Pyrrolo[2,3-d]pyrimidine compounds

p00001Pyrrolo [2,3-d] pyrimidine compounds

p00002Field of the Invention

p00003Herein, N-methyl (4- (methyl (7H-pyrrolo [2,3-d] pyrimidin-4-yl) amino) cyclohexyl) 5 methanesulfonamide, its analogues, its use as inhibitors of Janus kinases is described (JAK), pharmaceutical compositions containing these compounds and methods of preparing these compounds.

Background of the invention

p00004Protein kinases are families of enzymes that catalyze phosphorylation of specific moieties in proteins, and are generally classified into tyrosine and serine / threonine kinases. Inappropriate kinase activity, which arises from mutation, inappropriate overexpression or regulation, dysregulation or deregulation, as well as over- or underproduction of growth factors or cytokines, has been implicated in many diseases, including, but not limited to, cancer. , cardiovascular diseases, allergies, asthma and other respiratory diseases, autoimmune diseases, inflammatory diseases, bone diseases, metabolic disorders, and neurological and neurodegenerative disorders such as Alzheimer's disease. Inappropriate kinase activity

p00005fifteen it triggers a variety of biological cellular responses related to cell growth, cell differentiation, survival, apoptosis, mitogenesis, cell cycle control and cell mobility, implicated in the aforementioned diseases and related diseases.

p00006Therefore, protein kinases have become an important class of enzyme targets for therapeutic intervention. In particular, the JAK family of cell tyrosine kinase proteins (JAK-1, JAK-2, JAK-3 20 and Tyk-2) plays a central role in cytokine signaling (Kisseleva et al, "Gene", 2002, 285, 1; Yamaoka et al. "Genome Biology" 2004, 5, 253)). After binding to their receptors, the cytokines activate the JAKs, which then phosphorylate the cytokine receptor, thereby creating coupling sites for signaling molecules, in particular, members of the family of signal transducers and activators of the transcript (STAT) that ultimately leads to gene expression. Numerous cytokines are known that

p0000725 activate the JAK family.

p00008Therefore, there is still a need for alternative compounds that effectively inhibit JAK enzymes, including JAK-1, JAK-2, JAK-3 and / or Tyk-2.

p00009Summary of the invention

p00010The present invention provides a compound of formula I:

p00011or a pharmaceutically acceptable salt thereof, wherein R1 is linear, branched or cyclic saturated monovalent (C1-4) hydrocarbon, optionally substituted with hydroxy.

p00012Specifically, a compound of formula I, wherein R1 is methyl.

p00013Specifically, a compound of formula I, wherein R1 is ethyl or cyclobutyl.

p0001435 In other aspects, the present invention also provides:

p00015pharmaceutical compositions comprising a pharmaceutically acceptable carrier and a compound of formula I; pharmaceutical combinations comprising a pharmaceutically acceptable carrier, one or more additional agents that modulate a mammalian immune system or with anti-inflammatory agents, and a compound of formula I; compounds of formula I for use in therapy, more specifically, for use in the treatment of allergic reactions, allergic dermatitis, atopic dermatitis, eczema, pruritus, cancers, leukemia, lupus and multiple myeloma in a mammal; Crystalline form A of the compound of formula I which is maleic acid salt of N-methyl-1 {trans- (4- (methyl (7H-pyrrolo [2,3-d] pyrimidin-4-yl) amino) cyclohexyl} methanesulfonamide ; and a process for the preparation of a compound of formula I which is maleic acid salt of N-methyl-1- {trans- (4- (methyl (7H-pyrrolo [2,3d] pyrimidin-4-yl) amino)) cyclohexyl} methanesulfonamide.

p00016They are also described in this document:

p00017procedures for the control or treatment of a disorder or condition selected from rejection of organ transplantation, lupus, multiple sclerosis, rheumatoid arthritis, psoriasis, cancer, osteoarthritis and diabetes by administration to a mammal in need of a therapeutically amount effective of a compound of formula I or a pharmaceutically acceptable salt thereof;

p00018procedures for the control or treatment of a disorder or condition selected from diabetes, cancer, autoimmune thyroid disorders, ulcerative colitis, Crohn's disease, dry eyes, Alzheimer's disease, leukemia and other indications in which immunosuppression or immunomodulation would be desirable by administration to a mammal in need of a therapeutically effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof;

p00019procedures for the control or treatment of a disorder or condition selected from allergic reaction including allergic dermatitis, eczema, atopic dermatitis, pruritus and other pruritic conditions and inflammatory disease such as mammalian intestinal disease by administration to a mammal that it requires a therapeutically effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof;

p00020procedures for the control or treatment of a disorder or condition selected from asthma and other obstructive diseases of the respiratory tract, including chronic or inveterate asthma, late asthma, hypersensitivity of the respiratory tract, bronchitis, bronchial asthma, allergic asthma, asthma intrinsic, extrinsic asthma, dust asthma, recurrent airway obstruction and chronic obstructive pulmonary disease by administration to a mammal in need of a therapeutically effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof;

p00021methods for the inhibition of tyrosine kinase proteins or JAK-1, JAK-2, JAK-3 and / or Tyk-2 by administration to a mammal in need of a therapeutically effective amount of a compound of formula I or a pharmaceutically salt acceptable salt thereof;

p00022methods for the inhibition of tyrosine kinase proteins or JAK-1, JAK-2, JAK-3 and / or Tyk-2 by administration to a mammal in need of a therapeutically effective amount of a compound of formula I or a pharmaceutically salt acceptable salt thereof; and

p00023processes for the preparation of the compounds of the present invention.

p00024Brief description of the figures

p00025Fig. 1 is a powder X-ray diffraction pattern characteristic of the maleic acid salt of N-methyl-1 {trans- (4- (methyl (7H-pyrrolo [2,3-d] pyrimidin-4- il) amino) cyclohexyl} methanesulfonamide (Form A). Fig. 2 illustrates the VAS scores on day 27 for Example 1b in flea allergy dogs in the trial of reduction of pruritus and dermatitis associated with fleas. Fig. 3 illustrates the pruritus seconds per 4-hour record for Example 1b in flea allergy dogs in the trial of reduction of pruritus and dermatitis associated with fleas.

p00026Detailed description

p00027With respect to the above compound, and throughout the entire application and the claims, the following terms have the meanings defined below.

p00028The term "mammal" refers to humans or animals including livestock and companion animals. The term "companion animal" or "companion animals" refers to the animals that are cared for as pets. Examples of pets include cats, dogs and horses. The term "cattle" refers to animals raised in an agricultural environment to manufacture products such as food or fiber, or for their work. In some embodiments, cattle are suitable for consumption by mammals, for example, by humans. Examples of livestock animals include mammals such as cows, goats, horses, pigs, sheep, including lambs and rabbits, as well as birds such as chickens, ducks and turkeys.

p00029The term "control", "treat" or "treatment" of a disease includes: (1) preventing the disease, that is, causing the symptoms or clinical signs of the disease not to develop in a mammal that may be exposed or predisposed to the disease, but not yet experiencing or showing symptoms / signs of the disease; (2) inhibit the disease, that is, stop or reduce the development of the disease or its symptoms / clinical signs; or (3) alleviate the disease, that is, cause the regression of the disease or its symptoms / clinical signs.

p00030The term "therapeutically effective amount" means the amount of a compound that, when administered to a mammal to treat a disease, is sufficient to effect said treatment of the disease. The "therapeutically effective amount" will vary depending on the compound, the disease and its severity, and the age, the

p000315 weight, etc., of the mammal to be treated.

p00032The term "approximately", if used in the definition of a peak of a powder X-ray diffraction pattern, is defined as the indicated 2-theta value ± 0.2 degrees 2-theta. Any determination of whether a crystalline form is the polymorphic A form and is encompassed by the claims should be interpreted in light of the variability in this assay.

p0003310 "Pharmaceutically acceptable" means suitable for use in mammals, pets or livestock animals.

p00034The content of carbon atoms of various hydrocarbon-containing moieties is indicated by a prefix that indicates the minimum and maximum number of carbon atoms of the remainder, that is, the prefix Ci-j indicates a remainder of the integer number "i" to the number integer j of carbon atoms, both inclusive. Thus, for example, C1-4 alkyl refers to

p00035fifteen alkyl of one to four carbon atoms, both inclusive.

p00036The term "alkyl" refers to linear, branched and cyclic saturated monovalent hydrocarbon groups, but the reference to an individual radical such as "propyl" only encompasses the linear chain radical, a branched chain isomer such as "isopropyl" or a cyclic isomer such as cyclopropylmethyl or cyclopentyl which is specifically referenced.

