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

Field of the Invention

The present invention provides salts of (R) —3— (4— (7H-pyrrolo [2,3-d] pyrimidin-4-yl) -lH-pyrazol-l-yl) -3cyclopentylpropanitrile which are useful in the modulation of Janus kinase activity and are useful in the treatment of diseases related to kinase activity

Janus which include, for example, diseases related to the immune system, 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 procedures that include cell growth, survival and differentiation, organ donation and morphogenesis, neovascularization, tissue repair and regeneration, among others. Protein kinases exert their physiological functions through the channeling of the phosphorylation of proteins (or substrates) and therefore modulate the cellular activities of the substrates in various biological contexts. In addition to the function in normal tissues / organs, many protein kinases also play more specialized roles in a host of human disease ki including cancer. A subset of protein kinases (also referred to as oncogenic protein kinases), when deregulated, can cause tumor formation and growth, and also contribute to tumor maintenance and progression (Blume-Jensen P et al., Nature 2001, 41 (6835) : 355-365). In this way until now, oncogenic protein kinases represent one of the largest and most attractive groups of protein targets for intervention development and cancer drugs.

The Janus kinase family (JAK) plays a role in the cytosine-dependent regulation of proliferation and the function of the cells involved in an immune response. There are currently four members of the JAK family of known mammals: 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 are on a size scale of 120 to 140 kDa and comprise 7 conserved JAK (JH) homology domains; one of these is a functional catalytic kinase domain, and another is a pseudokinase domain that potentially serves a regulatory function and / or serves as a coupling site for STATs (Scott, Godshall et al., 2002, supra).

• 'in ·

The signal transduction block at the level of ¹ ’

<img file="CU20120155A7_D0001.tif" />

!, »!, 'JAK kinases keeps the promise of developing treatments for cancer in humans. The inhibition of JAK kinases also provides therapeutic benefits in patients suffering from immune disorders in the skin such as psoriasis, and skin sensitization. Therefore, Janus kinase inhibitors or related kinases are widely sought, and several publications report classes of effective compounds. For example, certain¹¹¹¹ JAK inhibitors, which include (R) -3- (4 - (7H-pyrrolo [2,310 d] pyrimidin-4-yl) -lH-pyrazol-l-yl) -3-cyclopentylpropanitrile described below, are reported in US No Series 11 / 637,545, filed on December 12, 2006.

<img file="CU20120155A7_D0002.tif" />

<img file="CU20120155A7_D0003.tif" />

In this way, new and improved forms of existing Janus kinase inhibitors are continually in need of developing new, improved and more effective pharmaceutical formulations for the treatment of cancer and other diseases. Salt forms and methods described herein address certain needs and others.

<img file="CU20120155A7_D0004.tif" />

terms

Brief Description of the Invention ¹ The present invention provides, inter alia, salts selected from the maleic acid salt of (R) -3- (4- (7H5 pyrrolo [2,3-d] pyrimidiη-4-yl) -lH-pyrazol-l-yl ) -3cyclopentylpropanitrile;

the sulfuric acid salt of (R) -3- (4- (7H-pyrrolo [2,3-d] pyrimidin-4-yl) -lH-pyrazol-l-yl) -3cyclopentylpropanitrile; and the phosphoric acid salt of (R) -3- (4- (7H-pyrrolo [2,3-dl pyrimidin-4-yl) -IH-pyrazol-l-yl) -3¹ cyclopentylpropanitrile.

The present invention further provides methods for preparing a salt of the invention comprising combining (R) -3- (4- (7H-pyrrolo [2,3-d] pyrimidin-4-yl) -lH-pyrazol-l-yl ) - 3cyclopentylpropanitrile with nucleic acid, sulfuric acid or phosphoric acid.

The present invention further provides compositions comprising a salt form of the invention -20 and at least one pharmaceutically acceptable carrier.

The present invention further provides methods for modulating a JAK activity comprising contacting the JAK with a salt of the invention.

