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

ES 2 575 797 T3

Janus (R) -3- (4- (7H-pyrrolo [2,3-d] pyrim idin-4-yl) -1 H-pyrazol-1-yl) -3-cyclopentylpropanonitrile salts

Description

FIELD OF THE INVENTION

The present invention provides salt forms of (R) -3- (4- (7H-pyrrolo [2,3-d] pyrimidin-4-yl) -1H-pyrazol-1-yl) -3-cyclopentylpropanenitrile that are useful in modulation of Janus kinase activity and are useful in treating diseases related to Janus kinase activity including, for example, immune-related diseases, skin disorders, myeloid proliferative disorders, cancer, and other diseases.

BACKGROUND OF THE INVENTION

Protein kinases (PK) are a group of enzymes that regulate various important biological processes including cell growth, survival and differentiation, organ formation and morphogenesis, neovascularization, tissue repair and regeneration, among others. Protein kinases exert their physiological functions by catalyzing the phosphorylation of proteins (or substrates) and thus modulating the cellular activities of substrates in various biological contexts. In addition to functions in normal tissues / organs, many protein kinases also perform more specialized functions in a receptor for human diseases including cancer. A subset of protein kinases (also called oncogenic protein kinases), when deregulated, can cause tumor formation and growth, and additionally contribute to tumor maintenance and progression (Blume-Jensen P et al., Nature 2001, 411 (6835): 355 -365). To date, oncogenic protein kinases represent one of the largest and most attractive groups of protein targets for cancer intervention and drug development.

The Janus kinase (JAK) family plays a role in the cytokine-dependent regulation of the proliferation and function of cells involved in the immune response. There are currently four known mammalian JAK family members: JAK1 (also known as Janus kinase 1), JAK2 (also known as Janus kinase 2), JAK3 (also known as Janus kinase leukocyte; JAKL; L-JAK, and Janus kinase 3) and TYK2 (also known as protein tyrosine kinase 2). JAK proteins range in size from 120 to 140 kDa and comprise seven conserved JAK (JH) homology domains; one of these is a functional catalytic kinase domain, and the other is a pseudo-kinase domain that possibly serves as a regulatory function and / or serves as a binding site for STAT (Scott, Godshall et al. 2002, supra).

Blocking signal transduction at the level of JAK kinases holds promise for developing treatments for human cancers. Inhibition of JAK kinases is also anticipated to have therapeutic benefits in patients suffering from immune skin disorders such as psoriasis, and skin sensitization. Accordingly, inhibitors of Janus kinases or related kinases are widely sought and several publications report effective classes of compounds. For example, certain JAK inhibitors, including (R) 3- (4- (7H-pyrrolo [2,3-d] pyrimidin-4-yl) -1 H-pyrazol-1-yl) -3-cyclopentylpropanonitr¡ The following are reported in U.S. Patent Serial No. 11 / 637,545 filed December 12, 2006.

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Thus, new or improved forms of existing Janus kinase inhibitors are continually needed to develop new, improved and more effective pharmaceutical formulations for the treatment of cancer and other diseases. The salt forms and procedures described herein are directed toward these needs and other purposes.

SUMMARY OF THE INVENTION

The present invention provides, among other things, salts selected from:

ES 2 575 797 T3 (R) -3- (4- (7H-pyrrolo [2,3-d] pyrimidin-4-yl) -1H-pyrazol-1-yl) -3-cyclopentylpropanenitrile maleic acid salt; (R) -3- (4- (7H-pyrrolo [2,3-d] pyrimidin-4-yl) -1H-pyrazol-1-yl) -3-cyclopentylpropanenitrile sulfuric acid salt; and (R) -3- (4- (7H-pyrrolo [2,3-d] pyrimidin-4-yl) -1H-pyrazol-1-yl) -3-cyclopentylpropanenitrile phosphoric acid salt;

in which the salt is crystalline.

The present invention further provides compositions comprising a crystalline salt form of the invention and at least one pharmaceutically acceptable carrier.

The present invention further provides a dosage form comprising a crystalline salt selected from the group consisting of:

(R) -3- (4- (7H-pyrrolo [2,3-d] pyrimidin-4-yl) -1H-pyrazol-1-yl) -3-cyclopentylpropanenitrile phosphoric acid salt; (R) -3- (4- (7H-pyrrolo [2,3-d] pyrimidin-4-yl) -1H-pyrazol-1-yl) -3-cyclopentylpropanenitrile sulfuric acid salt; and (R) -3- (4- (7H-pyrrolo [2,3-d] pyrimidin-4-yl) -1H-pyrazol-1-yl) -3-cyclopentylpropanitrile maleic acid salt.

DETAILED DESCRIPTION

The present invention provides, among other things, salts of the inhibitor of JAK (R) -3- (4- (7H-pyrrolo [2,3-d] pyrimidin-

4-yl) -1H-pyrazol-1-yl) -3-cyclopentylpropanenitrile selected from the maleic acid salt, sulfuric acid salt and phosphoric acid salt. These salts modulate the activity of one or more JAK and are useful, for example, in the treatment of diseases associated with the expression or activity of JAK.

