JP6310474B2 - Solid form of dihydro-pyrido-oxazine derivatives - Google Patents

Description

  The present invention relates to novel solid forms of dihydro-pyrido-oxazine derivatives, methods for their preparation and their use in pharmaceutical compositions.

International Patent Application No. PCT / IB2012 / 057554, published as WO2013 / 093849, discloses dihydro-benzo-oxazine and dihydro-pyrido-oxazine derivatives suitable for the treatment of disorders or diseases mediated by the activity of the PI3K enzyme. Yes. PCT / IB2012 / 057554 is (1,1-dioxo-hexahydro-1λ ⁶ -thiopyran-4-yl)-{(S) -3- [1- (6-methoxy-5-methyl-pyridin-3-yl) ) -2,3-dihydro-1H-pyrido [3,4-b] [1,4] oxazin-7-yloxy] -pyrrolidin-1-yl} -methanone and methods for making the compounds are disclosed Yes.

  These compounds, alone or in combination with one or more other pharmacologically active compounds, include but are not limited to autoimmune disorders, inflammatory diseases, allergic diseases, airway diseases such as asthma and COPD Useful for the treatment of transplant rejection, eg, diseases associated with PI3K, including cancer or solid tumors of hematopoietic origin.

  These compounds can also be used alone or in combination with one or more other pharmacologically active compounds to treat a condition, disease or disorder such as an autoimmune disorder, inflammatory disease, allergic disease, airway disease such as , Asthma and COPD, graft rejection; rheumatoid arthritis, pemphigus vulgaris, idiopathic thrombocytopenic purpura, systemic lupus erythematosus, multiple sclerosis, myasthenia gravis, Sjogren's syndrome, autoimmune hemolytic anemia, ANCA Related vasculitis, cold globulinemia, thrombotic thrombocytopenic purpura, chronic autoimmune urticaria, allergy (atopic dermatitis, contact dermatitis, allergic rhinitis), Goodpasture syndrome, AMR ( Antibody-mediated graft rejection), B cell-mediated hyperacute, acute and chronic graft rejection, and without limitation, multiple occurrences Myeloma; leukemia; acute myeloid leukemia; chronic myelogenous leukemia; lymphocytic leukemia; myeloid leukemia; non-Hodgkin's lymphoma; lymphoma; erythrocytosis; essential thrombocythemia; And cancers of hematopoietic origin, including Waldenstrom disease, with abnormal or undesirable antibody production, antigen presentation, cytokine production or lymphoid tissue formation; more suitably rheumatoid arthritis (RA), Pemphigus vulgaris (PV), idiopathic thrombocytopenic purpura (ITP), thrombotic thrombocytopenic purpura (TTP), autoimmune hemolytic anemia (AIHA), acquired hemophilia type A (AHA) Systemic lupus erythematosus (SLE), multiple sclerosis (MS), myasthenia gravis (MG), Sjogren's syndrome (SS), vasculitis associated with ANCA, cold globules Blood pressure, chronic autoimmune urticaria (CAU), allergy (atopic dermatitis, contact dermatitis, allergic rhinitis), Goodpasture syndrome, cancer of transplant rejection and hematopoietic origin, and disease or infection It is useful for the treatment of related immunopathologies such as severe and cerebral malaria, trypanosomiasis, leishmaniasis, toxoplasmosis and neurocystic disease.

The present invention relates to (1,1-dioxo-hexahydro-1λ ⁶ -thiopyran-4-yl)-{(S) -3- [1- (6-methoxy-5-methyl-pyridin-3-yl) -2 , 3-dihydro-1H-pyrido [3,4-b] [1,4] oxazin-7-yloxy] -pyrrolidin-1-yl} -methanone, an anhydrous crystalline form; relating to pharmaceutical compositions and combinations comprising this form . The invention further relates to methods of using this form, including pharmaceutical compositions and combinations thereof for the treatment of diseases mediated by the activity of the PI3K enzyme, preferably the activity of the PI3Kδ isoform.

  The crystalline form of an active pharmaceutical ingredient (API) of a particular drug is easy to prepare the drug, hygroscopic, stable, soluble, storage stability, ease of formulation, rate of dissolution in gastrointestinal fluid, and in vivo It is well known that it is often an important determinant of bioavailability. Crystal forms occur, where the same material composition crystallizes with different lattice arrangements, resulting in different thermodynamic properties and stability specific to a particular crystal form. Crystal forms can also include different hydrates or solvates of the same compound. In determining which form is preferred, multiple properties of the form are compared and the preferred form is selected based on the variable portion of the many physical properties. It is entirely possible that one form may be preferred in certain circumstances where certain aspects, such as ease of preparation, stability, etc. are deemed critical. In other situations, different forms may be preferred for faster dissolution rates and / or better bioavailability. Predicts whether a particular compound forms a polymorph, whether any such polymorph is suitable for commercial use in a therapeutic composition, or what polymorphs exhibit such desirable properties It is not yet possible to do.

Example F1 is an X-ray powder diffraction pattern of crystalline anhydrous form. It is a differential scanning calorimetry graph of Example F1 and a crystalline anhydrous form.

In one embodiment, the invention provides (1,1-dioxo-hexahydro-1λ ⁶ -thiopyran-4-yl)-{(S) -3- [1- (6-methoxy-5-methyl-pyridine-3 -Yl) -2,3-dihydro-1H-pyrido [3,4-b] [1,4] oxazin-7-yloxy] -pyrrolidin-1-yl} -methanone relates to the anhydrous crystalline form.

In another embodiment, (1,1-dioxo-hexahydro-1λ ⁶ -thiopyran-4-yl)-{(S) -3- [1- (6-methoxy-5-methyl-pyridin-3-yl) The anhydrous crystalline form of -2,3-dihydro-1H-pyrido [3,4-b] [1,4] oxazin-7-yloxy] -pyrrolidin-1-yl} -methanone has a degree of 2 theta +/- 0 The following peaks obtained at 2 degrees: 9.1, 10.2, 11.9, 13.0, 17.1, 17.7, 18.7, 20.3, 20.8, 26.0, Features X-ray powder diffraction patterns including 26.7, 23.2, 24.1, 24.8, 29.3, 27.4, and 21.4.

In another embodiment, (1,1-dioxo-hexahydro-1λ ⁶ -thiopyran-4-yl)-{(S) -3- [1- (6-methoxy-5-methyl-pyridin-3-yl) The anhydrous crystalline form of -2,3-dihydro-1H-pyrido [3,4-b] [1,4] oxazin-7-yloxy] -pyrrolidin-1-yl} -methanone is the X-ray powder shown in FIG. The X-ray diffraction spectrum is substantially the same as the diffraction spectrum.

In another embodiment, (1,1-dioxo-hexahydro-1λ ⁶ -thiopyran-4-yl)-{(S) -3- [1- (6-methoxy-5-methyl-pyridin-3-yl) The anhydrous crystalline form of -2,3-dihydro-1H-pyrido [3,4-b] [1,4] oxazin-7-yloxy] -pyrrolidin-1-yl} -methanone is the differential scanning calorific value shown in FIG. It has a differential scanning calorimetry graph that is substantially the same as the measurement graph.

Unless otherwise specified, the term “form of the invention” refers to (1,1-dioxo-hexahydro-1λ ⁶ -thiopyran-4-yl)-{(S) -3- [1- (6-methoxy-5 Anhydrous crystalline form of -methyl-pyridin-3-yl) -2,3-dihydro-1H-pyrido [3,4-b] [1,4] oxazin-7-yloxy] -pyrrolidin-1-yl} -methanone Point to.

  The general terms used above and below in this specification preferably have the following meanings within the context of this disclosure, unless otherwise indicated.

  The invention can be more fully understood with reference to the following description, including the following glossary of terms and concluding examples. As used herein, the terms “including”, “containing” and “comprising” are used herein in an open, non-limiting sense. The

  As used herein, the term “pharmaceutically acceptable carrier” includes any solvent, dispersion medium, coating agent, surfactant, antioxidant, known to those of skill in the art. Preservatives (eg, antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drug stabilizers, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, Dyes and the like and combinations thereof (see, for example, Remington's Pharmaceutical Sciences, 18th Edition Mack Printing Company, 1990, pages 1289-1329). Except insofar as any conventional carrier is incompatible with the active ingredient, its use in therapeutic or pharmaceutical compositions is contemplated. The term “therapeutically effective amount” of a compound of the present invention induces a biological or medical response in a subject, eg, a reduction or inhibition of enzyme or protein activity, or ameliorates symptoms, alleviates a condition, It refers to the amount of a compound of the present invention that slows or delays progression, or prevents disease or the like. In one non-limiting embodiment, the term “therapeutically effective amount” when administered to a subject is (1) (i) mediated by PI3K, or (ii) associated with PI3K activity, or (iii) PI3K. At least partially alleviate, inhibit, prevent and / or ameliorate a condition, or disorder or disease characterized by the activity (normal or abnormal) of (2) reduce or inhibit the activity of PI3K; Alternatively (3) refers to the amount of a compound of the invention effective to reduce or inhibit PI3K expression. In another non-limiting embodiment, the term “therapeutically effective amount” at least partially reduces the activity of PI3K when administered to a cell, or tissue, or a biological material that is not a cell, or medium. Refers to the amount of a compound of the invention effective to inhibit; or at least partially reduce or inhibit PI3K expression. The meaning of the term “therapeutically effective amount” as exemplified in the above embodiment for PI3K also applies to the same meaning to any other relevant protein / peptide / enzyme.

  As used herein, the term “subject” refers to an animal. Typically, the animal is a mammal. A subject also refers to, for example, primates (eg, humans, men or women), cows, sheep, goats, horses, dogs, cats, rabbits, rats, mice, fish, birds, and the like. In certain embodiments, the subject is a primate. In yet another embodiment, the subject is a human.

As used herein, the terms “inhibit”, “inhibition” or “inhibiting”
Refers to a reduction or suppression of a given condition, symptom, or disorder, or disease, or a significant decrease in the basic activity of a biological activity or process.

  As used herein, the term “treat”, “treating” or “treatment” of any disease or disorder, in one embodiment, improves the disease or disorder (ie, progression of the disease). Or delay or prevent or reduce at least one of its clinical symptoms). In another embodiment, “treating”, “treating” or “treatment” refers to reducing or improving at least one physical parameter, including those that may not be recognized by the patient. Point to. In yet another embodiment, “treat”, “treating” or “treatment” is either physical (eg, stabilization of recognizable symptoms) or physiological (eg, stabilization of physical parameters). Refers to modulating a disease or disorder. In yet another embodiment, “treating”, “treating” or “treatment” refers to preventing or delaying the onset or onset or progression of the disease or disorder.

  As used herein, a subject “needs” such treatment if the subject benefits from treatment in biological, medical or quality of life.

  As used herein, the terms “a”, “an”, “the” and similar terms used in the context of the present invention, particularly in the context of the claims, Unless otherwise indicated, or unless clearly contradicted by context, this should be interpreted to include both the singular and the plural.

  All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. Use of any and all examples provided herein or exemplary words, such as “such as”, is intended solely to make the present invention more clear and is set forth in the separate claims. It is not intended to limit the scope of the claimed invention.

  In another aspect, the present invention provides a pharmaceutical composition comprising a form of the present invention and a pharmaceutically acceptable carrier. The pharmaceutical composition can be formulated for a particular route of administration, such as oral, parenteral, and rectal administration. In addition, the pharmaceutical composition of the present invention can be in solid form (including, but not limited to, capsules, tablets, pills, granules, powders or suppositories) or in liquid form (unlimited, solutions, suspensions). Or an emulsion). The pharmaceutical composition can be subjected to conventional pharmaceutical processes such as sterilization and / or conventional inert diluents, lubricants or buffers, and adjuvants such as preservatives, stabilizers, wetting agents, An emulsifier, a buffer solution and the like can be contained.

Typically, the pharmaceutical composition is
a) diluents such as lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and / or glycine;
b) Lubricants such as silica, talcum, stearic acid, magnesium or calcium salts thereof and / or polyethylene glycol; further for tablets c) Binders such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose Carboxymethylcellulose sodium and / or polyvinylpyrrolidone; as appropriate d) disintegrants such as starch, agar, alginic acid or its sodium salts, or foaming mixtures; and / or e) absorbents, colorants, flavors and sweetness A tablet or gelatin capsule containing the active ingredient together with the agent.

  The tablets can be film coated or enteric coated according to methods known in the art.

  Compositions suitable for oral administration are effective in the form of tablets, lozenges, aqueous or oily suspensions, dispersible powders, or granules, emulsions, hard or soft capsules, or syrups or elixirs. A quantity of the form of the invention. Compositions intended for oral use are prepared according to any method known in the art for the manufacture of pharmaceutical compositions, and such compositions are formulated into pharmaceutically good and tasty formulations. To provide, one or more agents selected from the group consisting of sweeteners, flavoring agents, coloring agents and preservatives can be included. Tablets may contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients that are suitable for the manufacture of tablets. These excipients include, for example, inert diluents such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents such as corn starch, or alginic acid; binders such as starch Gelatin or acacia; and lubricants such as magnesium stearate, stearic acid or talc. Tablets are uncoated or coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate may be employed. Formulations for oral use are as hard gelatin capsules in which the active ingredient is mixed with an inert solid diluent such as calcium carbonate, calcium phosphate or kaolin, or the active ingredient is water or an oily medium such as peanut oil. Can be provided as soft gelatin capsules mixed with liquid paraffin or olive oil.

  Certain injectable compositions are aqueous isotonic solutions or suspensions, and suppositories are advantageously prepared from fatty emulsions or suspensions. The compositions can be sterilized and / or contain adjuvants such as preservatives, stabilizers, wetting or emulsifying agents, solution promoters, salts and / or buffers for regulating osmotic pressure. Can be contained. In addition, they can contain other therapeutically valuable substances. The compositions are prepared according to conventional mixing, granulating or coating methods, respectively, and contain about 0.1 to 75%, or about 1 to 50% active ingredient.

  Compositions suitable for transdermal administration include an effective amount of a form of the invention together with a suitable carrier. Suitable carriers for transdermal delivery include absorbable pharmacologically acceptable solvents to assist passage through the skin of the host. For example, transdermal devices include a reservoir containing a compound with a substrate, optionally a carrier, and optionally a rate-controlling barrier for delivering a compound of the host skin at a controlled rate over an extended period of time. And in the form of a bandage including means for securing the device to the skin.

  Compositions suitable for topical application, eg, skin and eyes, include aqueous solutions, suspensions, ointments, creams, gels, or sprayable formulations for delivery by, for example, an aerosol. Such topical delivery systems are particularly suitable for dermal application, for example for the treatment of skin cancer, for example prophylactic use in sunscreen creams, lotions, sprays and the like. They are therefore particularly suitable for use in topical formulations, including cosmetics, well known in the art. Such compositions can contain solubilizers, stabilizers, tonicity enhancing agents, buffers and preservatives.

  As used herein, topical application may belong to inhalation or intranasal application. They are dry powders (dry blends with mixtures, eg lactose) from aerosol spray presentations from dry powder inhalers or pressurized containers, pumps, sprays, atomizers or nebulizers, with or without the use of suitable propellants As such or in the form of, for example, mixed component particles with phospholipids).

  Since water can facilitate the degradation of certain compounds, the present invention further provides anhydrous pharmaceutical compositions and dosage forms comprising the forms of the present invention as active ingredients.

  Anhydrous pharmaceutical compositions and dosage forms of the invention can be prepared using anhydrous or low moisture containing ingredients and low moisture or low humidity conditions. An anhydrous pharmaceutical composition can be prepared and stored such that its anhydrous nature is maintained. Accordingly, anhydrous compositions are packaged using materials known to prevent exposure to water so that they can be included in suitable formulation kits. Examples of suitable packaging include, but are not limited to, sealed foils, plastics, unit dose containers (eg, vials), blister packs, and strip packs.

  The invention further provides pharmaceutical compositions and dosage forms that comprise one or more agents that reduce the rate by which the forms of the invention as an active ingredient degrade. Such agents referred to herein as “stabilizers” include, but are not limited to, antioxidants such as ascorbic acid, pH buffers, or salt buffers.

  Forms of the invention include rheumatoid arthritis, pemphigus vulgaris and related diseases, idiopathic thrombocytopenic purpura, systemic lupus erythematosus, multiple sclerosis, myasthenia gravis, Sjogren's syndrome, autoimmune hemolytic anemia, ANCA-related vasculitis, cold globulinemia, thrombotic thrombocytopenic purpura, chronic autoimmune urticaria, allergy (atopic dermatitis, contact dermatitis, allergic rhinitis), Goodpasture syndrome, AMR (Antibody mediated graft rejection), B cell mediated hyperacute, acute and chronic graft rejection, and without limitation, multiple myeloma; acute myeloid leukemia; chronic myeloid leukemia; lymphocytic Leukemia; myeloid leukemia; non-Hodgkin lymphoma; lymphoma; polycythemia vera; essential thrombocythemia; myelofibrosis with myeloid metaplasia; Associated with one or more of the functions of B cells, including cancers of hematopoietic origin including Nström's disease, for example, antibody production, antigen presentation, cytokine production or lymphoid tissue formation is abnormal or undesirable May be useful in the treatment of conditions, diseases or disorders involving immunopathology.

  The present invention relates to one or more of the functions of neutrophils, including superoxide release, stimulated, including rheumatoid arthritis, sepsis, lung or respiratory disorders such as asthma, inflammatory skin diseases such as psoriasis It includes methods of treating conditions in which exocytosis, or chemotaxis migration is abnormal or undesirable, diseases or disorders, and immunopathologies associated with the disease or infection.

  The present invention relates to one or more of the functions of basophils and mast cells, including allergic diseases (atopic dermatitis, contact dermatitis, allergic rhinitis), such as chemotactic migration or allergen-IgE- Including methods in which mediated degranulation is abnormal or undesirable, diseases or disorders, as well as other disorders such as COPD, asthma or emphysema.

  The present invention is mediated by one or more of the functions of T cells, including cytokine production or cells, including rheumatoid arthritis, multiple sclerosis, acute or chronic rejection of cellular tissue or organ transplantation, or cancer of hematopoietic origin. Includes methods of treating conditions, diseases or disorders where the cytotoxicity is abnormal or undesirable, as well as immune pathologies associated with the disease or infection.

  Furthermore, the present invention includes methods for treating neurodegenerative diseases, cardiovascular diseases and platelet aggregation. Furthermore, the present invention provides skin diseases such as late cutaneous porphyria, polymorphic sun rash, dermatomyositis, sun urticaria, lichen planus, panniculitis, scleroderma, urticaria-like vasculitis Including methods of treating.