p00037twenty Compounds that have the same molecular formula but differ in the nature or sequence of union of their atoms or in the arrangement of their atoms in space are called "isomers." Isomers that differ in the arrangement of their atoms in space are called "stereoisomers." Those skilled in the art will appreciate that the compound of formula I may exist as cis and trans aquatic diastereomers. In particular, the present invention provides a compound of formula IA having the chemical name N-methyl-1- {trans-4

p0003825 [methyl (7H-pyrrolo [2,3-d] pyrimidin-4-yl) amino] cyclohexyl} methanesulfonamide,

p00039or a pharmaceutically acceptable salt thereof.

p00040All isomers (for example, cis, trans or diastereomers) of the compounds described herein, alone, as well as any mixture are included within the scope of the described compounds. All

p0004130 These forms, including enantiomers, diastereomers, cis, trans, sin, anti, solvates (including hydrates), tautomers and mixtures thereof, are included in the compounds described.

p00042Stereoisomeric mixtures, for example, mixtures of diastereomers, can be separated into their corresponding isomers in a known manner by means of suitable separation procedures. Diastereomeric mixtures, for example, can be separated into their individual diastereomers by crystallization.

p0004335 fractional, chromatography, solvent distribution and similar procedures. This separation can take place either at the level of one of the starting compounds or in a compound of formula I itself. The enantiomers can be separated through the formation of diastereomeric salts, for example, by the formation of a salt with a pure enantiomeric chiral acid, or by chromatography, for example, by CLAR, using chromatographic substrates with chiral ligands.

p0004440 Administration routes

p00045In therapeutic use for the treatment of disorders in a mammal (i.e. humans and animals), a compound of the present invention or its pharmaceutical compositions can be administered orally, parenterally, topically, rectally, transmucosally or intestinally. Parenteral administrations include indirect injections

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p00046to generate a systemic effect or direct injections in the affected area. Topical administrations include the treatment of skin or organs easily accessible by local application, for example, eyes or ears. It also includes transdermal administration to generate a systemic effect. Rectal administration includes the form of suppositories. Preferred routes of administration are oral and parenteral.

p00047Pharmaceutical salts

p00048The compound of formula I can be used in its native form or in salt form. In cases where the formation of a stable non-toxic acid or base salt is desired, administration of the compound in the form of a pharmaceutically acceptable salt may be appropriate. Pharmaceutically acceptable salts of the compounds of formula I include the salts acetate, ascorbate, aspartate, benzoate, besylate, bicarbonate / carbonate, bisulfate / sulfate, borate, camsilate, citrate, edisilate, ethoglutarate, silate, formate, fumarate, gluceptate, gluconate , glucuronate, glycerophosphate, hexafluorophosphate, hibenzate, hydrochloride / chloride, hydrobromide / bromide, iodide / iodide, isethionate, lactate, malate, maleate, malonate, mesylate, methylsulfate, naphthylate, 2-napsilate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, hydrogen phosphate / phosphate / dihydrogen phosphate, sucrate, stearate, succinate, tartrate, tosylate and trifluoroacetate.

p00049Composition / formulation

p00050The pharmaceutical compositions of the present invention can be manufactured by methods well known in the art, for example, by conventional mixing, dissolution, granulation, dragee, levigation, emulsion, encapsulation, entrapment, lyophilization or spray drying procedures.

p00051Pharmaceutical compositions for use in accordance with the present invention can be formulated in a conventional manner using one or more pharmaceutically acceptable carriers comprising excipients and adjuvants that facilitate the processing of active compounds in preparations that can be used pharmaceutically. The appropriate formulation depends on the route of administration selected. Pharmaceutically acceptable excipients and carriers are generally known to those skilled in the art and, therefore, are included in the present invention. Such excipients and vehicles are described, for example, in "Remingtons Pharmaceutical Sciences" Mack Pub. Co., New Jersey (1991).

p00052The formulations of the invention can be designed to be short-acting, fast-release, long-acting and sustained-release. Therefore, pharmaceutical formulations can also be formulated for controlled release or for slow release.

p00053Dose

p00054Pharmaceutical compositions suitable for use in the present invention include compositions in which the active ingredients are contained in an amount sufficient to achieve the intended purpose, that is, the control or treatment of disorders or diseases. More specifically, a therapeutically effective amount means an amount of compound effective to prevent, alleviate or improve the symptoms / signs of a disease or prolong the survival of the subject being treated.

p00055The amount of active component, which is the compound of the present invention, in the pharmaceutical composition and unit dosage form thereof, can be varied or adjusted widely depending on the manner of administration, the potency of the particular compound and the concentration. desired. The determination of a therapeutically effective amount is the responsibility of those skilled in the art. In general, the amount of active component will vary from 0.01% to 99% by weight of the composition.

p00056In general, a therapeutically effective amount of active component dose will be in the range of about 0.01 to about 100 mg / kg body weight / day, preferably about 0.1 to about 10 mg / kg body weight / day, more preferably about 0.3 to 3 mg / kg body weight / day, even more preferably about 0.3 to 1.5 mg / kg body weight / day. It is to be understood that the doses may vary depending on the needs of each subject and the severity of the disorders or diseases being treated.

p00057The desired dose may conveniently be presented in a single dose or as divided doses administered at appropriate intervals, for example, as two, three, four or more sub-doses per day. The sub-dose itself can be further divided, for example, into a number of freely spaced differentiated administrations; such as multiple inhalations of an insufflator or by applying a plurality of drops in the eye.

p00058In addition, it should be understood that it is possible to increase the initial dose administered beyond the previous upper level in order to quickly reach the desired plasma concentration. On the other hand, the initial dose may be lower than the optimum and the daily dose may be progressively increased in the course of treatment depending on each situation. If desired, the daily dose can also be divided into multiple doses for administration, for example, two to four times per day.

p00059Medical and veterinary uses

p00060The compounds of the present invention are inhibitors of Janus kinases (JAK-i) effectively against Janus kinase-1 (JAK-1), Janus kinase-2 (JAK-2) and Janus kinase-3 (JAK- 3). Therefore, they are useful as therapeutic agents for organ transplants, lupus, multiple sclerosis, rheumatoid arthritis, psoriasis, type I diabetes and diabetes complications, cancer, asthma, atopic dermatitis, autoimmune thyroid disorders, ulcerative colitis, disease Crohn's disease, Alzheimer's disease, leukemia, osteoarthritis, pruritus control, chronic respiratory disease and other indications in which immunosuppression / immunomodulation would be desirable.

p00061In addition, there is a great need for safe and effective agents to control atopic dermatitis in animals. The market for the treatment of atopic dermatitis in animals is currently dominated by corticosteroids, which cause painful and undesirable side effects in animals, specifically in pets such as dogs. Antihistamines are also used, but they are ineffective. Currently, a canine cyclosporine formulation (ATOPICATM) is marketed for atopic dermatitis, but it is expensive and has a slow onset of its effectiveness. In addition, there are GI tolerance problems with ATOPICATM. The compounds of the present invention are JAK inhibitors effectively against JAK-1 and JAK-3. These compounds will be an alternative to the use of steroids and will provide a solution for chronic itching and inflammation that would either persist in atopic dermatitis or experience a slow setback after the removal of the allergen or the causative agent, such as fleas in allergic dermatitis from fleas

p00062The compounds of the present invention can be administered in a pharmaceutically acceptable form, either alone or in combination with one or more additional agents that modulate a mammalian immune system or with anti-inflammatory agents. These agents may include, but are not limited to, cyclosporin A (for example, Sandimmune.RTM. or Neoral.RTM., rapamycin, FK-506 (tacrolimus), leflunomide, deoxyspergualine, mycophenolate (for example, Cellcept.RTM.), azathioprine (for example, Imuran.RTM.), daclizumab (for example, Zenapax.RTM. ), OKT3 (for example, Orthocolone.RTM.), AtGam, aspirin, acetaminophen, ibuprofen, naproxen, piroxicam and anti-inflammatory steroids (for example, prednisolone or dexamethasone). These agents may be administered as part of the same dosage form or in separate dosage forms, by the same or different administration routes, and in the same or different administration guidelines according to the conventional pharmaceutical practice known to a person skilled in the art. .

p00063In one embodiment, the invention provides methods of treating or preventing a disease, condition or disorder associated with JAK in a subject, such as a human being or a mammalian animal, which comprises administering an effective amount of one or more compounds described in the Present the document to the subject. The disease, condition or disorder associated with JAK may be related to JAK-1, JAK-2, JAK-3 and / or Tyk-2. Suitable subjects that can be treated include domestic or wild animals, companion animals such as dogs, cats, horses and the like; cattle including cows and other ruminants, pigs, poultry, rabbits and the like; primates, for example, monkeys such as rhesus and cynomolgus monkeys (also known as crab or long-tailed macaque), marmosets, tamarins, chimpanzees, macaques and the like; and rodents such as rats, mice, gerbils, guinea pigs and the like. In one embodiment, the compound is administered in a pharmaceutically acceptable form, optionally in a pharmaceutically acceptable carrier.

