HK1217017B - Pyrimidine substituted purine compounds as kinase (s) inhibitors - Google Patents

Description

Pyrimidine substituted purine compounds as kinase inhibitors

The patent application of the invention is a divisional application of an invention patent application with the international application number of PCT/SG2009/000124, the international application date of 2009, 4 months and 3 days, the application number of 200980159441.3 entering the Chinese national stage and the invented name of "pyrimidine substituted purine compound as kinase inhibitor".

Technical Field

The present invention relates to 5- (9-isopropyl-8-methyl-2-morpholin-4-yl-9H-purin-6-yl) -pyrimidin-2-ylamine, processes for its preparation, pharmaceutical compositions containing the compound and the use of the compound in the treatment of certain kinase-associated diseases/disorders.

Background

The study of kinase inhibitors has proven to be a fruitful area for the development of useful pharmaceutically active substances. Kinases, otherwise known as phosphotransferases, are enzymes capable of transferring phosphate groups from high energy donor molecules (e.g., ATP) to specific target molecules (often referred to as substrates) in a process known as phosphorylation. The largest class of kinases is the protein kinases, which act on specific proteins and regulate their activity.

Since kinase inhibitors can be used as pharmaceutically active compounds, a great deal of research has been conducted to develop compounds with suitable activity for these targets. In the cancer field, two kinases of interest as potential targets for therapeutic compounds include mTOR and PI 3. One example of a study in this area is PCT/SG2008/000379, which discloses a number of compounds having kinase activity against both mTOR and PI 3.

Compounds that inhibit both mTOR and PI3K are expected to provide potent antiproliferative, antiangiogenic and antitumor activity because these compounds are able to act at multiple points in the PI3K/Akt/mTOR pathway. Currently, many of these inhibitors are undergoing first clinical studies (e.g., BEZ235, XL765, GDC0941, PX866, SF 1126).

In finding a suitable drug candidate, many factors are considered in ultimately determining whether a compound is a suitable drug candidate. Therefore, in evaluating potential compounds for further development, many other factors need to be considered in addition to the primary inhibitory activity of the compound itself. In the course of the evaluation, the skilled person looks at the "drug-like properties" of the molecule, including evaluating factors such as its activity on the target of interest, the solubility of the compound of interest (if they are insoluble, they are generally less suitable as a subsequent drug), the metabolic stability of the compound in vivo and in vitro, possible side effects of the compound on the human body, etc. The present application identifies compounds with significantly improved drug-like properties compared to other compounds in the art.

Summary of The Invention

The present invention provides compounds of formula (I):

or a pharmaceutically acceptable salt thereof.

In addition to the compounds of formula I, the disclosed embodiments are also directed to pharmaceutically acceptable salts, pharmaceutically acceptable N-oxides, pharmaceutically acceptable prodrugs and pharmaceutically active metabolites of the compounds, and pharmaceutically acceptable salts of the metabolites.

The invention also relates to pharmaceutical compositions comprising a compound of the invention and a pharmaceutically acceptable carrier, diluent or excipient.

Another aspect of the invention provides a method of inhibiting a protein kinase selected from the group consisting of: a serine/threonine protein kinase or a fragment or complex thereof or a functional equivalent thereof, and a PI3 kinase or a fragment or complex thereof or a functional equivalent thereof, the method comprising contacting the protein kinase or a fragment or complex thereof or a functional equivalent thereof and/or a cofactor thereof with an effective amount of a compound of the present invention.

The compounds disclosed herein can act directly and individually on kinase molecules or complexes or fragments thereof to inhibit biological activity. However, it is to be understood that the compounds may also act at least partially on cofactors involved in the phosphorylation process. Known kinase cofactors include ionic species (e.g., zinc and calcium), lipids (e.g., phosphatidylserine), and diacylglycerols.

In some embodiments, the protein kinase is a serine/threonine protein kinase or a fragment or complex thereof or a functional equivalent thereof. In some embodiments, the serine/threonine protein kinase or a fragment or complex thereof is an mTOR protein kinase or a fragment thereof, or a complex thereof or a functional equivalent thereof. In some embodiments, the serine/threonine protein kinase is mTORC1 or a fragment or complex thereof or a functional equivalent thereof. In some embodiments, the serine/threonine protein kinase is mTORC2 or a fragment or complex thereof or a functional equivalent thereof.

