mutant JAKs, for example, the JAK2V617F mutation seen in myeloid proliferative disorders. These experiments often use cytokine-dependent cells of the haematological lineage (for example, BaF / 3) in which wild type or mutant JAK kinases are expressed ectopically (James, C., et al. Nature 434: 1144-1148; Staerk, J., et al. JBC 280: 4189341899). The parameters include the effects of the compounds on cell survival, proliferation and phosphorylated JAK, STAT, Akt or Erk proteins.
[000125] Certain compounds here have been or can be evaluated
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45/50 regarding its T cell proliferation inhibiting activity. Like the assay, it can be considered a second proliferation assay conducted by the cytokine (ie, JAK) and also a simplistic assay of immune suppression or inhibition of immune activation. . What follows is a brief summary of how such experiments can be performed. Peripheral blood mononuclear cells (PBMCs) are prepared from human whole blood samples using the Ficoll Hypaque separation method and T cells (fraction 2000) can be obtained from PBMCs by elutriation. Freshly isolated human T cells can be maintained in culture medium (RPMI 1640 supplemented with 10% fetal bovine serum, 100 U / ml penicillin, 100 pg / ml streptomycin) at a density of 2 x 10 ⁶ cells / ml at 37 ° C for up to 2 days. For the analysis of IL-2-stimulated cell proliferation, T cells are first treated with Phytohemagglutinin (PHA) at a final concentration of 10 pg / ml for 72 h. After washing once with PBS, 6000 cells / well are plated onto 96 well plates and treated with compounds in different concentrations in the culture medium in the presence of 100 U / ml of human IL-2 (ProSpec-Tany TechnoGene; Rehovot, Israel). The plates are incubated at 37 ° C for 72 h and the proliferation index is evaluated using CelITiterGlo Luminescent reagents following the protocol suggested by the factory (Promega, Madison, Wl).
Example C: Anti-tumor efficacy in vivo [000126] The compounds of this invention can be evaluated in human models of tumor xenograft in immunocompromised mice. For example, a tumorigenic variant of the plasmacytoma cell line INA-6 can be used to inoculate SCID mice subcutaneously (Burger, R., et al. Hematol J. 2: 42-53, 2001). Tumor carrier animals can then be
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46/50 chosen at random from drug or vehicle treatment groups and different doses of compounds can be administered by any number of usual routes including oral, i.p., or continuous infusion using implantable pumps. The growth of the tumor is followed over time using calipers. In addition, tumor samples can be taken at any time after starting treatment for analysis as described above (Example B) to assess the effects of compound on JAK activity and downstream signaling pathways. In addition, the selectivity of the compounds can be assessed using xenograft tumor models that are driven by other known kinases (e.g., Bcr-Abl) such as the K562 tumor model.
Example D: Murine Skin Contact Delayed Hypersensitivity Response Test [000127] The compounds of this invention can also be tested for their efficacy (of inhibiting JAK targets) in the conducted murine delayed hypersensitivity test model by the T cell. The delayed murine skin contact hypersensitivity response (DTH) is considered to be a valid model of clinical contact dermatitis, and other immune disorders mediated by skin T lymphocytes, such as psoriasis (Immunol Today. 1998 Jan; 19 (1): 37-44). Multiple characteristics of murine DTH fractions with psoriasis, including the infiltrated immune system, the accompanying increase in inflammatory cytokines, and keratinocyte hyperproliferation. In addition, many classes of agents that are effective in treating psoriasis in the clinic are also effective inhibitors of the DTH response in mice (Agents Actions. 1993 Jan; 38 (12): 116-21).
[000128] On Day 0 and 1, Balb / c mice are sensitized with a topical application, on their abdomen scraped with the antigen
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2.4, dinitro-fluorobenzene (DNFB). On day 5, the ears are measured for thickness using an engineering micrometer. This measurement is recorded and used as a baseline. Both ears of the animals are then caused by a topical application of DNFB in a total of 20 μΙ_ (10 μΙ_ on the inner auricle and 10 μΙ_ on the outer auricle) at a concentration of 0.2%. Twenty-four to seventy-two hours after the challenge, the ears are measured again. Treatment with the test compounds was provided through sensitization and provocation phases (day 1 to day 7) or before and during the provocation phase (usually in the afternoon from day 4 to day 7). The treatment of the test compounds (in different concentration) was administered systemically or topically (topical application of the treatment to the ears). The effectiveness of the test compounds is indicated by a reduction in ear swelling compared to the situation without treatment. Compounds that cause a reduction of 20% or more have been found to be effective. In some experiments, mice are provoked, but not sensitized (negative control).
[000129] The inhibitory effect (activation of inhibition of the JAK-STAT pathways) of the test compounds can be confirmed by immunohistochemical analysis. The activation of the JAK-STAT pathways results in the formation and translocation of functional transcription factors. In addition, the influx of immune cells and increased proliferation of keratinocytes should also provide isolated changes in the expression profile in the ear, which can be investigated and quantified. The sections of the ear fixed with formalin and embedded with paraffin (collected after the provocation phase in the DTH model) are subjected to immunohistochemical analysis using an antibody that specifically interacts with phosphorylated STAT3 (clone 58E12, Cell Signaling Technologies). The mouse ears are treated with the test compounds,