p00064JAK / STAT signaling has been implicated in the mediation of many abnormal immune responses such as allergies, asthma, autoimmune diseases such as transplant rejection (allografts), rheumatoid arthritis, amyotrophic lateral sclerosis and multiple sclerosis, as well as solid malignant neoplasms. and hematological such as leukemia and lymphomas. For a review of the pharmaceutical intervention of the JAK / STAT pathway, see Frank, (1999), Mol. Med. 5: 432: 456 and Seidel et al., (2000), Oncogene 19: 2645-2656.

p00065JAK-3, in particular, has been involved in a variety of biological processes. For example, proliferation and survival of murine mast cells induced by IL-4 and IL-9 have been shown to be dependent on JAK-3 signaling and and chains. Suzuki et al., (2000), Blood 96: 2172-2180. JAK-3 also plays a crucial role in mast cell degranulation responses mediated by the IgE receptor (Malaviya et al., (1999), Biochem. Biophys. Res. Commun. 257: 807-813), and inhibition of JAK-3 kinase has been shown to prevent type I hypersensitivity reactions, including anaphylaxis (Malaviya et al., (1999), J. Biol. Chem. 274: 27028-27038) . Inhibition of JAK-3 has also been shown to produce immune suppression for allograft rejection (Kirken, (2001), Transpl. Proc. 33: 3268-3270). JAK-3 kinases have also been implicated in the mechanism that participates in the early and late stages of rheumatoid arthritis (Muller-Ladner et al., (2000), J. Immunal 164: 38943901); familial amyotrophic lateral sclerosis (Trieu et al., (2000), Biochem Biophys. Res. Commun. 267: 22-25); leukemia (Sudbeck et al., (1999), Clin. Cancer Res. 5: 1569-1582); Mycosis fungoides, a form of T lymphocyte lymphoma (Nielsen et al., (1997), Prac. Natl. Acad. Sci. USA. 94: 6764-6769); and abnormal cell growth (Yu et al., (1997), J. Immunol. 159: 5206-5210; Catlett-Falcone et al., (1999), Immunity 10: 105-115).

p00066JAK kinases, including JAK-3, are expressed in abundance in the primary leukemic cells of children with acute lymphoblastic leukemia, the most common form of childhood cancer, and there are studies that correlate STAT activation in certain cells with signs of regulation of apoptosis (Demoulin et al., (1996), Mol. Cell. Biol. 16: 4710-6; Jurlander et al., (1997), Blood 89: 4146-52; Kaneko et al., (1997) , Clin. Exp. Immun. 109: 185-193; and Nakamura et al., (1996), J. Biol. Chem. 271: 19483-8). They are also known to be important for the differentiation, function and survival of lymphocytes. JAK-3, in particular, plays an essential role in the function of lymphocytes, macrophages and mast cells. Given the importance of this JAK kinase, the compounds that modulate the JAK pathway, including those that are selective to JAK-3, may be useful for the treatment of diseases or conditions in which the function of lymphocytes, macrophages or mast cells (Kudlacz et al., (2004) Am.

p00067J. Transplant 4: 51-57; Changelian (2003), Science 302: 875-878).

p00068Conditions in which it is contemplated that the direction of the JAK pathway or the modulation of the JAK kinases, in particular JAK-3, are therapeutically useful include arthritis, asthma, autoimmune diseases, cancers or tumors, diabetes, certain diseases, disorders or eye conditions, inflammation, inflammations, allergies or intestinal conditions, neurodegenerative diseases, psoriasis, transplant rejection and viral infection. Conditions that may benefit from JAK-3 inhibition are discussed in more detail below.

p00069Accordingly, the compound of formula I or its pharmaceutically acceptable salts and pharmaceutical compositions can be used to treat a variety of conditions or diseases such as:

p00070arthritis, including rheumatoid arthritis, juvenile arthritis and psoriatic arthritis;

p00071asthma and other obstructive diseases of the respiratory tract, including chronic or inveterate asthma, late asthma, hypersensitivity of the respiratory tract, bronchitis, bronchial asthma, allergic asthma, intrinsic asthma, extrinsic asthma, dust asthma, recurrent airway obstruction and chronic obstructive pulmonary disease;

p00072autoimmune diseases or disorders such as those designated as single organ autoimmune disorders

p00073or individual cell type, for example, Hashimoto's thyroiditis, autoimmune hemolytic anemia, autoimmune atrophic gastritis of pernicious anemia, autoimmune encephalomyelitis, autoimmune orchitis, Goodpasture's disease, autoimmune thrombocytopenia, sympathetic ophthalmia, myasthenia gravis, Graves' disease, primary biliary cirrhosis , chronic aggressive hepatitis, ulcerative colitis and membranous glomerulopathy, those that include an autoimmune systemic disorder, for example, systemic lupus erythematosus, rheumatoid arthritis, Sjogren's syndrome, Reiter's syndrome, polymyositis-dermatomyositis, systemic sclerosis, polyarteritis nodosa, multiple sclerosis and bullous pemphigoid, and additional autoimmune diseases, which may be based on O (humoral) or T lymphocytes , including Cogan syndrome, ankylosing spondylitis, Wegener granulomatosis, autoimmune alopecia, juvenile or type I diabetes and thyroiditis;

p00074cancers or tumors, including cancer of the alimentary / gastrointestinal tract, colon cancer, liver cancer, skin cancer, including mast cell tumors and squamous cell carcinoma, breast and breast cancer, ovarian cancer, prostate cancer, lymphoma, leukemia, including acute myelogenous leukemia and chronic myelogenous leukemia, kidney cancer, lung cancer, muscle cancer, bone cancer, bladder cancer, brain cancer, melanoma including oral and metastatic melanoma, Kaposi's sarcoma, myelomas including multiple myeloma, myeloproliferative disorders, proliferative diabetic retinopathy and associated angiogenic disorders including solid tumors;

p00075diabetes, including type I diabetes and diabetes complications;

p00076eye diseases, disorders or conditions, including autoimmune eye diseases, keratoconjunctivitis, vernal conjunctivitis, uveitis including uveitis associated with Behcet's disease and lens-induced uveitis, keratitis, herpetic keratitis, conical keratitis, corneal epithelial dystrophy, keratoleucoma , ocular premphigus, Mooren's ulcer, scleritis, Graves ophthalmopathy, VogtKoyanagi-Harada syndrome, dry keratoconjunctivitis (dry eye), flicténula, iridocyclitis, sarcoidosis, endocrine ophthalmopathy, sympathetic ophthalmitis, allergic conjunctivitis and ocular neovascularization;

p00077inflammations, allergies or intestinal conditions including Crohn's disease and / or ulcerative colitis, inflammatory bowel disease, celiac diseases, proctitis, eosinophilic gastroenteritis and mastocytosis;

p00078neurodegenerative diseases, including motor neuron disease, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, Huntington's disease, cerebral ischemia or neurodegenerative disease caused by traumatic injury, stroke, glutamate neurotoxicity or hypoxia; ischemic injury / reperfusion in stroke, myocardial ischemia, renal ischemia, heart attacks, cardiac hypertrophy, atherosclerosis and arteriosclerosis, organ hypoxia and platelet aggregation;

p00079diseases, conditions or disorders of the skin, including atopic dermatitis, eczema, psoriasis, scleroderma, pruritus and other pruritic conditions;

p00080Allergic reactions including allergic dermatitis in mammals including equine allergic diseases such as hypersensitivity to bites, summer eczema and itching in horses.

p00081transplant rejection, including pancreatic islet transplant rejection, bone marrow transplant rejection, graft versus host disease, organ and cell transplant rejection, such as bone marrow, cartilage, cornea, heart, intervertebral disc, Islets, kidneys, limbs, liver, lung, muscle, myoblasts, nerve, pancreas, skin, small intestine or trachea, and xeno transplant.

p00082Another embodiment provides a method for inhibiting a JAK enzyme, including JAK-1, JAK-2, JAK-3 and / or Tyk-2, which includes contacting the JAK enzyme either with a non-therapeutic amount or with a therapeutically amount. effective of one or more of the present compounds. Such procedures can be produced in vivo or in vitro. In vitro contact may involve a detection assay to determine the efficacy of the one or more compounds against a selected enzyme in various amounts or concentrations. In vivo contact with a therapeutically effective amount of one or more compounds may involve the treatment of a described disease, disorder or condition, or prophylaxis of organ transplant rejection in the animal in which the

p0008310 Contact. The effect of one or more compounds on the JAK enzyme and / or host animal can also be determined or measured. Methods for determining JAK activity include those described in the Examples, as well as those described in WO 99/65908, WO 99/65909, WO 01/42246, WO 02/00661, WO 02/096909, WO 2004 / 046112 or WO 2007/012953.