In some embodiments, the protein kinase is PI3 kinase or a fragment thereof or a complex thereof or a functional equivalent thereof. In some embodiments, the PI3 kinase or a fragment thereof or a complex thereof or a functional equivalent thereof is class I PI3K or a fragment thereof or a complex thereof or a functional equivalent thereof.

In one embodiment of the method of contacting one or more protein kinases with the compound, the compound is administered to a mammal comprising one or more protein kinases.

In another aspect of the invention, there is provided the use of a compound of the invention to inhibit one or more protein kinases selected from the group consisting of: a serine/threonine protein kinase or a fragment or complex thereof or a functional equivalent thereof, and a PI3 kinase or a fragment or complex thereof or a functional equivalent thereof.

In some embodiments, the protein kinase is a serine/threonine protein kinase or a fragment or complex thereof or a functional equivalent thereof. In some embodiments, the serine/threonine protein kinase or a fragment or complex thereof is an mTOR protein kinase or a fragment thereof, or a complex thereof or a functional equivalent thereof. In some embodiments, the serine/threonine protein kinase is mTORC1 or a fragment or complex thereof or a functional equivalent thereof. In some embodiments, the serine/threonine protein kinase is mTORC2 or a fragment or complex thereof or a functional equivalent thereof.

In some embodiments, the protein kinase is PI3 kinase or a fragment thereof or a complex thereof or a functional equivalent thereof. In some embodiments, the PI3 kinase or a fragment thereof or a complex thereof or a functional equivalent thereof is class I PI3K or a fragment thereof or a complex thereof or a functional equivalent thereof.

In another aspect of the invention there is provided a method of treatment or prophylaxis of a disease in a mammal in which inhibition of one or more protein kinases selected from the group consisting of serine/threonine protein kinases or fragments or complexes thereof or functional equivalents thereof and PI3 kinase or fragments or complexes thereof or functional equivalents thereof prevents, inhibits or ameliorates the symptoms or symptoms of the disease which comprises administering a therapeutically effective amount of a compound of the invention.

In some embodiments, the protein kinase is a serine/threonine protein kinase or a fragment or complex thereof or a functional equivalent thereof. In some embodiments, the serine/threonine protein kinase or a fragment or complex thereof is an mTOR protein kinase or a fragment thereof, or a complex thereof or a functional equivalent thereof. In some embodiments, the serine/threonine protein kinase is mTORC1 or a fragment or complex thereof or a functional equivalent thereof. In some embodiments, the serine/threonine protein kinase is mTORC2 or a fragment or complex thereof or a functional equivalent thereof.

In some embodiments, the protein kinase is PI3 kinase or a fragment thereof or a complex thereof or a functional equivalent thereof. In some embodiments, the PI3 kinase or a fragment thereof or a complex thereof or a functional equivalent thereof is class I PI3K or a fragment thereof or a complex thereof or a functional equivalent thereof.

In some embodiments, the disorder is cancer. In some embodiments, the cancer is selected from the group consisting of: hematologic cancers such as myeloproliferative disorders (idiopathic myelofibrosis, polycythemia vera, essential thrombocythemia, chronic myelogenous leukemia), myeloid metaplasia, chronic myelomonocytic leukemia, acute lymphocytic leukemia, acute erythroblastic leukemia, hodgkin's and non-hodgkin's disease, B-cell lymphoma, acute T-cell leukemia, myelodysplastic syndrome, plasma cell disorders, hairy cell leukemia, kaposi's sarcoma, lymphoma, and hyperproliferative disorders such as psoriasis and restenosis; gynecological cancers, such as breast cancer, ovarian cancer, cervical cancer, vaginal and vulvar cancer, endometrial hyperplasia; gastrointestinal cancer such as colorectal cancer, polyp, liver cancer, gastric cancer, pancreatic cancer, gallbladder cancer; urinary tract cancers, such as prostate cancer, kidney and renal cancers; bladder cancer, urethral cancer, penile cancer; skin cancers, such as melanoma; brain tumors, such as glioblastoma, neuroblastoma, astrocytoma, ependymoma (ependonoma), brain stem glioma, medulloblastoma, meningioma (menigiomas), astrocytoma, mesothelioma; head and neck cancers, such as nasopharyngeal carcinoma, laryngeal carcinoma; respiratory tract cancers, such as lung cancer (NSCLC and SCLC), mesothelioma; eye diseases, such as retinoblastoma; muscular-skeletal diseases such as osteosarcoma, musculoskeletal tumors; squamous cell carcinoma and fibroma. In other embodiments, the compounds of the invention are useful for treating pre-cancer conditions (pre-cancer conditions) or hyperplasia, including: familial adenomatous polyposis, adenomatous polyposis of the colon, myelodysplasia, endometriosis, endometrial dysplasia, cervical dysplasia, vaginal intraepithelial neoplasia, benign prostatic hyperplasia, laryngeal chorioma, actinic and solar keratoses, seborrheic keratoses and keratoacanthoma.