The present invention further provides methods for treating a disease in a patient where the

<img file="CU20120155A7_D0005.tif" />

<img file="CU20120155A7_D0006.tif" />

The disease is associated with the activity of JAK, which comprises administering to a patient a therapeutically effective amount of a salt of the invention.

The present invention further provides methods for treating cancer, skin disorders or inflammation in a patient, which comprises administering to the patient a therapeutically effective amount of a salt of the invention.

The present invention further provides the salts of the invention for use in methods for treating the human or animal body by therapy.

The present invention further provides the salts of the invention for use in the treatment of cancer, skin disorders or inflammation.

........ The present invention further provides the use of the salts of the invention for the preparation of a medicament for use in the treatment of any of the diseases or disorders recited herein.

Detailed description of the invention

The present invention provides inter alia, salts of the inhibitor JAK (R) -3- (4- (7H-pyrrolo [2,3-d] pyrimidin-4-yl) lH-pyrazol-l-yl) -3-cyclopentylpropanitrile selected from the maleic acid salt, the sulfuric acid salt, and the phosphoric acid salt. These salts modulate the activity of one or more

JAK and are useful, for example, in the treatment of 25 diseases associated with the expression or activity of the

WIFr¹¹ ’

HOW

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 facilitates the preparation of pharmaceutical formulations and improves the general handling, storage manipulation of the active ingredient. The salts of the invention also have a higher aqueous solubility, a degree of dissolution, chemical stability (with a longer shelf life), compatibility with excipients, and degree of reproduction compared to the free base form.

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

The salts of the invention also include all isotopes of atoms that exist in the salts. Isotopes include those atoms that have the same atomic number but different mass numbers. For example, the isotopes of¹

<img file="CU20120155A7_D0007.tif" />

if

<img file="CU20120155A7_D0008.tif" />

wnii¹ ”2 Q hydrogen include tritium and deuterium.

The salts of the invention can be prepared using known techniques. Conventionally, a salt form is prepared by combining in solution the free base compound and an acid containing the anion of the desired salt form, and then the product is isolated from the solid salt of the reaction solution (for example, through crystallization, precipitation, evaporation, etc.). Other techniques to form salts can be used.

Methods of Use

The salts of the invention can modulate the activity of one or more Janus kinases (JAK). The term modular is intended to refer to an 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 for modulating a JAK by contacting the JAK with 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 JAK. In some embodiments, the compounds of the present invention may act to stimulate the activity of one or more JAK. 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

<img file="CU20120155A7_D0009.tif" />

Wllll * ¹¹¹ modulated amount of a salt of the invention.

The JAKs to which the salts herein bind and / or modulate include any member of the JAK family. In some modalities, the JAK is JAKl, JAK2, JAK3 or TYK2. In some modalities, the JAK is JAKl or JAK2. In some modalities the JAK is JAK2. In some modalities, the JAK is

JAK3.

The salts of the invention can be selective. By selective means that the compound binds to or inhibits a

JAK with a higher affinity or potency, respectively, compared to at least one other JAK. In some embodiments, the compounds of the invention are selective inhibitors of JAK1 or JAK2 over JAK3 and / or TYR2. In some embodiments the salts of the invention are selective inhibitors of JAK2 (for example, on JAK1, JAK3 and TYR2). Without wishing to be bound by any theory, because JAK3 inhibitors 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 a multiple myeloma, for example) can 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 100 times, at least about 200 times, at least about 500 times, or at least

<img file="CU20120155A7_D0010.tif" />

<img file="CU20120155A7_D0011.tif" />

<img file="CU20120155A7_D0012.tif" />

approximately 1000 times Selectivity can be measured through routine methods in the art. In some modalities, selectivity can be tested in the Km of each enzyme. In some embodiments, the selectivity of the salt of the invention for JAK2 over JAK3 can be determined through the cellular ATP concentration.

