Salts of the janus kinase inhibitor (r)-3-(4-(7h-pyrrolo(2,3-d)pyrimidin-4-yl)-1h-pyrazol-1-yl)-3-cyclopentylpropanenitrile

The invention relates to salt forms of (R) -3- (4- (7H-pyrrolo [2,3-d] pyrimidin-4-yl) -1H-pyrazol-1-yl) -3-cyclopentylpropanenitrile, which are useful in modulating kinase activity Janusowa and in the treatment of diseases associated with Janus kinase activity, which, for example, include diseases associated with the immune system, skin diseases, bone marrow proliferative disorders, cancers and other diseases.

Background of the invention [0002] Protein kinases (PKs) are a group of enzymes that regulate a variety of important biological processes, including cell growth, survival and differentiation, organ formation and morphogenesis, neovascularization, tissue repair and regeneration. Protein kinases perform their physiological functions by catalyzing the phosphorylation of proteins (or substrates), thus modulating the cellular activities of substrates under various biological conditions. In addition to their functions in normal tissues / organs, many protein kinases also play more specialized roles in the course of human diseases, including cancer. Disturbances in the activity of a subset of protein kinases (also referred to as oncogenic protein kinases) may be the cause of tumor formation and growth, subsequently contributing to its maintenance and development (Blume-Jensen P. et al., Nature 2001, 411 (6835): 355- 365). To date, oncogenic protein kinases are one of the largest and most attractive groups of target proteins for cancer therapy and drug development.

[0003] The Janus kinase (JAK) family plays a role in the cytokine-dependent regulation of cell proliferation and function involved in the immune response. There are currently four known members of the JAK kinase family found in mammals: JAK1 (also known as Janus kinase 1), JAK2 (also known as Janus kinase 2), JAK3 (also known as leukocyte Janus kinase; JAKL; LJAK and Janus kinase 3) and TYK2 (also known as protein tyrosine kinase 2). Proteins from the JAK kinase family range from 120 to 140 kDa and contain seven conserved JAK (JH) homologous domains; one of them is a functional, catalytic kinase domain, and the other is a pseudokinase domain, potentially having a regulatory function and / or serving as a STAT attachment site (Scott, Godshall et al. 2002, supra).

[0004] Blocking signal transduction at the JAK kinase level gives hope for the development of cancer therapy for humans. It is also anticipated that JAK kinase inhibition may have therapeutic benefits in patients suffering from skin immune diseases such as psoriasis and skin sensitization. Accordingly, inhibitors of Janus kinases or related kinases are widely sought after and classes of effective compounds are given in several publications. For example, certain JAK kinase inhibitors including (R) -3- (4- (7H-pyrrolo [2,3-d] pyrimidin-43-yl) -1H-pyrazol-1-yl) -3-cyclopentylpropanenitrile as shown below , described in US Ser. 11 / 637,545, filed December 12, 2006.

<img file="PL2173752T3_D0001.tif" />

[0005] Thus, there is a continuing need for new or improved forms of existing Janus kinase inhibitors to develop new, improved and more effective pharmaceutical forms for the treatment of cancer and other diseases. The salt forms and methods described herein are directed to these needs and other applications.

SUMMARY OF THE INVENTION [0006] The invention provides, inter alia, salts selected from:

(R) -3- (4- (7H-pyrrolo [2,3-d] pyrimidin-4-yl) -1H-pyrazol-1-yl) -3-cyclopentylpropanenitrile maleate;

(R) -3- (4- (7H-pyrrolo [2,3-d] pyrimidin-4-yl) -1H-pyrazol-1-yl) -3-cyclopentylpropanenitrile sulfate; and R) -3- (4- (7H-pyrrolo [2,3-d] pyrimidin-4-yl) -1H-pyrazol-1-yl) -3-cyclopentylpropanenitrile phosphate.

[0007] In addition, the invention provides methods for producing salts according to the invention which comprise a combination of (R) -3- (4- (7H-pyrrolo [2,3-d] pyrimidin-4-yl) -1H-pyrazol-1-yl) - Cyclopentylpropanenitrile with maleic acid, sulfuric acid or phosphoric acid.

[0008] In addition, the invention provides compositions comprising salt forms of a compound of the invention and at least one pharmaceutically acceptable carrier.

[0009] In addition, the invention provides methods for modulating JAK kinase activity, including contacting the JAK kinase with a salt of a compound of the invention.