p00084The following reaction schemes illustrate the general synthetic procedures of the compounds of the

p00085fifteen present invention All starting materials are prepared by the procedures described in these schemes or by procedures known to a person skilled in the art.

p00086Scheme I

p00087It will be apparent to those skilled in the art that it may be necessary to protect and deprotect sensitive functional groups (Pg or Pg1) during the synthesis of a compound of the invention. This can be achieved by conventional procedures, for example, as described in "Protective Groups in Organic Synthesis" by T.

p00088W. Greene and PGM Wuts, John Wiley &amp; Sons Inc (1999) and references therein.

p00089In Scheme I, 4-chloro-7H-pyrrolo [2,3-d] pyrimidine (a) can be obtained commercially. Trans-4 (methylamino) -cyclohexyl] methanol (b) can be obtained from the corresponding carboxylic acid, trans-4 [(tert-butoxycarbonyl) amino] cyclohexanecarboxylic acid, after treatment with a reducing agent such as lithium hydride and aluminum in an aprotic anhydrous solvent, such as tetrahydrofuran at temperatures between 0-60 ° C for several hours.

p00090As shown in Scheme I, a compound of structure (c) can be synthesized by the reaction of 4-chloro-7H-pyrrolo [2,3-d] pyrimidine (a) with trans-4- (methylamino) -cyclohexyl] methanol (b) in a suitable aprotic polar solvent, such as N, N-dimethylformamide, aqueous dioxane and dimethyl sulfoxide, in the presence of a suitable base such as triethylamine and potassium carbonate at elevated temperatures of up to 90 ° C for a maximum of a few hours .

p00091A compound of structure (d) could be synthesized in a two-stage process from a compound of structure (c). For example, a compound of structure (d) would be synthesized, first, by the use of bromination reagents such as thionyl bromide or phosphorus tribromide in an aprotic polar solvent such as methylene chloride, providing the cyclohexylmethyl bromide without protection, and secondly, by the addition of a suitable protective reagent such as tosyl chloride, giving the protected compound of structure (d).

p00092A compound of structure (e) can be prepared by the use of simple protection procedures from a compound of structure (c). For example, when Pg and Pg1 are both tosyl, this can be carried out in a one-stage reaction by treating the unprotected compound of structure (c) with tosyl chloride in the presence of an aprotic polar solvent such as methylene, a catalyst such as DMAP and a weak base such as triethylamine.

p00093A compound of structure (f) can be synthesized from a compound of structure (e) by S-alkylation using a suitable nucleophile. Therefore, compounds of structure (e) in which the protective group (Pg1) is a suitable hydroxyl protecting group, such as tosyl or mesyl, can be reacted with potassium thioacetate in a polar solvent such as dimethyl sulfoxide or N-methylpyrrolidine at elevated temperatures of up to 75 ° C for a maximum of 2 hours, giving compounds of structure (f).

p00094A compound of structure (g) can be synthesized by an oxidation process from compounds of formula (f). Those skilled in the art know many oxidizing conditions, for example, those described in "Handbook of Reagents for Organic Synthesis-Oxidising and Reducing Agents" edited by SD Burke and RL Danheiser. For example, a compound of structure (f), optionally moistened with water, can be treated with formic acid, followed by the slow addition of hydrogen peroxide while stirring at room temperature for approximately 15 hours, giving a compound of structure (g ). Alternatively, oxone can be used in a polar solvent such as acetic acid, and if the reaction is carried out in the presence of potassium acetate, the potassium salt of the compound of formula (g) is produced.

p00095It is envisioned that a compound of structure (g) can be synthesized directly from a compound of structure (e) after treatment with a suitable sulfur nucleophile, such as sodium sulphite in a polar solvent. Similarly, a compound of structure (g) could be synthesized from a compound of structure

p00096(d) after nucleophilic replacement with sodium sulphite.

p00097Treatment of the sulfonic acids of formula (g) with a chlorinating agent such as thionyl chloride in an aprotic polar solvent, such as methylene chloride, with a polar co-solvent, such as N, N-dimethylformamide at reflux gives the compounds chlorinated Then, the chlorinated compound reacts in an aprotic anhydrous solvent, such as tetrahydrofuran, with suitable amines in pure gaseous form, or dissolves in aprotic anhydrous solvents, such as tetrahydrofuran, at room temperature, producing a compound of structure (h). Optionally, a weak anhydrous base, such as triethylamine, can be used to clean the hydrochloric acid generated in the reaction.

p00098The compounds of formula I of the present invention can be prepared from compounds of formula (h), wherein Pg is a suitable protecting group by deprotection methods known to one skilled in the art. For example, when the protecting group (Pg) is tosyl, suitable deprotection conditions involve the reaction with a base such as lithium hydroxide or potassium hydroxide in a protic solvent such as methanol or isopropanol and, optionally, miscible co-solvents such as tetrahydrofuran and water at room temperature for several hours, producing the unprotected amine of formula I.

p00099The salts of the compounds of formula I can be formed by reacting the free base of the compounds of formula I with a suitable acid, such as maleic acid, in the presence of a protic solvent such as butanol and, optionally, a co-solvent such like water.

p00100Alternatively, the compounds of the present invention can be prepared according to Scheme II.

p00101Scheme II

p00102In Scheme II, 4-methyl-7 - [(4-methylphenyl) sulfonyl] -7H-pyrrolo [2,3-d] pyrimidine (j) can be obtained from (a) commercially available using a protective agent such as tosyl chloride by methods well known in the art. Trans-4- (methylamino) -cyclohexyl] methanol (b) can be obtained from the corresponding carboxylic acid, trans-4 - [(tert-butoxycarbonyl) amino] cyclohexanecarboxylic acid, after treatment with a reducing agent such as Vitride in an anhydrous solvent, such as toluene at temperatures between 0-110 ° C for several hours.

p00103As shown in Scheme II, a compound of structure (k) can be synthesized by the reaction of 4-chloro-7H-pyrrolo [2,3-d] pyrimidine (j) with trans-4- (methylamino) -cyclohexyl] methanol (b) in a suitable solvent such as acetone, in the presence of a suitable base such as triethylamine with a catalytic amount of potassium iodide at elevated temperatures of up to 60 ° C for a maximum of a few hours.

p00104A compound of the structure (k) could be synthesized by the addition of a suitable mesylation reagent such as mesyl chloride in the presence of a suitable base such as triethylamine or diethylisopropylamine in a suitable solvent such as acetone at elevated temperatures of up to 60 ° C , giving the methanesulfonyl compound of structure (k).

p00105A compound of structure (l) can be prepared by the use of simple S-alkylation procedures from a compound of structure (k) using a suitable nucleophile. Therefore, the structure compounds

p00106(k) they can be reacted with sodium sulphite in a solvent such as isopropyl alcohol or water or toluene at elevated temperatures of up to 90 ° C for up to 4 hours, giving compounds of structure (1).

p00107The treatment of sulfonic acids of formula (1) with a chlorinating agent, such as thionyl chloride, in an aprotic polar solvent, such as THF or methylene chloride, with a polar co-solvent such as N, N-dimethylformamide at temperatures between 0 and 40 ° C gives the chlorinated compounds. Next, the chlorinated compound reacts in an aprotic anhydrous solvent, such as tetrahydrofuran, with suitable amines, such as methylamine, cyclobutylamine or 2-hydroxyazetidine, preferably in pure gaseous form, or is dissolved in aprotic anhydrous solvents such as tetrahydrofuran, at temperature environment, producing a compound of structure (h). Optionally, a weak anhydrous base, such as triethylamine, can be used to clean the hydrochloric acid generated in the reaction.