Another aspect of the present invention pertains to methods for treating a disease or disorder associated with JAK in an individual (eg, patient) by administration to the individual in need of treatment of the therapeutically effective amount or dose of a salt of the present invention or an e its pharmaceutical compositions. A disease associated with JAK may include any disease, disorder or condition that is directly or indirectly linked to the expression or activity of JAK, which includes overexpression and / or abnormal activity levels. A disease associated with JAK can also include any disease, disorder or condition that can be avoided, mitigated or cured through the modulation of JAK activity.

Examples of diseases associated with JAK that include diseases that involve the immune system, for example, organ transplant rejection (for example, allograft rejection and graft against host disease).

Additional examples of diseases associated with JAK include autoimmune diseases such as multiple sclerosis, rheumatoid arthritis, juvenile arthritis, type I diabetes, lupus, psoriasis, inflammatory bowel disease, ulcerative colitis, Crohn's disease, severe miastemia, immunoglobulin nephropathies , autoimmune thyroid disorders, and the like. In some embodiments, autoimmune disease is a skin disorder of autoimmune blisters such as blister or 10 pemphigus vulgaris (PV) or pemphigoid blister (BP).

Additional examples of diseases associated with JAK include allergic conditions such as asthma, food allergies, dermatitis and atopic rhinitis. Another 15 examples of diseases associated with JAK include viral diseases such as Epstein Barr virus (EBV), Hepatitis B, Hepatitis C, HIV, HTLC 1, virus

Chickenpox-Zoster (VZV) and Human Papillomavirus (HPV).

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

<td>In</td><td>modalities</td><td>additional,</td><td>the</td><td>associated disease</td>

<td>with the JAK</td><td>it's cancer</td><td>what includes</td><td>the</td><td>characterized by</td>

solid tumors (e.g. prostate cancer, kidney cancer, liver cancer, pancreatic cancer, gastric cancer, breast cancer, lung cancer, head and neck cancer, thyroid cancer, glioblastoma, Kaposi's sarcoma, Castleman disease, melanoma, etc.), hematologic cancers (for example, leukemia lymphoma 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 lymphoma include Sezary syndrome and fungoid mycosis.

The diseases associated with JAK may also include those characterized by the expression of a JAK2 mutant such as that which has at least one mutation in the pseudo-kinase domain (eg, JAK2V617F).

The diseases associated with JAK may also include myeloproliferative disorders (MPD), such as polycythemia vera (PV), essential thrombocythemia (ET), myeloid metaplasia with myelofibrosis (MMM), chronic myelogenous leukemia (CML), chronic myelomonocytic leukemia (CMML), hypereosinophilic syndrome (HES), Systemic mast cell disease (SMCD), and the like.

Additional diseases associated with JAK include inflammation and inflammatory diseases. Illustrative inflammatory diseases include inflammatory diseases of the eye (for example, iritis, uveitis, scleritis, conjunctivitis or related diseases, inflammatory diseases of the respiratory tract (for example, the upper respiratory tract that includes the nose and sinuses such as rhinitis or sinusitis or the lower respiratory tract that includes bronchitis, chronic obstructive pulmonary disease and the like), inflammatory myopathy such as myocarditis and other inflammatory diseases. Other inflammatory diseases treatable through the compounds of the invention include systemic inflammatory response syndrome (SIRS) and septic shock.

The JAK inhibitors described herein can also be used to treat reperfusion damage from ischemia or a disease or condition related to an inflammatory ischemic event such as stroke or cardiac arrest. The JAK inhibitors described herein can also be used to treat anorexia, cachexia, or fatigue such as those resulting from or associated with cancer. The JAK inhibitors described herein can also be used to treat restenosis, sclerodormitis, or fibrosis. The JAK inhibitors described herein can also be used to treat conditions associated with hypoxia and 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. Epub 2004 March 2.