In some embodiments, the disorder is an autoimmune or inflammatory disease or a disease resulting from excessive neovascularization. Diseases that are due in part to autoimmune etiology or involve pathological inflammatory and neovascularization reactions include the following: acute disseminated encephalomyelitis, Addison's disease, agammaglobulinemia, agranulocytosis, allergic asthma, allergic encephalomyelitis, allergic rhinitis, alopecia areata, senile alopecia, erythropoiesis inability, ankylosing spondylitis, antiphospholipid antibody syndrome, aortic inflammation syndrome, aplastic anemia, atopic dermatitis, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune oophoritis, Barlow's disease, Basedow's disease, Ocular-oral-genital triple syndrome, bronchial asthma, Cassman's syndrome, celiac disease, Chagas disease, chronic inflammatory demyelinating polyneuropathy, Church-Schneider syndrome, Cogans syndrome, keratoconus, leukoplakia (kernalysis), leukoplakia kernala), and Crohn's syndrome, Coxsackie viral myocarditis, CREST disease, crohn's disease, epidermal eosinophilia, epidermal T cell lymphoma, dermatitis diversities, erythrocytosis myelomatosis, dermatomyositis, diabetic retinopathy, post-myocardial infarction syndrome, corneal epithelial dystrophy, eczematous dermatitis, eosinophilic fasciitis, eosinophilic gastroenteritis, epidermolysis bullosa, Evans syndrome, fibrosing alveolitis, pemphigoid gestationis, glomerulonephritis, goodpasture's syndrome, graft-versus-host disease, grave's disease, Guillain-Barre syndrome (Guillain-Barre syndrome), hashimoto's disease, hemolytic uremic syndrome, herpetic keratitis, psoriasis vulgaris, idiopathic interstitial pneumonia, idiopathic thrombocytopenic purpura, inflammatory bowel disease, teratocarcinosis, keratitis, keratoconjunctivitis, lambert-eaton syndrome, inflammatory bowel disease, teratocarcinoma syndrome, Leukoplakia vulgaris, lichen planus, lichen sclerosus, lyme disease, linear IgA bullous disease (linear IgAdisease), macular degeneration, megaloblastic anemia, Meniere's disease, Moren's ulcer, Muren-Harberman's disease, polymyositis, multiple sclerosis, myasthenia gravis, necrotizing enterocolitis, encephalomyelopathy, ocular pemphigus, ocular myoclonus syndrome, Alder's thyroiditis (Ord 'sthyroidis), paroxysmal nocturnal hemoglobinuria, Bart-Turner syndrome, pemphigus, periodontitis, pernicious anemia, pollen allergy, polyacrine autoimmune syndrome, postuveitis, simple cirrhosis lobule, proctitis, pseudomembranous colitis, psoriasis, emphysema, pyoderma, Reiter's syndrome, reversible fever, psoriasis, emphysema, multiple sclerosis, acute conjunctivitis, chronic conjunctivitis, rheumatoid arthritis, sarcoidosis, scleritis, sezary syndrome, sjogren's syndrome, subacute bacterial endocarditis, systemic lupus erythematosus, takayasu arteritis, temporal arteritis, Toyoh-Huntington's syndrome, type I diabetes, ulcerative colitis, urticaria, vernal conjunctivitis, vitiligo, Wagner-Taurus-Protozoa syndrome (Vogy-Koyanagi-Haradasyn drome), and Wechner's granulomatosis. In some embodiments, the disorder is endometriosis.

In another aspect of the invention there is provided the use of a compound of the invention in the manufacture of a medicament for the treatment of a disease in an animal, wherein inhibition of one or more protein kinases selected from serine/threonine protein kinase or fragments or complexes thereof or functional equivalents thereof and PI3 kinase or fragments or complexes thereof or functional equivalents thereof prevents, inhibits or ameliorates the condition or symptoms of the disease.