  Furthermore, the present invention includes methods for treating chronic inflammatory diseases such as sarcoidosis, cyclic granulomas.

  In other embodiments, the condition or disorder (eg, mediated by PI3K) is polycythemia vera, essential thrombocythemia, myelofibrosis with myeloid metaplasia, asthma, COPD, ARDS, Leffler syndrome, favorable Eosinophilic pneumonia, parasites (especially metazoans) parasites (including tropical eosinophilia), bronchopulmonary aspergillosis, polyarteritis nodosa (including Churg-Strauss syndrome), eosinophils Granulomas, disorders involving eosinophils that affect the respiratory tract caused by drug reaction, psoriasis, contact dermatitis, atopic dermatitis, alopecia areata, polymorphic erythema, herpetic dermatitis, scleroderma , Vitiligo, irritable vasculitis, hives, bullous pemphigoid, lupus erythematosus, pemphigus, acquired epidermolysis bullosa, autoimmune blood disorders (eg, hemolytic anemia, aplastic anemia, genuine red blood cells) Sexual anemia Idiopathic thrombocytopenia), systemic lupus erythematosus, polychondritis, scleroderma, Wegener's granulomatosis, dermatomyositis, chronic active hepatitis, myasthenia gravis, Stevens-Johnson syndrome, idiopathic sprue, autoimmunity Inflammatory bowel disease (eg, ulcerative colitis and Crohn's disease), endocrine eye disorders, Graves' disease, sarcoidosis, alveolitis, chronic hypersensitivity pneumonitis, multiple sclerosis, primary biliary cirrhosis, uvea Inflammation (anterior and posterior), interstitial pulmonary fibrosis, psoriatic arthritis, glomerulonephritis, cardiovascular disease, atherosclerosis, hypertension, deep vein thrombosis, stroke, myocardial infarction, unstable angina Thromboembolism, pulmonary embolism, thrombolysis, acute arterial ischemia, peripheral thrombotic occlusion, and coronary artery disease, reperfusion injury, retinopathy, eg diabetic retinopathy or hyperbaric oxygen Retinopathy emitted, and elevated intraocular pressure or a condition characterized by secretion of aqueous humor, for example, is selected from the group consisting of glaucoma.

  In another embodiment, the forms of the present invention comprise an autoimmune disease and an inflammatory condition, particularly an inflammatory condition with an etiology that includes an autoimmune component, such as arthritis (eg, rheumatoid arthritis, chronic progressive arthritis and osteoarthritis). Arthritis), and rheumatic diseases including inflammatory conditions, and rheumatic diseases involving bone loss, inflammatory pain, spondyloarthropathy including ankylosing spondylitis, Reiter syndrome, reactive arthritis, psoriatic arthritis, and enteropathic Useful in the treatment, prevention, or amelioration of arthritis, hypersensitivity (including both airway hypersensitivity and cutaneous hypersensitivity), and allergies. In contrast, certain autoimmune diseases that may use the forms of the invention include autoimmune blood disorders (eg, including hemolytic anemia, aplastic anemia, genuine erythrocytic anemia and idiopathic thrombocytopenia), Acquired hemophilia A, cold agglutinin disease, cold globulinemia, thrombotic thrombocytopenic purpura, Sjogren's syndrome, systemic lupus erythematosus, inflammatory myopathy, polychondritis, scleroderma, antineutrophil cytoplasm Antibody-related vasculitis, IgM-mediated neuropathy, ocular clonusmyoclonus syndrome, Wegener's granulomatosis, dermatomyositis, chronic active hepatitis, myasthenia gravis, psoriasis, Stevens-Johnson syndrome, pemphigus vulgaris, decidual pemphigus, idiopathic Sprue, autoimmune inflammatory bowel disease (including ulcerative colitis, Crohn's disease and irritable bowel syndrome), endocrine eye disorders, Graves , Sarcoidosis, multiple sclerosis, neuromyelitis optica, primary biliary cirrhosis, juvenile diabetes (type 1 diabetes mellitus), uveitis (anterior, middle and posterior, and panuveitis), dry keratoconjunctivitis And spring keratoconjunctivitis, interstitial pulmonary fibrosis, psoriatic arthritis and glomerulonephritis (eg, with and without idiopathic nephrotic syndrome or nephrotic syndrome including minimal change nephropathy), tumor, skin and corneal inflammation Sexual diseases, myositis, loose bone grafts, metabolic disorders such as atherosclerosis, diabetes, and dyslipidemia.

  In another embodiment, the form of the invention comprises primary cutaneous B-cell lymphoma, autoimmune bullous disease, pemphigus vulgaris, deciduous pemphigus, a unique form of Brazilian pemphigus (Brazilian pemphigus), tumor Concomitant pemphigus, bullous pemphigoid, mucocele pemphigoid, acquired epidermolysis bullosa, chronic graft-versus-host disease, dermatomyositis, systemic lupus erythematosus, vasculitis, small vessel inflammation, hypocomplementary renal disease Measles-like vasculitis, antineutrophil cytoplasmic antibody-vasculitis, cold globulinemia, Schnitzler syndrome, Waldenstrom macroglobulinemia, angioedema, vitiligo, systemic lupus erythematosus, idiopathic thrombocytopenic purpura Selected from the group consisting of multiple sclerosis, cold agglutinin disease, autoimmune hemolytic anemia, anti-neutrophil cytoplasmic antibody-related vasculitis, graft-versus-host disease, cold globulinemia and thrombocytopenia Useful in the treatment of conditions or disorders.

Thus, as a further embodiment, the present invention relates to (1,1-dioxo-hexahydro-1λ ⁶ -thiopyran-4-yl)-{(S) -3- [1- (6-methoxy-5) in therapy Anhydrous crystalline form of -methyl-pyridin-3-yl) -2,3-dihydro-1H-pyrido [3,4-b] [1,4] oxazin-7-yloxy] -pyrrolidin-1-yl} -methanone Provide the use of. In a further embodiment, the treatment is selected from diseases that can be treated by inhibition of PI3K. In another embodiment, the disease is as listed above, suitably an autoimmune disorder, inflammatory disease, allergic disease, airway disease such as asthma and COPD, graft rejection; rheumatoid arthritis, pemphigus vulgaris, Idiopathic thrombocytopenic purpura, systemic lupus erythematosus, multiple sclerosis, myasthenia gravis, Sjogren's syndrome, autoimmune hemolytic anemia, ANCA-related vasculitis, cold globulinemia, thrombotic thrombocytopenic purpura Disease, chronic autoimmune urticaria, allergy (atopic dermatitis, contact dermatitis, allergic rhinitis), Goodpasture syndrome, AMR (antibody mediated graft rejection), B cell mediated hyperacute, Acute and chronic transplant rejection, and without limitation, multiple myeloma; leukemia; acute myeloid leukemia; chronic myeloid leukemia; lymphocytic leukemia Non-Hodgkin lymphoma; lymphoma; polycythemia vera; essential thrombocythemia; myelofibrosis with myeloid metaplasia; and antibody production, antigens, including cancers of hematopoietic origin including Waldenstrom disease From presentations where cytokine production or lymphoid tissue formation is abnormal or undesirable; more suitably, rheumatoid arthritis (RA), pemphigus vulgaris (PV), idiopathic thrombocytopenic purpura (ITP), Thrombotic thrombocytopenic purpura (TTP), autoimmune hemolytic anemia (AIHA), acquired hemophilia type A (AHA), systemic lupus erythematosus (SLE), multiple sclerosis (MS), myasthenia gravis Disease (MG), Sjogren's syndrome (SS), vasculitis associated with ANCA, cold globulinemia, chronic autoimmune urticaria (CAU), allergy (atopic skin) Inflammation, contact dermatitis, allergic rhinitis), Goodpasture syndrome, cancer of graft rejection and hematopoietic origin, and immunopathologies associated with the disease or infection, such as severe and cerebral malaria, trypanosomiasis, leishmaniasis, Selected from Toxoplasmosis and Neurocysticosis.

In another embodiment, the present invention provides a therapeutically acceptable amount of (1,1-dioxo-hexahydro-1λ ⁶ -thiopyran-4-yl)-{(S) -3- [1- (6- Of methoxy-5-methyl-pyridin-3-yl) -2,3-dihydro-1H-pyrido [3,4-b] [1,4] oxazin-7-yloxy] -pyrrolidin-1-yl} -methanone A method of treating a disease treated by inhibition of PI3K comprising administering an anhydrous crystalline form is provided. In a further embodiment, the disease is from the above list, suitably an autoimmune disorder, inflammatory disease, allergic disease, airway disease such as asthma and COPD, graft rejection; rheumatoid arthritis, pemphigus vulgaris, Idiopathic thrombocytopenic purpura, systemic lupus erythematosus, multiple sclerosis, myasthenia gravis, Sjogren's syndrome, autoimmune hemolytic anemia, ANCA-related vasculitis, cold globulinemia, thrombotic thrombocytopenic purpura Disease, chronic autoimmune urticaria, allergy (atopic dermatitis, contact dermatitis, allergic rhinitis), Goodpasture syndrome, AMR (antibody mediated graft rejection), B cell mediated hyperacute, Acute and chronic graft rejection, and without limitation, multiple myeloma; leukemia; acute myeloid leukemia; chronic myeloid leukemia; lymphocytes Antibody production, including leukemia; myeloid leukemia; non-Hodgkin lymphoma; lymphoma; polycythemia vera; essential thrombocythemia; myelofibrosis with myeloid metaplasia; and cancers of hematopoietic origin including Waldenstrom's disease From abnormal or undesirable antigen presentation, cytokine production or lymphoid tissue formation; more suitably rheumatoid arthritis (RA), pemphigus vulgaris (PV), idiopathic thrombocytopenic purpura (ITP) ), Thrombotic thrombocytopenic purpura (TTP), autoimmune hemolytic anemia (AIHA), acquired hemophilia type A (AHA), systemic lupus erythematosus (SLE), multiple sclerosis (MS), severe Myasthenia gravis (MG), Sjogren's syndrome (SS), ANCA-related vasculitis, cold globulinemia, chronic autoimmune urticaria (CAU), allergy (at -Dermatitis, contact dermatitis, allergic rhinitis), Goodpasture syndrome, cancer of graft rejection and hematopoietic origin, and immunopathologies associated with the disease or infection, eg severe and cerebral malaria, trypanosomiasis, leash Selected from maniasis, toxoplasmosis and neurocysticosis.

Thus, as a further embodiment, the present invention provides (1,1-dioxo-hexahydro-1λ ⁶ -thiopyran-4-yl)-{(S) -3- [1- (6 -Methoxy-5-methyl-pyridin-3-yl) -2,3-dihydro-1H-pyrido [3,4-b] [1,4] oxazin-7-yloxy] -pyrrolidin-1-yl} -methanone The use of the anhydrous crystalline form of In a further embodiment, the medicament is for the treatment of a disease that can be treated by inhibition of PI3K. In another embodiment, the disease is from the above list, suitably an autoimmune disorder, inflammatory disease, allergic disease, airway disease such as asthma and COPD, graft rejection; rheumatoid arthritis, pemphigus vulgaris , Idiopathic thrombocytopenic purpura, systemic lupus erythematosus, multiple sclerosis, myasthenia gravis, Sjogren's syndrome, autoimmune hemolytic anemia, ANCA-related vasculitis, cold globulinemia, thrombocytopenia Purpura, chronic autoimmune urticaria, allergy (atopic dermatitis, contact dermatitis, allergic rhinitis), Goodpasture syndrome, AMR (antibody mediated graft rejection), B cell mediated hyperacute Acute and chronic transplant rejection, and without limitation, multiple myeloma; leukemia; acute myeloid leukemia; chronic myelogenous leukemia; Disease; myeloid leukemia; non-Hodgkin lymphoma; lymphoma; polycythemia vera; essential thrombocythemia; myelofibrosis with myeloid metaplasia; and cancers of hematopoietic origin including Waldenstrom's disease From abnormal or undesirable antigen presentation, cytokine production or lymphoid tissue formation; more suitably rheumatoid arthritis (RA), pemphigus vulgaris (PV), idiopathic thrombocytopenic purpura (ITP) ), Thrombotic thrombocytopenic purpura (TTP), autoimmune hemolytic anemia (AIHA), acquired hemophilia type A (AHA), systemic lupus erythematosus (SLE), multiple sclerosis (MS), severe Myasthenia gravis (MG), Sjogren's syndrome (SS), ANCA-related vasculitis, cold globulinemia, chronic autoimmune urticaria (CAU), allergy (atopy) Dermatitis, contact dermatitis, allergic rhinitis), Goodpasture syndrome, cancer of graft rejection and hematopoietic origin, and immunopathologies associated with the disease or infection, eg severe and cerebral malaria, trypanosomiasis, leishmaniasis Selected from toxoplasmosis and neurocysticosis.

  A pharmaceutical composition or combination of the present invention provides a subject of about 50-70 kg with about 1-1000 mg of active ingredient, or about 1-500 mg or about 1-250 mg or about 1-150 mg or about 0.5-100 mg, or It may be a unit dose of about 1-50 mg of active ingredient. The therapeutically effective dosage of a compound, pharmaceutical composition thereof, or combination thereof will depend on the species, weight, age and individual condition of the subject, the disorder or disease or the severity of the disorder or disease to be treated. The ordinary skill physician, clinician or veterinarian can readily determine the effective amount of each of the active ingredients required to prevent, treat or inhibit progression of the disorder or disease.

The above administration characteristics can be demonstrated in in vitro and in vivo studies using advantageous mammals such as mice, rats, dogs, monkeys or isolated organs, tissues and preparations thereof. The forms of the present invention are in solution, for example in the form of an aqueous solution, in vitro, and for example as a suspension or in aqueous solution, enterally, parenterally, preferably intravenously. Can be applied. In vitro doses can vary between about ^10-3 molar and ^10-9 molar. The therapeutically effective amount in vivo depends on the route of administration and can range from about 0.1 to 500 mg / kg, or between about 1 to 100 mg / kg.

  Forms of the invention can be administered simultaneously with one or more other therapeutic agents or before or after one or more other therapeutic agents. The forms of the invention may be administered separately, by the same or different routes of administration, or together in the same pharmaceutical composition as other drugs.

In one embodiment, the present invention provides (1,1-dioxo-hexahydro-1λ ⁶ -thiopyran-4-yl)-{(as a combined preparation for simultaneous, separate or sequential use in therapy. S) -3- [1- (6-Methoxy-5-methyl-pyridin-3-yl) -2,3-dihydro-1H-pyrido [3,4-b] [1,4] oxazin-7-yloxy ] -Pyrrolidin-1-yl} -methanone in anhydrous crystalline form and at least one other therapeutic agent is provided. In one embodiment, the treatment is treatment of a disease or condition mediated by the activity of the PI3K enzyme. The products provided as a combined preparation are together in the same pharmaceutical composition, (1,1-dioxo-hexahydro-1λ ⁶ -thiopyran-4-yl)-{(S) -3- [1- (6-Methoxy-5-methyl-pyridin-3-yl) -2,3-dihydro-1H-pyrido [3,4-b] [1,4] oxazin-7-yloxy] -pyrrolidin-1-yl} A composition comprising an anhydrous crystalline form of methanone and other therapeutic agent (s), or in separate forms, for example in the form of a kit, (1,1-dioxo-hexahydro-1λ ⁶ -thiopyran-4-yl )-{(S) -3- [1- (6-Methoxy-5-methyl-pyridin-3-yl) -2,3-dihydro-1H-pyrido [3,4-b] [1,4] oxazine -7-yloxy] -pyrrolidin-1-yl} -meta Including emissions of anhydrous crystalline forms and other therapeutic agent (s).

In one embodiment, the invention provides (1,1-dioxo-hexahydro-1λ ⁶ -thiopyran-4-yl)-{(S) -3- [1- (6-methoxy-5-methyl-pyridine-3 -Yl) -2,3-dihydro-1H-pyrido [3,4-b] [1,4] oxazin-7-yloxy] -pyrrolidin-1-yl} -methanone in anhydrous crystalline form and another therapeutic agent ( A pharmaceutical composition is provided. Optionally, the pharmaceutical composition may comprise a pharmaceutically acceptable carrier as described above.

In one embodiment, the present invention provides a kit comprising two or more separate pharmaceutical compositions, at least one of which is (1,1-dioxo-hexahydro-1λ ⁶ -thiopyran-4-yl) -{(S) -3- [1- (6-Methoxy-5-methyl-pyridin-3-yl) -2,3-dihydro-1H-pyrido [3,4-b] [1,4] oxazine- Contains the anhydrous crystalline form of 7-yloxy] -pyrrolidin-1-yl} -methanone. In one embodiment, the kit comprises a means for holding the compositions separately, such as a container, a divided bottle, or a divided foil packet. An example of such a kit is a blister pack such as is typically used for the packaging of tablets, capsules and the like.

  The kits of the invention are used to administer different dosage forms, e.g. oral and parenteral, to administer separate compositions at different dosing intervals, or to titrate separate compositions against each other. obtain. In order to assist compliance, the kit of the invention typically includes instructions for administration.

  In the combination therapy of the present invention, the forms of the present invention and other therapeutic agents may be manufactured and / or formulated by the same or different manufacturers. Further, the forms of the invention and other therapeutic agents may be (i) prior to release of the combination product to a physician (eg, in the case of a kit comprising the forms of the invention and other therapeutic agents); (ii) administration (Iii) For example, during the sequential administration of the forms of the invention and other therapeutic agents, the patient can be put together in a combination therapy immediately before (or under the supervision of the physician).

The present invention also relates to (1,1-dioxo-hexahydro-1λ ⁶ -thiopyran-4-yl)-{(S) -3- [1- (6-methoxy-5-methyl-) for use as a medicament. Use of the anhydrous crystalline form of pyridin-3-yl) -2,3-dihydro-1H-pyrido [3,4-b] [1,4] oxazin-7-yloxy] -pyrrolidin-1-yl} -methanone provide.