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

As used herein, the term "contact" refers to gathering identical fractions in an in vitro system or in an in vivo system. For example, contacting a JAK with a salt of the invention includes the administration of a salt of the present invention to a

<img file="CU20120155A7_D0013.tif" />

individual or patient, such as a human, who has a JAK as well as for example, introducing a salt of the invention into a sample containing a cellular or purified preparation containing a JAK.

_ 5 As used herein, the term ^{1111 1} individual or patient is used interchangeably, refers to any animal, including mammals, preferably mice, rats, other rodents, rabbits, dogs, cats, pigs, cattle, sheep, horses, or primates and more preferably humans.

As used herein, the phrase "therapeutically effective amount" refers to the amount of the active salt or pharmaceutical agent that elicits the response.

W ....... biological or medicinal that is being sought in a tissue, system, animal, individual or human through the researcher, veterinarian, medical doctor or other doctor.

As used herein, the term "treat" or "treatment" refers to one or more of (1) preventing the disease; for example, preventing a disease, condition or disorder of an individual who may be predisposed to the disease, condition or disorder but does not yet experience or display the disease pathology or symptomatology; (2) inhibit the disease; for example inhibiting a disease, condition or disorder in an individual who is experiencing or displaying the disease pathology or symptomatology,

<img file="CU20120155A7_D0014.tif" />

<img file="CU20120155A7_D0015.tif" />

<img file="CU20120155A7_D0016.tif" />

'i'¹ condition or disorder; and (3) mitigate the disease; for example, to mitigate a disease, condition or disorder in an individual who is experiencing or displaying the pathology or symptomatology of the disease, condition or disorder (for example, reversing the pathology and / or symptomatology) such as decreasing the severity of the disease.

Combination Therapies _ One or more of the additional pharmaceutical agents

..... - such as, for example, chemotherapeutic agents, anti-inflammatory agents, spheroids, immunosuppressants, as well as Bcr-Abl, Flt, 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 present invention for the treatment of diseases, disorders or conditions associated with JAK. The one or more additional pharmaceutical people can be administered to a patient - simultaneously or sequentially.

Illustrative chemotherapeutics include proteasome inhibitors (e.g., bertezomib), thalidomide, revlimid and DNA damaging agents such as melphalan, doxorubicin, cyclophosphamide, vincristine, ethoposide, carmustine, and the like.

Example spheroids include corticosteroids such as dexamethasone or prednisone.

Bcr-Abl inhibitors include the compounds, and their pharmaceutically acceptable salts, of the genus and species described in US Patent No. 5,521,184, WO

WIF¹¹

04/005281, EP2005 / 009967, EP2005 / 010408, and USA No. of

Series 60 / 578,491.

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

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

............. Suitable example FAK inhibitors include ^........ -&gt; χ to compounds, and their pharmaceutically acceptable salts, as described in WO 04/080980, WO 04/056786, WO 03/024967, WO 01/064655, WO 00/053595, and 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.

*·'^SG ~ ^ 2 0 In some embodiments, one or more salt forms of the invention may be used in combination with a chemotherapeutic agent in the treatment of cancer, such as multiple myeloma, and may improve the response to treatment when compared to the response to the agent. Chemotherapeutic alone, without exacerbating its toxic effects. The examples of

<img file="CU20120155A7_D0017.tif" />

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 also used in the treatment of multiple myeloma include Bcr-Abl, Flt3, RAF and FAK kinase inhibitors. The additive or synergistic effects are desirable results of combining a JAK inhibitor of the present invention with an additional agent. In addition, resistance to multiple myeloma cells to agents such as dexamethasone can be reversed after treatment with a JAK inhibitor of the present invention. The agents may be combined with the compounds herein in an individual or continuous dosage form, or the agents may 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 wherein dexamethasone is administered intermittently as continuously opposed.