Examples

p00108Preparation 1: N-methyl-1- [trans-4- (methyl {7 - [(4-methylphenyl) sulfonyl] -7H-pyrrolo [2,3-d] pyrimidin-4-yl} amino) cyclohexyl] methanesulfonamide

p001095 Procedure (a). To a solution of the compound of Preparation 2 (308.0 g wet weight, 214.5 g dry weight, 0.41 mol) in tetrahydrofuran (1.01), methylamine (2 M in tetrahydrofuran, 687 ml) is added ) for 1 h. After stirring at room temperature for 30 min, more methylamine (2 M in tetrahydrofuran, 53 ml) is added and the reaction mixture is stirred at room temperature for 18 h. The mixture is reduced in volume to 600 ml, through vacuum distillation, and tetrahydrofuran (300 ml) is added, before reducing the mixture again in

p0011010 volume up to approximately 750 ml. To the mixture, heated to 45 ° C, 2-propanol (247 ml) and water (693 ml) are added. After cooling to room temperature, the solid material is collected by filtration, washed with water (2 x 250 ml) and dried under vacuum at 65 ° C, giving the title compound (180.4 g).

p001111H NMR (d6-DMSO): 1.17-1.32 (2H), 1.57-1.73 (4H), 1.76-1.92 (1H), 1.93-2.08 ( 2H), 2.30-2.39 (3H), 2.53-2.62 (3H), 2.87-2.98 (2H), 3.07-3.17 (3H), 4.53 -4.75 (1H), 6.81-6.94 (1H), 7.38-7.47 (2H), 7.56-7.65 (1H), 7.92-8.02 (2H ),

fifteen 8.15-8.27 (1H).

p00112Procedure (b). To a solution of the compound of Preparation 2 (165 g, 0.34 mol) in THF (1.65 l) and N, N-dimethylformamide (5.0 ml) at 0-5 ° C, thionyl chloride (125 ml) is added , 17 mol), for 25 min. The reaction mixture was stirred for 30 minutes at 0-5 ° C and then slowly heated to 40 ° C for 8 h. After cooling to room temperature, the solvent was evaporated under reduced pressure and azeotropically distilled with THF

p00113twenty to remove thionyl chloride. To the sulfonyl chloride obtained, freshly prepared THF (1.65 L) was added and the mixture was cooled to 0 ° C. The dry N-methylamine gas was purged for 30 minutes and the reaction was stirred for another 4 hours at room temperature. The solvent was evaporated to half its volume (800 ml), and heptane (1.5 L) was added. The product precipitated, and was filtered and washed with water (1 L), giving the title compound (80 g).

p001141H NMR (d6-DMSO): 1.17-1.32 (2H), 1.57-1.73 (4H), 1.76-1.92 (1H), 1.93-2.08 ( 2H), 2.30-2.39 (3H), 2.53-2.62

25 (3H), 2.87-2.98 (2H), 3.07-3.17 (3H), 4.53-4.75 (1H), 6.81-6.94 (1H), 7, 38-7.47 (2H), 7.56-7.65 (1H), 7.92-8.02 (2H), 8.15-8.27 (1H).

p00115Preparation 2: [trans-4- (methyl {7 - [(4-methylphenyl) sulfonyl] -7H-pyrrolo [2,3-d] pyrimidin-4-yl} amino) cyclohexyl] methanesulfonyl chloride

p00116To a solution of the compound of Preparation 3 (210.0 g, 0.42 mol) in dichloromethane (1.2 L) and N, N

p001175 dimethylformamide (4.1 ml), thionyl chloride (151.0 ml, 2.1 mol) is added for 25 min. The reaction mixture is heated at reflux for 18 h and then reduced in volume to 800 ml by vacuum distillation. To the mixture, heated to approximately 30 ° C, ethyl acetate (1.1 L) is added for 1 h, followed by heptane (546 ml), for 20 min at room temperature. The mixture is cooled to 0 ° C and stirred for 1 h, and the resulting precipitate is collected by filtration under a nitrogen atmosphere. The solid is washed with heptane (2 x 125 ml), giving the

p0011810 title compound (308.0 g wet weight), which is stored under a nitrogen atmosphere and used directly.

p00119Preparation 3: [trans-4- (methyl {7 - [(4-methylphenyl) sulfonyl] -7H-pyrrolo [2,3-d] pyrimidin-4-yl} amino) cyclohexyl] methanesulfonic acid

p00120Procedure (a). To a mixture of the compound of Preparation 4 (100.0 g wet weight, 23.5 g dry weight,

p00121fifteen 47.8 mmol) and formic acid (82.0 g, 68.0 ml, 1.8 mol), hydrogen peroxide (35% by weight in water, 21.0 ml, 0.26 mol) is added over 10 min. The reaction mixture is stirred at room temperature for 15 h and then quenched by the addition to an aqueous solution of sodium metabisulfate or metabisulfite (33% by weight, 35 ml). Water is added to the mixture (5 ml), 2-propanol (50 ml) and aqueous sodium hydroxide solution (33% by weight, 161 ml), and the suspension is stirred at room temperature for 1 h. The solid material is collected by

p00122twenty filtration, washed with water (100 ml) and dried in vacuo at 60 ° C, giving the title compound (26.0 g).

p001231H NMR (d6-DMSO): 0.98-1.18 (2H), 1.55-1.76 (5H), 1.99-2.13 (2H), 2.29-2.39 ( 5H), 3.05-3.15 (3H), 4.47-4.76 (1H), 6.77-6.92 (1H), 7.38-7.48 (2H), 7.54 -7.62 (1 H), 7.91-8.02 (2H), 8.17-8.25 (1H).

p00124Procedure (b). To a solution of the compound of Preparation 3 in IPA-water (585 ml of each, 1: 1, v / v), sodium sulfate was added and the mixture was heated to 80-90 ° C for 24 hours. After allowing to cool to room temperature, the solvent was evaporated to 50% and the pH of the reaction mixture was adjusted in the range 3-4 by the addition of acetic acid. Toluene (1 L) was added and the mixture was evaporated to 80%. More toluene (1 L) was added, and the mixture was heated at reflux for 4 hours. Toluene was decanted and the resulting title compound was obtained by vacuum drying (168 g).

p001251H NMR (d6-DMSO): 0.98-1.18 (2H), 1.55-1.76 (5H), 1.99-2.13 (2H), 2.29-2.39 ( 5H), 3.05-3.15 (3H), 4.47-4.76 (1H), 6.77-6.92 (1H), 7.38-7.48 (2H), 7.54 -7.62 (1H), 7.91-8.02 (2H), 8.17-8.25 (1H)

p00126Preparation 4. S - {[trans-4- (methyl {7 - [(4-methylphenyl) sulfonyl] -7H-pyrrolo [2,3-d] pyrimidin-4-yl} amino) cyclohexyl] methyl} ethanethioate

p00127To a solution of potassium thioacetate (11.4 g, 99.4 mmol) in dimethyl sulfoxide (30 ml), the compound of Preparation 5 (50.0 g, 87.9 mmol) in dimethyl sulfoxide is added (130 ml). The reaction mixture is heated at 55 ° C for 3 h, cooled to room temperature and quenched by the addition to an aqueous solution of sodium bicarbonate (0.1 M, 640 ml). The mixture is cooled to 13 ° C, and the resulting precipitate is collected by

p00128fifteen filtration and wash with water (250 ml), giving the title compound (204.0 g wet weight).

p001291H NMR (d6-DMSO): 1.06-1.23 (2H), 1.39-1.51 (1H), 1.51-1.70 (4H), 1.74-1.88 ( 2H), 2.30-2.40 (6H), 2.73-2.84 (2H), 3.06-3.14 (3H), 4.44-4.76 (1H), 6.76 -6.94 (1H), 7.36-7.49 (2H), 7.56-7.62 (1H), 7.90-8.02 (2H), 8.17-8.26 (1H ).

p00130Preparation 5. [trans-4- (methyl {7 - [(4-methylphenyl) sulfonyl] -7H-pyrrolo [2,3-d] pyrimidin-4-yl} amino) cyclohexyl] methyl-4-methylbenzenesulfonate

p00131To a solution of the compound of Preparation 6 (42.0 g, 0.16 mol) in dichloromethane (1 L), triethylamine (68.3 g, 0.68 mol) and 4-dimethylaminopyridine (1.0 g) are added , 8.2 mmol), followed by p-toluenesulfonyl chloride (62 g, 0.33 mol). The reaction mixture is stirred at room temperature for 2 h, before the addition of more p-toluenesulfonyl chloride (45.5 g, 0.24 mol). After stirring for 18 h, the mixture is concentrated in vacuo and a portion of the residue (approximately 309 g) is suspended in methanol (758 ml) for 15 min. Water is added to the suspension (600 ml) and saturated aqueous sodium bicarbonate solution (142 ml), and the mixture is stirred for 1 h. The solid material is collected by filtration and washed with methanol: water [1: 1, 50 ml], water (50 ml) and hexanes (50 ml). The solid is dried in vacuo at 60 ° C, giving the title compound (87.1 g).

p001321H NMR (d6-DMSO): 1.02-1.20 (2H), 1.53-1.75 (7H), 2.31-2.39 (3H), 2.39-2.47 ( 3H), 3.04-3.12 (3H), 3.80-3.91 (2H), 4.34-4.76 (1H), 6.78-6.93 (1H), 7.37 -7.54 (4H), 7.54-7.64 (1H), 7.75-7.84 (2H), 7.91-8.01 (2H), 8.14-8.25 (1H ).