The salts of the invention have numerous advantageous properties over the free base form and other salt forms. In particular, these salts were highly crystalline which would facilitate the preparation of pharmaceutical formulations and improve the general transport, handling and storage of the active principle. The salts of the invention also have superior aqueous solubility, dissolution rate, chemical stability (with long shelf life), excipient compatibility, and reproducibility compared to the free base form.

In some embodiments, the salts of the invention are substantially isolated. By "substantially isolated" it is meant that the salt is at least partially or substantially separated from the environment in which it was formed or detected. The partial separation can include, for example, a composition enriched in the salt of the invention. Substantial separation can include compositions containing at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, or at least about 99%, by weight of the salt.

The salts of the invention also include all isotopes of atoms that occur in salts. Isotopes include those atoms that have the same atomic number but different mass numbers. For example, isotopes of hydrogen include tritium and deuterium.

The salts of the invention can be prepared using known techniques. Conventionally, a salt form is prepared by combining the free base compound and an acid containing the anion of the desired salt form in solution, and then isolating the solid salt product from the reaction solution (e.g., by crystallization, precipitation, evaporation, etc.). Other salt-forming techniques can be employed.

Procedures for use

The salts of the invention can modulate the activity of one or more Janus kinases (JAK). The term "modulate" is intended to refer to an ability to increase or decrease the activity of one or more members of the JAK family of kinases. Accordingly, the compounds of the invention can be used in methods of modulating a JAK by contacting the JAK with any one or more of the compounds or compositions described herein. In some embodiments, the salts of the present invention can act as inhibitors of one or more JAKs. In some embodiments, the compounds of the present invention can act by stimulating the activity of one or more JAKs. In other embodiments, the compounds of the invention can be used to modulate the activity of a JAK in an individual in need of receptor modulation by administering a modulating amount of a salt of the invention.

JAK with which the present salts bind and / or modulate include any member of the JAK family. In some embodiments, the JAK is JAK1, JAK2, JAK3, or TYK2. In some embodiments, the JAK is JAK1 or JAK2. In some embodiments, the JAK is JAK2. In some embodiments, the JAK is JAK3.

The salts of the invention can be selective. By "selective" it is meant that the compound binds to or inhibits one JAK with higher affinity or potency, respectively, compared to at least one other JAK. In some embodiments, the salts of the invention are selective inhibitors of JAK1 or JAK2 over JAK3 and / or TYK2.

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In some embodiments, the compounds of the invention are selective inhibitors of JAK2 (eg, relative to JAK1, JAK3, and TYK2). Without wishing to be bound by theory, because inhibitors of jAK3 can lead to immunosuppressive effects, a compound that is selective for JAK2 over JAK3 and that is useful in the treatment of cancer (such as multiple myeloma, for example) may offer the added benefit of having fewer immunosuppressive side effects. The selectivity can be at least about 5 times, 10 times, at least about 20 times, at least about 50 times, at least about 100 times, at least about 200 times, at least about 500 times, or at least about 1000 times. Selectivity can be measured by routine procedures in the art. In some embodiments, selectivity can be tested at the Km of each enzyme. In some embodiments, the selectivity of salts of the invention for JAK2 over JAK3 can be determined by the cellular ATP concentration.

The salts of the invention can be used in methods of treating a JAK-associated disease or disorder in an individual (eg, patient) by administering to the individual in need of such treatment an amount or dose of a salt of the present invention or a composition. Pharmaceutical of the same. A JAK-associated disease can include any disease, disorder, or condition that is directly or indirectly linked to JAK expression or activity, including overexpression and / or abnormal activity levels. A JAK-associated disease can also include any disease, disorder or condition that can be prevented, ameliorated or cured by modulating JAK activity.

Examples of JAK-associated diseases include diseases involving the immune system including, for example, organ transplant rejection (eg, allograft rejection and graft versus host disease).

Other examples of JAK-associated 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, immunoglobulin nephropathies, thyroid disorders. autoimmune, and the like. In some embodiments, the autoimmune disease is an autoimmune bullous skin disorder such as pemphigus vulgaris (PV) or bullous pemphigoid (PB).

Other examples of JAK-associated diseases include allergic conditions such as asthma, food allergies, atopic dermatitis, and rhinitis. Other examples of JAK-associated diseases include viral diseases such as Epstein Barr virus (EBV), hepatitis B, hepatitis C, HIV, HTLV 1, varicella zoster virus (VZV), and human papilloma virus (HPV).

Other examples of JAK-associated diseases or conditions include skin disorders such as psoriasis (eg, psoriasis vulgaris), atopic dermatitis, rash, skin irritation, skin sensitization (eg, contact dermatitis or allergic dermatitis. contact). For example, certain substances included in some pharmaceuticals when applied topically can cause skin sensitization. In some embodiments, the co-administration or sequential administration of at least one JAK inhibitor of the invention together with the agent causing unwanted sensitization may be useful in treating such unwanted sensitization or dermatitis. In some embodiments, the skin disorder is treated by topical administration of at least one JAK inhibitor of the invention.

In other embodiments, the JAK-associated disease is cancer that includes those characterized by solid tumors (eg, prostate cancer, kidney cancer, liver cancer, pancreatic cancer, gastric cancer, breast cancer, lung cancer, head cancers. and neck, thyroid cancer, glioblastoma, Kaposi's sarcoma, Castleman's disease, melanoma etc.), hematological cancers (eg lymphoma, leukemia such as acute lymphoblastic leukemia, acute myelogenous 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.