Accordingly, the present invention relates to (1,1-dioxo-hexahydro-1λ ⁶ -thiopyran-4-yl)-{(S) -3- [1- (6-methoxy-5-methyl) for treating diseases. Use of the anhydrous crystalline form of -pyridin-3-yl) -2,3-dihydro-1H-pyrido [3,4-b] [1,4] oxazin-7-yloxy] -pyrrolidin-1-yl} -methanone I will provide a. The present invention also provides (1,1-dioxo-hexahydro-1λ ⁶ -thiopyran-4-yl)-{(S) -3- [1-] for the treatment of diseases or conditions mediated by the activity of the PI3K enzyme. (6-Methoxy-5-methyl-pyridin-3-yl) -2,3-dihydro-1H-pyrido [3,4-b] [1,4] oxazin-7-yloxy] -pyrrolidin-1-yl} -Providing the use of the anhydrous crystalline form of methanone, the medicament is prepared for administration with another therapeutic agent. The present invention also provides the use of another therapeutic agent for the treatment of a disease or condition mediated by the activity of the PI3K enzyme, wherein the medicament is (1,1-dioxo-hexahydro-1λ ⁶ -thiopyran-4-yl )-{(S) -3- [1- (6-Methoxy-5-methyl-pyridin-3-yl) -2,3-dihydro-1H-pyrido [3,4-b] [1,4] oxazine -7-yloxy] -pyrrolidin-1-yl} -methanone is administered with an anhydrous crystalline form.

The present invention also provides (1,1-dioxo-hexahydro-1λ ⁶ -thiopyran-4-yl)-{(S) -3- [1- (6-methoxy-5-methyl) for use in therapy. An anhydrous crystalline form of -pyridin-3-yl) -2,3-dihydro-1H-pyrido [3,4-b] [1,4] oxazin-7-yloxy] -pyrrolidin-1-yl} -methanone To do. The present invention also provides (1,1-dioxo-hexahydro-1λ ⁶ -thiopyran-4-yl)-{(S) -3 for use in a method of treating a disease or condition mediated by the activity of the PI3K enzyme. -[1- (6-Methoxy-5-methyl-pyridin-3-yl) -2,3-dihydro-1H-pyrido [3,4-b] [1,4] oxazin-7-yloxy] -pyrrolidine- An anhydrous crystalline form of 1-yl} -methanone is provided and (1,1-dioxo-hexahydro-1λ ⁶ -thiopyran-4-yl)-{(S) -3- [1- (6-methoxy-5- The anhydrous crystal form of methyl-pyridin-3-yl) -2,3-dihydro-1H-pyrido [3,4-b] [1,4] oxazin-7-yloxy] -pyrrolidin-1-yl} -methanone is For administration with another therapeutic agent It is prepared. The present invention also provides another therapeutic agent for use in a method of treating a disease or condition mediated by the activity of the PI3K enzyme, wherein the other therapeutic agent is (1,1-dioxo-hexahydro-1λ ^6- Thiopyran-4-yl)-{(S) -3- [1- (6-methoxy-5-methyl-pyridin-3-yl) -2,3-dihydro-1H-pyrido [3,4-b] [ Prepared for administration with the anhydrous crystalline form of 1,4] oxazin-7-yloxy] -pyrrolidin-1-yl} -methanone. The present invention also provides (1,1-dioxo-hexahydro-1λ ⁶ -thiopyran-4-yl)-{(S) -3 for use in a method of treating a disease or condition mediated by the activity of the PI3K enzyme. -[1- (6-Methoxy-5-methyl-pyridin-3-yl) -2,3-dihydro-1H-pyrido [3,4-b] [1,4] oxazin-7-yloxy] -pyrrolidine- An anhydrous crystalline form of 1-yl} -methanone is provided and (1,1-dioxo-hexahydro-1λ ⁶ -thiopyran-4-yl)-{(S) -3- [1- (6-methoxy-5- The anhydrous crystal form of methyl-pyridin-3-yl) -2,3-dihydro-1H-pyrido [3,4-b] [1,4] oxazin-7-yloxy] -pyrrolidin-1-yl} -methanone is Administered with another therapeutic agent . The present invention also provides another therapeutic agent for use in a method of treating a disease or condition mediated by the activity of the PI3K enzyme, wherein the other therapeutic agent is (1,1-dioxo-hexahydro-1λ ^6- Thiopyran-4-yl)-{(S) -3- [1- (6-methoxy-5-methyl-pyridin-3-yl) -2,3-dihydro-1H-pyrido [3,4-b] [ It is administered with an anhydrous crystalline form of 1,4] oxazin-7-yloxy] -pyrrolidin-1-yl} -methanone.

The present invention also provides (1,1-dioxo-hexahydro-1λ ⁶ -thiopyran-4-yl)-{(S) -3- [1- (6-methoxy-5-methyl-) for the treatment of diseases. Use of the anhydrous crystalline form of pyridin-3-yl) -2,3-dihydro-1H-pyrido [3,4-b] [1,4] oxazin-7-yloxy] -pyrrolidin-1-yl} -methanone provide. The present invention also provides (1,1-dioxo-hexahydro-1λ ⁶ -thiopyran-4-yl)-{(S) -3- [1-] for the treatment of diseases or conditions mediated by the activity of the PI3K enzyme. (6-Methoxy-5-methyl-pyridin-3-yl) -2,3-dihydro-1H-pyrido [3,4-b] [1,4] oxazin-7-yloxy] -pyrrolidin-1-yl} -Providing the use of the anhydrous crystalline form of methanone, the patient has been previously treated (eg within 24 hours) with another therapeutic agent. The invention also provides for the use of another therapeutic agent to treat a disease or condition mediated by the activity of the PI3K enzyme, wherein the patient has previously (eg, within 24 hours) (1,1-dioxo-hexahydro −1λ ⁶ -thiopyran-4-yl)-{(S) -3- [1- (6-methoxy-5-methyl-pyridin-3-yl) -2,3-dihydro-1H-pyrido [3,4 -B] [1,4] oxazin-7-yloxy] -pyrrolidin-1-yl} -methanone has been treated with the anhydrous crystalline form.

(1,1-dioxo-hexahydro-1λ ⁶ -thiopyran-4-yl)-{(S) -3- [1- (6-methoxy-5-methyl-pyridin-3-yl) -2,3-dihydro The anhydrous crystalline form of -1H-pyrido [3,4-b] [1,4] oxazin-7-yloxy] -pyrrolidin-1-yl} -methanone can be used as the only active ingredient or, for example, as an adjuvant, eg Other drugs, such as immunosuppressants or immunomodulators or other anti-inflammatory agents, for the treatment or prevention of acute or chronic rejection of allografts or xenografts, or inflammatory or autoimmune disorders Alternatively, it can be administered in conjunction with a chemotherapeutic agent, such as a malignant cell antiproliferative agent. For example, (1,1-dioxo-hexahydro-1λ ⁶ -thiopyran-4-yl)-{(S) -3- [1- (6-methoxy-5-methyl-pyridin-3-yl) -2,3 An anhydrous crystalline form of -dihydro-1H-pyrido [3,4-b] [1,4] oxazin-7-yloxy] -pyrrolidin-1-yl} -methanone is a calcineurin inhibitor such as cyclosporin A or FK506; mTOR Inhibitors such as rapamycin, 40-O- (2-hydroxyethyl) -rapamycin, CCI779, ABT578, AP23573, TAFA-93, biolimus-7 or biolimus-9; ascomycin with immunosuppressive properties such as ABT-281, ASM981, etc .; corticosteroids; cyclophosphamide; azathioprene; Leflunomide; mizoribine; mycophenolic acid or salt; mycophenolate mofetil; 15-deoxyspergualin or an immunosuppressive analog, analog or derivative thereof; A compound of Example 56 or 70; a JAK3 kinase inhibitor such as N-benzyl-3,4-dihydroxy-benzylidene-cyanoacetamide α-cyano- (3,4-dihydroxy)-] N-benzylcinnamamide (Tyrphostin AG 490), prodigiosin 25-C (PNU156804), [4- (4′-hydroxyphenyl) -amino-6,7-dimethoxyquinazoline] (WHI-P131), [4- (3′-bromo-4 '-Hydroxypheny ) -Amino-6,7-dimethoxyquinazoline] (WHI-P154), [4- (3 ′, 5′-dibromo-4′-hydroxyphenyl) -amino-6,7-dimethoxyquinazoline] WHI-P97, KRX-211, a free form or a pharmaceutically acceptable salt form, such as 3-{(3R, 4R) -4-methyl-3- [methyl- (7H-pyrrolo [2,3-d] in monocitrate Pyrimidin-4-yl) -amino] -piperidin-1-yl} -3-oxo-propionitrile (also referred to as CP-690,550), or a compound disclosed in WO 04/052359 or WO 05/066156; Inhibitory monoclonal antibodies, eg, monoclonal antibodies to leukocyte receptors, eg, MHC, CD2, CD3, CD4, CD7, CD8, D25, CD28, CD40, CD45, CD52, CD58, CD80, CD86 or their ligands; other immunomodulatory compounds, eg, recombinant binding molecules or variants having at least a portion of the extracellular domain of CTLA4, eg, non-CTLA4 At least one extracellular portion of CTLA4 bound to the protein sequence or a variant thereof, eg CTLA4Ig (eg named ATCC 68629) or a variant thereof eg LEA29Y; an adhesion molecule inhibitor such as an LFA-1 antagonist, ICAM- 1 or -3 antagonists, VCAM-4 antagonists or VLA-4 antagonists; or antihistamines; or antitussives or bronchodilators; or angiotensin receptor blockers; or anti-infectious agents (anti -infectious agent).

(1,1-dioxo-hexahydro-1λ ⁶ -thiopyran-4-yl)-{(S) -3- [1- (6-methoxy-5-methyl-pyridin-3-yl) -2,3-dihydro The anhydrous crystalline form of -1H-pyrido [3,4-b] [1,4] oxazin-7-yloxy] -pyrrolidin-1-yl} -methanone has other immunosuppressive / immunomodulatory and anti-inflammatory properties. When administered in conjunction with a chemotherapeutic or anti-infective treatment, the dose of co-administered immunosuppressant, immunomodulatory, anti-inflammatory, chemotherapeutic or anti-infective compound will be Depending on the type, eg, whether it is a steroid or calcineurin inhibitor, it will of course vary depending on the particular drug used, the condition being treated, etc.

(1,1-dioxo-hexahydro-1λ ⁶ -thiopyran-4-yl)-{(S) -3- [1- (6-methoxy-5-methyl-pyridin-3-yl) -2,3-dihydro The anhydrous crystalline form of -1H-pyrido [3,4-b] [1,4] oxazin-7-yloxy] -pyrrolidin-1-yl} -methanone is also useful for other therapeutic agents, particularly other antiproliferative agents. Can be advantageously used in combination. Such antiproliferative agents include, but are not limited to, aromatase inhibitors; antiestrogens; topoisomerase I inhibitors; topoisomerase II inhibitors; microtubule activators; alkylating agents; histone deacetylase inhibitors; Compounds that induce the process; cyclooxygenase inhibitors; MMP inhibitors; mTOR inhibitors; anti-neoplastic antimetabolites; platin compounds; compounds that target / reduce protein or lipid kinase activity, and additional anti-angiogenic compounds; Compounds that target, decrease or inhibit the activity of protein or lipid phosphatases; gonadorelin agonists; antiandrogens; methionine aminopeptidase inhibitors; bisphosphonates; biological response modifiers; antiproliferative antibodies; Ras carcinogenic isoform Pesticides; telomerase inhibitors; proteasome inhibitors; drugs used in the treatment of hematological malignancies; compounds that target, decrease or inhibit the activity of Flt-3; Hsp90 inhibitors; temozolomide (TEMODAL®) As well as leucovorin. The term “aromatase inhibitor” as used herein relates to a compound that inhibits estrogen production, ie, conversion of the substrates androstenedione and testosterone to estrone and estradiol, respectively. This term includes, but is not limited to, steroids, especially atamestan, exemestane and formestane; Fadrozole, anastrozole and letrozole are included. Exemestane can be administered, eg, in the form as it is marketed, eg under the trademark AROMASIN. Formestane can be administered, eg, in the form as it is marketed, eg under the trademark LENTARON. Fadrozole can be administered, eg, in the form as it is marketed, eg under the trademark AFEMA. Anastrozole can be administered, eg, in the form as it is marketed, eg under the trademark ARIMIDEX. Letrozole can be administered, eg, in the form as it is marketed, eg under the trademark FEMARA or FEMAR. Aminoglutethimide can be administered, eg, in the form as it is marketed, eg under the trademark ORIMETEN. A combination of the invention comprising a chemotherapeutic agent that is an aromatase inhibitor is particularly useful for the treatment of hormone receptor positive tumors, eg, breast tumors.

  The term “antiestrogenic agent” as used herein relates to a compound that antagonizes the effect of estrogen at the estrogen receptor level. The term includes, but is not limited to tamoxifen, fulvestrant, raloxifene and raloxifene hydrochloride. Tamoxifen can be administered, eg, in the form as it is marketed, eg under the trademark NOLVADEX. Raloxifene hydrochloride can be administered, eg, in the form as it is marketed, eg under the trademark EVISTA. Fulvestrant can be formulated as disclosed in US Pat. No. 4,659,516, or it can be administered, eg, in the form as it is marketed, eg, under the trademark FASLODEX. Can do. A combination of the invention comprising a chemotherapeutic agent that is an anti-estrogen agent is particularly useful for the treatment of estrogen receptor positive tumors, eg, breast tumors.

  The term “antiandrogen” as used herein relates to any substance capable of inhibiting the biological effects of androgen, including but not limited to bicalutamide (CASODEX), which includes For example, as disclosed in US Pat. No. 4,636,505.

  The term “gonadorelin agonist” as used herein includes, but is not limited to, abarelix, goserelin and goserelin acetate. Goserelin is disclosed in US Pat. No. 4,100,274 and can be administered, eg, in the form as it is marketed, eg under the trademark ZOLADEX. Abarelix can be formulated, for example, as disclosed in US Pat. No. 5,843,901.

  The term “topoisomerase I inhibitor” as used herein includes, but is not limited to, topotecan, gimatecan, irinotecan, camptothecin and its analogs, 9-nitrocamptothecin and the macromolecular camptothecin conjugate PNU-166148 ( Compounds A1) in WO 99/17804 are included. Irinotecan can be administered, eg, in the form as it is marketed, eg under the trademark CAMPTOSAR. Topotecan can be administered, eg, in the form as it is marketed, eg under the trademark HYCAMTIN.

  The term “topoisomerase II inhibitor” as used herein includes, but is not limited to, anthracyclines, eg, liposomal formulations, eg, doxorubicin including CAELYX; daunorubicin; epirubicin; idarubicin; nemorubicin; Mitoxantrone and rosoxantrone; and the podophyllotoxins etoposide and teniposide. Etoposide can be administered, eg, in the form as it is marketed, eg under the trademark ETOPOPHOS. Teniposide can be administered, eg, in the form as it is marketed, eg under the trademark VM26-BRISTOL. Doxorubicin can be administered, eg, in the form as it is marketed, eg under the trademark ADRIBLASTIN or ADRIAMYCIN. Epirubicin can be administered, eg, in the form as it is marketed, eg under the trademark FARMORUBICIN. Idarubicin can be administered, eg, in the form as it is marketed, eg under the trademark ZAVEDOS. Mitoxantrone can be administered, eg, in the form as it is marketed, eg under the trademark NOVANTRON.

  The term “microtubule active agent” includes, but is not limited to, taxanes such as paclitaxel and docetaxel; vinca alkaloids such as vinblastine sulfate, especially vinblastine sulfate; vincristine, particularly vincristine sulfate and vinorelbine; discodermolide; colchicine; And microtubule stabilizers, microtubule destabilizers and microtubule polymerization inhibitors, including epothilone and derivatives thereof, such as epothilone B or D or derivatives thereof. Paclitaxel can be administered, eg, in the form as it is marketed, eg, TAXOL. Docetaxel can be administered, eg, in the form as it is marketed, eg under the trademark TAXOTERE. Vinblastine sulfate is, for example, the trademark VINBLASTIN R.I. P can be administered, eg, in the form as it is marketed. Vincristine sulfate can be administered, eg, in the form as it is marketed, eg under the trademark FARMISTIN. Discodermride can be obtained, for example, as disclosed in US Pat. No. 5,010,099. Also included are the epothilone derivatives disclosed in WO 98/10121, US Pat. No. 6,194,181, WO 98/25929, WO 98/08849, WO 99/36553, WO 98/22461 and WO 00/31247. Particularly preferred is epothilone A and / or B.

  The term “alkylating agent” as used herein includes, but is not limited to, cyclophosphamide, ifosfamide, melphalan or nitrosourea (BCNU or Gliadel). Cyclophosphamide can be administered, eg, in the form as it is marketed, eg under the trademark CYCLOSTIN. Ifosfamide can be administered, eg, in the form as it is marketed, eg under the trademark HOLOXAN.

  The term “histone deacetylase inhibitor” or “HDAC inhibitor” relates to a compound that inhibits histone deacetylase and has a growth inhibitory action. This is the case with compounds disclosed in WO 02/22577, in particular N-hydroxy-3- [4-[[(2-hydroxyethyl) [2- (1H-indol-3-yl) ethyl] -amino] methyl. ] Phenyl] -2E-2-propenamide, N-hydroxy-3- [4-[[[2- (2-methyl-1H-indol-3-yl) -ethyl] -amino] methyl] phenyl] -2E 2-propenamide and pharmaceutically acceptable salts thereof. This further includes, among others, suberoylanilide hydroxamic acid (SAHA).

  The term “anti-neoplastic antimetabolite” includes, but is not limited to, 5-fluorouracil or 5-FU; capecitabine; gemcitabine; DNA demethylating agents such as 5-azacytidine and decitabine; methotrexate and edatrexate; Antagonists such as pemetrexed are included. Capecitabine can be administered, eg, in the form as it is marketed, eg under the trademark XELODA. Gemcitabine can be administered, eg, in the form as it is marketed, eg under the trademark GEMZAR. Also included are monoclonal antibody trastuzumab, which can be administered, eg, in the form as it is marketed, eg under the trademark HERCEPTIN.

  The term “platin compound” as used herein includes, but is not limited to carboplatin, cisplatin, cisplatinum and oxaliplatin. Carboplatin can be administered, eg, in the form as it is marketed, eg under the trademark CARBOPLAT. Oxaliplatin can be administered, eg, in the form as it is marketed, eg under the trademark ELOXATIN.