[0010] In addition, the invention provides compounds for use in methods of treating a disease associated with JAK kinase activity in a patient, comprising administering to the patient a therapeutically effective amount of a salt of a compound of the invention.

[0011] Furthermore, the invention provides compounds for use in methods of treating cancer, skin diseases or inflammation in a patient, comprising administering to the patient a therapeutically effective amount of a salt of a compound of the invention.

[0012] In addition, the invention provides salts of a compound of the invention for use in methods of treating the human or animal body by therapy.

[0013] In addition, the invention provides salts of a compound of the invention for use in the treatment of cancer, skin diseases or inflammation.

[0014] In addition, the invention provides the use of a salt of a compound of the invention for the manufacture of a medicament used in the treatment of any disease or disorder mentioned herein.

DETAILED DESCRIPTION OF THE INVENTION [0015] The invention relates, among others, to (R) -3- (4- (7H-pyrrolid [2,3-d] pyrimidin-4-yl) -1H-pyrazol-1-yl) -3-cyclopentylpropanenitrile as JAK kinase inhibitor selected from maleic acid, sulfuric acid and phosphoric acid salts. These salts modulate the activity of one or more JAK kinases and are useful, for example, in the treatment of diseases associated with kinase expression or activity

HOW.

[0016] Salts of the compound of the invention have more advantageous properties than the free base form and other salts. In particular, they are high-crystalline salts, which may facilitate the production of pharmaceutical preparations and facilitate the preparation, processing and storage of the active substance. Compared to the free base form, the salts of the invention also have better water solubility, dissolution rate, chemical stability (longer shelf life), are compatible with excipients and reproducibility.

[0017] In some embodiments, the salts of the compound of the invention are substantially isolated. By "substantially isolated" it is meant that the salt is at least partially or mostly separated from the environment in which it was formed or detected. Partial separation may include, for example, the salt enriched composition of the invention. Substantial separation may include compositions comprising 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 about 97% or at least about 99% by weight of salt.

[0018] Salts of the compound of the invention also include all isotopes of atoms occurring in the salts. Isotopes include atoms that have the same atomic number but different mass numbers. For example, isotopes of hydrogen include tritium and deuterium.

[0019] Salts of a compound of the invention can be prepared using known techniques. Typically, the salt form can be prepared by combining the free base compound and the anion containing acid in solution with the desired salt form, and then isolating the salt product in solid form from the reaction solution (e.g., by crystallization, precipitation, evaporation under reduced pressure, etc.). Other techniques for salt production may be used.

Methods of application [0020] Salts of the compound of the invention can modulate the activity of one or more Janus kinases (JAKs). The term "modulate" means the ability to increase or decrease the activity of one or more members of the JAK kinase family. Accordingly, the compounds of the invention can be used in methods of modulating JAK kinase, by contacting JAK kinase with one or more compounds or compositions described herein. In some embodiments, the salts of the invention may act as inhibitors of one or more JAK kinases. In some embodiments, compounds of the invention can stimulate the activity of one or more JAK kinases. In further embodiments, compounds of the invention can be used to modulate JAK kinase activity in a patient in need of receptor modulation by administering a salt of the invention in an amount that will induce this modulation.

[0021] JAK kinases to which they bind and / or which are modulated by the present salts include each member of the JAK kinase family. In some embodiments, the JAK kinase is JAK1, JAK2, JAK3 or TYK2 kinase. In some embodiments, the JAK kinase is JAK1 or JAK2 kinase. In some embodiments, the JAK kinase is JAK2 kinase. In some embodiments, the JAK kinase is JAK3 kinase.

[0022] The salts of the invention may be selective. By "selective" is meant that the compound binds to or inhibits JAK kinase with correspondingly greater affinity or potency as compared to at least one other JAK kinase. In some embodiments, the compounds of the invention are more potent selective JAK1 or JAK2 kinase inhibitors than JAK3 and / or TYK2. In some embodiments, the salts of the compound of the invention are selective JAK2 kinase inhibitors (e.g. stronger than JAK1, JAK3 and TYK2 kinases). Without being limited by theory, since JAK3 inhibitors can cause immunosuppressive effects, a compound that is more selective for JAK2 than JAK3 and which is useful in treating cancer (such as, for example, multiple myeloma) may have the added benefit of being less the amount of side effects caused by immunosuppression. The selectivity may be at least 5 times, 10 times, at least 20 times, at least 50 times, at least 100 times, at least 200 times, at least 500 times or at least 1000 times. Selectivity can be measured by conventional methods used in the art. In some embodiments, selectivity can be tested at the Km value of each enzyme. In some embodiments, greater selectivity of a salt of a compound of the invention relative to JAK2 over JAK3 can be determined by determining the cellular ATP concentration.