JAK-associated diseases can further include those characterized by expression of a mutant JAK2 such as those that have at least one mutation in the pseudo-kinase domain (eg, JAK2V617F).

JAK-associated diseases may additionally include myeloproliferative disorders (PMD) such as polycythemia vera (PV), essential thrombocythemia (TE), myeloid metaplasia with myelofibrosis (MMM), chronic myelogenous leukemia (CML), chronic myelomonocytic leukemia (CML), syndrome hypereosinophilic (HES), systemic mast cell disease (ESM), and the like.

Other diseases associated with JAK include inflammation and inflammatory diseases. Examples of inflammatory diseases include inflammatory diseases of the eye (for example, iritis, uveitis, scleritis, conjunctivitis, or related disease), inflammatory diseases of the respiratory tract (for example, the upper respiratory tract including the nose and sinuses such as rhinitis or sinusitis or the lower respiratory tract including bronchitis, chronic obstructive pulmonary disease, and the like), inflammatory myopathy such as myocarditis, and other inflammatory diseases. Other inflammatory diseases treatable by

ES 2 575 797 T3 compounds of the invention include systemic inflammatory response syndrome (SIRS) and septic shock.

The JAK inhibitors described herein can be further used to treat ischemia-reperfusion injury or a disease or condition related to an inflammatory ischemic event such as stroke or cardiac arrest. The JAK inhibitors described herein can further be used to treat anorexia, cachexia, or fatigue such as that resulting from or associated with cancer. The JAK inhibitors described herein can further be used to treat restenosis, sclerodermatitis, or fibrosis. The JAK inhibitors described herein can be further used to treat conditions associated with hypoxia or astrogliosis such as, for example, diabetic retinopathy, cancer, or neurodegeneration. See, for example, 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. Electronic publication of March 2, 2004.

The JAK inhibitors described herein can be further used to treat gout and enlarged prostate due to, for example, benign prostatic hypertrophy or benign prostatic hyperplasia.

As used herein, the term "contacting" refers to bringing the indicated moieties together in an in vitro system or an in vivo system. For example, "contacting" a JAK with a salt of the invention includes administering a salt of the present invention to an individual or patient, such as a human, who has a JAK, in addition to, for example, introducing a salt of the invention in a sample containing a purified or cellular preparation containing JAK.

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

As used herein, the term "therapeutically effective amount" refers to the amount of active salt or pharmaceutical agent that elicits the biological or medicinal response that is being sought in a tissue, system, animal, individual, or human by a researcher, veterinarian, medical doctor, or other clinical professional.

As used herein, the term "treat" or "treatment" refers to one or more of (1) preventing disease; for example, preventing a disease, condition or disorder in an individual who may have a predisposition to the disease, condition or disorder, but who does not yet experience or display the pathology or symptomatology of the disease; (2) inhibit disease; for example, inhibiting a disease, condition or disorder in an individual who is experiencing or exhibiting the pathology or symptomatology of the disease, condition or disorder; and (3) ameliorate the disease; for example, ameliorating a disease, condition or disorder in an individual who is experiencing or exhibiting the pathology or symptomatology of the disease, condition or disorder (ie, reversing the pathology and / or symptomatology) such as decreasing the severity of the disease.

Combination therapies

One or more additional pharmaceutical agents such as, for example, chemotherapeutics, anti-inflammatory agents, steroids, immunosuppressants, in addition to Bcr-Abl, Flt-3, RAF and FAK kinase inhibitors such as, for example, those described in WO 2006 / 056399, or other agents, can be used in combination with the salts of the present invention for the treatment of diseases, disorders or conditions associated with JAK. The one or more additional pharmaceutical agents can be administered to a patient simultaneously or sequentially.

Examples of chemotherapeutics include proteasome inhibitors (eg, bortezomib), thalidomide, revlimid, and DNA damaging agents such as melphalan, doxorubicin, cyclophosphamide, vincristine, etoposide, carmustine, and the like.

Examples of steroids include corticosteroids such as dexamethasone or prednisone.

Examples of Bcr-Abl inhibitors include the compounds, and pharmaceutically acceptable salts thereof, of the genera and species disclosed in US Patent No. 5,521,184, WO 04/005281, EP2005 / 009967, EP2005 / 010408 and US Patent Serial No. 60 / 578,491.

Examples of suitable Flt-3 inhibitors include compounds, and their pharmaceutically acceptable salts, as disclosed in WO 03/037347, WO 03/099771 and WO 04/046120.

Examples of suitable RAF inhibitors include compounds, and their pharmaceutically acceptable salts, as disclosed in WO 00/09495 and WO 05/028444.

Examples of suitable FAK inhibitors include compounds, and their pharmaceutically acceptable salts, as disclosed in WO 04/080980, WO 04/056786, WO 03/024967, WO 01/064655, WO 00/053595 and

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WO 01/014402.

In some embodiments, the salt forms of the invention can be used in combination with other kinase inhibitors such as imatinib, particularly for the treatment of patients resistant to imatinib or other kinases.