p00133Preparation 6. {trans-4- [methyl (7H-pyrrolo [2,3-d] pyrimidin-4-yl) amino] cyclohexyl} -methanol

p0013410 A mixture of the trans-4- (methylamino) cyclohexyl] methanol compound is heated at 90 ° C for 15 h (can be prepared according to the procedure described in WO 2002/14267) (50.0 g, 0.35 mol), 4-chloro-7Hpyrrolo [2,3-d] pyrimidine (commercially available, 42.9 g, 0.27 mol) and potassium carbonate (57.3 g, 0.42 mol) in water ( 1 l) and 1,4-dioxane (100 ml). The compound of Preparation 7 (2.0 g, 14.0 mmol) is added to the mixture, and the reaction mixture is heated at 90 ° C for a further 1 h. After cooling to room temperature, stir the

p00135fifteen Mix for 1 h and collect the solid material by filtration, wash with water (150 ml) and dry under vacuum at 65 ° C, giving the title compound (72.7 g).

p001361H NMR (d6-DMSO): 1.00-1.19 (2H), 1.30-1.45 (1H), 1.52-1.77 (4H), 1.77-1.91 ( 2H), 3.09-3.20 (3H), 3.20-3.29 (2H), 4.37-4.51 (1H), 6.45-6.57 (1H), 7.06 -7.17 (1H), 8.01-8.14 (1H).

p00137Preparation 7. [trans-4- (methyl {7 - [(4-methylphenyl) sulfonyl] -7H-pyrrolo [2,3-d] pyrimidin-4-yl} amino) cyclohexyl] methanesulphinate

p00138To a solution of the compound of Preparation 4 (60.0 g, 0.418 mol) in acetone (600 ml), triethylamine (117.5 ml, 0.837 mol) and catalytic potassium iodide (3.4 g, 0, 05 mol), followed by 4-chloro-7H-pyrrolo [2,3-d] pyrimidine (commercially available, 102.8 g, 0.335 mol). The resulting mixture was heated to 60 ° C for 22 h. After allowing to cool to room temperature, acetone (300 ml) was added, followed by triethylamine (146.9 ml, 1.04 mol) and

p0013925 then mesyl chloride (81.7 ml, 1.047 mol). After stirring for 4 hours at room temperature, water (1.8 L) was added, after which the product precipitated. The product was filtered, dried and triturated with a mixture of MTBE-heptane (6: 4, 600 ml). A second crushing was carried out in MTBE-heptane and the resulting title compound (120 g) was obtained. 1H NMR (d6-DMSO):

p00140Preparation 8: [trans-4- (methylamino) cyclohexyl] methanol

p00141Vitride solution (65%, 767 ml, 2,465 mol) was added dropwise over 1 h to a solution of trans-4 [(tert-butoxycarbonyl) amino] cyclohexanecarboxylic acid (commercially available, 100 g, 0.4109 mol ) in toluene (1 l).

p001425 After the addition was completed, the reaction mixture was heated at reflux for 100-110 ° C. The reaction mixture was quenched with aqueous sodium sulfate solution (800 ml) at temperatures below 10 ° C. The reaction mixture was filtered through celite and the filter cake was washed with DCM (500 ml) followed by water (100 ml). The organic layer was separated and the aqueous layer was extracted twice with DCM (600 ml and then 400 ml). The combined organic layers were dried over sodium sulfate and concentrated in vacuo to give the title compound (62 g).

p0014310 1H NMR (CD3OD): 1.08-1.31 (4H), 1.51-1.64 (1H), 1.93-2.05 (2H), 2.10-2.22 (2H) , 2.38-2.50 (1H), 2.50-2.54 (3H), 3.48-3.55 (2H).

p00144Example 1a. Preparation of N-methyl-1- {trans-4- [methyl (7H-pyrrolo [2,3-d] pyrimidin-4-yl) amino] cyclohexyl} methanesulfonamide.

p00145fifteen To a solution of the compound of Preparation 1 (250.0 g, 0.48 mol) in 2-propanol (1.2 L), lithium hydroxide (48.7 g, 2.03 mol) in water ( 1.2 l). The reaction mixture is heated to 40 ° C for 8 h and then stirred at room temperature for 18 h. The mixture is filtered, washing through with 2-propanol: water (1: 1, 100 ml), and the filtrate is adjusted to pH 7.5 by the addition of hydrochloric acid (6 N). After stirring for 1 h, the solid material is collected by filtration, washed with 2-propanol: water (1: 2, 240 ml) and dried in vacuo at 60 ° C, giving the compound of

p00146twenty title (148.7 g) in the form of a free base (Example 1a).

p001471H NMR (d6-DMSO): 1.20-1.39 (2H), 1.62-1.75 (4H), 1.77-1.91 (1H), 1.97-2.11 ( 2H), 2.54-2.63 (3H), 2.89-2.99 (2H), 3.10-3.21 (3H), 4.44-4.86 (1 H), 6, 43-6.61 (1H), 7.01-7.19 (1H), 7.94-8.16 (1H).

p00148Example 1b Preparation of N-methyl-1- {trans-4- [methyl (7H-pyrrolo [2,3-d] pyrimidin-4-yl) amino] cyclohexyl} methanesulfonamide maleic acid salt

p0014925 A mixture of the compound of Example 1a (212.0 g, 628.3 mmol) and maleic acid (67.2 g, 579.0 mmol) in 1-butanol (3,200 ml) and water (400 ml) is stirred at temperature ambient for 18 h. The mixture is reduced in volume until

p001501,600 ml, by vacuum distillation (55 ° C, 10 kPa) and then cooled to 0 ° C. The resulting solid is collected by filtration, washed with heptane (500 ml) and dried in vacuo at 35 ° C, giving the N-methyl-1 {trans-4- [methyl (7H-pyrrolo [2,3] maleate salt. -d] pyrimidin-4-yl) amino] -cyclohexyl} methanesulfonamide (253.0 g) as a crystalline form

p0015130 known as the experimental A. MH + Form: 338.2; Expected: 338.2.

p001521H NMR (d6-DMSO): 1.24-1.38 (2H), 1.68-1.92 (5H), 2.00-2.11 (2H), 2.56-2.61 ( 3H), 2.91-3.00 (2H), 3.15-3.27 (3H), 4.39-4.70 (1H), 6.53-6.73 (1H), 7.16 -7.36 (1H), 8.07-8.29 (1H).

p00153Example 1c Process for collecting X-ray powder diffraction of maleic acid salt of N-methyl-1 {trans-4- [methyl (7H-pyrrolo [2,3-d] pyrimidin-4-yl) amino] cyclohexyl} -methanesulfonamide (Form A)

p00154X-ray powder diffraction patterns for Form A, maleic acid salt of N-methyl-1- {trans-4 [methyl (7H-pyrrolo [2,3-d] pyrimidin-4-yl) amino] cyclohexyl} -methanesulfonamide, were collected using a D4 5 x-ray powder diffractometer from Bruker-AXS Ltd. equipped with an automatic sample changer, a thetatheta goniometer, automatic beam divergence slit and a Vantec-1 PSD detector. The sample was prepared for analysis by mounting it on a silicon wafer sample holder with a low bottom cavity. The sample was rotated while irradiating with copper K-C1 X-rays (wavelength = 1.5406 Angstroms) with the X-ray tube running at 40 kV / 35 mA. The analyzes were performed with the goniometer operating in continuous mode

p0015510 set at a count of 0.2 seconds per stage at 0.018 º in a two theta interval from 2 º to 55 º. The results are summarized in Table 1 and Table 2.

p00156Table 1

<dl><dt>X-ray powder diffraction peaks expressed in 2-theta degrees, ± 0.2 degrees approximately </dt><dd /></dl>

<dl><dt>2-theta angle </dt><dd>Intensity (% of I) 2-theta angle Intensity (% of I) 2-theta angle Intensity (% of I) </dd></dl>

<dl><dt>6,178 </dt><dd> 72,6 17,997 32,3 24,86 19,8 </dd></dl>

<dl><dt>8,519 </dt><dd> 24,5 18,539 44,5 25,602 10,7 </dd></dl>

<dl><dt>12,601 </dt><dd> 88,4 20,298 18,2 26,582 13 </dd></dl>

<dl><dt>13,819 </dt><dd> 38 20,659 27,8 27,02 30,6 </dd></dl>

<dl><dt>15,478 </dt><dd> 34,3 21,583 11,1 27,721 18,7 </dd></dl>

<dl><dt>15,719 </dt><dd> 100 22,642 12,7 28,161 23,7 </dd></dl>

<dl><dt>16,32 </dt><dd> 27,1 23,08 12,1 28,38 28,9 </dd></dl>

p00157Table 2

<dl><dt>Selected X-ray powder diffraction peaks expressed in 2-theta degrees, ± 0.2 degrees approximately </dt><dd /></dl>