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

<img file="CU20120155A7_D0018.tif" />

<img file="CU20120155A7_D0019.tif" />

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 through a variety of routes, which depend on whether local or systemic treatment is desired and on the area to be treated. Administration can be topical (including transdermal, epidermal, ophthalmic and to mucous membranes that include intranasal, vaginal and rectal distribution), pulmonary (for example, through inhalation or insufflation of powders or aerosols, including through nebulizer, intrhaecal or intranasal), oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal, intramuscular or injection or infusion; or intracranial, for example, intrahecal or intraventricular administration. Parenteral administration may be in the form of an individual bolus dose or may be, for example, through a continuous perfusion gum. Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Pharmaceutically conventional carriers, aqueous, powdered or oily bases, binders and the like can be

<img file="CU20120155A7_D0020.tif" />

<img file="CU20120155A7_D0021.tif" />

Necessary or desirable. Coated condoms, gloves and the like are also useful.

This invention also includes pharmaceutical compositions containing, as active ingredient, one or more of the above compounds of the invention in combination with or more pharmaceutically acceptable carriers (excipients). In the manufacture of the compositions of the invention, the active ingredient is typically mixed with an excipient, diluted by an excipient or wrapped in a carrier in the form of, for example, a capsule, pad, paper to another container. When the excipient serves as a diluent, it can be a solid, semi-solid or liquid material, which acts as a vehicle, carrier or medium for the active ingredient. In this way, the compositions may be in the form of tablets, pills, powders, troches, pads, seals, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium), ointments containing, for example, up to 10% by weight of the active compound, soft or hard '20 gelatin capsules, sterile injectable solutions and sterile packaged powders.

In the preparation of a formulation, the active compound can be milled to provide the appropriate particle size before combining it with other ingredients. If the active compound is substantially insoluble, it can be milled to a particle size smaller than a 200 mesh. If the active compound is substantially water soluble, the particle size can be adjusted by grinding to provide a substantially uniform distribution in the formulation, for example, approximately 40 mesh.

The compounds of the invention can be milled using known milling methods such as wet milling to obtain an appropriate particle size for tablet formation and for other types of formulation. The finely divided preparations (nanoparticles) of the compounds of the invention can be prepared through 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 gum, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup and methylcellulose.

The formulations may additionally include: lubricating agents such as talc, magnesium stearate and mineral oil; wetting agents; emulsifying agents and iffWi¹ suspension; preservatives such as methyl and polypropylhydroxybenzoates; sweetening agents; and flavoring agents. The compositions of the invention can be formulated so as to provide a rapid, sustained or delayed release of the active ingredient after administration to the patient using methods known in the art.

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

The active compound can be effective over a wide range of doses and is generally administered in a pharmaceutically effective amount. It will be understood, however, that the amount of the compound currently administered will usually be determined through a physician, in accordance with the relevant circumstances, including the condition to be treated, the route of administration selected, the current compound administered, age. , the weight and response of the individual patient, the severity of the patient's symptoms, and the like.

the severity of is

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 referring to these formulation compositions as homogeneous, the active ingredient is typically dispersed uniformly throughout the composition such 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 covered or conversely formed into compounds to provide a dosage form that of the advantage of prolonged action. For example, the tablet or pill may

-20 comprising an alternate dosing component and an external dosing component, the former being in the form of a wrap on the main. The two components can be separated through an enteric layer that serves to resist disintegration in the stomach and allow the inner component to pass intact to the duodenum or retract into the

I

<img file="CU20120155A7_D0022.tif" />

release. A variety of materials can be used for such enteric layers or coatings, such as materials that include a number of polymeric acids and mixtures of polymeric acids such as shellac, cetyl alcohol and cellulose acetate materials.

Liquid forms wherein the compounds and compositions of the present invention can be incorporated for oral or injection administration include aqueous solutions, suitable flavored syrups, aqueous or oily suspensions, and flavored emulsions with edible oils such as cottonseed oils. , sesame oil, coconut oil, or peanut oil, as well as elixirs and similar pharmaceutical vehicles.