In another aspect of the invention there is provided the use of a compound of the invention, or a pharmaceutically acceptable salt, N-oxide or prodrug thereof, in the treatment of a disease wherein inhibition of one or more protein kinases selected from the group consisting of serine/threonine protein kinases or fragments or complexes thereof or functional equivalents thereof and PI3 kinase or fragments or complexes thereof or functional equivalents thereof prevents, inhibits or ameliorates the symptoms or symptoms of the disease.

In some embodiments, the protein kinase is a serine/threonine protein kinase or a fragment or complex thereof or a functional equivalent thereof. In some embodiments, the serine/threonine protein kinase or a fragment or complex thereof is an mTOR protein kinase or a fragment thereof, or a complex thereof or a functional equivalent thereof. In some embodiments, the serine/threonine protein kinase is mTORC1 or a fragment or complex thereof or a functional equivalent thereof. In some embodiments, the serine/threonine protein kinase is mTORC2 or a fragment or complex thereof or a functional equivalent thereof.

In some embodiments, the protein kinase is PI3 kinase or a fragment thereof or a complex thereof or a functional equivalent thereof. In some embodiments, the PI3 kinase or a fragment thereof or a complex thereof or a functional equivalent thereof is class I PI3K or a fragment thereof or a complex thereof or a functional equivalent thereof.

In another aspect of the invention, there is provided a method of preventing or treating a proliferative disease in a subject, the method comprising administering a therapeutically effective amount of a compound of the invention.

In another aspect of the invention, there is provided the use of a compound of the invention in the manufacture of a medicament for the treatment of a proliferative disease in a subject.

In some embodiments, the disorder is cancer. In some embodiments, the cancer is selected from the group consisting of: hematologic cancers such as myeloproliferative disorders (idiopathic myelofibrosis, polycythemia vera, essential thrombocythemia, chronic myelogenous leukemia), myeloid metaplasia, chronic myelomonocytic leukemia, acute lymphocytic leukemia, acute erythroblastic leukemia, hodgkin's and non-hodgkin's disease, B-cell lymphoma, acute T-cell leukemia, myelodysplastic syndrome, plasma cell disorders, hairy cell leukemia, kaposi's sarcoma, lymphoma; gynecological cancers, such as breast cancer, ovarian cancer, cervical cancer, vaginal and vulvar cancer, endometrial hyperplasia; gastrointestinal cancer such as colorectal cancer, polyp, liver cancer, gastric cancer, pancreatic cancer, gallbladder cancer; urinary tract cancers, such as prostate cancer, kidney and renal cancers; bladder cancer, urethral cancer, penile cancer; skin cancers, such as melanoma; brain tumors, such as glioblastoma, neuroblastoma, astrocytoma, ependymoma, brain stem glioma, medulloblastoma, meningioma, astrocytoma, mesothelioma; head and neck cancers, such as nasopharyngeal carcinoma, laryngeal carcinoma; respiratory tract cancers, such as lung cancer (NSCLC and SCLC), mesothelioma; eye diseases, such as retinoblastoma; muscular-skeletal diseases such as osteosarcoma, musculoskeletal tumors; squamous cell carcinoma and fibroma. In some embodiments, the disorder is endometriosis.

Furthermore, formula (I) shall cover solvated as well as unsolvated forms of the compounds, if applicable. Accordingly, each formula includes compounds having the indicated structure, including hydrated as well as non-hydrated forms.

These and other features of the invention are as follows.

Detailed Description

A number of terms well known to the skilled person are used in this specification. However, a number of terms will be defined for clarity.