The term “protein or lipid kinase activity; or compound that targets / reduces protein or lipid phosphatase activity; or further anti-angiogenic compounds” as used herein includes, but is not limited to protein tyrosine kinases. And / or serine and / or threonine kinase inhibitors or lipid kinase inhibitors, for example
a) a compound that targets, decreases or inhibits the activity of platelet derived growth factor receptor (PDGFR), eg, a compound that targets, decreases or inhibits the activity of PDGFR, in particular a compound that inhibits PDGF receptor; For example, N-phenyl-2-pyrimidin-amine derivatives such as imatinib, SU101, SU6668 and GFB-111;
b) a compound that targets, decreases or inhibits the activity of fibroblast growth factor receptor (FGFR);
c) Compounds that target, decrease or inhibit the activity of insulin-like growth factor receptor I (IGF-IR), eg compounds which target, decrease or inhibit the activity of IGF-IR, in particular IGF-IR Compounds that inhibit the receptor, such as those compounds disclosed in WO 02/092599;
d) compounds that target, decrease or inhibit the activity of the Trk receptor tyrosine kinase family;
e) a compound that targets, decreases or inhibits the activity of the Axl receptor tyrosine kinase family;
f) a compound that targets, decreases or inhibits the activity of the c-Met receptor;
g) a compound that targets, decreases or inhibits the activity of the Kit / SCFR receptor tyrosine kinase;
h) compounds that target, decrease or inhibit the activity of C-kit receptor tyrosine kinases (parts of the PDGFR family), eg compounds which target, decrease or inhibit the activity of the c-Kit receptor tyrosine kinase family In particular a compound that inhibits the c-Kit receptor, such as imatinib;
i) c-Abl family members and compounds of these gene fusion products, such as compounds that target, decrease or inhibit the activity of BCR-Abl kinase, such as c-Abl family members and the activity of these gene fusion products Compounds targeted, reduced or inhibited, for example N-phenyl-2-pyrimidin-amine derivatives such as imatinib, PD180970, AG957, NSC680410 or PD173955 from Parkedavi;
j) Raf family members of protein kinase C (PKC) and serine / threonine kinases, MEK, SRC, JAK, FAK, PDK members and Ras / MAPK family members, or PI (3) kinase family, or PI (3) -Kinases-related kinase families, and / or compounds that target, decrease or inhibit the activity of members of the cyclin dependent kinase family (CDK), and in particular, disclosed in US Pat. No. 5,093,330 Examples of these staurosporine derivatives, such as midostaurin; further compounds include, for example, UCN-01; Safingaol; BAY 43-9006; Bryostatin 1; Perifosine; Ilmofosin; RO318220 and RO3204 2; GO6976; Isis3521; LY333531 / LY379196; isoquinoline compounds, such as those disclosed in WO00 / 09495; including or QAN697 (P13K inhibitor); FTI; PD184352;
k) Targeting, reducing or inhibiting the activity of protein-tyrosine kinase inhibitors, including compounds which target, decrease or inhibit the activity of protein-tyrosine kinase inhibitors, for example imatinib mesylate (GLEEVEC) or tyrphostin Compound. Tylphostin is preferably a low molecular weight (Mr <1500) compound, or a pharmaceutically acceptable salt thereof, in particular a compound selected from the benzylidene malonitrile class or the S-arylbenzenemalonitrile or bisubstrate quinoline class of compounds, More particularly, tyrphostin A23 / RG-50810, AG99, tyrphostin AG213, tyrphostin AG1748, tyrphostin AG490, tyrphostin B44, tyrphostin B44 (+) enantiomer, tyrphostin AG555, AG494, tyrphostin AG556, AG957 and adaphostin (4-{[(2, 5-dihydroxyphenyl) methyl] amino} -benzoic acid adamantyl ester, NSC680410, any compound selected from the group consisting of adaphostin And 1) compounds that target, decrease or inhibit the activity of the epidermal growth factor family of receptor tyrosine kinases (EGFR, ErbB2, ErbB3, ErbB4 as homodimers or heterodimers), such as epithelial growth Compounds that target, decrease or inhibit the activity of the factor receptor family specifically inhibit members of the EGF receptor tyrosine kinase family, such as the EGF receptor, ErbB2, ErbB3 and ErbB4, or EGF or EGF-related ligands Compounds, proteins or antibodies that bind to, in particular those compounds, proteins or monoclonal antibodies generally and specifically disclosed in WO 97/02266, eg of Example 39 or EP 0564409; WO 99 EP0520722; EP0566226; EP0877722; EP0837063; U.S. Pat. No. 5,747,498; WO98 / 10767; WO97 / 30034; WO97 / 49688; WO97 / 38983, and in particular known as compounds in WO96 / 30347, such as CP358774. Compounds of WO 96/33980, eg, compound ZD1839; and WO 95/03283, eg, compound ZM105180, eg, trastuzumab (HERCEPTIN), cetuximab, Iressa, Tarceva, OSI-774, CI-1033, EKB-569, GW-2016. E1.1, E2.4, E2.5, E6.2, E6.4, E2.11, E6.3 or E7.6.3; and 7H-pyrrolo- [2,3-d] pyrimidine derivatives disclosed in WO 03/013541 are included.

  Additional anti-angiogenic compounds include, for example, compounds having another mechanism for these activities independent of protein or lipid kinase inhibition, such as thalidomide (THALOMID) and TNP-470.

  Compounds that target, decrease or inhibit the activity of protein or lipid phosphatases are, for example, inhibitors of phosphatase 1, phosphatase 2A, PTEN or CDC25, such as okadaic acid or derivatives thereof.

  Compounds that induce cell differentiation processes are, for example, retinoic acid, α-, γ- or δ-tocopherol or α-, γ- or δ-tocotrienol.

  The term cyclooxygenase inhibitor as used herein includes, but is not limited to, for example, Cox-2 inhibitors, 5-alkyl substituted 2-arylaminophenylacetic acids and derivatives such as celecoxib (CELEBREX), rofecoxib ( VIOXX), etoroxixib, valdecoxib or 5-alkyl-2-arylaminophenylacetic acid, such as 5-methyl-2- (2′-chloro-6′-fluoroanilino) phenylacetic acid or lumiracoxib.

  The term “bisphosphonate” as used herein includes, but is not limited to, etidronic acid, clodronic acid, tiludronic acid, pamidronic acid, alendronic acid, ibandronic acid, risedronic acid and zoledronic acid. “Etridonic acid” can be administered, eg, in the form as it is marketed, eg under the trademark DIDRONEL. “Clodronic acid” can be administered, eg, in the form as it is marketed, eg under the trademark BONEFOS. “Tiludronic acid” can be administered, eg, in the form as it is marketed, eg under the trademark SKELID. “Pamidronic acid” can be administered, eg, in the form as it is marketed, eg under the trademark AREDIA ™. “Alendronic acid” can be administered, eg, in the form as it is marketed, eg under the trademark FOSAMAX. “Ibandronic acid” can be administered, eg, in the form as it is marketed, eg under the trademark BONDRANAT. “Risedronic acid” can be administered, eg, in the form as it is marketed, eg under the trademark ACTONEL. “Zoledronic acid” can be administered, eg, in the form as it is marketed, eg under the trademark ZOMETA.

  The term “mTOR inhibitor” refers to compounds that inhibit mammalian rapamycin target protein (mTOR) and have antiproliferative effects, such as sirolimus (Rapamune®), everolimus (Certican ™), CCI-779 and It relates to ABT578.

  The term “heparanase inhibitor” as used herein refers to a compound that targets, decreases or inhibits the degradation of heparan sulfate. This term includes, but is not limited to, PI-88.

  The term “biological response modifier” as used herein refers to lymphokines or interferons, eg, interferon γ.

  The term “inhibitor of Ras carcinogenic isoform”, eg, H-Ras, K-Ras or N-Ras, as used herein, targets, decreases or inhibits the oncogenic activity of Ras. A compound, for example a “farnesyltransferase inhibitor”, such as L-744832, DK8G557 or R115777 (Zarnestra).

  The term “telomerase inhibitor” as used herein refers to a compound that targets, decreases or inhibits the activity of telomerase. Compounds that target, decrease or inhibit the activity of telomerase are in particular compounds which inhibit the telomerase receptor, for example telomestatin.

  The term “methionine aminopeptidase inhibitor” as used herein refers to a compound that targets, decreases or inhibits the activity of methionine aminopeptidase. A compound that targets, decreases or inhibits the activity of methionine aminopeptidase is, for example, benamide or a derivative thereof.

  The term “proteasome inhibitor” as used herein refers to a compound that targets, decreases or inhibits the activity of the proteasome. Compounds that target, decrease or inhibit the activity of the proteasome include, for example, PS-341 and MLN341.

  The term “matrix metalloprotease inhibitor” or “MMP inhibitor” as used herein includes, but is not limited to, collagen peptidomimetic and non-peptidomimetic inhibitors, tetracycline derivatives such as hydroxamate peptides. The mimetic inhibitor batimastat and its orally bioavailable analogs marimastat (BB-2516), purinomastert (AG3340), metastat (NSC683551) BMS-279251, BAY12-9566, TAA211, MMI270B or AAJ996 is included.

  The term “agent used in the treatment of hematological malignancies” as used herein includes, but is not limited to, an FMS-like tyrosine kinase inhibitor, eg, an FMS-like tyrosine kinase receptor (Flt-3R). Compounds that target and decrease or inhibit activity; interferon, 1-bD-arabinofuranosylcytosine (ara-c) and bisulfan; and ALK inhibitors such as anaplastic lymphoma kinase Or a compound that inhibits.

  Compounds that target, decrease or inhibit the activity of the FMS-like tyrosine kinase receptor (Flt-3R) are in particular compounds, proteins or antibodies that inhibit members of the Flt-3R receptor kinase family, eg PKC412, midostaurin , Staurosporine derivatives, SU11248 and MLN518.

  The term “HSP90 inhibitor” as used herein includes, but is not limited to, HSP90 client protein via the ubiquitin proteasome pathway, which targets, decreases or inhibits the endogenous ATPase activity of HSP90. Compounds that degrade, target, reduce or inhibit are included. Compounds that target, decrease or inhibit the endogenous ATPase activity of HSP90 are in particular compounds, proteins or antibodies that inhibit the ATPase activity of HSP90, such as 17-allylamino, 17-demethoxygeldanamycin (17AAG ), Geldanamycin derivatives, other geldanamycin related compounds, radicicol and HDAC inhibitors.

  The term “antiproliferative antibody” as used herein includes, but is not limited to, trastuzumab (Herceptin ™), trastuzumab-DM1, erlotinib (Tarceva ™), bevacizumab (Avastin ™) ), Rituximab (Rituxan®), PRO64553 (anti-CD40) and 2C4 antibody. By antibody is meant, for example, intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies formed from at least two intact antibodies, and antibody fragments so long as they exhibit the desired biological activity.

For the treatment of acute myeloid leukemia (AML), (1,1-dioxo-hexahydro-1λ ⁶ -thiopyran-4-yl)-{(S) -3- [1- (6-methoxy-5-methyl) The anhydrous crystalline form of -pyridin-3-yl) -2,3-dihydro-1H-pyrido [3,4-b] [1,4] oxazin-7-yloxy] -pyrrolidin-1-yl} -methanone is It can be used in combination with standard treatment of leukemia, in particular in combination with the treatment used for the treatment of AML. In particular, (1,1-dioxo-hexahydro-1λ ⁶ -thiopyran-4-yl)-{(S) -3- [1- (6-methoxy-5-methyl-pyridin-3-yl) -2,3 Anhydrous crystalline forms of dihydro-1H-pyrido [3,4-b] [1,4] oxazin-7-yloxy] -pyrrolidin-1-yl} -methanone are for example farnesyl transferase inhibitors and / or AML Can be administered in combination with other drugs useful for the treatment of, for example, daunorubicin, adriamycin, Ara-C, VP-16, teniposide, mitoxantrone, idarubicin, carboplatinum and PKC412.

(1,1-dioxo-hexahydro-1λ ⁶ -thiopyran-4-yl)-{(S) -3- [1- (6-methoxy-5-methyl-pyridin-3-yl) -2,3-dihydro The anhydrous crystalline form of -1H-pyrido [3,4-b] [1,4] oxazin-7-yloxy] -pyrrolidin-1-yl} -methanone is also useful for other therapeutic agents, particularly other antimalarial agents. Can be advantageously used in combination. Such antimalarial agents include, but are not limited to, proguanil, chlorproguanil, trimethoprim, chloroquine, mefloquine, lumefantrin, atovacon, pyrimethamine-sulfadoxine, pyrimethamine-dapsone, halophanthrin, quinine, quinidine , Amodiaquine, amopyroquine, sulfonamide, artemisinin, arteflene, artemether, artesunate, primaquine, inhaled NO, L-arginine, dipropylenetri-amine NONOate (NO donor), rosiglitazone (PPARγ agonist), activated carbon, erythropoietin, levamisole And pyronalidine.

(1,1-dioxo-hexahydro-1λ ⁶ -thiopyran-4-yl)-{(S) -3- [1- (6-methoxy-5-methyl-pyridin-3-yl) -2,3-dihydro The anhydrous crystalline form of -1H-pyrido [3,4-b] [1,4] oxazin-7-yloxy] -pyrrolidin-1-yl} -methanone is also exemplified by, for example, leishmaniasis, trypanosomiasis, toxoplasmosis and It can be advantageously used in combination with other therapeutic agents used for the treatment of neurocystic disease. Such drugs include, but are not limited to, chloroquine sulfate, atovacon-proguanil, artemether-lumefantrin, quinine sulfate, artesunate, quinine, doxycycline, clindamycin, meglumine antimonate, sodium stibogluconate , Miltefosine, ketoconazole, pentamidine, amphotericin B (AmB), liposomal-AmB, paromomycin, eflornitine, niflutimox, suramin, melarsoprol, prednisolone, benznidazole, sulfadiazine, pyrimethamine, clindamycin, trimetropine, sulfamethoxy Sazole, azithromycin, atovacon, dexamethasone, praziquantel, albendazole, beta-lactam, fluoroquinolone, macrola De, aminoglycosides include sulfadiazine and pyrimethamine.

  The structure of the active agent identified by code number, generic name or trade name can be obtained from current editions or databases of the standard overview “The Merck Index”, eg Patents International, eg IMS World Publications.

(1,1-dioxo-hexahydro-1λ ⁶ -thiopyran-4-yl)-{(S) -3- [1- (6-methoxy-5-methyl-pyridin-3-yl) -2,3-dihydro The above compounds that can be used in combination with the anhydrous crystalline form of -1H-pyrido [3,4-b] [1,4] oxazin-7-yloxy] -pyrrolidin-1-yl} -methanone include, for example: They can be prepared and administered as described in the art in the references cited above.

(1,1-dioxo-hexahydro-1λ ⁶ -thiopyran-4-yl)-{(S) -3- [1- (6-methoxy-5-methyl-pyridin-3-yl) -2,3-dihydro The anhydrous crystalline form of -1H-pyrido [3,4-b] [1,4] oxazin-7-yloxy] -pyrrolidin-1-yl} -methanone is also known from known therapeutic methods such as hormones, or in particular, It can be advantageously used in combination with the administration of radiation.

(1,1-dioxo-hexahydro-1λ ⁶ -thiopyran-4-yl)-{(S) -3- [1- (6-methoxy-5-methyl-pyridin-3-yl) -2,3-dihydro The anhydrous crystalline form of -1H-pyrido [3,4-b] [1,4] oxazin-7-yloxy] -pyrrolidin-1-yl} -methanone is particularly useful for the treatment of tumors that show poor sensitivity to radiation therapy In particular, it can be used as a radiosensitizer.

“Combination” means a fixed combination in one dosage unit form, or a kit of parts for combined administration, (1,1-dioxo-hexahydro-1λ ⁶ -thiopyran-4-yl)- {(S) -3- [1- (6-Methoxy-5-methyl-pyridin-3-yl) -2,3-dihydro-1H-pyrido [3,4-b] [1,4] oxazine-7 An anhydrous crystalline form of -yloxy] -pyrrolidin-1-yl} -methanone and a combination partner is a time interval that specifically allows the combination partner to exhibit a synergistic effect, such as a synergistic effect, or the effect of any of these combinations. Within, may be administered independently, simultaneously or separately. The terms “simultaneous administration” or “combined administration” and the like as used herein include administration of a selected combination partner to a single subject (eg, a patient) in need thereof. Is meant to include therapeutic regimens that are not necessarily administered by the same route of administration or simultaneously. The term “pharmaceutical combination” as used herein results from mixing or combining a plurality of active ingredients, and both fixed and non-fixed combinations of active ingredients Means a product containing The term “fixed combination” refers to active ingredients such as (1,1-dioxo-hexahydro-1λ ⁶ -thiopyran-4-yl)-{(S) -3- [1- (6-methoxy-5- Anhydrous crystalline form of methyl-pyridin-3-yl) -2,3-dihydro-1H-pyrido [3,4-b] [1,4] oxazin-7-yloxy] -pyrrolidin-1-yl} -methanone and It means that the combination partner is administered to the patient both simultaneously in the form of a single entity or dose. The term “unfixed combination” refers to active ingredients such as (1,1-dioxo-hexahydro-1λ ⁶ -thiopyran-4-yl)-{(S) -3- [1- (6-methoxy-5 -Methyl-pyridin-3-yl) -2,3-dihydro-1H-pyrido [3,4-b] [1,4] oxazin-7-yloxy] -pyrrolidin-1-yl} -methanone) It means that the form and combination partner are both administered to the patient as separate entities, simultaneously, concurrently or sequentially, without a specific deadline, and such administration is Realize therapeutically effective levels of certain compounds. The latter also applies to cocktail therapy, eg the administration of 3 or more active ingredients.

Experimental details:
The following examples are intended to illustrate the invention and are not to be construed as limitations thereon. Temperature is expressed in degrees Celsius. Unless otherwise stated, all evaporations are performed under reduced pressure, typically between about 15 mmHg and 100 mmHg (= 20-133 mbar). The structure of the final products, intermediates and starting materials is confirmed by standard analytical methods such as microanalysis and spectroscopic features such as MS, IR, NMR. Abbreviations used are those conventional in the art.

  All starting materials, building blocks, reagents, acids, bases, dehydrating agents, solvents, and catalysts utilized to synthesize the forms of the present invention are either commercially available or produced by organic synthesis methods known to those skilled in the art. (Houben-Weyl 4th edition 1952, Methods of Organic Synthesis, Thieme, 21). Furthermore, the forms of the present invention can be produced by organic synthesis methods known to those skilled in the art, as shown in the examples below.