In some embodiments, one or more salt forms of the invention can be used in combination with a chemotherapeutic in the treatment of cancer, such as multiple myeloma, and can improve the response of treatment relative to the response to the chemotherapeutic agent alone, without exacerbation. of its toxic effects. Examples of additional pharmaceutical agents used in the treatment of multiple myeloma, for example, may include, without limitation, melphalan, melphalan plus prednisone [MP], doxorubicin, dexamethasone, and Velcade (bortezomib). Additional agents used in the treatment of multiple myeloma include Bcr-Abl, Flt3, RAF, and FAK kinase inhibitors. Additive or synergistic effects are desirable outcomes of combining a JAK inhibitor of the present invention with an additional agent. Furthermore, the resistance of multiple myeloma cells to agents such as dexamethasone may be reversible after treatment with a JAK inhibitor of the present invention. The agents can be combined with the present compounds in a single or continuous dosage form, or the agents can be administered simultaneously or sequentially as separate dosage forms.

In some embodiments, a corticosteroid such as dexamethasone is administered to a patient in combination with at least one JAK inhibitor when dexamethasone is administered intermittently as opposed to continuously.

In some other embodiments, combinations of one or more JAK inhibitors of the invention with other therapeutic agents can be administered to a patient before, during, and / or after a bone marrow transplant or stem cell transplant.

Pharmaceutical Formulations and Dosage Forms

When used as pharmaceuticals, the salts of the invention can be administered in the form of pharmaceutical compositions. These compositions can be prepared in a manner well known in the pharmaceutical art, and can be administered by a variety of routes, depending on whether local or systemic treatment is desired and the area to be treated. Administration can be topical (including transdermal, epidermal, ophthalmic, and on mucous membranes including intranasal, vaginal, and rectal administration), pulmonary (for example, by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal or intranasal), oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal, intramuscular injection, or infusion; or intracranial, eg, intrathecal or intraventricular administration. Parenteral administration can be in the form of a single bolus dose, or it can be, for example, by a continuous infusion pump. Pharmaceutical compositions and formulations for topical administration can include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, and powders. Conventional pharmaceutical carriers, aqueous, powdered or oily bases, thickeners, and the like may be necessary or desirable. Coated condoms, gloves, and the like may also be useful.

The present invention also includes pharmaceutical compositions containing, as active principle, one or more of the salts of the above invention in combination with one or more pharmaceutically acceptable carriers (excipients). In preparing the compositions of the invention, the active ingredient is usually mixed with an excipient, diluted with an excipient or enclosed within such a carrier in the form of, for example, a capsule, envelope, paper and other container. If the excipient serves as a diluent, it can be a solid, semi-solid or liquid material, which acts as a vehicle, support or medium for the active principle. Thus, the compositions may be in the form of tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium), ointments containing, for example, up to 10% by weight of the active compound, soft and hard gelatin capsules, suppositories, sterile injectable solutions and sterile packaged powders.

In preparing a formulation, the active compound can be ground to provide the appropriate particle size before combining with the other components. If the active compound is substantially insoluble, it can be ground to a particle size of less than 200 mesh. If the active compound is substantially water soluble, the particle size can be adjusted by milling to provide a substantially uniform distribution in the formulation, eg, about 40 mesh.

The salts of the invention can be ground using known milling procedures such as wet milling to obtain an appropriate particle size for tableting and other types of formulation. Finely divided (nanoparticle) preparations of the compounds of the invention can be prepared by procedures known in the art, for example, see International Patent Application No. WO 2002/000196.

Some examples of suitable excipients include lactose, dextrose, sucrose, sorbitol, mannitol, starches, acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose,

ES 2 575 797 T3 polyvinylpyrrolidone, cellulose, water, syrup and methyl cellulose. The formulations may additionally include: lubricating agents such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifiers and suspending agents; preserving agents such as methyl- and propylhydroxybenzoates; sweeteners; and flavorings. The compositions of the invention may be formulated so as to provide rapid, sustained or delayed release of the active ingredient after administration to the patient using procedures known in the art.

The compositions can be formulated in unit dosage form, each dosage containing from about 5 to about 1000 mg (1 g), more usually about 100 to about 500 mg, of the active ingredient. The term "unit dosage forms" refers to physically discrete units suitable as unit dosages for human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient.

The active compound can be effective over a wide dosage range and is generally administered in a pharmaceutically effective amount. It will be understood, however, that the amount of the compound actually administered will normally be determined by a physician, based on the relevant circumstances, including the condition to be treated, the chosen route of administration, the actual compound administered, age, weight. and individual patient response, severity of the patient's symptoms, and the like.

To prepare solid compositions such as tablets, the main active ingredient is mixed with a pharmaceutical excipient to form a solid preformulation composition containing a homogeneous mixture of a compound of the present invention. When these preformulation compositions are referred to as homogeneous, the active ingredient is normally uniformly dispersed throughout the composition so that the composition can easily be subdivided into equally effective unit dosage forms such as tablets, pills, and capsules. This solid preformulation is then subdivided into unit dosage forms of the type described above containing, for example, about 0.1 to about 1000 mg of the active ingredient of the present invention.