[0023] Another aspect of the invention relates to compounds used in methods of treating a JAK kinase related disease or disorder in an individual (e.g., a patient), by administering to the subject in need of such treatment, a salt of the invention or a pharmaceutical composition thereof in a therapeutically effective amount or dose. JAK kinase associated disease can include any disease, disorder or condition that is directly or indirectly associated with JAK kinase expression or activity, including overexpression and / or abnormal levels of activity. JAK kinase associated disease can also include any disease, disorder or condition that can be prevented, alleviated or cured by modulation of JAK kinase activity.

[0024] Examples of JAK kinase-related diseases include diseases associated with the immune system including, for example, organ transplant rejection (e.g. allograft rejection and graft versus host disease).

[0025] Further examples of JAK kinase related diseases include autoimmune diseases such as multiple sclerosis, rheumatoid arthritis, juvenile arthritis, type I diabetes, lupus, psoriasis, inflammatory bowel disease, ulcerative colitis, Crohn's disease, myasthenia gravis, IgA nephropathy , autoimmune thyroid disorders and the like. In some embodiments, the autoimmune disease is an autoimmune bullous skin disease such as pemphigus vulgaris (PV) or bullous pemphigoid (BP).

[0026] Further examples of JAK kinase related diseases include allergic conditions such as asthma, food allergies, atopic dermatitis and rhinitis. Further examples of JAK kinase related diseases include diseases caused by viruses such as Epstein-Barr virus (EBV), hepatitis B, hepatitis C, HIV, HTLV 1, varicella and zoster virus (VZV) and human papillomavirus ( HPV).

[0027] Further examples of JAK kinase related diseases or conditions include skin diseases such as psoriasis (e.g. psoriasis vulgaris), atopic dermatitis, skin rash, skin irritation, allergic reaction (e.g. contact dermatitis or allergic contact dermatitis). For example, certain substances, including some pharmaceutical agents, administered topically may cause an allergic reaction. In some embodiments, the simultaneous or sequential administration of at least one JAK kinase inhibitor of the invention together with an agent causing an adverse allergic reaction may be helpful in treating such an allergic reaction or dermatitis. In some embodiments, skin lesions are treated with topical administration of at least one JAK kinase inhibitor of the invention.

[0028] In further embodiments, the JAK kinase-related disease is cancer, including that characterized by solid tumors (e.g., prostate cancer, kidney cancer, liver cancer, pancreatic cancer, stomach cancer, breast cancer, lung cancer, head and neck cancers, thyroid cancer, glioma, Kaposi's sarcoma, Castleman's disease, melanoma, etc.), hematological cancers (e.g. 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. Examples of cutaneous T cell lymphomas include Sezary syndrome and mycosis fungoides.

[0029] Diseases associated with JAK kinase may further include those that are characterized by the expression of a mutated JAK2 kinase, such as those having at least one mutation in the pseudokinase domain (eg, JAK2V617F).

[0030] In addition, JAK kinase related diseases may include myeloproliferative disorders (MPD) such as polycythemia vera (PV), essential thrombocythaemia (ET), bone marrow metaplasia (MMM), chronic myelogenous leukemia (CML), chronic myelomonocytic leukemia (CMML), hypereosinophilic syndrome (HES), systemic mastocytosis (SMCD) and the like.

[0031] Other JAK kinase related diseases include inflammation and inflammatory diseases. Exemplary inflammatory diseases include inflammatory eye diseases (e.g., iritis, uveitis, scleritis, conjunctivitis or related diseases), inflammatory diseases of the respiratory tract (e.g. upper respiratory tract (including nasal and sinus such as rhinitis or sinusitis or lower respiratory tract (including bronchitis, chronic obstructive pulmonary disease and the like), inflammatory myopathy such as myocarditis and other inflammatory diseases. Other inflammatory diseases that can be treated with the compounds of the invention include systemic inflammatory response syndrome (SIRS) and septic shock.