Compositions for inhalation and insufflation include solutions or suspensions in pharmaceutically acceptable aqueous or organic solvents or mixtures thereof, and powders. The liquid or solid compositions may contain suitable pharmaceutically acceptable excipients as described supra. In some embodiments, the compositions are administered through the oral or nasal respiratory route for local or systemic effect. The compositions can be nebulized through the use of inert gases. The nebulized solutions can be breathed directly from the nebulizer device or the nebulizer device can be attached one plus 25 tent, or positive pressure breathing machine

IN¹¹¹¹¹ intermittent. The solution, suspension or powder compositions may be administered orally or nasally from devices that provide the formulation in an appropriate form.

The nasal amount or composition administered to a patient will vary depending on what is being administered, the purpose of the administration, such as prophylaxis or therapy, the patient's condition, the form of administration and

....... Similar. In therapeutic applications, the compositions can be administered to a patient who already suffers from a disease in an amount sufficient to cure or at least stop the symptoms of the disease and its complications. The effective doses will depend on the disease condition being treated as well as through the judgment of the attending physician depending on factors such as disease severity, age, weight and general condition of the patient and the like.

Wffp¹¹ '

The compositions administered to a patient may be in the form of pharmaceutical compositions described above. These compositions can be sterilized through conventional sterilization techniques, or they can be sterile filtered. Aqueous solutions can be packaged for use as such, or lyophilized, the lyophilized preparation is combined with a sterile aqueous carrier before administration. The pH of the preparations

<img file="CU20120155A7_D0023.tif" />

of a compound will typically be between 3 and 11, more preferably 5 to 9 and more preferably 7 to 8. He ,_{! Lb? for}......... will understand that the use of certain excipients, carriers or stabilizers above will result in the formation of pharmaceutical salts.

The therapeutic dosage of the salts of the invention may vary according to, for example, the particular use for which the treatment is made, the method 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 h'w ....... may vary depending on a number of factors including dosage, chemical characteristics (e.g. hydrophobicity), and the route of administration. . For example, the salts of the invention may be provided in a physiological to aqueous pH regulatory solution containing from about 0.1 to about 10% w / v of the compound for parenteral administration. Some typical dose ranges are between 1 pg / kg to approximately 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 will probably depend on variables such as the type and degree of progression of the disease or disorder, the general 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 test systems or from the animal model.

The compositions of the invention may further include one or more additional pharmaceutical agents such as chemotherapeutics, steroids, an anti-inflammatory compound, or immunosuppressants, examples of which are listed here above.

Marked Compounds and Test Methods

Another aspect of the present invention relates to the labeled salts of the invention (radiolabelled with fluorescence, etc.) that could be useful not only in imaging techniques but also in vitro and in vivo assays to locate and quantify JAK in tissue samples, including humans, and to identify JAK ligands through inhibition of binding of a labeled compound. Accordingly, the present invention includes JAK assays containing the labeled compounds.

The present invention also includes isotopically labeled salts of the invention. An isotopically or radiolabeled compound is a salt of the invention wherein one more atoms are replaced or substituted by an atom that has an atomic mass or a mass number different from the atomic mass or mass number typically found in nature (it is say of natural existence).

Suitable radionuclides that can be incorporated into the compounds of the present invention include but are not limited to, ²H (also written as D for deuterium), ³H (also

13,

14,

13,, 15,

15, written as T for tritium), ¹¹C, ^iJC, ¹⁴C, ^{± J}N, ¹⁰N, ^so0, ^±'OR,

The, ⁱ⁸F, ^3üS ^3bCl, ⁸²Br, ^{/ b}Br, ^{/ b}Br, ^{WITH /}Br,

123

I

124_T 125 z

I and ¹³¹I.