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48/50 vehicle, or dexamethasone (a clinically effective treatment for psoriasis), or without any treatment, in the DTH model for comparisons. Test compounds and dexamethasone can produce similar transcriptional changes both qualitatively and quantitatively, and both test compounds and dexamethasone can reduce the number of infiltrating cells. Both systemic and topical administration of the test compounds can produce inhibitory effects, that is, a reduction in the number of infiltrating cells and inhibition of transcriptional changes.
Example E: Anti-inflammatory activity in vivo [000130] The compounds of this invention can be evaluated in rodent or non-rodent models designed to replicate an isolated or complex inflammation response. For example, rodent models of arthritis can be used to assess the therapeutic potential of the dosed compounds preventively or therapeutically. These models include, but are not limited to, collagen-induced arthritis in mice or rats, adjuvant-induced arthritis in mice, and antibody-induced arthritis with collagen. Autoimmune diseases including, but not limited to, multiple sclerosis, type I diabetes mellitus, uveoretinitis, thyroiditis, myasthenia gravis, immunoglobulin nephropathies, myocarditis, airway sensitization (asthma), lupus, or colitis, can also be used for evaluate the therapeutic potential of the compounds described herein. These models are well established in the research community and are familiar with those instructed in the technique (Current Protocols in Immunology, Vol 3., Coligan, JE et al, Wiley Press .; Methods in Molecular Biology: Vol. 225, Inflammation Protocols., Winyard, PG and Willoughby, DA, Humana Press, 2003.).
Example F: Animal Models for the Treatment of Dry Eye, Uveitis and Conjunctivitis
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49/50 [000131] The compounds can be evaluated in one or more preclinical dry eye models known to those instructed in the art including, but not limited to, the rabbit concanavalin A (ConA) lacrimal gland model, the model of mouse scopolamine (subcutaneous or transdermal), the Botulinumn mouse lacrimal gland model, or any of several autoimmune models of spontaneous rodents that result in dysfunction of the eye glands (for example, NOD-SCID, MRL / lpr, or NZB / NZW ) (Barabino et al., Experimental Eye Research 2004, 79, 613-621 and Schrader et al., Developmental Opthalmology, Karger 2008, 41, 298312, each of which is incorporated herein by reference in its entirety). The parameters in these models may include histopathology of the eye and eye glands (cornea, etc.) and possibly the classic Schirmer test or its modified versions (Barabino et al.) That measure tear production. Activity can be assessed by dosing through multiple routes of administration (for example, systemic or topical) that can begin before or after measurable disease exists.
[000132] The compounds can be evaluated in one or more preclinical models of uveitis known to those skilled in the art. These include, but are not limited to, the models of experimental autoimmune uveitis (UAE) and endotoxin-induced uveitis (UAE). UAE experiments can be performed on rabbits, rats or mice and may involve passive or active immunization. For example, any one of several retinal antigens can be used to sensitize animals to a relevant immunogen, after which animals can be challenged ocularly with the same antigen. The EIU model is more acute and involves local or systemic administration of lipopolysaccharide in sublethal doses. The parameters for both the EIU and UAE models may include an examination of
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50/50 fundscopy, histopathology, among others. These models are analyzed by Smith et al. (Immunology and Cell Biology 1998, 76, 497-512, which is incorporated herein by reference in its entirety). Activity is assessed by dosing through multiple routes of administration (for example, systemic or topical) that can begin before or after the measurable disease exists. Some models listed above may also develop scleritis / episcleritis, chorioditis, cyclitis, or iritis and are therefore useful in investigating the potential activity of compounds for the therapeutic treatment of these diseases.
[000133] The compounds can also be evaluated in one or more preclinical models of conjunctivitis known to those skilled in the art. These include, but are not limited to, rodent models that use guinea pig, rat, or mouse. Guinea pig models include those that use active or passive immunization and / or immune challenge protocols with antigens such as ovalbumin or rag (reviewed in Groneberg, DA, et al., Allergy 2003, 58, 1101-1113, which is incorporated herein by reference in its entirety). The rat and mouse models are similar in general structure to those in the guinea pig (also reviewed by Groneberg). Activity can be assessed by dosing through multiple routes of administration (for example, systemic or topical) that can begin before or after the measurable disease exists. The criteria for such studies may include, for example, histological, immunological, biochemical or molecular analysis of ocular tissues such as the conjunctiva.
[000134] Various modifications of the invention, in addition to those described herein, will be evident to those skilled in the art from the description above. Each reference cited in this application is hereby incorporated by reference in its entirety.