The tablets or pills of the present invention may be coated or otherwise combined to provide a dosage form that provides the long-acting benefit. For example, the tablet or pill may comprise an internal dosage and an external dosage component, the latter being in the form of an envelope over the former. The two components can be separated by an enteric layer that serves to resist disintegration in the stomach and allow the internal component to pass intact into the duodenum or to be delayed-release. A variety of materials can be used for such enteric layers or coatings, such materials including various polymeric acids and mixtures of polymeric acids with materials such as Shellac, cetyl alcohol, and cellulose acetate.

Liquid forms into which the compounds and compositions of the present invention can be incorporated for oral administration or by injection include aqueous solutions, suitably flavored syrups, aqueous or oil suspensions, and emulsions flavored with edible oils such as kernel oil. cotton wool, sesame oil, coconut oil or peanut oil, as well as elixirs and similar pharmaceutical vehicles.

Compositions for inhalation or insufflation include solutions and suspensions in pharmaceutically acceptable aqueous or organic solvents, or mixtures thereof, and powders. Liquid or solid compositions may contain suitable pharmaceutically acceptable excipients as described above. In some embodiments, the compositions are administered by the oral or nasal respiratory route for local or systemic effect. The compositions can be nebulized using inert gases. Nebulized solutions can be breathed directly from the nebulizer device or the nebulizer device can be attached to a face shield, or intermittent positive pressure respirator. The solution, suspension, or powder compositions can be administered orally or nasally from devices that deliver the formulation in an appropriate manner.

The amount of salt or composition administered to a patient will vary depending on what is being administered, the purpose of administration, such as prophylaxis or therapy, the condition of the patient, the mode of administration, and the like. In therapeutic applications, the compositions can be administered to a patient already suffering from a disease in an amount sufficient to cure or at least partially arrest the symptoms of the disease and its complications. Effective dosages will depend on the disease condition being treated, in addition to the judgment of the adjunct clinician depending on factors such as the severity of the disease, the age, weight, and general condition of the patient, and the like.

Compositions administered to a patient can be in the form of pharmaceutical compositions as described above. These compositions can be sterilized by conventional sterilization techniques, or they can be filter sterilized. Aqueous solutions can be packaged for use as such, or lyophilized, the lyophilized preparation being combined with a sterile aqueous vehicle prior to administration. The pH of compound preparations will usually be between 3 and 11, more preferably 5 to 9, and most

ES 2 575 797 T3 preferably 7 to 8. It will be understood that the use of certain of the foregoing excipients, carriers or stabilizers will result in the formation of pharmaceutical salts.

The therapeutic dosage of the salts of the present invention may vary, for example, according to the particular use for which the treatment is made, the mode of administration of the compound, the health and condition of the patient and the judgment of the prescribing physician. The proportion or concentration of a salt of the invention in a pharmaceutical composition can vary depending on several factors including dosage, chemical characteristics (eg, hydrophobicity) and the route of administration. For example, the salts of the invention may be provided in an aqueous physiological buffer solution containing from about 0.1 to about 10% weight / volume of the compound for parenteral administration. Some typical dose ranges are from about 1 pg / kg to about 1 g / kg of body weight per day. In some embodiments, the dose range is from about 0.01 mg / kg to about 100 mg / kg of body weight per day. The dosage is likely to depend on variables such as the type and degree of progression of the disease or disorder, the overall health status of the particular patient, the relative biological efficacy of the selected compound, the formulation of the excipient and its route of administration. Effective doses can be extrapolated from dose response curves derived from in vitro or animal model test systems.

Compositions of the invention may further include one or more additional pharmaceutical agents such as a chemotherapeutic, steroid, anti-inflammatory or immunosuppressive compound, examples of which are listed hereinabove.

Labeled compounds and test procedures

The salts of the invention can be labeled (radiolabeled, fluorescently labeled, etc.) and can be useful not only in imaging techniques, but also in assays, both in vitro and in vivo, to localize and quantify JAK in samples of tissue, including human, and to identify JAK ligands by binding inhibition of a labeled compound. Accordingly, the present invention includes JAK assays containing such labeled compounds.

The salts of the invention can be isotopically labeled. An "isotopically" or "radiolabeled" compound is a salt of the invention in which one or more atoms are replaced or substituted with an atom having an atomic mass or mass number different from the atomic mass or mass number normally found in nature. (that is, it occurs naturally). Suitable radionuclides that can be incorporated into compounds of the present invention include, but are not limited to,²H (also written as D for deuterium), ³H (also written as T for tritium), ¹¹C, ¹³C, ¹⁴C, ¹³N, ¹⁵N, ¹⁵OR,¹⁷OR, ¹⁸OR, ¹⁸F, ³⁵Yes, ³⁶Cl, ⁸²Br, ⁷⁵Br, ⁷⁶Br, ⁷⁷Br, ¹²³I, ¹²⁴I, ¹²⁵I and ¹³¹I. The radionuclide that is incorporated into the present radiolabeled compounds will depend on the specific application of that radiolabeled compound. For example, for in vitro metalloprotease labeling and competition assays, compounds incorporating³H, ¹⁴C, ⁸²Br, ¹²⁵I, ¹³¹I, ³⁵S o will generally be the most useful. In order to^{1 1 1 1} ή ή ή Q ή OC ή OQ ή ΟΛ ή Οή 7C 7yes? ~ l ~ l CF, I, I, I, I, Br, Br, or Br radio-imaging applications will generally be the most useful.