[0032] In addition, the JAK kinase inhibitors described herein can be used to treat an ischemia-reperfusion injury or disease or condition associated with an inflammatory process and associated ischemic event, such as stroke or cardiac arrest. In addition, the JAK kinase inhibitors described herein can be used to treat anorexia, cachexia or fatigue that may be caused or associated with cancer. In addition, the JAK kinase inhibitors described herein can be used to treat restenosis, scleroderma or fibrosis. In addition, the JAK kinase inhibitors described herein can be used to treat conditions associated with hypoxia or astrogliosis such as, for example, diabetic retinopathy, cancer, or neurodegeneration. See, e.g., Dudley AC 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.

[0033] Furthermore, the JAK kinase inhibitors described herein can be used to treat gout and enlarged prostate due to, for example, benign hyperplasia or hyperplasia.

[0034] The term "contacting" as used herein refers to the combination of indicated fragments in an in vitro or in vivo system. For example, "contacting" the JAK kinase with a salt of a compound of the invention includes administering a salt of a compound of the invention to a subject or patient such as a human having a JAK kinase, as well as, for example, introducing a salt of the compound of the invention into a sample containing a cellular or purified preparation containing JAK kinase.

[0035] As used interchangeably herein, the term "subject" or "patient" refers to any animal, including mammals, preferably mice, rats, other rodents, rabbits, dogs, cats, pigs, cattle, sheep, horses or primates, and most preferably to people.

[0036] The term "therapeutically effective amount" as used herein refers to that amount of active salt or pharmaceutical agent that elicits the biological or therapeutic response sought by the researcher, veterinarian, physician or other clinician in the tissue, system, animal, and in the subject or human.

[0037] The term "treat" or "treatment" as used herein refers to one or more of the following: (1) preventing disease; for example, preventing a disease, condition or disorder in an individual that may be predisposed to the disease, condition or disorder but who does not yet experience or display the pathology or symptoms of the disease; (2) inhibiting the disease; for example, inhibiting the disease, condition or disorder in an individual who is experiencing or displaying the pathology or symptoms of the disease, condition or disorder; and (3) relieving the disease; for example, ameliorating a disease, condition or disorder in an individual who is experiencing or displaying the pathology or symptoms of the disease, condition or disorder (i.e., reversing the pathology and / or symptoms) by, e.g., reducing the severity of the disease.

Combination Treatment [0038] One or more additional pharmaceutical agents such as, for example, chemotherapeutic agents, 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 agents may be used in combination with the salts of the compound of the invention for the treatment of diseases, disorders or conditions associated with JAK kinase. One or more additional pharmaceutical agents may be administered to the patient simultaneously or sequentially.

[0039] Exemplary chemotherapeutic agents include proteasome inhibitors (e.g., bortezomib), thalidomide, revlimid, and DNA damaging agents such as melphalan, doxorubicin, cyclophosphamide, vincristine, etoposide, carmustine and the like.

[0040] Exemplary steroids include corticosteroids such as dexamethasone or prednisone.

[0041] Exemplary Bcr-Abl inhibitors include types and types of compounds and their pharmaceutically acceptable salts, disclosed in US Patent 5,521,184, WO 04/005281, EP2005 / 009967, EP2005 / 010408 and US Ser. 60 / 578.491.

[0042] Exemplary suitable Flt-1 inhibitors include compounds and pharmaceutically acceptable salts thereof, such as those disclosed in WO 03/037347, WO 03/099771 and WO 04/046120. [0043] Exemplary suitable RAF inhibitors include compounds and pharmaceutically acceptable salts thereof, such as those disclosed in WO 00/09495 and WO 05/028444.

[0044] Exemplary suitable FAK inhibitors include compounds and pharmaceutically acceptable salts thereof, such as those disclosed in WO 04/080980, WO 04/056786, WO 03/024967, WO 01/064655, WO 00/053595 and WO 01/014402 .

[0045] In some embodiments, the salts of the compound of the invention can be used in combination with other kinase inhibitors such as imatinib, in particular for the treatment of patients resistant to imatinib or other kinases.

[0046] In some embodiments, in the treatment of a cancer such as multiple myeloma, one or more salts of a compound of the invention may be used in combination with chemotherapeutic agents, and compared to the response to the chemotherapeutic agent itself, may improve the response to treatment without exacerbating its toxic effects. . Examples of additional pharmaceutical agents used in the treatment of multiple myeloma may include, but are not limited to, for example, melphalan, melphalan together with prednisone [MP], doxorubicin, dexamethasone and Velcade (bortezomib). In addition, additional agents used to treat multiple myeloma include Bcr-Abl, Flt-3, RAF and FAK kinase inhibitors. Additive or synergistic effects are the desired effects of combining the JAK kinase inhibitor of the invention with an additional agent. In addition, the resistance of multiple myeloma cells to agents such as dexamethasone may be inhibited after treatment with a JAK kinase inhibitor of the invention. The agents may be combined with the compounds of the invention in a single dose or administered continuously or they may be administered simultaneously or sequentially as separate doses.