<dl><dt>2-theta angle </dt><dd>Dimension of the crystal lattice (d (A)) Intensity (% of I) </dd></dl>

<dl><dt>6,179 </dt><dd> 14,3 73 </dd></dl>

<dl><dt>12,601 </dt><dd> 7,01 88 </dd></dl>

<dl><dt>15,719 </dt><dd> 5,63 100 </dd></dl>

<dl><dt>18,539 </dt><dd> 4,78 45 </dd></dl>

<dl><dt>27,02 </dt><dd> 3,29 30,6 </dd></dl>

<dl><dt>28,38 </dt><dd> 3,14 28,9 </dd></dl>

p00158As is evident to any person skilled in the art, the results of any powder X-ray diffraction

p00159fifteen they may vary, and subsequent DRXPs will not be identical, even in the same batch of material. This variation may be due to the preparation of the test sample, the temperature, the particular X-ray diffractometer model used, the operator technique, etc. The term "approximately", if used in the definition of a peak of a powder X-ray diffraction pattern, is defined as the indicated value 28 ± 0.2 º28. Any determination of whether a crystalline form is Polymorphic Form A and is encompassed by the claims should be

p00160twenty interpret in light of the variability of this essay.

p00161This variability is demonstrated in Figure 1. The two different batches of Form A maleic acid salt of Nmethyl-1- {trans-4- [methyl (7H-pyrrolo [2,3-d] pyrimidin-4-yl) amino] cyclohexyl} -methanesulfonamide were subjected to the same DRXP diffractometer. The characteristic peaks of Figure 1 confirm that it is the polymorphic A form. However, the relative intensity of these peaks, as well as the other identification peaks varied slightly.

p0016225 Example 2. Preparation of N-cyclobutyl-1- {trans-4- [methyl (7H-pyrrolo [2,3-d] pyrimidin-4-yl) amino] cyclohexyl} methanesulfonamide

p00163Following the general procedure of Example 1 and making minor variations, but replacing the cyclobutamine precursor, the title compound is provided. Experimental MH +: 378.0; Expected: 378.2.

p001641H NMR (d6-DMSO): 1.22-1.32 (2H), 1.47-1.70 (6H), 1.78-2.04 (5H), 2.16-2.24 ( 2H), 2.85-2.86 (2H), 3.15 (3H). 3.68-3.78 (1H), 4.60-4.72 (1H), 6.51-6.54 (1H), 7.11-7.12 (1H), 7.44-7, 49 (1H), 8.08 (1H), 11.60 (1H).

p00165Example 3. Preparation of N-ethyl-1- {trans-4- [methyl (7H-pyrrolo [2,3-d] pyrimidin-4-yl) amino] cyclohexyl} methanesulfonamide

p0016610 Following the general procedure of Example 1 and making minor variations, but substituting the ethanamine precursor, the title compound is provided.

p00167Experimental MH +: 352.0; Expected: 352.2.

p001681H NMR (CDCl3): 1.24-1.44 (5H), 1.64-1.74 (2H), 1.87-2.09 (3H), 2.15-2.21 (2H) , 2.97-2.99 (2H), 3.18-3.27 (5H), 4.46-4.52 (1H), 4.74-4.86 (1H), 6.55 (1H ), 7.04 (1H), 8.28 (1H).

p00169fifteen Example 4. JAK Enzymatic Assay

p00170Materials: Recombinant JAK-2 (Catalog number PV4210) and JAK-3 (Catalog number PV3855) were purchased from (Invitrogen Corporation, Madison, WI). The recombinant JAK-1 (GST-JAK-1 (852-1142)) and Tyk-2 (GST-Tyk2 (870-1187, C1187S)) used in the present study were expressed and purified in Pfizer laboratories. Adenosine 5'-triphosphate (ATP) was obtained from Sigma Chemical Company, St. Louis, MO. The JAKtide Peptide

p00171twenty (peptide sequence, FITC-KGGEEEEYFELVKK (SEQ ID NO: 1)) used for the JAK-2 and JAK-3 assays and the IRS-1 peptide (peptide sequence, 5-FAM-KKSRGDYMTMQIG (SEQ ID NO. : 2)) used for the JAK-1 and Tyk-2 assays were purchased from (American Peptide Company, Sunnyvale, CA). Coating reagent 3 was purchased from (Caliper Life Sciences, Hopkinton, MA).

p00172Procedures: a peptide mobility change assay was used to quantify phosphorylation of the

p0017325 JAKtide peptide (JAK-2 and JAK-3) or IRS-1 peptide (JAK-1 and Tyk-2). The reactions were carried out in a 384-well plate (matrix MP-101) in a total volume of 10 microliters. The reaction mixtures contained 20 mM HEPES, pH 7.4, 10 mM magnesium chloride, 0.01% bovine serum albumin (ASB), 0.0005% Tween-20, ATP (4 micromolar for JAK-2 and JAK-3, 40 micromolar for JAK-1 and 7 micromolar for Tyk-2)), 2% DMSO and 1 micromolar peptide substrate (JAKtide peptide for JAK-2 and JAK-3 or IRS-1 for JAK-1 and Tyk-2). The

p0017430 Compounds were serially diluted in 100% dimethylsulfoxide and tested in a dose response of 11 points in duplicate or quadruplicate (200 nL of compound / DMSO was added per 10 microliter reaction). The reactions were initiated by the addition of enzyme to the final concentration of 2 nM JAK-2, 1 nM JAK-3, 7 nM Tyk2 or 20 nM JAK-1. The test was performed for 240 minutes for JAK-1, 150 minutes for JAK-2, 90 minutes for JAK-3 and 60 minutes for Tyk-2. The tests were stopped at the specified times with 20 microliters of 140 mM HEPES, 22.5 mM EDTA and 0.15% coating reagent 3. Plates were placed on a LabChip 3000 (LC3000) instrument (Caliper Life Sciences) to measure phosphorylated peptide formation. The data were analyzed using the Hits Well Analyzer software program (Caliper Life Sciences), obtaining the quantity of product formed.

p00175Next, the data was imported into an internal application, where each data point was expressed as% inhibition based on non-inhibited and non-enzymatic controls. Next, the dose response data was adjusted using a 4-parameter logistic equation (Equation 1) to determine an IC50 value.

p00176Where max is the adjusted uninhibited value, min is the adjusted full inhibition value and s is the factor of 10 pending. Using this protocol, the following results were generated for the title compounds of Example 1 and 2. Table 3

<dl><dt>Results of the JAK enzyme test </dt><dd /></dl>

<dl><dt>Ex. Nº </dt><dd>JAK-1 IC50 JAK-2 IC50 JAK-3 IC50 Tyk-2 IC50 </dd></dl>

<dl><dt>1st </dt><dd>9.53 nM 17.5 nM 95.1 nM 75.1 nM </dd></dl>

<dl><dt>2 </dt><dd>45.0 nM 101.0 nM 742 nM -</dd></dl>

p00177Example 5. In vitro canine T-cell proliferation assay

p00178Activation of T lymphocytes plays a key role in a variety of inflammatory disorders and

p00179fifteen autoimmune, as well as in asthma, allergies and pruritus. Since the activation of T lymphocytes, in part, can be triggered by cytokines that signal through the JAK-STAT pathway, a JAK inhibitor could be effective against such diseases that involve aberrant activation of T lymphocytes.

p00180Procedures: Complete canine blood was collected in sodium heparin tubes from 29 Beagle dogs and 23 mongrel dogs. Whole blood (20 µl) was seeded in 96-well plates (Costar 3598) with 180 µl of medium 20 (RPMI 1640, Gibco No. 21870-076, with 1% heat-inactivated fetal bovine serum, Gibco No. 10082 -39, 292 µg / ml L-glutamine, Gibco No. 250030-081, 100 U / ml penicillin and 100 µg streptomycin per ml, Gibco No. 15140122) containing the vehicle control or test compound (0.001 at 10 µM), concanavalin A (ConA; 1 µg / ml, Sigma C5275) and canine interleukin-2 (IL-2; fifty ng / ml, R&amp;D Systems 1815-CL / CF). Wells containing whole blood, medium with vehicle control and without ConA or IL-2 were used as background controls. The 25 plates were incubated at 37 ° C for 48 hours. Valued thymidine, 0.4! Ci / well (Perkin Elmer, Net027A-005MC) was added for a further 20 hours. The plates were frozen, and then thawed, washed and filtered using a Brandel MLR-96 cell harvester and pre-moistened filter mats (Wallac 1205-401, Perkin Elmer). The filters were dried at 60 ° C for one hour (Precision 16EG convection oven) and placed in filter sample bags (Wallac 1205-411, Perkin Elmer) with 10 ml scintillation liquid (Wallac 1205-440, Perkin

p0018130 Elmer) The counting was performed on the filters sealed in a Betaplate LKB Wallac 1205 liquid scintillation counter. The data was collected using the Gterm Betaplate v 1.1 program (Wallac copyright 1989-1990) and transformed into inhibition percentages, calculated using the following formula:

p00182Data were plotted as percent inhibition using GraphPad Prism 4.0, and IC50 curves 35 were adjusted using a point-to-point analysis.