A "radiolabeled" or "labeled" compound is understood to be a salt that has incorporated at least one radionuclide. In some embodiments, the radionuclide is selected from the group consisting of³H,¹⁴C, ¹²⁵I, ³⁵S and ⁸²Br.

Synthetic methods for incorporating radioisotopes into compounds of the invention are also considered. Synthetic procedures for incorporating radioisotopes into organic compounds are well known in the art, and one of ordinary skill in the art will readily recognize the procedures applicable to the compounds of the invention.

A labeled salt of the invention can be used in a screening assay to identify / evaluate compounds. For example, a newly synthesized or identified compound (ie test compound) that is labeled can be evaluated for its ability to bind to JAK by monitoring its variation in concentration when contacted with JAK, by monitoring the label. For example, a test compound (labeled) can be evaluated for its ability to reduce the binding of another compound that is known to bind to a JAK (ie, conventional compound). Accordingly, the ability of a test compound to compete with the conventional compound to bind JAK directly correlates with its binding affinity. In contrast, in some other screening assays, the conventional compound is labeled and the test compounds are not labeled. Accordingly, the concentration of the labeled conventional compound is monitored in order to evaluate the competition between the conventional compound and the test compound, and thus the relative binding affinity of the test compound is determined.

Kits

The crystalline salts of the invention can be included in pharmaceutical kits useful, for example, in the treatment or prevention of diseases or disorders associated with JAK, such as cancer, inflammation or skin disorders,

ES 2 575 797 T3 which include one or more containers containing a pharmaceutical composition comprising a therapeutically effective amount of a salt of the invention. Such kits may further include, if desired, one or more of various components of conventional pharmaceutical kits such as, for example, containers with one or more pharmaceutically acceptable carriers, additional containers, etc., as will be readily apparent to those skilled in the art. matter. Instructions, both as brochures and labels, indicating amounts of the components to be administered, guidelines for administration, and / or guidelines for mixing the components, may also be included in the kit.

The invention will be described in more detail by way of specific examples. The following examples are offered for illustrative purposes and are not intended to limit the invention in any way. Those skilled in the art will readily recognize a variety of non-critical parameters that can be changed or modified giving essentially the same results.

EXAMPLES

Example 1: Preparation of the maleic acid salt of (R) -3- (4- (7H-pyrrolo [2,3-d] pyrimidin-4-yl) -1H-pyrazol-1-yl) 3-cyclopentylpropanonitrile

To a test tube was added (R) -3- (4- (7H-pyrrolo [2,3-d] 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 until clear, cooled to room temperature, and then stirred for another 2.5 hours. The precipitate was collected by filtration and the cake was washed with 0.8 ml of cold IPA. The cake was dried under vacuum at constant weight to provide the final salt product (173 mg).

The maleic acid salt was shown to be a 1: 1 salt by NMR ¹H and crystallinity was confirmed by X-ray powder diffraction (XRPD). Differential Scanning Calorimetry (DSC) gave a sharp melting peak at approximately 175.96 ° C (appearance at 175.67 ° C). The product only showed a slight weight loss up to 150 ° C by thermogravimetric analysis (TGA).

Example 2: Preparation of the phosphoric acid salt of (R) -3- (4- (7H-pyrrolo [2,3-d] pyrimidin-4-yl) -1H-pyrazol-1-yl) 3-cyclopentylpropanenitrile

To a test tube was added (R) -3- (4- (7H-pyrrolo [2,3-d] 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 until clear, cooled to room temperature, and then stirred for another 2 hours. The precipitate was collected by filtration and the cake was washed with 0.6 ml of cold IPA. The cake was dried under vacuum at constant weight to provide the final salt product (171.7 mg).

The phosphoric acid salt was shown to be a 1: 1 salt by NMR ¹H and crystallinity was confirmed by X-ray powder diffraction (XRPD). Differential Scanning Calorimetry (DSC) gave a sharp melting peak at about 198.66 ° C. The product showed little weight loss up to 200 ° C by TGA.

Example 3: Preparation of the sulfuric acid salt of (R) -3- (4- (7H-pyrrolo [2,3-d] pyrimidin-4-yl) -1H-pyrazol-1-yl) 3-cyclopentylpropanonitrile

To a test tube was added (R) -3- (4- (7H-pyrrolo [2,3-d] 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 until clear, cooled to room temperature, and then stirred for another 2 hours. The precipitate was collected by filtration and the cake was washed with 0.8 ml of cold acetonitrile. The cake was dried under vacuum at constant weight to provide the final salt product (180 mg).

The sulfuric acid salt was shown to be a 1: 1 salt by NMR ¹H and crystallinity was confirmed by X-ray powder diffraction (XRPD). Differential Scanning Calorimetry (DSC) gave a sharp melting peak at approximately 186.78 ° C. The product showed little weight loss up to 175 ° C by TGA.