The term "pharmaceutically acceptable salts" refers to salts that retain the desired biological activity of the compounds identified above, including pharmaceutically acceptable acid addition salts and base addition salts. Suitable pharmaceutically acceptable acid addition salts of the compounds of formula (I) may be prepared from inorganic or organic acids. Examples of these inorganic acids include hydrochloric acid, sulfuric acid, and phosphoric acid. Suitable organic acids may be selected from the aliphatic, cycloaliphatic, aromatic, heterocyclic carboxylic and sulfonic classes of organic acids, examples of which are formic acid, acetic acid, propionic acid, succinic acid, glycolic acid, gluconic acid, lactic acid, malic acid, tartaric acid, citric acid, fumaric acid, maleic acid, alkyl sulfonic acids, aryl sulfonic acids. Additional information on pharmaceutically acceptable salts is found in Remington's pharmaceutical sciences, 19 th edition, Mark Publishing Co., Mack Publishing Co., Iston, Pa., 1995. In the case of a drug that is a solid, the skilled artisan will recognize that the compounds, agents and salts of the present invention may exist in various crystalline or polymorphic forms, and all such materials are intended to be within the scope of the present invention and the structural formulae shown.

The term "therapeutically effective amount" or "effective amount" is an amount sufficient to achieve a beneficial or desired clinical result. An effective amount may be administered in one or more divided doses. An effective amount is generally sufficient to reduce, ameliorate, stabilize, reverse, slow or delay the progression of the disease state.

The term "functional equivalent" shall include variants of the specific protein kinase classes described herein. It will be appreciated that the kinases can have various isoforms such that although the primary, secondary, tertiary or quaternary structure of a given kinase isoform is different from the prototype kinase, the molecule retains the biological activity of the protein kinase. Isoforms may arise from normal allelic mutations within the human population, including mutations such as amino acid substitutions, deletions, insertions, truncations, or repeats. The term "functional equivalents" also includes variants produced at the transcriptional level. Other functional equivalents include kinases in which post-translational modifications (e.g., glycosylation) are altered.

The compounds of the present invention have superior drug-like properties compared to compounds of similar structure in the art, as described in more detail below. These excellent properties suggest that the compounds of the present invention may be suitable as candidates for drug development in the art. As a first observation, the compounds of the invention showed comparable, if not superior, activity in inhibiting both kinases of interest, mTOR and PI 3. The compounds of the invention are more active than all of the comparative compounds tested, their activity against mTOR is comparable to the comparative compounds and at levels acceptable for therapeutic use.

Although enzyme activity tests indicate that almost all of the comparative compounds have acceptable activity levels, further testing of the compounds suggests that many of the compounds cannot be used for drug development for other reasons. For example, the compounds of the present invention have suitable levels of aqueous solubility (178 μ M) and can therefore be formulated into pharmaceutical formulations for oral absorption, whereas many of the comparative compounds do not have acceptable solubility. Thus, the compounds of the present invention have both excellent activity and acceptable solubility characteristics.

Among compounds having both activity and solubility, the compounds of the present invention are more excellent in metabolic stability. The compounds of the invention have excellent stability in human liver microsome studies, indicating that they are robust in physiological environments and relatively resistant to degradation. In contrast, other compounds with activity and solubility were not stable at all in these studies. Thus, the compounds of the present invention possess a unique combination of activity, solubility and stability, making them excellent candidates as compared to related compounds in the art, despite the very significant structural similarity of some of these compounds.

The compounds of the present invention are capable of inhibiting the activity of certain protein kinases. The ability to inhibit kinase activity is a result of the compounds of the invention acting directly on the kinase molecule alone to inhibit biological activity. However, it will be appreciated that the compound may also act at least in part on a cofactor for the kinase involved in the phosphorylation process. The compounds have activity on a PI3 protein kinase or a fragment or complex thereof or a functional equivalent thereof. The compounds may be active against certain serine/threonine kinases such as mTOR or a fragment or complex thereof or a functional equivalent thereof.

Protein kinase inhibition can be performed in a number of ways known in the art. For example, if in vitro inhibition of a protein kinase is desired, an appropriate amount of a compound of the invention can be added to a solution containing the purified kinase. Where inhibition of kinase activity in a mammal is desired, inhibition of the kinase will generally involve administering the compound to the mammal containing the kinase.

Thus, the compounds of the present invention may have a variety of applications, taking advantage of their ability to inhibit protein kinases of the type described above. For example, the compounds can be used to inhibit serine/threonine protein kinases. Also useful for treating or preventing a disorder in a mammal in which inhibition of its protein kinase and/or cofactor prevents, inhibits or ameliorates the condition or symptoms of the disease.

The disclosed compounds have the ability to be used in the treatment of proliferative diseases. An example of such a disease is cancer. The compounds are expected to be capable of treating solid tumors and liquid tumors. In some embodiments, examples of cancers that can be treated by the compounds of the present invention include solid tumors and hematological cancers.