p00183Results: The mean IC50 values ​​obtained when using whole blood of Beagle dogs were 66.3 nM for the compound of Example 1a; 410 nM for Example 2; and 83 nM for Example 3. The average IC50 value obtained when whole blood of mixed breed dogs was used was 138 nM for the compound of Example 1a.

p00184These data suggest that the compounds of the present invention are effective in inhibiting proliferation of T lymphocytes, a key feature in many diseases.

p00185Example 6. Trial of reduction of pruritus and dermatitis associated with fleas

p00186Pruritus and dermatitis associated with fleas are common skin conditions in dogs. Itching is one of the most serious clinical signs associated with flea-associated dermatitis, and continued scratching, rubbing of the face and nibbling of the hooves can lead to a variety of skin changes such as erythema, edema, alopecia, lichenification and hyperpigmentation. Pruritus and dermatitis associated with fleas can be induced experimentally. In these models, it has been observed that inflammatory cells and cytokines mediate immune reactions to allergens. Therefore, a JAK inhibitor that inhibits signal transduction of pruritogenic and proinflammatory cytokine receptors could be effective in inhibition, reduction

p00187or minimization of pruritus and dermatitis associated with fleas.

p00188Study design

p00189Twenty-eight male and female dogs of mixed breeds with a variable weight of 5 to 35 kg and over one year of age were infested with approximately 100 unleaded adult cat fleas (Ctenocephalides felis) 14 days before the start of dosing, and they were infested again with 30 fleas per dog every 4 days during the study. Seven days before dosing, twenty-four dogs were randomly distributed in three different treatment groups, placebo, 0.5 mg / kg or 0.25 mg / kg of the compound of Example 1b, based on the scores of a visual analog scale (EAV) of skin lesions. The treatments were administered orally twice a day for 28 days, and pruritic behavior was evaluated, as well as erythema and skin lesions during the study. Itching behavior was recorded by placing the dogs in pens with video recording capabilities, and recording their activity for 4 hours. Itching activity was quantified by determining how many seconds the dogs spent scratching. Cutaneous lesions were recorded by capturing images of the abdominal inguinal region, and severity was classified according to a visual analogue scale (VAS).

p00190Statistic analysis:

p00191The elapsed time of pruritic behavior captured by video was analyzed using a mixed linear model for repeated measurements. The model included the fixed effects of the treatment and the day of the study, and the interaction of the treatment and the day of the study. Random effects included the block, the interaction between the block and the treatment, and the error. Reference data for pruritic behavior (day -1) were used as a covariate in the analysis of pruritic behavior. The least squares means were used as estimates of the treatment means. The standard errors of the means were estimated by least squares and confidence intervals of 90% were constructed. The geometric means were calculated from the means by least squares for the logarithmically transformed data. A priori contrasts were used to evaluate the treatment. The differences in treatment were evaluated at the level of significance of 10% (P: 0.10).

p00192Results

p00193The results of the treatment are shown in Figure 2 and Figure 3. The lesions and erythema were significantly reduced in the 0.5 mg / kg group. Figure 2 illustrates the EAV scores on day 27 for Example 1b in dogs allergic to fleas (means by least squares). Significant reductions in pruritus were observed compared to placebo at a significance level of 10% at various time points during the study for both groups (days 1, 4 and 12 for the dose of 0.25 mg / kg and days 1 and 20 for the dose of 0.5 kg / mg). Figure 3 illustrates the pruritus seconds during the 4-hour recording for Example 1b in dogs allergic to fleas (Long geometric mean).

p00194Example 7. Cell Proliferation Inhibitor Assay

p00195Feline cell lines. MYA-1 and FETJ are feline T lymphoblast cell lines obtained from ATCC (Manassas, VA). These cells were cultured in complete RPMI 1640 medium supplemented with 10% SFB at 37 ° C in a humidified incubator with 5% CO2.

p00196Lymphoma tissue of canine lymphoma ex vivo

p00197The malignant lymph nodes were removed by the veterinary staff of the Michigan State University School of Veterinary Medicine (MSU), and placed in means of transport (Advanced RPMI 1640 complete medium supplemented with 10% fetal bovine serum (SFB), 100 U / ml of penicillin, 100 ug / ml of streptomycin and 0.25 ug / ml of amphotericin B (Invitrogen / Gibco®)). The nodes were processed within 24 hours of the extraction, chopping them into small pieces and passing them through a tissue screen. The cell suspensions were centrifuged at 200 xg, the supernatant was removed and the cell pellet was resuspended in NH4Cl for 10 minutes at room temperature. The cell suspension was pelleted by centrifugation; NH4Cl was removed and washed once with Hanks equilibrated saline solution (HBSS), followed by resuspension in proliferation medium (complete RPMI medium Advanced, 1% SFB, 50 nM 2-mercaptoethanol, 100 U / ml penicillin, 100 ug / ml streptomycin and 0.25 ug / ml amphotericin B). Next, the cell suspension was passed through a sieve for 100 µm nylon cells (BD-Falcon) and counting was performed using a hemocytometer. Cells were cultured well in proliferation medium alone, proliferation medium supplemented with 0.005% Pansorbin® (Staphylococcus aureus (CSA) cells fixed in heat-inactivated formalin, Calbiochem) and 10 ng / ml of canine IL-2 (R &amp; D Systems), or proliferation medium supplemented with 125 ng / ml of

p001985 concavalin A (Sigma) and 125 ng / ml of lipopolysaccharide (LPS; Calbiochem).

p00199Test procedure against in vitro proliferation

p00200Cultured cells were seeded in the medium described above in Costar 96-well plates (Coming) at a density of 1 x 103 cells / well (feline cell lines) or 2 x 105 cells / well (lymph node cells) and exposed at various concentrations of test compounds for up to 5 days at 37 ° C in a humidified incubator with 5% CO2. The effects on proliferation were determined using the CellTiter 96® AQueous non-radioactive cell proliferation assay (Promega) according to the manufacturer's instructions. In general, proliferation was measured indirectly using a soluble tetrazolium salt (MTS) and an electron coupling agent. The bioreduction of MTS in a soluble formazan product in tissue culture medium was monitored by absorbance at 490 nM in a Spectramax plate reader using the program

p00201fifteen Pro 4.6 Softmax software (Molecular Devices). The data was plotted as the percentage of DMSO control using GraphPad Prism 4.00, and the IC50 curves were adjusted using a nonlinear regression model with a sigmoid dose response.

p00202Results

p00203Table 4 demonstrates that the compound of Example 1 can inhibit the proliferation of the feline lymphoid cell line.

p00204twenty MYA-1 that depends on IL-2 for proliferation, but not an independent line of IL-2 (FETJ). The compound of formula IA or its salt can also inhibit the proliferation of canine ganglion tissue obtained from dogs diagnosed with T or B lymphocyte. These results suggest that a JAK inhibitor may be effective in the treatment of canine and feline lymphomas.

p00205Table 4

<dl><dt>Species </dt><dd>Cell line or lymph node Description Stimulant in culture medium IC50 (nM) </dd></dl>

<dl><dt>Feline </dt><dd>MYA-1 Lymphoid line 122 (n = 2) </dd></dl>

<dl><dt>Feline </dt><dd>FETJ Lymphoid line > 1000 </dd></dl>

<dl><dt>Canine </dt><dd>MSU LN 8 De novo T lymphocyte lymphoma LPS + ConA 357 </dd></dl>

<dl><dt>Canine </dt><dd>MSU LN 8 De novo T lymphocyte lymphoma SAC + IL-2 38 </dd></dl>

<dl><dt>Canine </dt><dd>MSU LN 9 De novo B lymphocyte lymphoma LPS + ConA 147 </dd></dl>

<dl><dt>Canine </dt><dd>MSU LN 9 De novo B lymphocyte lymphoma SAC + IL-2 100 </dd></dl>

<dl><dt>Canine </dt><dd>MSU LN 10 Chemotherapy-resistant B lymphocyte lymphoma LPS + ConA 687 </dd></dl>

<dl><dt>Canine </dt><dd>MSU LN 11 De novo B lymphocyte lymphoma LPS + ConA 64</dd></dl>