Radiolabelled compounds are incorporated herein will depend on the specific application of the radiolabeled compound. For example, for labeling and competition assays of in vitro metalloprotease, the compounds that incorporate³H ¹⁴C, ⁸²Br, ^lz5I ¹³¹I ³⁵S will generally be more useful. For radio applications The radionuclide that imaging^ORC, ¹⁸F, ¹²⁵I ⁱ²³I ¹²⁴I ¹³¹I ⁷⁵Br,

Br o

77_r

Br will generally be more useful.

It is understood that a radiolabelled or labeled compound is a salt that has at least one radionuclide incorporated. In some embodiments, radionuclides are selected from the group consisting of³H ^14/

The present invention may also include synthetic methods for incorporating radio-i sotopes in the compounds of the invention. Synthetic methods for incorporating radio-isotopes into organic compounds are well known in the art and one skilled in the art will readily recognize the applicable methods for the compounds of the invention.

A labeled salt of the invention can be used in ⁴C, ¹²⁵I ³⁵S and ⁸²Br.

bt

a classification test to identify / evaluate compounds. For example, a newly synthesized or identified compound (i.e., a test compound) that is labeled can be evaluated for its ability to bind to a JAK by monitoring its variation in concentration when it contacts the JAK through tracking the dial. For example, a test compound (labeled) can be evaluated for its ability to reduce the binding of another compound that is known to bind a JAK (i.e., a standard compound). Therefore, the ability of a test compound to compete with the standard compound for binding to JAK directly correlates with its binding affinity. Conversely, in some other classification tests, the standard compound is labeled and the test compounds are unchecked. Accordingly, the concentration of the labeled standard compound is monitored in order to assess the competition between the standard compound and the test compound, and the relative affinity for the test compound.

<img file="CU20120155A7_D0024.tif" />

IN this way it is evaluated.

Ki ts

The present invention also includes pharmaceutical kits useful, for example, in the treatment or prevention of diseases or disorders associated with JAK, such as cancer, inflammation, or skin disorders, which include or more containers containing a pharmaceutical composition comprising a therapeutically effective amount of a salt of the invention. Such kits may also include, if desired, one or more of the 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 an expert. in the technique Instructions, either as inserts or as labels, indicate¹¹¹ The quantities of components to be administered, instructions for administration and / or instructions for mixing the components, can also be included in the kit.

The invention is described in greater detail through the specific examples. The following specific examples are presented 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 to produce essentially the same results.

EXAMPLES

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

To a test tube (R) -3 - (4 - (7H-pyrrolo [2,3-d] pyrimidin-4-yl) -lH-pyrazol-l-yl) - 325 cyclopropanitrile (153.7 mg, 0.5) was added mmoles) maleic acid (61.7

..... ¹ ~ mg) followed by isopropyl alcohol (IPA) (4 ml). The resulting mixture was heated to 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 then dried under vacuum at a constant weight to provide the final salt product (173 mg).

The maleic acid salt proved to be a 1: 1 salt per H¹ NMR and crystallinity was confirmed by X-ray powder diffraction (XRPD). Differential scanning calorimetry (DSC) gave a precise melting peak at approximately 175.96 ° C (starting at 175.67 ° C). The product showed only slight weight loss up to 150 ° C by thermogravimetric analysis (TGA).

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

To a test tube (R) -3- (4- (7H. 2 0 pyrrolo [2,3-d] pyrimidin-4-yl) -lH-pyrazol-l-i1) -3cyclopropanitriium (153.5 mg) was added ) and phosphoric acid (56.6 mg) followed by isopropyl alcohol (IPA) (5.75 ml). The resulting mixture was heated to become transparent, cooled to room temperature, and then stirred for another 2 hours. The precipitate was collected by filtration and the cake was

<img file="CU20120155A7_D0025.tif" />

W ”'15 washed with 0.6 ml of cold IPA. The cake was then dried under vacuum at a constant weight to provide the final salt product (171.7 mg). The phosphoric acid salt proved to be a 1: 1 salt by N NMR and the crystallinity was confirmed by X-ray powder diffraction (XRPD). Differential scanning calorimetry (DSC) gave a precise melting peak at approximately 198.66 ° C. The product showed a slight weight loss up to 200 ° C by TGA.