Example A

In vitro assay of JAK kinases

The inhibitory activity of test compounds on JAK targets can be tested according to the following in vitro assay described in Park et al., Analytical Biochemistry 1999, 269, 94-104. The catalytic domains of human JAK1 (aa 837-1142), Jak2 (aa 828-1132) and Jak3 (aa 781-1124) with an N-terminal His tag are expressed using baculovirus on insect cells and purified. The catalytic activity of JAK1, JAK2 or JAK3 is tested by measuring the phosphorylation of a biotinylated peptide. The phosphorylated peptide was detected by time resolved homogeneous fluorescence (HTRF). The IC50 of the compounds are measured for each kinase in reactions containing the enzyme, aTp and 500 nM peptide in 50 mM Tris buffer (pH 7.8) with 100 mM NaCl, 5 mM DTT and 0.1

ES 2 575 797 T3 mg / ml (0.01%) of BSA. The ATP concentration in the reactions is 90 μΜ for Jak1, 30 μΜ for Jak2 and 3 μΜ for Jak3. Reactions are carried out at room temperature for 1 h and then stopped with 20 µl 45 mM EDTA, 300 nM SA-APC, 6 nM Eu-Py20 in Assay Buffer (Perkin Elmer, Boston, MA). Binding to the europium-labeled antibody occurs for 40 minutes and the HTRF signal is measured on a Fusion plate reader (Perkin Elmer, Boston, MA). Both the phosphoric acid salt of the invention and the corresponding free base compound were found to have IC50 values ​​less than 50 nM for each of JAK1, JAK2 and JAK3.

Reference realizations

1. A salt selected from:

(R) -3- (4- (7H-pyrrolo [2,3-d] pyrimidin-4-yl) -1H-pyrazol-1-yl) -3-cyclopentylpropanitrile maleic acid salt;

(R) -3- (4- (7H-pyrrolo [2,3-d] pyrimidin-4-yl) -1H-pyrazol-1-yl) -3-cyclopentylpropanenitrile sulfuric acid salt; and (R) -3- (4- (7H-pyrrolo [2,3-d] pyrimidin-4-yl) -1H-pyrazol-1-yl) -3-cyclopentylpropanenitrile phosphoric acid salt.

two. The salt of embodiment 1 which is the maleic acid salt of (R) -3- (4- (7H-pyrrolo [2,3-d] pyrimidin-4-yl) -1H-pyrazol-1-yl) 3 -cyclopentylpropanitrile.

3. The salt of embodiment 1 which is the sulfuric acid salt of (R) -3- (4- (7H-pyrrolo [2,3-d] pyrimidin-4-yl) -1H-pyrazol-1-yl) -3- cyclopentylpropanenitrile.

Four. The salt of embodiment 1 which is the phosphoric acid salt of (R) -3- (4- (7H-pyrrolo [2,3-d] pyrimidin-4-yl) -1H-pyrazol-1-yl) -3- cyclopentylpropanenitrile.

5. The salt of embodiment 1 that is substantially isolated.

6. A method of preparing a salt of any one of embodiments 1 to 5 which comprises combining (R) 3- (4- (7H-pyrrolo [2,3-d] pyrimidin-4-yl) -1H-pyrazol-1 -yl) -3-cyclopentylpropanenitrile with maleic acid, sulfuric acid or phosphoric acid.

7. A composition comprising at least one salt of any one of embodiments 1 to 5 and at least one pharmaceutically acceptable carrier.

8. The composition of embodiment 7 that is suitable for oral or topical administration.

9. The composition of embodiment 7 that is suitable for topical administration.

10. A method of modulating a JAK activity comprising contacting JAK with a salt of any one of embodiments 1 to 5.

eleven. The method of embodiment 10, wherein said modulation is inhibition.

12. A method of treating a disease in a patient, wherein said disease is associated with JAK activity, comprising administering to said patient a therapeutically effective amount of a salt of any one of embodiments 1 to 5.

13. The method of embodiment 12, wherein said disease is allograft rejection or graft versus host disease.

14. The method of embodiment 12, wherein said disease is an autoimmune disease.

fifteen. The method of embodiment 14, wherein said autoimmune disease is a skin disorder, multiple sclerosis, rheumatoid arthritis, juvenile arthritis, type I diabetes, lupus, inflammatory bowel disease, Crohn's disease, myasthenia gravis, immunoglobulin nephropathies , myocarditis or autoimmune thyroid disorder.

16. The method of embodiment 12, wherein said autoimmune disease is bullous skin disorder.

17. The method of embodiment 16, wherein said bullous skin disorder is pemphigus vulgaris (PV) or bullous pemphigoid (BP).

18. The method of embodiment 12, wherein said disease is a skin disorder.

19. The method of embodiment 18, wherein said skin disorder is atopic dermatitis, psoriasis, skin sensitization, skin irritation, skin rash, contact dermatitis, or allergic contact sensitization.

twenty. The method of embodiment 12, wherein said disease is a viral disease.

ES 2 575 797 T3

twenty-one. The method of embodiment 20, wherein said viral disease is Epstein Barr virus (EBV), hepatitis B, hepatitis C, HIV, HTLV 1, varicella zoster virus (VZV) or human papilloma virus (HPV).

22. The method of embodiment 12, wherein said disease is cancer.

2. 3. The method of embodiment 22, wherein said cancer is a solid tumor

24. The method of embodiment 22, wherein said cancer is prostate cancer, kidney cancer, liver cancer, breast cancer, lung cancer, thyroid cancer, Kaposi's sarcoma, Castleman's disease, or pancreatic cancer.