Abbreviations ACN Acetonitrile AcOH Acetic acid aq. Aqueous solution Boc tert-butoxycarbonyl Boc ₂ O di-tert-butyl dicarbonate tBu tert-butyl tBuOH tert-butanol BrettPhos 2- (dicyclohexylphosphino) -3,6-dimethoxy-2′-4′-6′-triisopropyl -1,1′-biphenyl br s broad singlet COMU (1-cyano-2-ethoxy-2-oxoethylideneaminooxy) dimethylamino-morpholino-carbenium hexafluorophosphate conc. Concentrated d d Doublet dd Doublet doublet dba Dibenzylideneacetone DCM Dichloromethane DEA Diethylamine DEAD Diethyl azodicarboxylate DEAP Diethylaminopyridine DIPEA Diisopropylethylamine DMF Dimethylformamide DMME Dimethoxymethane DMSO Dimethylsulfoxide DPPA Diphenylphosphoryl azide DPPF 1 , 1′-bis (diphenylphosphino) ferrocene EDC 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride eq. Equivalent ESI Electrospray ionized Et ₃ N Triethylamine Et ₂ O Diethyl ether EtOAc Ethyl acetate EtOH Ethanol h Time HATU O- (7-azabenzotriazol-1-yl) -N, N, N ′, N′-Tetramethyluronium Hexafluorophosphate HBTU O- (1H-benzotriazol-1-yl) -N, N, N ′, N′-tetramethyluronium hexafluorophosphate HMDS hexamethyldisilazane HOBT 1-hydroxy-benzotriazole HPLC high performance liquid chromatography Chromatography IPA isopropanol LCMS liquid chromatography mass spectrometry mCPBA meta-chloroperbenzoic acid MeOH methanol m multiplet min min MS mass spectrometry mw microwave NMR nuclear magnetic resonance analysis N aOtBu sodium tert-butoxide NP normal phase OBD optimal bed density Pd ₂ (dba) ₃ tris (dibenzylideneacetone) dipalladium PL-HCO ₃ MP SPE polymer supported bicarbonate cartridge for acid removal prep. Preparative PPh ₃ Triphenylphosphine q Quartet Rac-BINAP Racemic 2,2′-bis (di-p-tolylphosphino) -1,1′-binaphthyl RP Reversed phase Rt Retention time rt Room temperature RuPhos 2-dicyclohexylphosphino -2 ', 6'-di-isopropoxy-1,1'-biphenyl sat. Saturated SCX-2 polymer-supported sulfonic acid macroporous polystyrene soln. Solution t triplet TBME tert-butyl methyl ether TBAF tetrabutylammonium fluoride TBDMSCl tert-butyldimethylsilyl chloride tetramethyl-t-butyl-XPhos 2-di-t-butylphosphino-3,4,5,6-tetra Methyl-2 ′, 4 ′, 6′-triisopropylbiphenyl TFA trifluoroacetic acid THF tetrahydrofuran TLC thin layer chromatography UPLC ultrahigh performance liquid chromatography XPhos 2-dicyclohexylphosphino-2 ′, 4 ′, 6′-triisopropylbiphenyl Pd [RuPhos] (2-dicyclohexylphosphino-2′6′-diisopropyl-11′-biphenyl) (2- (2-aminoethyl) phenyl) palladium (II)
The microwave device used is Biotage Initiator (registered trademark).
All compounds are named using AutoNom.

General Chromatographic Information LCMS Method M1 (Rt _M1 )
HPLC-column dimensions: 2.1 x 50 mm
HPLC-column type: Acquity UPLC HSS T3, 1.8 [mu] m
HPLC-eluent: A) Water + 0.05% by volume formic acid + 3.75 mM ammonium acetate, B) ACN + 0.04% by volume formic acid HPLC-Gradient: 2-98% B, 98% B, 0. 45 minutes, flow rate = 1.2 ml / min HPLC-column temperature: 50 ° C
LCMS method M2 (Rt _M2 )
HPLC-column dimensions: 2.1 x 30 mm
HPLC-column type: Ascentis Express C18, 2.7 μm
HPLC-eluent: A) Water + 0.05% by volume formic acid + 3.75 mM ammonium acetate, B) ACN + 0.04% by volume formic acid HPLC-Gradient: 2-98% B, 98% B, 0. 75 minutes, flow rate = 1.2 ml / min HPLC-column temperature: 50 ° C
LCMS method M3 (Rt _M3 )
HPLC-column dimensions: 2.1 x 30 mm
HPLC-column type: Ascentis Express C18, 2.7 μm
HPLC-eluent: A) water + 0.05% by volume formic acid + 3.75 mM ammonium acetate, B) ACN + 0.04% by volume formic acid HPLC-gradient: 2-98% B, 98% B, 1 min at 8.5 min. , Flow rate = 1.2 ml / min HPLC-column temperature: 50 ° C
LCMS method M4 (Rt _M4 )
HPLC-column dimensions: 4.6 x 50 mm
HPLC-column type: SunFire C18, 5 μm
HPLC-eluent: A) water + 0.1% by volume TFA, B) ACN + 0.1% by volume TFA
HPLC-gradient: 5-100% BB at 8.0 min, flow rate = 2 ml / min HPLC-column temperature: 40 ° C.
LCMS method M5 (Rt _M5 )
HPLC-column dimensions: 0.46 × 25 cm
HPLC-column type: Chiralcel OJ-H (1189)
HPLC-eluent: EtOH / MeOH, 60:40
HPLC-gradient: uniform concentration, flow rate = 0.5 ml / min Detector: UV 220 nm
LCMS method M6 (Rt _M6 )
HPLC-column dimensions: 2.1 x 30 mm
HPLC-column type: Ascentis Express C18, 2.7 μm
HPLC-eluent: A) water + 0.05% TFA, B) ACN + 0.04% TFA
HPLC-gradient: 2-98% B in 1.4 minutes, 98% B, 0.75 minutes, flow rate = 1.2 ml / min HPLC-column temperature: 50 ° C.
LCMS method M7 (Rt _M7 )
HPLC-column dimensions: 2.1 x 30 mm
HPLC-column type: Ascentis Express C18, 2.7 μm
HPLC-eluent: A) water + 0.05% TFA, B) ACN + 0.04% TFA
HPLC-gradient: 10-95% B, 95% B, 3.0 min at 3.0 min, flow rate = 1.2 ml / min HPLC-column temperature: 50 °
LCMS method M8 (Rt _M8 )
HPLC-column dimensions: 2.1 x 30 mm
HPLC-column type: Ascentis Express C18, 2.7 μm
HPLC-eluent: A) water + 0.05% formic acid + 3.75 mM ammonium acetate, B) acetonitrile + 0.04% formic acid HPLC-gradient: 10-95% B in 3.0 minutes, flow rate = 1.2 ml / min. LCMS method M9 (Rt _M9 )
HPLC-column dimensions: 2.1 x 30 mm
HPLC-column type: Ascentis Express C18, 2.7 μm
HPLC-eluent: A) water + 0.05% formic acid + 3.75 mM ammonium acetate, B) acetonitrile + 0.04% formic acid HPLC-gradient: 10% B, 0.0-0.5 min, then gradient 10 95% B, 0.5 min to 3.0 min, flow rate = 1.2 ml / min LCMS method M10 (Rt _M10 )
HPLC-column dimensions: 2.1 x 50 mm
HPLC-column type: Acquity UPLC BEH C18, 1.7 [mu] m
HPLC-eluent: A) water + 0.1% by volume formic acid, B) acetonitrile HPLC-gradient: 20-25% B at 1.00 min, then 25-95% B at 3.20 min, then 0. 95-100% B in 10 minutes, then 100% 0.20 minutes, flow rate = 0.7 ml / min LCMS method M11 (Rt _M11 )
HPLC-column dimensions: 2.1 x 50 mm
HPLC-column type: Acquity UPLC BEH C18, 1.7 [mu] m
HPLC-eluent: A) water + 0.1% by volume formic acid, B) acetonitrile HPLC-gradient: 5-10% B at 1.00 min, then 10-90% B at 3.00 min, then 0. 90-100% B in 10 minutes, then 100% 0.40 minutes, flow rate = 0.7 ml / min LCMS method M12 (Rt _M12 )
HPLC-column dimensions: 2.1 x 30 mm
HPLC-column type: Ascentis Express C18, 2.7 μm
HPLC-eluent: A) water + 0.1 vol% TFA, B) acetonitrile HPLC-gradient: 10-95% B over 1.7 min and 1.2 mL / min as solvent flow rate then over 0.7 min 955B, flow rate = 1.4 mL / min.
LCMS method M13 (Rt _M13 )
HPLC-column dimensions: 2.1 x 30 mm
HPLC-column type: Ascentis Express C18, 2.7 μm
HPLC-eluent: A) water + 0.05% formic acid + 3.75 mM ammonium acetate, B) acetonitrile + 0.04% formic acid HPLC-gradient: 10-95% B in 3.7 minutes, flow rate = 1.2 ml / min. LCMS method M14 (Rt _M14 )
HPLC-column dimensions: 2.1 x 30 mm
HPLC-column type: Ascentis Express C18, 2.7 μm
HPLC-eluent: A) water + 0.05% formic acid + 3.75 mM ammonium acetate, B) acetonitrile + 0.04% formic acid HPLC-gradient: 10-95% B, 95% B, 1 min in 1.5 min. Flow rate = 1.2 ml / min LCMS method M15 (Rt _M15 )
HPLC-column dimensions: 0.46 × 25 cm
HPLC-column type: Chiralcel OD-H (1194)
HPLC-eluent: Hexan / EtOH50: 50 + 0.05% DEA
HPLC-gradient: uniform concentration, flow rate = 0.5 ml / min Detector: UV 220 nm
LCMS method M16 (Rt _M16 )
HPLC-column dimensions: 2.1 x 50 mm
HPLC-column type: Acquity UPLC HSS T3, 1.8 [mu] m
HPLC-eluent: A) water + 0.05% by volume formic acid + 3.75 mM ammonium acetate, B) ACN + 0.04% by volume formic acid HPLC-gradient: 5-98% B in 1.4 min, 0.4 min, 98 % B, flow rate = 1.0 ml / min HPLC-column temperature: 60 ° C

X-ray powder diffractometers:
Method X1
Equipment Bruker D8 GADDS Discover
Irradiation CuKα (40 kV, 40 mA)
Detector HI-STAR Area detector scanning range, 6 ° to 39 ° (2-theta value)

Determination of melting point:
The melting point was determined by differential scanning calorimetry (DSC). The DSC was recorded on a TA Instruments DSC Q2000 using a heating rate of 10 ° C./min. A 0.6 mg sample was weighed into a standard aluminum pan (pan + lid, TA9000078.901, 900779.901). The instrument is a Thermal Advantage Q-series software V. Operated using 2.6.0.367 and Thermal Advantage software V4.6.9. The Thermal Event was characterized using Universal Analysis V4.3A Build 4.3.0.6. Samples were measured on sample pans without pinholes. Samples were treated according to the following protocol.
Step 1: Equilibrate at 0 ° C Step 2: 10 ° C / min ramp to 300 ° C

Example F1: (1,1-dioxo-hexahydro-1λ ⁶ -thiopyran-4-yl)-{(S) -3- [1- (6-methoxy-5-methyl-pyridin-3-yl) -2 , 3-Dihydro-1H-pyrido [3,4-b] [1,4] oxazin-7-yloxy] -pyrrolidin-1-yl} -methanone

a) 7-Chloro-2,3-dihydro-1H-pyrido [3,4-b] [1,4] oxazine 7-Chloro-1H-pyrido [3,4-b] [1,4] oxazine-2 - one (CAS registration 928118-43-8) (3.70g, 20mmol) and THF (63 ml) solution of _was treated with ^BH 3 * THF (THF in 1M, 47 ml, 47 mmol). The reaction mixture was stirred at 75 ° C. for 1 hour, then cooled to room temperature and quenched with methanol (24 ml, 600 mmol). The reaction mixture was concentrated under reduced pressure and the residue was dissolved with EtOAc and washed with saturated aqueous NaHCO ₃ . The organic layer was dried over MgSO ₄ , filtered and concentrated under reduced pressure to give the title product as a pale yellow solid (3.3 g, 96% yield).
UPLC Rt _M1 = 0.47 min; ESIMS: 171 [(M + H) ⁺ ].
¹ H NMR (400 MHz, DMSO-d ₆ ): δ 7.53 (s, 1H), 7.11 (br s, 1H), 6.47 (s, 1H), 4.09 (t, 2H), 3.17-3.38 (m, 2H ).
b) 2,3-Dihydro-1H-pyrido [3,4-b] [1,4] oxazin-7-ol 7-chloro-2,3-dihydro-1H-pyrido [32.5 ml] in dioxane [32.5 ml] 3,4-b] [1,4] oxazine (1.08 g, 6.33 mmol), aqueous KOH (1.07 g in 5.4 ml water, 19 mmol KOH), 2-di-t-butylphosphino-3 , 4,5,6-tetramethyl-2 ′, 4 ′, 6-tri-i-propylbiphenyl 98% (0.30 g, 0.63 mmol) and Pd ₂ (dba) ₃ (0.29 g, 0.32 mmol) ) Was degassed three times with nitrogen, the tube was sealed and the reaction mixture was stirred at 100 ° C. for 5 h. After cooling to room temperature, the reaction mixture was filtered through hyflo and rinsed with EtOAc and methanol. The filtrate was concentrated and the title compound was obtained as an orange residue after flash chromatography on silica gel (DCM / MeOH, 98: 2 to 75:25) (660 mg, 69% yield).
UPLC Rt _M1 = 0.34 min; ESIMS: 153 [(M + H) ⁺ ].
¹ H NMR (400 MHz, DMSO-d ₆ ): δ 10.33 (br s, 1H), 7.03 (br s, 1H), 6.71 (s, 1H), 5.15 (s, 1H), 3.95 (t, 2H) , 3.25 (m, 2H).
c) (S) -3- (2,3-dihydro-1H-pyrido [3,4-b] [1,4] oxazin-7-yloxy) -pyrrolidine-1-carboxylic acid tert-butyl ester dried 2 , 3-Dihydro-1H-pyrido [3,4-b] [1,4] oxazin-7-ol (0.66 g, 4.34 mmol) and (R) -3-methanesulfonyloxy-pyrrolidine-1-carvone Treatment of acid tert-butyl ester (CAS Registry 127423-61-4) (1.73 g, 6.51 mmol) in DMF (40 ml) with sodium hydride (60% in mineral oil, 0.21 g, 8.68 mmol) And the reaction mixture was stirred at 80 ° C. for 18 hours. After cooling to room temperature, the reaction mixture was diluted with TBME and washed with saturated aqueous NaHCO ₃ solution. The organic layer was dried over MgSO ₄ , filtered and concentrated to give the title compound as a yellow oil after flash chromatography on silica gel (cyclohexane / EtOAc, 95: 5 to 30:70) (1.035 g, 75% purity, 56% yield)
UPLC Rt _M1 = 0.65 min; ESIMS: 322 [(M + H) ⁺ ].
¹ H NMR (400 MHz, CDCl ₃ ): δ 7.54 (s, 1H), 5.86 (s, 1H), 5.42 (br s, 1H), 4.25-4.41 (m, 1H), 4.19 (t, 2H), 3.38-3.66 (m, 6H), 2.00-2.18 (m, 2H), 1.46 (d, 9H).
d) (S) -3- [1- (6-Methoxy-5-methyl-pyridin-3-yl) -2,3-dihydro-1H-pyrido [3,4-b] [1,4] oxazine- 7-yloxy] -pyrrolidine-1-carboxylic acid tert-butyl ester (S) -3- (2,3-dihydro-1H-pyrido [3,4-b] [1,4] oxazine in dioxane (6 ml) -7-yloxy) -pyrrolidine-1-carboxylic acid tert-butyl ester (254 mg, 0.79 mmol), 5-bromo-2-methoxy-3-methylpyridine (CAS Registry 760207-87-2) (208 mg, 1. 03mmol), XPhos (30mg, 0.06mmol ), and NaOtBu (167 mg, degassed with argon for 5 minutes a mixture of 1.74 mmol), then, Pd _{(Dba) 3 (29mg, 0.03mmol} ) was added. The tube was filled with argon, sealed, and the reaction mixture was stirred at 100 ° C. for 2 hours. After cooling to room temperature, the reaction mixture was filtered through hyflo, rinsed with EtOAc, and the filtrate was washed with saturated aqueous NaHCO ₃ solution. The aqueous layer was re-extracted twice with EtOAc, the combined organic layers were dried over Na ₂ SO ₄ , filtered and concentrated, and the title compound was flash chromatographed on silica gel (heptane / EtOAc, 100: 0 50:50) and obtained as a clear gum (274 mg, 78% yield). UPLC Rt _M1 = 1.20 min; ESIMS: 443 [(M + H) ⁺ ].
¹ H NMR (400 MHz, CDCl ₃ ): δ 7.93 (d, 1H), 7.61 (br s, 1H), 7.30-7.35 (m, 1H), 5.71 (s, 1H), 5.34-5.46 (m, 1H ), 4.31 (br s, 2H), 3.99 (s, 3H), 3.68 (t, 2H), 3.34-3.62 (m, 4H), 2.23 (s, 3H), 2.01-2.09 (m, 2H), 1.44 (s, 9H).
e) 1- (6-Methoxy-5-methyl-pyridin-3-yl) -7-((S) -pyrrolidin-3-yloxy) -2,3-dihydro-1H-pyrido [3,4-b] [1,4] Oxazine (S) -3- [1- (6-Methoxy-5-methyl-pyridin-3-yl) -2,3-dihydro-1H-pyrido [3,4-b] [1, 4] A solution of oxazin-7-yloxy] -pyrrolidine-1-carboxylic acid tert-butyl ester (364 mg, 0.82 mmol) in DCM (6 ml) was treated with TFA (0.63 ml, 8.23 mmol) and the reaction mixture Was stirred at room temperature for 18 hours, then quenched with saturated aqueous NaHCO ₃ and extracted with DCM. The organic layer was dried over MgSO ₄ , filtered and concentrated under reduced pressure to give the title product as a red oil that was used in the next step without further purification (313 mg, 90% purity). , Quantitative yield).
UPLC Rt _M1 = 0.65 min; ESIMS: 343 [(M + H) ⁺ ].
¹ H NMR (400 MHz, CDCl ₃ ): δ 7.93 (d, 1H), 7.62 (s, 1H), 7.32 (d, 1H), 5.70 (s, 1H), 5.26-5.36 (m, 1H), 4.31 (t, 2H), 3.99 (s, 3H), 3.67 (t, 2H), 2.95-3.15 (m, 3H), 2.81-2.92 (m, 1H), 2.22 (s, 3H), 1.98-2.10 (m , 1H), 1.79-1.90 (m, 1H).
f) (1,1-dioxo-hexahydro-1λ ⁶ -thiopyran-4-yl)-{(S) -3- [1- (6-methoxy-5-methyl-pyridin-3-yl) -2,3 -Dihydro-1H-pyrido [3,4-b] [1,4] oxazin-7-yloxy] -pyrrolidin-1-yl} -methanone 1,1-dioxo-hexahydro-1λ ⁶ -thiopyran-4-carboxylic acid A solution of (CAS Registry 64096-87-3) (106 mg, 0.59 mmol) in DMF (4 ml) was treated with HBTU (225 mg, 0.59 mmol) and DIPEA (0.24 ml, 1.37 mmol). The orange solution thus obtained is stirred at room temperature for 5 minutes and then 1- (6-methoxy-5-methyl-pyridin-3-yl) -7-((S) -pyrrolidin-3- A solution of (Iloxy) -2,3-dihydro-1H-pyrido [3,4-b] [1,4] oxazine (156 mg, 0.46 mmol) in DMF (2 ml) was added. The reaction mixture was stirred at room temperature for 1 h then concentrated under reduced pressure and the residue was dissolved in DCM and washed with saturated aqueous NaHCO ₃ . The organic layer was dried by passing through a phase separation cartridge, concentrated and SFC chromatography (column DEAP (250 mm × 30 mm, 60A, 5 μm) Princeton, gradient 11-16% of methanol in supercritical CO ₂ in 6 minutes) The title compound was obtained as a slightly colored solid later (112 mg, 49% yield).
UPLC Rt _M1 = 0.81 min; ESIMS: 503 [(M + H) ⁺ ].
¹ H NMR (400 MHz, DMSO-d ₆ ): δ 8.01 (s, 1H), 7.61 (m, 1H), 7.52 (d, 1H), 5.52 (d, 1H), 5.24-5.43 (m, 1H) , 4.26 (br s, 2H), 3.89 (s, 3H), 3.59-3.79 (m, 3H), 3.41-3.56 (m, 2H), 3.21-3.39 (m, 1H), 2.98-3.21 (m, 4H ), 2.67-2.83 (m, 1H), 1.84-2.20 (m, 9H).
¹ H NMR (600 MHz, DMSO-d ₆ ): δ 8.01 (s, 1H), 7.63-7.59 (m, 1H), 7.55-7.51 (m, 1H), 5.55-5.51 (m, 1H), 5.43- 5.24 (m, 1H), 4.29-4.22 (m, 2H), 3.90 (s, 3H), 3.80-3.60 (m, 2H), 3.56-3.37 (m, 3H), 3.28-2.99 (m, 5H), 2.89-2.66 (m, 1H), 2.19-2.09 (m, 4H), 2.08-1.98 (m, 2H), 1.98-1.86 (m, 3H).