[0047] In some embodiments, a corticosteroid, such as dexamethasone, is administered to a patient in combination with at least one JAK kinase inhibitor, wherein dexamethasone is not administered continuously, but intermittently.

[0048] In certain further embodiments, the combination of one or more JAK kinase inhibitors of the invention with other therapeutic agents can be administered to a patient before, during and / or after a bone marrow or stem cell transplant.

Pharmaceutical Forms and Dosage Forms [0049] When the salts of the invention are used as pharmaceuticals, they may be administered in the form of a pharmaceutical composition. These compositions can be prepared in a manner well known in the pharmaceutical art and can be administered by various routes depending on whether local or systemic treatment is desired and the area being treated. Administration can be local (including transdermal, epidermal, intraocular and mucosal administration, including intranasal, vaginal and rectal administration), pulmonary (e.g. by inhalation or insufflation of powders or aerosols, including through a nebulizer; intratracheal or intranasal) , oral or parenteral. Parenteral administration includes, intravenous, intraarterial, subcutaneous, intraperitoneal, intramuscular or intravenous injection or infusion; or intranasal administration, e.g. intrathecal or intraventricular administration. Parenteral administration can be as a single bolus dose or can be carried out, for example, by means of a continuous perfusion perfusion pump. Pharmaceutical compositions and topical dosage forms may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Typical pharmaceutical carriers, aqueous, powder or oil bases, thickeners and the like may be necessary or desirable. Coated condoms, gloves and the like may also be useful.

[0050] The invention also includes pharmaceutical compositions which contain as active substance one or more of the above compounds of the invention in combination with one or more pharmaceutically acceptable carriers (excipients). When preparing the compositions of the invention, the active substance is usually mixed with the excipient, diluted with the excipient, or enclosed in an excipient carrier in the form of, for example, a capsule, sachet, paper or other packaging. When the excipient serves as a diluent, it may be in solid, semi-solid or liquid form, acting as a vehicle, carrier or medium for the active substance. Thus, the compositions may be in the form of tablets, pills, powders, lozenges, sachets, capsules, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium), ointments containing, for example, 10% by weight of the active substance, soft and hard gelatin capsules, suppositories, sterile injectable solutions and sterile packaged powders.

[0051] During the preparation of the dosage form, before being combined with the other ingredients, the active substance may be ground to obtain the appropriate particle size. If the active substance is substantially insoluble, it can be ground to particles smaller than 200 mesh. If the active substance is substantially water-soluble, the particle size can be adjusted by milling to obtain a substantially uniform distribution in the form of the drug, e.g. about 40 mesh.

[0052] The compounds of the invention can be milled using known procedures, such as wet milling, to obtain particles of a size suitable for making tablets and other types of drug forms. Ground formulations (nanoparticles) of the compounds of the invention can be prepared by methods known in the art, for example, see International Patent Application WO 2002/000196.

[0053] Some examples of suitable excipients include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup and methyl. In addition, dosage forms may include: lubricants such as talc, magnesium stearate, and mineral oil; moisturizers; emulsifying and suspending agents; preservatives such as methyl and propylhydroxybenzoates; sweeteners; and flavorings. The compositions of the invention may be formulated such that, when administered to a patient using methods known in the art, rapid, long-lasting or delayed release of the active ingredient is obtained.

[0054] The compositions may be formulated into a unit dosage form, each dose containing from about 5 to about 1000 mg (1 g), more typically about 100 to about 500 mg of active ingredient. The term "unit dosage forms" refers to physically discrete units used as unitary dosages for humans and other mammals, each unit containing a predetermined quantity of active substance calculated so that, when combined with the appropriate pharmaceutical excipient, produces the desired therapeutic effect .

[0055] The active substance may be effective over a wide dosage range and is generally administered in a pharmaceutically effective amount. However, it will be understood that the amount of compound properly administered will typically be determined by the physician according to the circumstances including the condition being treated, the mode of administration chosen, the particular compound being administered, the age, weight and individual response of the patient, the severity of the patient's symptoms and the like.