权利要求:
Claims (12)
[1]
1. Compound, characterized by the fact that it is 3-cyclobutyl-3 [4- (7H-pyrrolo [2,3-d] pyrimidin-4-yl) -1H-pyrazol-1-yl] propanonitrile, or a pharmaceutically salt acceptable of this.
[2]
2. Compound according to claim 1, characterized in that it is (R) -3-cyclobutyl-3- [4- (7H-pyrrolo [2,3-d] pyrimidin-4-yl) 1H-pyrazole- 1-yl] propanonitrile, or a pharmaceutically acceptable salt thereof.
[3]
A compound according to claim 1, characterized in that it is (S) -3-cyclobutyl-3- [4- (7H-pyrrolo [2,3-d] pyrimidin-4-yl) 1H-pyrazole- 1-yl] propanonitrile, or a pharmaceutically acceptable salt thereof.
[4]
4. Compound characterized by the fact that it is 3- (4- (7Hpyrrolo [2,3-d] pyrimidin-4-yl) -1H-pyrazol-1-yl) -3- (3methylcyclobutyl) propanonitrile, or a pharmaceutically salt acceptable of this.
[5]
Compound according to claim 4, characterized in that it is 3- (4- (7H-pyrrolo [2,3-d] pyrimidin-4-yl) -1H-pyrazol-1-yl) -3 ( (trans) -3-methylcyclobutyl) propanonitrile or a pharmaceutically acceptable salt thereof.
[6]
Compound according to claim 5, characterized in that it is (3R) -3- (4- (7H-pyrrolo [2,3-d] pyrimidin-4-yl) -1H-pyrazol1-yl) - 3 - ((trans) -3-methylcyclobutyl) propanonitrile or a pharmaceutically acceptable salt thereof.
[7]
Compound according to claim 5, characterized in that it is (3S) -3- (4- (7H-pyrrolo [2,3-d] pyrimidin-4-yl) -1H-pyrazol1-yl) - 3 - ((trans) -3-methylcyclobutyl) propanonitrile or a pharmaceutically acceptable salt thereof.
[8]
A compound according to claim 4, characterized in that it is 3- (4- (7H-pyrrolo [2,3-d] pyrimidin-4-yl) -1H-pyrazol-1-yl) -3-
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2/2 ((cis) -3-methylcyclobutyl) propanonitrile, or a pharmaceutically acceptable salt thereof.
[9]
Compound according to claim 8, characterized in that it is (3R) -3- (4- (7H-pyrrolo [2,3-d] pyrimidin-4-yl) -1H-pyrazol1-yl) - 3 - ((cis) -3-methylcyclobutyl) propanonitrile or a pharmaceutically acceptable salt thereof.
[10]
A compound according to claim 8, characterized in that it is (3S) -3- (4- (7H-pyrrolo [2,3-d] pyrimidin-4yl) -1H-pyrazol-1-yl) - 3 - ((cis) -3-methylcyclobutyl) propanonitrile or a pharmaceutically acceptable salt thereof.
[11]
11. Salt, characterized by the fact that it is the phosphoric acid salt of the compound as defined in any one of claims 1 to 10.
[12]
12. Composition, characterized in that it comprises the compound as defined in any one of claims 1 to 10, or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable carrier.

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AU2011217961A1|2012-09-06|

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法律状态:
2017-06-27| B25G| Requested change of headquarter approved|Owner name: INCYTE CORPORATION (US) |

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2017-07-11| B25A| Requested transfer of rights approved|Owner name: INCYTE HOLDINGS CORPORATION (US) |

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2018-01-23| B07D| Technical examination (opinion) related to article 229 of industrial property law|

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2018-04-10| B06F| Objections, documents and/or translations needed after an examination request according art. 34 industrial property law|

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2019-05-28| B07E| Notice of approval relating to section 229 industrial property law|Free format text: NOTIFICACAO DE ANUENCIA RELACIONADA COM O ART 229 DA LPI |

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2019-08-27| B06U| Preliminary requirement: requests with searches performed by other patent offices: suspension of the patent application procedure|

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2020-03-10| B09A| Decision: intention to grant|

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2020-05-12| B16A| Patent or certificate of addition of invention granted|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 18/02/2011, OBSERVADAS AS CONDICOES LEGAIS. |

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