Crystallization of Example F1 by heating and cooling in isopropanol / diethyl ether 474 mg of amorphous Example F1 was suspended in 1.4 mL of isopropanol. The mixture was heated to 70 ° C. and stirred at 70 ° C. to allow complete dissolution of Example F1. The solution was cooled to room temperature and an adhesive residue was formed. 2 mL of diethyl ether was added and the slurry was stirred for 48 hours. A white suspension was formed. The suspension was filtered and the solid was dried at 40 ° C. and 15 mbar. A fine white powder was obtained. The material contains only a few residual solvents (<0.5%). The crystalline anhydrous form of Example F1 with the onset of melting at 148.77 ° C. was obtained.

  A list of the most significant 2 theta peaks from the X-ray powder diffraction pattern with tolerance ± 0.5 for the anhydrous form of Example F1 (Method M1), including low / weak peaks for information. Note: This list of peaks is not exhaustive, but merely “among others”.

Biological Evaluation The activity of a compound can be evaluated by the following in vitro and in vivo methods.

Biological Assay 1 Measurement of Enzymatic PI3K Alpha and PI3K Delta Isoform Inhibition 1.1 Testing Lipid Kinase Activity The efficacy of compounds as PI3 kinase inhibitors can be demonstrated as follows.
The kinase reaction is performed in a final volume of 50 μl per well of a half area COSTAR, 96 well plate. The final concentrations of ATP and phosphatidylinositol in the assay are 5 μM and 6 μg / mL, respectively. The reaction is initiated by the addition of PI3 kinase, for example PI3 kinase δ.
p110δ. Assay components are added per well as follows.
-10 [mu] l test compound in 5% DMSO per well in column 2-1.
Total activity is measured by addition of 10 μl of 5% volume / volume DMSO in the first 4 wells of column 1 and the last 4 wells of column 12.
Background is measured by the addition of 10 μM control compound to the last 4 wells of column 1 and the first 4 wells of column 12.
-Prepare 2 mL "assay mix" per plate.
1.912 mL HEPES assay buffer 8.33 μl of a 3 mM stock of ATP resulting in a final concentration of 5 μM per well
1 μl of [ ³³ P] ATP on the day of activity resulting in 0.05 μCi per well
30 μl of 1 mg / mL PI stock resulting in a final concentration of 6 μg / mL per well
5 μl of 1M stock MgCl ₂ resulting in a final concentration of 1 mM per well
-Add 20 μl assay mix per well.
Prepare 2 mL “enzyme mix” per plate (x ^* μl PI3 kinase p110β in 2 mL kinase buffer). The “enzyme mix” is kept on ice during the addition to the assay plate.
-Add 20 μl “Enzyme Mix” per well to start the reaction.
-The plate is then incubated for 90 minutes at room temperature.
Stop the reaction by adding 50 μl WGA-SPA bead (scintillation proximity assay beads coated with wheat germ agglutinin) suspension per well.
Seal the assay plate using TopSeal-S (heat seal for polystyrene microplates, PerkinElmer LAS [Deutschland] GmbH, Rodgau, Germany) and incubate at room temperature for at least 60 minutes.
-The assay plate is then centrifuged at 1500 rpm for 2 minutes using a Jouan tabletop centrifuge (Jouan Inc., Nantes, France).
-Using Packard TopCount, count assay plate and count each well for 20 seconds.
^* The amount of enzyme depends on the enzyme activity of the batch used.

  In one more preferred assay, the kinase reaction is performed in a final volume of 10 μl per well of a small volume non-binding CORNING, 384 well black plate (Cat # 3676). The final concentrations of ATP and phosphatidylinositol (PI) in the assay are 1 μM and 10 μg / mL, respectively. The reaction is initiated by the addition of ATP.

Assay components are added per well as follows.
50 nl test compound in 90% DMSO per well in columns 1-20 at 8 concentrations one by one (1/3 and 1 / 3.33 serial dilution steps).
Low control: 50 nl of 90% DMSO (final 0.45%) in half wells of columns 23-24.
High control: 50 nl (final 2.5 μM) of the reference compound (eg compound of example 7 in WO 2006/122806) in the other half of columns 23-24.
Standard: 50 nl reference compound just mentioned, diluted as test compound in columns 21-22.
-Prepare 20 mL "buffer" per assay.
200 μl of 1M TRIS HCl pH 7.5 (final 10 mM)
60 μl of 1M MgCl ₂ (final 3 mM)
500 μl of 2M NaCl (final 50 mM)
100 μl of 10% CHAPS (final 0.05%)
200 μl of 100 mM DTT (1 mM final)
18.94 mL of nanopure water—10 mL “PI” is prepared per assay.
200 μl of 1 mg / mL 1-alpha-phosphatidylinositol prepared in 3% octyl glucoside (Live Bovine, Avanti Polar Lipids catalog number 840042C MW = 909.12) (final 10 μg / mL)
9.8 mL of “buffer”
-Prepare 10 mL "ATP" per assay.
6.7 μl of 3 mM stock of ATP resulting in a final concentration of 1 μM per well 10 mL of “buffer”
Prepare 2.5 mL of each PI3K construct per assay in “PI” at the following final concentrations:
10 nM PI3K Alpha EMV B1075
25nM Beta EMV BV949
10nM Delta EMV BV1060
150nM Gamma EMV BV950
Add 5 μl of “PI / PI3K” per well.
-Add 5 [mu] l "ATP" per well to start the reaction.
-The plate is then incubated at room temperature for 60 minutes (alpha, beta, delta) or 120 minutes (gamma).
Stop the reaction by the addition of 10 μl kinase-Glo (Promega catalog number 6714).
-Read assay plate after 10 minutes with Synergy 2 reader (BioTek, Vermont USA) with integration time of 100 ms and sensitivity set at 191.
-Output: The high control is about 60'000 counts and the low control is 30'000 or less.
This luminescence assay gives useful Z ′ ratios between 0.4 and 0.7. The Z ′ value is a universal measure of the robustness of the assay. A Z 'between 0.5 and 1.0 is recognized as an excellent assay.

  For this assay, the described PI3K construct is prepared as follows.

1.2 Gene Construct Generation Two different constructs, BV 1052 and BV 1075, are used to generate PI3 kinase alpha protein for compound screening.

PI3Kα BV-1052 p85 (iSH2) -Gly linker-p110a (D20aa) -C-terminal His tag Inter-SH2 domain (iSH2) of p85 subunit and p110-a subunit (with deletion of the first 20 amino acids) PCR product for) and fused by overlap PCR. The iSH2 PCR product was first prepared with primers gwG130-p01 (5′-CGAGAATATGATAGATTATAGAGAAAT-3 ′) (SEQ ID NO: 1) and gwG130-p02 (5′-TGGTTT-AATGCTGTTCATACGTTGTCAAT-3 ′) (SEQ ID NO: 2)
From single stranded cDNA. Subsequently, in a secondary PCR reaction, Gateway (Invitrogen AG, Basel, Switzerland) recombinant AttB1 site and linker sequence were combined with primers gwG130-p03 (5′-GGGACAAGTTTGTACAAAAAAAGCAGGCTACGAGAGAGAATACAT-3) p04 (5′-TACCATAATTCCACCACCACCACCCGGAAAATTCCCCCTGGTT-AATGCTGTTCATACGTTTGTCAAT-3 ′) (SEQ ID NO: 4)
To the 5 'and 3' ends of the p85 iSH2 fragment, respectively.

Similarly, the p110-a fragment was first prepared with primers gwG152-p01 (5′-CTAGTGGAATGTTTACTACCAAATGG-3 ′) (SEQ ID NO: 5) and gwG152-p02 (5′-GTTCAATG-CATGCTGTTTAATTGTGT-3 ′) (SEQ ID NO: 6).
Is used to generate from single-stranded cDNA.

In the subsequent PCR reaction, the linker sequence and the histidine tag were used as primers gw152-p03 (5′-GGGGGAATTTCCCGGTGTGTGGTGGTGGAATTAGGGTAC-TAGTGGAATGTGTACTACC-AAATGGTC-3GTGATGCT-TGATTGCT (SEQ ID NO: 8)
To the 5 ′ and 3 ′ ends of the p110-a fragment, respectively.

The p85-iSH2 / p110-a fusion protein is recombined with the above-described gwG130-p03 primer and overlapping histidine tag and AttB2 recombination with overlapping linkers at the 3 ′ end of the iSH2 fragment and the 5 ′ end of the p110-a fragment. Sequence (5'-GGGACCACTTTGTTACAAGAAAGCTGGGTTTAAGCTCCGTGATGGTGATGGTGAT-GTGCTCCC-3 ') (SEQ ID NO: 9)
Are assembled in a third PCR reaction using primers containing

  This final product is incorporated into the donor vector pDONR201 in an (Invitrogen) OR reaction to generate an ORF318 entry clone. This clone is verified by sequencing and used in the Gateway LR reaction to transfer the insert to the Gateway adopted pBlueBac4.5 (Invitrogen) vector for generation of the baculovirus expression vector LR410.

PI3Kα BV-1075 p85 (iSH2) -12 XGly linker-p110a (D20aa) -C-terminal His tag The construct for baculovirus BV-1075 is composed of the p85 fragment and the p110-a fragment cloned into the vector pBlueBac4.5. Generated by the three-part ligation. The p85 fragment is derived from plasmid p1661-2 digested with Nhe / Spe. The p110-a fragment is derived from LR410 (see above) as a SpeI / HindIII fragment. The cloning vector pBlueBac4.5 (Invitrogen) is digested with Nhe / HindIII. This results in the construct PED 153.8.

The p85 component (iSH2) consists of ORF 318 (described above) as a template and one forward primer KAC1028 (5′-GCTAGCATGCGAGAATATGATATAGATTATGAGAAATATACC) (SEQ ID NO: 10) and two reverse primers KAC1029 (5′-GCCCTCTACTGTCTGTCTGTCTGTCTGTCTGTCTG
(SEQ ID NO: 11) and KAC1039 (5′-TACTAGTCCCCCTCCCACCACCTCCCGCTCCCACCACCTCCCCC)
(SEQ ID NO: 12)
To generate by PCR.

  The two reverse primers overlap and incorporate the 12x Gly linker and the N-terminal sequence of the p110a gene into the SpeI site. The 12x Gly linker replaces the linker in the BV1052 construct. The PCR fragment is cloned into pCR2.1 TOPO (Invitrogen). In the resulting clone, p1661-2 is determined to be correct. This plasmid is digested with Nhe and SpeI and the resulting fragment is gel isolated and purified for subcloning. The p110-a cloning fragment is generated by enzymatic digestion of clone LR410 (see above) with Spe I and HindIII. The SpeI site is in the coding region of the p110a gene. The resulting fragment is gel isolated and purified for subcloning.

  A cloning vector, pBlueBac4.5 (Invitrogen) is prepared by enzymatic digestion with Nhe and HindIII. The cleaved vector is purified using a Qiagen (Qiagen NV, Venlo, Netherlands) column and then dephosphorylated using calf intestinal alkaline phosphatase (CIP) (New England BioLabs, Ipswich, Mass.). After completion of the CIP reaction, the cleaved vector is again column purified to produce the final vector. A three-part ligation is performed using Roche Rapid ligase and vendor specifications.

PI3Kβ BV-949 p85 (iSH2) -Gly linker-p110b (full length) -C-terminal His tag generates PCR products for the interSH2 domain (iSH2) of p85 subunit and full length p110-b subunit , Fusing by overlapping PCR.

The iSH2 PCR product was first prepared with primers gwG130-p01 (5′-CGAGAATATGATAGATTATAGAGAAAT-3 ′) (SEQ ID NO: 1) and gwG130-p02 (5′-TGGTTT-AATGCTGTTCATACGTTGTCAAT-3 ′) (SEQ ID NO: 2)
Is used to generate from single-stranded cDNA. Subsequently, in the secondary PCR reaction, Gateway (Invitrogen) recombinant AttB1 site and linker sequence were combined with primers gwG130-p03 (5′-GGGACAAGTTTGTACAAAAAAGCAGGCTACGAAGGAGAATA-TACATATGCGAGAATATGATAGATAGTAT-3GA) ACTGAAGCATCCTCTCTCCTCCTCCTCCTGGTTTAAT-GCTGTTCATACGTTTGCTC-3 ′) (SEQ ID NO: 13)
To the 5 'and 3' ends of the p85 iSH2 fragment, respectively.

The p110-b fragment is similarly a primer gwG130-p04 containing the linker sequence and the 5 ′ end of p110-b (5′-ATTAAACCAGGAGGAGGAGGGAGGAGGATGCTTCAGTTTCATAATGGCC-TCCTTGCT-3 ′) (SEQ ID NO: 4)
And primer gwG130-p06 (5′-AGCTCCGTGATGTGGATGGGTGATGTGCTCCCAGATCTGTTCCTTT-CCGAACTGTTGTG-3 ′) (SEQ ID NO: 14) containing the sequence at the 3 ′ end of p110-b fused to a histidine tag
Is first used to generate from single-stranded cDNA.

  p85-iSH2 / p110-b fusion protein with the above-described gwG130-p03 primer and overlapping histidine tag and AttB2 recombination sequence (5′-GGGACCACTTTTGTACAAGAAAGCTGGGTTTT-AAGCTCCGTGATGGTGATGGTCCAT ′ containing the sequence SEQ ID No.) Used to assemble by overlapping PCR, which is a linker reaction at the 3 ′ end of the iSH2 fragment and the 5 ′ end of the p110-b fragment.

  The final product is incorporated into the donor vector pDONR201 in a Gateway (Invitrogen) OR reaction to generate an ORF253 entry clone. This clone is verified by sequencing and used in the Gateway LR reaction to transfer the insert to the Gateway adopted pBlueBac4.5 (Invitrogen) vector for the generation of the baculovirus expression vector LR280.

PI3Kδ BV-1060 p85 (iSH2) -Gly linker-p110d (full length) -C-terminal His tag generates PCR products for the interSH2 domain (iSH2) and full length p110-d subunit of the p85 subunit , Fusing by overlapping PCR.

The iSH2 PCR product was first prepared with primers gwG130-p01 (5′-CGAGAATATGATAGATTATAGAGAAAT-3 ′) (SEQ ID NO: 1) and gwG130-p02 (5′-TGGTTT-AATGCTGTTCATACGTTGTCAAT-3 ′) (SEQ ID NO: 2)
Is used to generate from single-stranded cDNA. Subsequently, in the secondary PCR reaction, the Gateway (Invitrogen) recombinant AttB1 site and linker sequence were converted to primers gwG130-p03 (5′-GGGACAAGTTTGTACAAAAAAGCAGGCTACGAAGGAGAATATACAT-ATGCGAGAATATGATAGAATTATA′G4AT3G) TCCTCCTCCTCTCCTCCTCCTGGTTTAATGCTGTTCATACGTTTGTC-3 ′) (SEQ ID NO: 16)
To the 5 'and 3' ends of the p85 iSH2 fragment, respectively.

The pi lO-a fragment is similarly first prepared with primers gwG154-p01 (5'-ATGCCCCCTGGGGGTGACTGCCCCAT-3 ') (SEQ ID NO: 17) and gwG154-p02 (5'-CACTTG-CCTGTTGTTCTTTGACACGT-3') (SEQ ID NO: 18)
Is used to generate from single-stranded cDNA.

In the subsequent PCR reaction, the linker sequence and the histidine tag were combined with primers gw154-p03 (5′-ATTAACCAGGAGGAGGAGGGAGGAGGACCCCCTGGGGTGGAC-TGCCCATGGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTC (SEQ ID NO: 20)
To the 5 ′ and 3 ′ ends of the p110-d fragment, respectively.

  p85-iSH2 / p110-d fusion protein in the third PCR reaction was combined with the gwG130-p03 primer and the overlapping histidine tag and Gateway (Invitrogen) AttB2 recombination sequence (5'-GGGACCACTTTTGTA-CAAGAAGCTGGGTGTT-AAGCTGG 3 ′) (SEQ ID NO: 21) is used to assemble by overlapping the linker at the 3 ′ end of the iSH2 fragment and the 5 ′ end of the p110-d fragment.