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

To a test tube (R) - 3 - (4 - (7II- pyrrolo [2,3-d] pyrimidin-4-yl) -lH-pyrazol-l-yl) -3-cyclopropanitrile (153.0 mg) and acid was added sulfuric acid (56.1 mg) followed by acetonitrile (7.0 ml). The resulting mixture was heated to become transparent, 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 then dried under vacuum at a constant weight to provide the final salt product (180 mg).

<img file="CU20120155A7_D0026.tif" />

<img file="CU20120155A7_D0027.tif" />

The sulfuric acid salt proved to be a salt of

1: 1 by NMR and crystallinity was confirmed by X-ray powder diffraction (XRPD). Differential scanning calorimetry (DSC) gave a precise melting peak at

<img file="CU20120155A7_D0028.tif" />

<img file="CU20120155A7_D0029.tif" />

'W ......

approximately 186.78 ° C. The product showed a slight weight loss up to 175 ° C by TGA.

Example A

In vitro JAK Kinase Assay

The inhibitory activity of test compounds in JAK targets can be tested according to the following

<td>rehearsal in</td><td>I saw</td><td>described in</td><td>Park</td><td>and</td><td>others Analytical</td>

<td>Biochemistry</td><td> 1999,</td><td> 269, 94-104.</td><td>The</td><td colspan="2">catalytic domains of</td>

<td>JAK1 human</td><td>(a. a.</td><td> 837-1 142),</td><td>Jak2 (</td><td>to. to.</td><td>828-1 132) and Jak3</td>

<img file="CU20120155A7_D0030.tif" />

(aa 781-1124) with an N-terminal His tag were expressed using baculovirus in insect cells and purified.

The catalytic activity of JAK1, JAK2 or JAK3 was tested by measuring the phosphorylation of a biotened peptide. The phosphorylated peptide was detected by homogeneous time resolved fluorescence (HTRF). The IC50 of the compounds were measured for each kinase in the reactions

<td></td><td>containing</td><td>the</td><td colspan="2">enzyme,</td><td colspan="2">ATP and 500</td><td>nM</td><td>from</td><td>peptide</td><td>in</td><td> 50</td><td>mM of</td>

<td>'' In .....</td><td>regulator</td><td>pH</td><td>Three</td><td>(pH</td><td> 7.8)</td><td>with</td><td> 100</td><td>mM</td><td>of NaCI,</td><td> 5</td><td>mM</td><td>of DTT,</td>

<td></td><td>and 0.1 mg / ml</td><td> (0</td><td> . 01%)</td><td>from</td><td>BSA</td><td>The</td><td colspan="3">concentration</td><td>from</td><td>ATP</td><td>in the</td>

<img file="CU20120155A7_D0031.tif" />

reactions is 90 μΜ for Jakl, 30 μΜ for Jak2 and 3 μΜ for Jak3. The reactions were carried out at room temperature for 1 hour and then stopped with 20 μΐ of 45 mM EDTA, 300 nM SA-APC, 6 nM Eu-Py20 in assay pH regulator (Perkin Elmer, Boston, MA). Binding to the labeled Europium antibody took place in 40 minutes and the HTRF signal was^11,1

<img file="CU20120155A7_D0032.tif" />

measured in 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 IC values.₅₀ less than 50 nM for each JAK1, JAK2, and JAK3.

Various modifications of the invention, in addition to those described herein, will be apparent to those skilled in the art from the above description. Such modifications are also intended to fall within the scope of the appended claims. Each reference cited in this application is incorporated herein by reference in its entirety.

<img file="CU20120155A7_D0033.tif" />

<img file="CU20120155A7_D0034.tif" />

Havana City, October 30, 2012

Dánice Vázquez D'Alvaré Official Agent