[0056] To form solid compositions such as tablets, the main active substance is mixed with the pharmaceutical excipient to form a solid consistency preformulation composition that contains a homogeneous mixture of a compound of the invention. When the pre-formulation compositions are referred to as homogeneous, it means that the active substance is usually dispersed evenly throughout the composition in such a way that the composition can easily be divided into equally effective unit dosage forms such as tablets, pills and capsules. The solid preformulation is then divided into unit dosage forms as described above, which contains, for example, from about 0.1 to about 1000 mg of the active ingredient of the invention.

[0057] The tablets or pills of the invention may be coated or otherwise prepared so as to produce a dosage form that allows for obtaining a beneficial, prolonged effect. For example, the tablet or pill may comprise a dose of the component in the inner and outer parts, the latter surrounding the first. The two components can be separated by a layer which dissolves in the intestine, which prevents disintegration in the stomach and allows the internal component to pass intact into the duodenum or for sustained release. Many materials including several polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol and cellulose acetate can be used to form such coatings or layers that dissolve in the intestine.

[0058] The compounds and compositions of the invention for oral or injectable administration may be in liquid form including aqueous solutions, suitably flavored syrups, aqueous or oily suspensions, and flavored emulsions with edible oils, such as cottonseed oil, sesame oil, oil coconut or peanut oil, as well as elixirs and similar pharmaceuticals.

[0059] Compositions for inhalation or insufflation include solutions and suspensions in pharmaceutically acceptable, aqueous or organic solvents, or mixtures thereof, and powders. As described above, the compositions in liquid or solid form may contain suitable pharmaceutically acceptable excipients. In some embodiments, the compositions are administered orally or nasally for local or systemic effect. The compositions can be nebulized by the use of inert gases. Nebulized solutions can be inhaled directly from the nebulizing device or the nebulized device can be attached to a face mask tent or an intermittent positive pressure ventilation apparatus. Solution, suspension or powder compositions can be administered orally or nasally by devices that deliver the form of the drug in an appropriate manner.

[0060] The amount of salt or composition administered to a patient will vary, depending on what is administered, the purpose of administration, such as prophylactic or therapeutic, the condition of the patient, method of administration and the like. In therapeutic applications, the compositions may be administered to a patient who is already sick in an amount sufficient to cure or at least partially inhibit the symptoms of the disease and its complications. Effective doses will depend on the condition being treated, as well as the opinion of the treating physician based on factors such as severity, age, weight, and general condition of the patient, and the like.

[0061] The compositions administered to a patient may take the form of the pharmaceutical compositions described above. The compositions can be sterilized by classical sterilization methods or by sterile filtration. Aqueous solutions for use may be packaged unchanged or may be lyophilized, wherein such lyophilized preparation is combined with a sterile aqueous carrier prior to administration. The pH of the combined preparations will usually be from 3 to 11, more preferably from 5 to 9, and most preferably from 7 to

8. It will be understood that the use of some of the above excipients, carriers or stabilizers will result in the formation of pharmaceutical salts.

[0062] The therapeutic dose of the salt of the invention may vary depending on, for example, the particular application for which the treatment is being conducted, the method of administering the compound, the patient's health and condition, and the judgment of the attending physician. The ratio or concentration of the salts of the invention in the pharmaceutical composition may vary depending on many factors, including dosage, chemical properties (e.g., hydrophobicity) and route of administration. For example, the salts of the invention may be provided in a physiological buffered aqueous solution containing from about 0.1 to about 10% by weight of the compound for parenteral administration.

Some typical dosage ranges are from about 1 μg / kg to about 1 g / kg body weight per day. In some embodiments, the dosage range is from about 0.01 mg / kg to about 100 mg / kg body weight per day. The dosage may depend on variables such as the type and degree of disease or disorder progression, the patient's overall health, the relative biological effectiveness of the selected compound, the form of the excipient, and the route of administration. Effective doses can be extrapolated from dose-response curves derived from in vitro systems or animal test models.

[0063] In addition, the compositions of the invention may contain one or more additional pharmaceutical agents such as a chemotherapeutic, steroid, anti-inflammatory or immunosuppressive compound, examples of which are mentioned above.

Labeled Compounds and Assay Methods [0064] Another aspect of the invention relates to the labeled salts of the invention (radiolabelled, fluorescent, etc.) that would be useful not only in imaging techniques, but also in in vitro and in vivo assays, in localization and quantitative assay JAK kinases in tissue samples, including human, and in identifying JAK kinase ligands by inhibiting binding of the labeled compound. Accordingly, the invention includes JAK kinase assays that utilize such labeled compounds.