  This final product is incorporated into the donor vector pDONR201 in a Gateway (Invitrogen) OR reaction to generate an ORF319 entry clone. This clone is verified by sequencing and used in the Gateway LR reaction to transfer the insert to the Gateway adopted pBlueBac4.5 (Invitrogen) vector for generation of the baculovirus expression vector LR415.

PI3Kγ BV-950 p110g (D144aa) -C-terminal His tag This construct is obtained from Roger Williams lab, MRC Laboratory of Molecular Biology, Cambridge, UK (November 2003). Description of the construct: Pacold ME et al. (2000) Cell 103, 931-943.

1.3 Protein Expression and Purification Methods for Producing Recombinant Baculoviruses and Proteins for PI3K Isoforms:
PBlue-Bac4.5 (for a, b, and d isoforms) or pVL1393 (for g) plasmids containing different PI3 kinase genes were transformed into BaculoGold WT genomic DNA (BD Biosciences) using the method recommended by the vendor. , Franklin Lakes, NJ, USA). Subsequently, the recombinant baculovirus obtained from transfection is plaque purified on Sf9 insect cells to obtain several isolates expressing the recombinant protein. Positive clones are selected by anti-HIS or anti-isotype antibody western. For PI3K alpha and delta isoforms, secondary plaque purification is performed on the first clonal virus stock of PI3K. Amplification of all baculovirus isolates is performed at a low multiplicity of infection (moi) to produce a high titer low passage stock for protein production. The baculoviruses are referred to as BV1052 (α) and BV1075 (α), BV949 (β), BV1060 (δ) and BV950 (γ).

Protein production was observed in suspended Tn5 (Trichoplusia ni in protein-free medium at 2-10 moi in a 2 liter glass Erlenmyer flask (110 rpm) or wave-bioreactor (22-25 rpm) for 39-48 hours. ) Or TiniPro (Expression Systems, LLC, Woodland, CA, USA) cell infection (passage 3 and below). Initially, a 10 l working volume wave-bioreactor is seeded at a density of 3e5 cells / mL at half capacity (5 L). The reactor is rocked for 72 hours at 15 rpm during the cell growth phase to make up for 5% oxygen mixed with air (0.2 l / min). Immediately prior to infection, wave-reactor cultures are analyzed for density, viability and diluted to approximately 1.5e6 cells / mL. 100-500 mL of high titer, low passage virus is added 2-4 hours after additional culture. During the 39-48 hour infection period, the oxygen is increased to 35% and the shaking table rpm is increased to 25. During infection, cells are monitored for viability, diameter and density bioprocesses by a Vicell viability analyzer (Beckman Coulter, Inc, Fullerton, CA, USA). Various parameters and metabolites (pH, O ₂ saturation, glucose, etc.) are read by Nova Bioanalyzer (NOVA Biomedical Corp., Waltham, Mass., USA) every 12-18 hours until recovery. Wave-bioreactor cells are collected within 40 hours after infection. Cells are collected by centrifugation (4 ° C at 1500 rpm) and subsequently kept on ice during pellet pooling for lysis and purification. A small amount of cold, non-supplemented Grace medium (without protease inhibitors) is used to make the pellet pool.

PI3K alpha purification protocol for HTS (BV1052) PI3K alpha has three chromatography steps: immobilized metal affinity chromatography on Ni Sepharose resin (GE Healthcare, General Electric Company, Fairfield, CT, USA), Superdex 200 26 Purify by gel filtration using a / 60 column (GE Healthcare) and finally a cation exchange step on an SP-XL column (GE Healthcare). All buffers are cooled to 4 ° C. and lysis is performed while cooling on ice. Column fractionation is performed rapidly at room temperature.

  Typically, frozen insect cells are lysed in hypertonic lysis buffer and applied to a preparative IMAC column. The resin is washed with 3-5 column volumes of lysis buffer, then with 3-5 column volumes of wash buffer containing 45 mM imidazole, and the protein of interest is then washed with a buffer containing 250 mM imidazole. Elute. Fractions are analyzed on a Coomassie stained SDS-PAGE gel and fractions containing the protein of interest are pooled and applied to a preparative GFC column. Fractions from the GFC column are analyzed on a Coomassie stained SDS-PAGE gel and fractions containing the protein of interest are pooled. The pool from the GFC column is diluted in low salt buffer and applied to a preparative SP-XL column. The column is washed with low salt buffer until a stable A280 baseline absorbance is achieved and eluted using a 20 column volume gradient from 0 mM NaCl to 500 mM NaCl. Again, fractions from the SP-XL column are analyzed on a Coomassie-stained SDS-PAGE gel, and fractions containing the protein of interest are pooled. The final pool is dialyzed into a storage buffer containing 50% glycerol and stored at -20 ° C. The final pool is evaluated for activity in a phosphoinositol kinase assay.

PI3Kbeta purification protocol for HTS (BV949) PI3Kbeta is a two chromatographic step: immobilized metal affinity chromatography (IMAC) and Superdex 200 26/60 column (GE Healthcare) on Ni Sepharose resin (GE Healthcare). Purify by gel filtration (GFC) using All buffers are cooled to 4 ° C. and lysis is performed while cooling on ice. Column fractionation is performed rapidly at room temperature.

  Typically, frozen insect cells are lysed in hypertonic lysis buffer and applied to a preparative IMAC column. The resin is washed with 3-5 column volumes of lysis buffer, then with 3-5 column volumes of wash buffer containing 45 mM imidazole, and the protein of interest is then washed with a buffer containing 250 mM imidazole. Elute. Fractions are analyzed on a Coomassie stained SDS-PAGE gel and fractions containing the protein of interest are pooled and applied to a preparative GFC column. Fractions from the GFC column are analyzed on a Coomassie stained SDS-PAGE gel and fractions containing the protein of interest are pooled. The final pool is dialyzed into a storage buffer containing 50% glycerol and stored at -20 ° C. The final pool is evaluated for activity in a phosphoinositol kinase assay.

PI3K gamma purification protocol for HTS (BV950) PI3K gamma is a two chromatographic step: immobilized metal affinity chromatography (IMAC) on Ni Sepharose resin (GE Healthcare) and Superdex 200 26/60 column (GE Healthcare). Purify by gel filtration (GFC) using All buffers are cooled to 4 ° C. and lysis is performed while cooling on ice. Column fractionation is performed rapidly at room temperature. Typically, frozen insect cells are lysed in hypertonic lysis buffer and applied to a preparative IMAC column. The resin is washed with 3-5 column volumes of lysis buffer, then with 3-5 column volumes of wash buffer containing 45 mM imidazole, and the protein of interest is then washed with a buffer containing 250 mM imidazole. Elute. Fractions are analyzed on a Coomassie stained SDS-PAGE gel and fractions containing the protein of interest are pooled and applied to a preparative GFC column. Fractions from the GFC column are analyzed on a Coomassie stained SDS-PAGE gel and fractions containing the protein of interest are pooled. The final pool is dialyzed into a storage buffer containing 50% glycerol and stored at -20 ° C. The final pool is evaluated for activity in a phosphoinositol kinase assay.

PI3K delta purification protocol for HTS (BV1060) PI3K delta uses three chromatography steps: immobilized metal affinity chromatography on Ni Sepharose resin (GE Healthcare), Superdex 200 26/60 column (GE Healthcare). Purification by gel filtration and finally an anion exchange step on a Q-HP column (GE Healthcare). All buffers are cooled to 4 ° C. and lysis is carried out while cooling on ice. Perform column fractionation rapidly at room temperature. Typically, frozen insect cells are lysed in hypertonic lysis buffer and applied to a preparative IMAC column. The resin is washed with 3-5 column volumes of lysis buffer, then with 3-5 column volumes of wash buffer containing 45 mM imidazole, and the protein of interest is then washed with a buffer containing 250 mM imidazole. Elute. Fractions are analyzed on a Coomassie stained SDS-PAGE gel and fractions containing the protein of interest are pooled and applied to a preparative GFC column. Fractions from the GFC column are analyzed on a Coomassie stained SDS-PAGE gel and fractions containing the protein of interest are pooled. The pool from the GFC column is diluted in low salt buffer and applied to a preparative Q-HP column. The column is washed with low salt buffer until a stable A280 baseline absorbance is achieved and eluted using a 20 column volume gradient from 0 mM NaCl to 500 mM NaCl. Again, fractions from the Q-HP column are analyzed on a Coomassie-stained SDS-PAGE gel, and fractions containing the protein of interest are pooled. The final pool is dialyzed into a storage buffer containing 50% glycerol and stored at -20 ° C. The final pool is evaluated for activity in a phosphoinositol kinase assay.

IC ₅₀ is determined by a four parameter curve fitting routine in combination with “excel fit”. A four parameter logistic equation is used to calculate the percent inhibition of each compound at eight concentrations (usually 10, 3.0, 1.0, 0.3, 0.1, 0.030, 0.010 and 0.003 μM). Used to calculate IC ₅₀ values (IDBS XLfit). Alternatively, IC ₅₀ values are calculated using the idbsXLfit model 204, which is a four parameter logistic model.

Further alternatively, for the ATP depletion assay, the compound to be tested is dissolved in DMSO and dispensed directly into white 384 well plates at 0.5 μl per well. To initiate the reaction, 10 μl of 10 nM PI3 kinase and 5 μg / mL 1 alpha-phosphatidylinositol (PI) are added to each well followed by 10 μl of 2 μM ATP. The reaction is performed until approximately 50% of ATP is depleted and then stopped by the addition of 20 μl of Kinase-Glo solution (Promega Corp., Madison, Wis., USA). The stopped reaction is incubated for 5 minutes and the remaining ATP is then detected by luminescence. IC ₅₀ is then determined.

In one embodiment of the invention, a PI3K inhibitor, said inhibitor having an inhibitory effect on the PI3K delta isoform, the range of activities expressed as IC ₅₀ in the enzymatic PI3K delta assay is from 1 nM Between 500 nM.

In another embodiment of the invention, a PI3K inhibitor, said inhibitor having an inhibitory effect on the PI3K delta isoform, the range of activity expressed as IC ₅₀ in the enzymatic PI3K delta assay is 1 nM To 100 nM.

In another embodiment of the invention a PI3K inhibitor, said inhibitor having an inhibitory effect on the PI3K delta isoform, the range of activity expressed as IC ₅₀ in the enzymatic PI3K delta assay is 0 Between 5 nM and 10 nM.

In one embodiment of the invention, a PI3K inhibitor, said inhibitor having an inhibitory effect on the PI3K delta isoform, the range of activity expressed as IC ₅₀ in the PI3K delta assay of cells is from 1 nM Between 1000 nM.

In another embodiment of the invention, a PI3K inhibitor, said inhibitor having an inhibitory effect on the PI3K delta isoform, the range of activity expressed as IC ₅₀ in the PI3K delta assay of cells is 1 nM To 500 nM.

  In another embodiment of the invention, a PI3K inhibitor, wherein the inhibitor has an inhibitory effect on the PI3K delta isoform, and the inhibitor is more than one or more of the other isoforms into the PI3K delta isoform. The selectivity is at least 10 times.

  In another embodiment of the invention, a PI3K inhibitor, wherein the inhibitor has an inhibitory effect on the PI3K delta isoform, and the inhibitor is more than one or more of the other isoforms into the PI3K delta isoform. The selectivity is at least 20 times.

  In another embodiment of the present invention, a PI3K inhibitor, said inhibitor having an inhibitory effect on the PI3K delta isoform, wherein the inhibitor has selectivity for the PI3K delta isoform over the different paralogs PI3Kα and β. This selectivity is at least 10 times.

  In another embodiment of the present invention, a PI3K inhibitor, said inhibitor having an inhibitory effect on the PI3K delta isoform, wherein the inhibitor has selectivity for the PI3K delta isoform over the different paralogs PI3Kα and β. As shown, this selectivity is at least 20 times.

In another embodiment of the invention, a PI3K inhibitor, said inhibitor having an inhibitory effect on the PI3K delta isoform, the range of activity expressed as IC ₅₀ in the PI3K delta assay of cells is 1 nM Between 500 and 500 nM, the inhibitor has an inhibitory effect on the PI3K delta isoform, which exhibits selectivity to the PI3K delta isoform over the different paralogs PI3Kα and β, this selectivity being at least 10 times It is.

In another embodiment of the invention, a PI3K inhibitor, said inhibitor having an inhibitory effect on the PI3K delta isoform, the range of activity expressed as IC ₅₀ in the PI3K delta assay of cells is 1 nM Between 500 and 500 nM, the inhibitor has an inhibitory effect on the PI3K delta isoform, which exhibits selectivity to the PI3K delta isoform over the different paralogs PI3Kα and β, this selectivity being at least 20 times It is.

2. Cellular assay 2.1 Phosphoinositide-3 kinase (PI3K) -mediated Akt1 / 2 (S473) phosphorylation in Rat-1 cells Alpha, beta or delta of the catalytic subunit of human phosphoinositide-3 kinase (PI3K) Rat-1 cells stably overexpressing the myristoylated form were added to 30 ul complete growth medium (10% (v / v) fetal calf serum, 1% (v / v) MEM non-essential amino acids, 10 mM HEPES. 7500 (PI3K alpha), 6200 in Dulbecco's modified Eagle's medium (DMEM high glucose) supplemented with 2 mM L-glutamine, 10 μg / mL puromycin and 1% (v / v) penicillin / streptomycin) Pieces (PI3K beta) or 4000 pieces (PI3K delta) Were plated in 384 well plates and incubated at 37% C / 5% CO ₂ /95% humidity for 24 hours. Compounds were diluted in 384 well compound plates to obtain 8 point serial dilutions for 40 test compounds in 90% DMSO and 4 reference compounds plus 16 high and 16 low (inhibited) controls. Predilution plates were prepared by pipetting 250 nl of compound solution into 384 well polypropylene plates using a Hummingwell nanoliter dispenser. Compounds were prediluted by the addition of 49.75 ul of complete growth medium. 10 ul of prediluted compound solution was transferred to the cell plate using a 384 well pipetter resulting in a final DMSO concentration of 0.11%.

Cells were incubated for 1 hour at 37% C / 5% CO ₂ /95% humidity. The supernatant was removed and cells were lysed in 20 ul lysis buffer for AlphaScreen® SureFire® detection.

  A SureFire® p-Akt 1/2 (Ser473) assay kit (PerkinElmer, USA) was used for the detection of p-AKT (Ser473). 5 ul of cell lysate was transferred to a 384 well small volume Proxiplate for detection using a 384 well pipettor. Addition of AlphaScreen® SureFire® reagent was performed according to the manufacturer's protocol. First, activation buffer mix containing 5 ul of reaction buffer plus AlphaScreen® acceptor beads was added, the plate was sealed and incubated on a plate shaker for 2 hours at room temperature. Second, 2 ul of dilution buffer containing AlphaScreen® donor beads was added and the plate was incubated on the plate shaker as described above for an additional 2 hours. Plates were read on an AlphaScreen® compatible plate reader using standard AlphaScreen® settings.

2.2 Determination of B cell activation in mice PI3Kδ has been recognized to modulate B cell function when cells are stimulated via the B cell receptor (BCR) (Okkenhaug et al. Science 297: 1031). (2002)). To assess the inhibitory properties of compounds on B cell activation, the upregulation of activation markers CD86 and CD69 on mouse B cells derived from mouse spleen antibodies is measured following stimulation with anti-IgM. CD69 is a well-known activation marker for B and T cells (Sancho et al. Trends Immunol. 26: 136 (2005)). CD86 (also known as B7-2) is predominantly expressed on antigen presenting cells including B cells. Resting B cells express low levels of CD86 but, for example, upregulate CD86 following stimulation of BCR or IL-4 receptors. CD86 on B cells interacts with CD28 on T cells. This interaction is required for optimal T cell activation and for generating an optimal IgG1 response (Carreno et al. Annu Rev Immunol. 20:29 (2002)).

Spleens from Balb / c mice are collected and splenocytes are isolated and washed twice with RPMI containing 10% fetal bovine serum (FBS), 10 mM HEPES, 100 units / mL penicillin / streptomycin. The RPMI thus supplemented is then referred to as the medium. Cells are adjusted to 2.5 × 10 ⁶ cells / mL in media and 200 μl of cell suspension (5 × 10 ⁶ cells) is added to the appropriate wells of a 96 well plate.

  Cells are then stimulated by adding 50 μl of anti-IgM mAb (final concentration: 30 μg / mL) in the medium. After 24 hours incubation at 37 ° C., the cells are stained with the following antibody cocktail. Anti-mouse CD86-FITC, anti-mouse CD69-PerCP-Cy5.5, anti-mouse CD19-PerCP for B-cell assessment, and anti-mouse CD3-FITC, anti-mouse CD69-PE for T-cell assessment (2 μl Of each antibody / well). After 1 hour at room temperature (rt) in the dark, the cells are transferred to a 96 deep well plate. Cells are washed once with 1 mL PBS containing 2% FBS and after resuspension in 200 μl, samples are analyzed on a FACS Calibur flow cytometer. Lymphocytes are gated in FSC / SSC dot plots by size and granularity and further analyzed for expression of CD19, CD3 and activation markers (CD86, CD69). Data is calculated from the dot blot as the percentage of cells positively stained for activation markers within the CD19 + or CD3 + population using BD CellQuest software.

  To assess the inhibitory properties of a compound, the compound is first dissolved, diluted in DMSO, followed by 1:50 dilution in media. Spleen cells from Balb / c mice are isolated, resuspended and transferred to a 96 well plate as described above (200 μl / well). Diluted compound or solvent is added to the plate (25 μl) and incubated at 37 ° C. for 1 hour. Cultures were then stimulated with 25 μl anti-IgM mAb / well (final concentration 30 μg / mL) for 24 hours at 37 ° C. and stained with anti-mouse CD86-FITC and anti-mouse CD19-PerCP (2 μl of each antibody / well) To do. CD86 expression on CD19 positive B cells is quantified by flow cytometry as described above.

2.3 Determination of rat B cell activation PI3Kδ has been recognized to modulate B cell function when cells are stimulated via the B cell receptor (BCR) (Okkenhaug et al. Science 297: 1031 (2002)). To assess the inhibitory properties of compounds on B cell activation, the upregulation of activation marker CD86 on rat B cells derived from whole blood is measured after stimulation with anti-IgM and recombinant IL-4. The CD86 molecule (also known as B7-2) is predominantly expressed on antigen presenting cells including B cells. Resting B cells express low levels of CD86 but, for example, upregulate CD86 following stimulation of BCR or IL-4 receptors. CD86 on B cells interacts with CD28 on T cells. This interaction is required for optimal T cell activation and for generating an optimal IgG1 response (Carreno et al. Annu Rev Immunol. 20:29 (2002)).