25. The method of embodiment 24, wherein said cancer is prostate cancer.

26. The method of embodiment 22, wherein said cancer is hematological.

27. The method of embodiment 26, wherein said cancer is lymphoma, leukemia, or multiple myeloma.

28. The method of embodiment 22, wherein said cancer is skin cancer.

29. The method of embodiment 28, wherein said skin cancer is cutaneous T-cell lymphoma or cutaneous B-cell lymphoma.

30. The method of embodiment 22, wherein said cancer is multiple myeloma.

31. The method of embodiment 12, wherein said disease is characterized by a mutant JAK2.

32. The method of embodiment 31, wherein at least one mutation of said mutant JAK2 resides in the pseudo-kinase domain of said JAK2.

33. The method of embodiment 12, wherein said disease is a myeloproliferative disorder.

3. 4. The method of embodiment 33, wherein said myeloproliferative disorder (MPD) is polycythemia vera (PV), essential thrombocythemia (TE), myeloid metaplasia with myelofibrosis (MMM), chronic myelogenous leukemia (CML), chronic myelomonocytic leukemia (CMML) , hypereosinophilic syndrome (HES) or systemic mast cell disease (SMCD).

35. The method of embodiment 12, wherein said disease is an inflammatory disease.

36. The method of embodiment 35, wherein said disease is an inflammatory disease of the eye.

37. The method of embodiment 36, wherein said disease is iritis, uveitis, scleritis, or conjunctivitis.

38. The method of embodiment 35, wherein said disease is an inflammatory airway disease.

39. The method of embodiment 35, wherein said inflammatory disease relates to the upper respiratory tract.

40. The method of embodiment 35, wherein said inflammatory disease relates to the lower respiratory tract.

41. The method of embodiment 35, wherein said inflammatory disease is an inflammatory myopathy.

42. The method of embodiment 35, wherein said inflammatory disease is myocarditis.

43. The method of embodiment 12, wherein said disease is ischemia-reperfusion or related to an ischemic event.

44. The method of embodiment 12, wherein said disease is anorexia or cachexia resulting from or associated with cancer.

Four. Five. The method of embodiment 12, wherein said disease is fatigue resulting from or associated with cancer.

46. A method of treating cancer in a patient, comprising administering to said patient a therapeutically effective amount of a salt of any one of embodiments 1 to 5.

ES 2 575 797 T3

47. A method of treating a skin disorder in a patient comprising topically administering to said patient a therapeutically effective amount of a salt of any one of embodiments 1 to 5.

48. A method of treating inflammation in a patient comprising topically administering to said patient a therapeutically effective amount of a salt of any one of embodiments 1 to 5.

49. A method of treating rheumatoid arthritis in a patient comprising administering to said patient a therapeutically effective amount of a compound of any one of embodiments 1 to 5, or a pharmaceutically acceptable salt thereof.

fifty. A method of treating prostate cancer in a patient comprising administering to said patient a therapeutically effective amount of a compound of any one of embodiments 1 to 5, or a pharmaceutically acceptable salt thereof.

51. A method of treating psoriasis in a patient comprising administering to said patient a therapeutically effective amount of a compound of any one of embodiments 1 to 5, or a pharmaceutically acceptable salt thereof.

52. A method of treating multiple myeloma in a patient comprising administering to said patient a therapeutically effective amount of a compound of any one of embodiments 1 to 5, or a pharmaceutically acceptable salt thereof.

53. A method of treating myeloid metaplasia with myelofibrosis (MMM) in a patient comprising administering to said patient a therapeutically effective amount of a compound of any one of embodiments 1 to 5, or a pharmaceutically acceptable salt thereof.

54. A method of treating polycythemia vera (PV) in a patient comprising administering to said patient a therapeutically effective amount of a compound of any one of embodiments 1 to 5, or a pharmaceutically acceptable salt thereof.

55. A method of treating essential thrombocythemia (ET) in a patient comprising administering to said patient a therapeutically effective amount of a compound of any one of embodiments 1 to 5, or a pharmaceutically acceptable salt thereof.

56. A method of treating mycosis fungoides in a patient comprising administering to said patient a therapeutically effective amount of a compound of any one of embodiments 1 to 5, or a pharmaceutically acceptable salt thereof.

57. A method of treating a hematological cancer in a patient comprising administering to said patient a therapeutically effective amount of a compound of any one of embodiments 1 to 5, or a pharmaceutically acceptable salt thereof.

58. A method of treating chronic myelogenous leukemia (CML) in a patient comprising administering to said patient a therapeutically effective amount of a compound of any one of embodiments 1 to 5, or a pharmaceutically acceptable salt thereof.

59. A method of treating acute lymphoblastic leukemia (ALL) in a patient comprising administering to said patient a therapeutically effective amount of a compound of any one of embodiments 1 to 5, or a pharmaceutically acceptable salt thereof.

60. A method of treating chronic myelomonocytic leukemia (CML) in a patient comprising administering to said patient a therapeutically effective amount of a compound of any one of embodiments 1 to 5, or a pharmaceutically acceptable salt thereof.