[0065] In addition, the invention includes isotope-labeled salts of the invention. A "isotope" or "radiologically labeled" compound is a salt of the invention in which one or more atoms are replaced or substituted with an atom whose atomic mass or number is different from those typically found in nature (i.e. naturally occurring).

Suitable radionuclides that can be included in the compounds of the invention include, but are not limited to ²H also referred to as D, deuterium), ³H (also known as T, tritium) ¹¹C ¹³C ¹⁴C ¹³N ¹⁵N ³⁶Cl, ⁸²br, ⁷⁵br, ⁷⁶br, ⁷⁷br, ¹²³AND, ¹²⁴AND,

The radionuclide that is incorporated into exemplary radiolabeled compounds will depend on the particular use of the compound. For example, in general, compounds containing will be most useful for in vitro metalloprotease labeling and competition assays³H ¹⁴C ⁸²br, ¹²⁵AND,

17 18 18 35 ¹⁵ABOUT, ¹⁷ABOUT, ¹⁸ABOUT, ¹⁸F ³⁵S

125 131 ¹²⁵And and ¹³¹AND.

131

And / or ³⁵S. For use in radio imaging, they will generally be most useful ¹¹C ¹⁸F

AND,

AND,

AND,

AND, ⁷⁵br, ⁷⁶Br or ⁷⁷Br.

[0066] It is understood that "radiolabeled" or "labeled compound" means a salt that contains at least one radionuclide. In some embodiments, the radionuclide is selected from the group consisting of³H ¹⁴C ¹²⁵AND, ³⁵S and ⁸²Br. [0067] In addition, the invention may include synthetic methods for incorporating radioisotopes in compounds of the invention. Synthetic methods for incorporating radioisotopes into organic compounds are well known in the art, and those skilled in the art will readily recognize those methods that are suitable for the compounds of the invention.

[0068] The labeled salt of the invention can be used in a screening assay to identify / evaluate compounds. For example, a newly-synthesized or identified compound (i.e., a test compound) that has been labeled can be evaluated for its ability to bind to JAK kinase by controlling its concentration change by tracking the label when contacting with JAK kinase. For example, a test (labeled) compound may be evaluated for its ability to reduce the binding of another compound that is known to bind to JAK kinase (i.e., reference compound). Accordingly, the ability of a test compound to compete with a reference compound for binding to JAK kinase is directly correlated with its affinity. In contrast, in other screening tests, the reference compound is labeled, while the test compound is unlabelled. Accordingly, the concentration of the labeled reference compound is controlled to determine the competition between the reference compound and the test compound, and thus the relative affinity of the test compound is determined.

Kits [0069] The invention also includes pharmaceutical kits comprising one or more containers with a pharmaceutical composition containing a therapeutically effective amount of a salt according to the invention that is useful, for example, in the treatment or prevention of diseases or disorders associated with JAK kinases, such as cancer, inflammation or skin diseases. It will be apparent to those skilled in the art that, if desired, such kits may additionally contain one or more different, typical components of a pharmaceutical kit such as, for example, containers with one or more pharmaceutically acceptable carriers, additional containers, etc. The kit may also contain instructions or in the form of leaflets or labels indicating the amounts of ingredients administered, administration guidelines and / or mixing guidelines.

[0070] The invention will be described in more detail based on specific examples. The following examples are given to illustrate the invention and are not intended to be limiting in any way. Those skilled in the art will easily recognize a number of non-critical parameters that can be changed or modified, in fact giving the same results.

EXAMPLES

Example 1: Preparation of (R) -3- (4- (7H-pyrrolo [2,3-d] pyrimidin-4-yl) -1H-pyrazol-1-yl) -3-cyclopentylpropanenitrile maleate.

[0071] To the tube was added (R) -3- (4- (7H-pyrrolo [2,3d] pyrimidin-4-yl) -1H-pyrazol-1-yl) -3-cyclopentylpropanenitrile (153.7 mg, 0 , 5 mmol) and maleic acid (61.7 mg) followed by isopropyl alcohol (IPA) (4 ml). The resulting mixture was heated to obtain a clear solution, cooled to room temperature, and then stirred for another 2.5 hours. The precipitate was filtered off and then rinsed with 0.8 ml cold IPA. dried in vacuo to constant weight (173 mg) to obtain the final product as a salt.