Rat blood collection Whole blood was collected from the abdominal aorta of adult male Lewis rats (LEW / HanHsd) using a 10 ml syringe with hypodermic needle pre-coated with sodium heparin. The blood was transferred into a 50 ml Falcon tube and the anticoagulant concentration was adjusted to 100 U / ml.

Stimulation of rat B cells and treatment with specific inhibitors For assessment of the in vitro effects of immunosuppressive drugs, heparinized blood was prediluted to 50% in medium. The medium served was 100 U / ml penicillin, 100 mg / ml streptomycin, 2 mM L-glutamine, 50 mg / ml dextran 40 and 5% fetal calf serum (FCS, Fetalclone I, Gibco # 10270-106) DMEM high glucose (Animed catalog # 1-26F01-I). 190 μl of pre-diluted blood was then spiked with 10 μl of pre-diluted test compound in a 96 well U-bottom microtiter plate (Nunc), resulting in a 3-fold serial dilution with a concentration range of 20-0.0003 μM. Control wells were pretreated with DMSO to obtain a final concentration of 0.5% DMSO. Cultures were set up in duplicate, mixed well by agitation on a plate shaker (Heidolph Titramax 101; 30 seconds, speed 900), sucked up by a pipette, spit out, and again agitated on the plate shaker. Cultures were incubated for 1 hour at 37 ° C., 5% CO ₂ . Then 20 μl of polyclonal goat anti-rat IgM Ab (Serotec, catalog # 302001) and 10 μl of diluted recombinant rIL-4 (Immunotools # 340085) were added to give final concentrations of 30 μg / ml and 5 ng / ml, respectively. . Plates were mixed by agitation on a plate shaker as described above and incubated at 37 ° C., 5% CO ₂ for 24 hours.

Determination of B cell activation by flow cytometry After incubation, 15 μl of 25 mM EDTA solution was added per well and shaken for 15 minutes to detach adherent cells. Samples were then stained with PE-Cy5-labeled anti-rat CD45RA (BD catalog # 557015) for analysis of surface activation markers, allowing gating on B cells in FACS analysis. In addition, the samples were stained with PE-labeled anti-rat CD86 (BD catalog # 551396). All staining procedures were performed for 30 minutes at room temperature in the dark. Following incubation, the samples were transferred to 96 deep well V-bottom microtiter plates (Corning # 396096) containing 2 ml / well BD Lysing Solution (BD # 349202). After lysis of red blood cells, the sample was washed with 2 ml of CellWASH (BD # 349524). Data were acquired on an LSRII or FACScalibur flow cytometer (BD Biosciences) using Cellquest Plus or DIVA (version 6.1.1) software, respectively. Lymphocytes were gated in FSC / SSC dot plots by size and granularity and further analyzed for expression of CD45RA and activation markers. Data was calculated from dot blots or histograms as the percentage of cells positively stained for activation markers within the CD45RA + population.

Statistical evaluation The percentage inhibition of B cell activation after drug exposure was calculated by the following formula:

ORIGIN 7 software (OriginLab Corporation, Northampton, Mass.) Was used for non-linear regression curve fitting. The drug concentration resulting in 50% inhibition (IC ₅₀ ) was obtained by fitting Hill's equation to the inhibition data.

2.4 Measurement of TLR9-induced IL-6 in mouse spleen cells Preparation of single cell suspension from mouse spleen Immediately after euthanasia, spleen was excised from C57BL / 6 mice. Excess fat was trimmed from the spleen before grinding the spleen through a 0.4 μM cell strainer using a plunger from a 5 ml syringe. A single cell suspension was prepared and the volume adjusted to 15 ml in a 50 ml Falcon tube using cold PBS. Prior to removal of the supernatant and resuspension in 5 ml of red blood cell lysis buffer per spleen and 5 minutes of room temperature incubation, the cells were centrifuged at 1500 rpm for 5 minutes at 4 ° C. Ice-cold PBS (30 ml) was added to the cells prior to centrifugation at 1500 rpm for 5 minutes at 4 ° C. The supernatant was removed and the cells were washed twice with 40 ml of mouse spleen cell culture medium (MSCM). MSCM was supplemented with 100 units / ml penicillin and 100 μg / ml streptomycin, 1 × nonessential amino acid, 1 mM sodium pyruvate, 0.05 mM β-mercaptoethanol, and 10% heat-inactivated fetal bovine serum (FBS). Made of RPMI. Cells were resuspended in 10-20 ml MSCM and counted using a Countess cell counter. Approximately 60 × 10 ⁶ splenocytes were obtained from a single C57BL / 6 mouse spleen.

Stimulation of mouse splenocytes and treatment with specific inhibitors Splenocytes were plated in 96-well flat-bottomed plates in a volume of 100 μl at a final density of 2 × 10 ⁵ cells / well, and 2-4 in a humidified 37 ° C. incubator. Incubated for hours. The compound to be tested was then dispensed using an automated liquid handling machine using the previously prepared compound stock plate. Stock plates consisted of compounds arranged in 8-10 points (in 90% / 10% DMSO / ddH ₂ O) using 2-fold or 3-fold dilutions. The liquid handling machine dispensed 1 μl of each dilution from the previously prepared compound source plate into the appropriate target well in a 96 well plate. The final starting concentration of the compound in the cell culture was 10 μM. The final concentration of DMSO in the cell culture was 0.5%. Cells were incubated with compound for 1 hour prior to addition of TLR ligand. Then 10 × EC ₈₀ concentration of CpG1826 was added in an amount of 20 μl (for a final culture volume of 200 μl), after which the culture was incubated overnight in a humidified 37 ° C. incubator.

Measurement of interleukin 6 by ELISA After overnight culture, the plates were centrifuged at 2000 rpm for 5 minutes at room temperature. Subsequently, 150 μl of each culture was transferred to a 96-well V-bottom plate and IL-6 levels were measured using a commercially available mouse IL-6 sandwich ELISA kit. Temporarily, plates were coated with capture antibody overnight before blocking with PBS / 0.1% BSA for 1 hour. Samples and standards were added in a volume of 50 μl and the plates were incubated for 2 hours at room temperature. After removal of the standard / sample, before addition of 50 μl of biotinylated detection antibody, the plate was washed with PBS / 0.05% Tween, and then the plate was incubated for 2 hours at room temperature with agitation. Plates were washed again for 20 minutes prior to the addition of 50 μl streptavidin-horseradish peroxidase per well. Following the additional plate wash, 50 μl TMB substrate was added to each well and the plate was incubated for 20 minutes prior to the addition of 25 μl / well stop solution. IL-6 levels were measured using a SpectraMax 190 Plate Reader (450 nm) and analyzed using SoftMax Pro and GraphPad Prism software.

2.5 Measurement of TLR9-induced IFN alpha in human peripheral blood mononuclear cells (PBMC) Preparation of PBMC from fresh human blood Human blood (approximately 75 ml) was obtained from Heparin (S-Monovette 7.5 mL NH Heparin 16 IU / mL blood. Collected in a 10S-Monovette tube containing Starstedt). Leucosep ™ tubes (30 mL # 227290; Greiner Bio-one) were prepared by addition of 15 ml lymphocyte separation medium LSM1077 ™ (# J15-004; PAA Laboratories) per tube and centrifugation at 1000 g for 30 seconds. . Following dilution with an equal volume of PBS (without Ca2 + / Mg2 +; # 14190-094), approximately 25 ml of blood was transferred to a Leucosep ™ tube. Samples were centrifuged at 800 g for 20 minutes at 22 ° C. using an Eppendorf 5810R centrifuge without braking. The PBMC layer was carefully removed from the plasma: separation media interface and transferred to a clean 50 ml tube. The cells were washed once by the addition of PBS (up to 45 ml) and centrifuged (1400 rpm at 22 ° C., 10 min) using the Eppendorf 5810R with braking (set at speed 9). The pellet cells were carefully resuspended in medium (RPMI 1640 + GlutaMAX-I, 0.05 mM 2-mercaptoethanol, 10 mM HEPES and 5% v / v FCS) and the samples were pooled. Media components 2-mercaptoethanol (# 31350-010; 50 mM), Hepes (# 15630-056, 1M) and RPMI 1640 (1 ×) + GlutaMAX-I (# 61870-010) were obtained from Gibco. FCS (# 2-01F36-1) was obtained from Aimed. PBMC were counted using a Countess® automated cell counter (samples were prediluted 1:10 in medium prior to addition of equal volume (10 μl) Trypan Blue). Cells were diluted to 4 × 10 ⁶ cells / ml and seeded in 384-well plates (# 353936; Becton Dickinson AG) to give a final volume of 25 μl (ie 1 × 10 ⁵ cells / well).

Stimulation of PBMC and treatment with specific inhibitors Compounds were prediluted in 100% v / v DMSO (# 41640-100 mL; Sigma-Aldrich) and then transferred to media (final DMSO concentration of 0.25%) To achieve). Cells were treated with the appropriate compound dilution (5 μl) or vehicle control (5 μl) and incubated at 37 ° C. for 30 minutes in a humidified incubator in air containing 5% (v / v) CO ₂ . Cells were stimulated with CpG2216 (0.3 μM; # tlrl-hodna; Invivogen) or vehicle control (10 μl / well) and incubated for 20 hours. Plates were briefly centrifuged (200 × g, 2 minutes at 22 ° C.) and supernatant samples (30 μl) were removed for quantification of IFNα levels.

Quantification of IFNα using AlphaLisa technology A human interferon AlphaLISA kit (# AL264F) from PerkinElmer was used for quantification of IFNalpha. An antibody mix containing anti-IFNα acceptor beads (final 5 μg / ml) and biotinylated antibody anti-IFNα (final 0.5 nM) is freshly prepared and dispensed into a 384 well Optiplate ™ (# 600007299; PerkinElmer). (5 μl). A dilution of a known IFNα standard (human IFNα B (2b)) was prepared and added to the plate along with the cell supernatant (5 μl). Plates were briefly centrifuged (pulse at 200 g), covered with adhesive sealing film, vortexed and incubated for 1 hour at room temperature in the dark. Streptavidin-coated donor beads (final 20 μg / ml) were prepared and added to each well (5 μl) in a dim place (light sensitive mix). Plates were incubated for 30 minutes at room temperature (plates should not be centrifuged or covered). After incubation, “AlphaScreen standard setting” of the device itself (for example, total measurement time: 550 ms, laser 680 nm excitation time: 180 ms, mirror: D640 as, emission filter: M570w, central wavelength 570 nm, bandwidth 100 nm, transmittance 75%) Plates were read on an EnVision ™ multiplate reader with an ALPHA option using. Data was collected for analysis and quantification of IFNα levels.

Data Evaluation and Analysis Data were analyzed using Excel XL fit 4.0 (Microsoft) with XLfit add-in (IDBS; version 4.3.2). Specific IFNα concentrations were determined according to an extrapolation to a standard curve using human IFNα B (2b). Individual IC ₅₀ values for compounds were determined by non-linear regression after fitting the curves to experimental data.

3 Determination of antibody production against sheep erythrocytes (SRBC) Briefly, OFA rats are given i. v. They were injected and treated orally with the compound under study for 4 consecutive days (d0 to d3). A spleen cell suspension was prepared at d4 and lymphocytes were plated on soft agar in the presence of indicator cells (SRBC) and complement. Plaques were caused by the lysis of indicator cells by the secretion of SRBC-specific antibodies (mainly the IgM subclass) and the presence of complement. The number of plaques per plate was counted and expressed as the number of plaques per spleen.

Immunization: Groups of 5 female OFA rats were administered i.v. in a volume of 0.5 ml per rat. v. Immunization was performed on day 0 with 2 × 10 ⁸ cells / ml SRBC (obtained from Laboratory Animal Services LAS, Novartis Pharma AG) by injection.

Compound treatment: Animals were suspended in 0.5% CMC, 0.5% Tween 80 for 4 consecutive days (Day 0, Day 1, Day 2 and Day 3 starting on the day of immunization) Eye) Treated. The compound was administered orally twice daily at an applied volume of 5 ml / kg body weight with a 12 hour interval between doses.

Preparation of spleen cell suspension:
On the fourth day, the animals were euthanized by CO _2. The spleen was removed, weighed and placed in a plastic tube containing 10 ml of cold (4 ° C.) Hanks balanced salt solution (HBSS; Gibco, pH 7.3, containing 1 mg phenol red / 100 ml) for each rat spleen. . The spleen was homogenized with a glass potter, left on ice for 5 minutes, and 1 ml of the supernatant was transferred into a new tube. The cells were washed once in 4 ml HBSS, then the supernatant was discarded and the pellet was resuspended in 1 ml HBSS. The number of lymphocytes per spleen was determined by an automated cell counter and the spleen cell suspension was adjusted to a cell concentration of 30 × 10 ⁶ cells / ml.

Plaque formation assay:
Soft agar petri dishes were prepared with 0.7% agarose (SERVA) in HBSS.

In addition, 1 ml of 0.7% agarose was prepared in a plastic tube and kept at 48 ° C. in a water bath. 50 μl of spleen cell suspension at 30 × 10 ⁶ cells / ml and 50 μl of SRBC at 40 × 10 ⁸ cells / ml were added, mixed rapidly (vortex) and poured into the prepared agarose dish. The Petri dish was tilted slightly to achieve a uniform distribution of the cell mixture on the agarose layer. The dish was left at room temperature for 15 minutes and then incubated at 37 ° C. for 60 minutes. Then 1.4 ml of guinea pig complement (Harlan; 10%) was added and incubation continued at 37 ° C. for an additional 60 minutes. SRBC-specific antibodies were released by plated-out B cells that bound antigen (SRBC) in their vicinity. These antigen-antibody complexes activated complement, resulting in SRBC lysis, leaving a bright spot (plaque) in the red blood cell layer. Plaques were counted with a microscope.

The following formula for determination of inhibition of plaque formation was used.
% Inhibition = C ^* 100 / V-100
Where V = mean number of plaques for vehicle group / spleen; C = mean number of plaques for compound treatment group / spleen

References:

Biological data enzyme assay

Cell assay

The present invention includes the following embodiments.
[1] (1,1-dioxo-hexahydro-1λ ⁶ -Thiopyran-4-yl)-{(S) -3- [1- (6-methoxy-5-methyl-pyridin-3-yl) -2,3-dihydro-1H-pyrido [3,4-b] Anhydrous crystalline form of [1,4] oxazin-7-yloxy] -pyrrolidin-1-yl} -methanone.
[2] The following peaks obtained at 2 theta degrees +/− 0.2 degrees: 9.1, 10.2, 11.9, 13.0, 17.1, 17.7, 18.7, 20. Characterized by X-ray powder diffraction patterns including 3, 20.8, 26.0, 26.7, 23.2, 24.1, 24.8, 29.3, 27.4, and 21.4, The anhydrous crystal form according to [1].
[3] The anhydrous crystal form according to [1], having an X-ray diffraction spectrum that is substantially the same as the X-ray powder diffraction spectrum shown in FIG.
[4] The anhydrous crystal form according to [1], having a differential scanning calorimetry graph that is substantially the same as that shown in FIG.
[5] The form according to any one of [1] to [4], for use as a pharmaceutical product.
[6] A combination comprising a therapeutically effective amount of the form according to any one of [1] to [4] and one or more therapeutically active agents.
[7] Use of the form according to any one of [1] to [4] for the manufacture of a medicament for the treatment of a disease or disorder mediated by the activity of the PI3K enzyme, preferably the activity of the PI3Kδ isoform .
[8] The form of any one of [1] to [4] for use in the treatment of a disease or disorder mediated by the activity of the PI3K enzyme, preferably the activity of the PI3Kδ isoform.
[9] A pharmaceutical composition comprising a therapeutically effective amount of the form according to any one of [1] to [4] and one or more pharmaceutically acceptable carriers.
[10] A method of modulating the activity of a PI3K enzyme, preferably a PI3Kδ isoform in a subject, comprising administering to the subject a therapeutically effective amount of the form according to any one of [1] to [4] .
[11] Use of the form according to any one of [1] to [4] for the treatment of a disorder or disease mediated by the activity of a PI3K enzyme, preferably the activity of a PI3Kδ isoform, in a subject.

Claims (9)

(1,1-dioxo-hexahydro-1λ ⁶ -thiopyran-4-yl)-{(S) -3- [1- (6-methoxy-5-methyl-pyridin-3-yl) -2,3-dihydro An anhydrous crystalline form of -1H-pyrido [3,4-b] [1,4] oxazin-7-yloxy] -pyrrolidin-1-yl} -methanone.   The following peaks obtained at 2 degrees +/− 0.2 degrees: 9.1, 10.2, 11.9, 13.0, 17.1, 17.7, 18.7, 20.3, 20 , 26.0, 26.7, 23.2, 24.1, 24.8, 29.3, 27.4, and 21.4, characterized by an X-ray powder diffraction pattern. An anhydrous crystalline form as described in 1. Figure 1 :
Having an X-ray powder diffraction spectrum and the same is X-ray diffraction spectrum shown in anhydrous crystalline form of claim 1. Figure 2 :
It has a differential scanning calorimetry chart and is the same as that shown in, anhydrous crystalline form of claim 1.   A form according to any one of claims 1 to 4 for use as a medicament. A combination comprising a therapeutically effective amount of the form according to any one of claims 1 to 4 and one or more therapeutically active agents . Rheumatoid arthritis (RA); pemphigus vulgaris (PV); idiopathic thrombocytopenic purpura (ITP); thrombotic thrombocytopenic purpura (TTP); autoimmune hemolytic anemia (AIHA); acquired hemophilia Disease type A (AHA); systemic lupus erythematosus (SLE); multiple sclerosis (MS); myasthenia gravis (MG); Sjogren's syndrome (SS); vasculitis associated with ANCA; Immune Urticaria (CAU); allergy selected from the group consisting of atopic dermatitis, contact dermatitis and allergic rhinitis; Goodpasture syndrome; transplant rejection; cancer of hematopoietic origin; and severe and cerebral malaria A disease selected from the group consisting of trypanosomiasis, leishmaniasis, toxoplasmosis and neurocystiasis or an immunopathology associated with an infection It is selected from the group, for the manufacture of a medicament for the treatment of diseases or disorders mediated activity of PI3K enzymes, used in the form of any one of claims 1 to 4.   Use of the form according to claim 7, wherein the activity of the PI3K enzyme is that of the PI3Kδ isoform.   A pharmaceutical composition comprising a therapeutically effective amount of the form according to any one of claims 1 to 4 and one or more pharmaceutically acceptable carriers.