[0072] Thanks ¹H NMR showed that the signal to salt ratio of maleic acid was 1: 1, and its crystallinity was confirmed by X-ray powder diffraction (XRPD). Differential scanning calorimetry (DSC) gave a sharp melting peak at about 175.96 ° C (onset at

175.67 ° C). During thermogravimetric analysis (TGA), the product showed only slight weight loss up to 150 ° C.

Example 2: Preparation of (R) -3- (4- (7H-pyrrolo [2,3d] pyrimidin-4-yl) -1H-pyrazol-1-yl) -3-cyclopentylpropanenitrile phosphate [0073] (R) - 3- (4- (7H-pyrrolo [2,3d] pyrimidin-4-yl) -1H-pyrazol-1-yl) -3-cyclopentylpropanenitrile (153.5 mg) and phosphoric acid (56.6 mg) followed by isopropyl alcohol (IPA) (5.75 ml). The resulting mixture was heated to obtain a clear solution, cooled to room temperature, and then stirred for another 2 hours. The precipitate was filtered off and then rinsed with 0.6 ml cold IPA. the precipitate was dried under reduced pressure to a constant weight (171.7 mg) to obtain the final product as a salt.

[0074] Thanks ¹H NMR showed that the signal to salt ratio of phosphoric acid was 1: 1, and its crystallinity was confirmed by X-ray powder diffraction (XRPD). Differential scanning calorimetry (DSC) gave a sharp melting peak at about 198.66 ° C. During TGA analysis it was shown that up to 200 ° C the product showed only slight weight loss.

Example 3: Preparation of (R) -3- (4- (7H-pyrrolo [2,3d] pyrimidin-4-yl) -1H-pyrazol-1-yl) -3-cyclopentylpropanenitrile sulfate [0075] (R ) -3- (4- (7H-pyrrolo [2,3d] pyrimidin-4-yl) -1H-pyrazol-1-yl) -3-cyclopentylpropanenitrile (153.0 mg) and sulfuric acid (56.1 mg) followed by acetonitrile (7.0 mL). The resulting mixture was heated to obtain a clear solution, cooled to room temperature, and then stirred for another 2 hours. The precipitate was filtered off and then rinsed with 0.8 ml cold acetonitrile. The resulting precipitate was dried under reduced pressure to a constant weight (180 mg) to obtain the final product as a salt.

[0076] Thanks ¹H NMR showed that the signal to salt ratio of sulfuric acid was 1: 1, and its crystallinity was confirmed by X-ray powder diffraction (XRPD). Differential scanning calorimetry (DSC) gave a sharp melting peak at about 186.78 ° C. The product showed only slight weight loss up to 175 ° C.

Example A

In vitro JAK kinase assay [0077] The inhibitory effect of test compounds on JAK target kinases was tested according to the in vitro assay described by Park et al., Analytical Biochemistry 1999, 269, 94-104. The human kinase JAK1 (837-1142 aa), JAK2 (828-1132 aa) and JAK3 (781-1124 aa) catalytic domains with His tag at the N-terminus are expressed using baculovirus in insect cells and then purified. By measuring the phosphorylation of a biotinylated peptide, the catalytic activity of JAK1, JAK2 or JAK3 kinases is determined. The phosphorylated peptide was determined by HTRF (

homogenous time resolved fluorescence. IC50 values ​​of compounds were measured for each kinase in reaction mixtures containing; enzyme, ATP and 500 nM peptide, suspended in 50mM Tris buffer (pH 7.8) with 100 mM NaCl, 5 mM DTT and 0.1 mg / ml (0.01%) BSA. The ATP concentration in the reaction mixtures was 90 μΜ for Jaki kinase, 30 μΜ for Jak2 kinase and 3 μΜ for Jak3 kinase. Reactions were carried out at room temperature for 1 h and then inhibited by the addition of assay buffer containing 20 µl 45 mM EDTA, 300 nM SA-APC, 6 nM Eu-Py20 (Perkin Elmer, Boston, MA). Binding to europium-labeled antibody takes 40 minutes and HTRF signal is measured using a Fusion plate reader (Perkin Elmer, Boston, MA). It was found that for each JAK1, JAK2 and JAK3 kinase, the IC50 value was less than 50 nM, both for the phosphoric acid salt of the invention and the corresponding free base compound.

[0078] From the above description, various modifications of the invention in addition to those described herein will be apparent to those skilled in the art. Such modifications are intended to fall within the scope of the claims. All literature cited in the application is fully included in the literature here.