As used herein, the term "cancer" is a generic term intended to encompass a number of disorders characterized by uncontrolled abnormal growth of cells. The compounds of the present invention are expected to be useful in the treatment of various cancers, including but not limited to: bone cancer, brain and CNS tumors, breast cancer, colorectal cancer, endocrine adenocarcinomas including adrenocortical cancer, pancreatic cancer, pituitary cancer, thyroid cancer, parathyroid cancer, thymus cancer, gastrointestinal cancer, liver cancer, extra-biliary cancer, gastrointestinal carcinoid tumor, bladder cancer, genitourinary cancer, gynecological cancer, head and neck cancer, leukemia, myeloma, hematological disorders, lung cancer, lymphoma, eye cancer, skin cancer, soft tissue sarcoma, adult soft tissue sarcoma, kaposi's sarcoma, urinary system cancer.

Examples of cancers that may be treated by the compounds of the present invention include: hematologic cancers such as myeloproliferative disorders (idiopathic myelofibrosis, polycythemia vera, essential thrombocythemia, chronic myelogenous leukemia), myeloid metaplasia, chronic myelomonocytic leukemia, acute lymphocytic leukemia, acute erythroblastic leukemia, hodgkin's and non-hodgkin's disease, B-cell lymphoma, acute T-cell leukemia, myelodysplastic syndrome, plasma cell disorders, hairy cell leukemia, kaposi's sarcoma, lymphoma and hyperproliferative disorders such as psoriasis and restenosis; gynecological cancers, such as breast cancer, ovarian cancer, cervical cancer, vaginal and vulvar cancer, endometrial hyperplasia; gastrointestinal cancer such as colorectal cancer, polyp, liver cancer, gastric cancer, pancreatic cancer, gallbladder cancer; urinary tract cancers, such as prostate cancer, kidney and renal cancers; bladder cancer, urethral cancer, penile cancer; skin cancers such as melanoma; brain tumors, such as glioblastoma, neuroblastoma, astrocytoma, ependymoma, brain stem glioma, medulloblastoma, meningioma, astrocytoma, mesothelioma; head and neck cancers, such as nasopharyngeal carcinoma, laryngeal carcinoma; respiratory tract cancers, such as lung cancer (NSCLC and SCLC), mesothelioma; eye diseases, such as retinoblastoma; muscular-skeletal diseases such as osteosarcoma, musculoskeletal tumors; squamous cell carcinoma and fibroma. The compounds of the invention are also useful in the treatment of precancerous conditions or hyperplasia, including familial adenomatous polyposis, adenomatous polyposis of the colon, myelodysplasia, endometriosis, endometrial dysplasias, cervical dysplasias, vaginal intraepithelial neoplasias, benign prostatic hyperplasia, laryngeal chorioma, actinic and solar keratoses, seborrheic keratoses, and keratoacanthoma.

It is also contemplated that the compounds of the invention may be useful in the treatment of autoimmune or inflammatory diseases or diseases caused by excessive neovascularization. Diseases that are attributable in part to autoimmune etiology, or that are involved in pathological inflammation and neovascularization reactions, include, but are not limited to: acute disseminated encephalomyelitis, Addison's disease, agammaglobulinemia, agranulocytosis, allergic asthma, allergic encephalomyelitis, allergic rhinitis, alopecia areata, senile alopecia, erythropoiesis inability, ankylosing spondylitis, antiphospholipid antibody syndrome, aortic inflammation syndrome, aplastic anemia, atopic dermatitis, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune oophoritis, Barlow's disease, Palygod's disease, Ocular-oral-genital triple syndrome, bronchial asthma, Cassman syndrome, celiac disease, Chagas' disease, chronic inflammatory demyelinating polyneuropathy, Church-Shidi's syndrome, Cotogen syndrome, keratoconus keratoderma, Coxsackie viral myocarditis, CREST disease, Crohn's disease, epidermal eosinophilia, epidermal T-cell lymphoma, Dermatitis diversities polycythemia, dermatomyositis, diabetic retinopathy, post-myocardial infarction syndrome, corneal epithelial dystrophy, eczematous dermatitis, eosinophilic fasciitis, eosinophilic gastroenteritis, epidermolysis bullosa, evans syndrome, fibrosing alveolitis, pemphigus gestationis, glomerulonephritis, goodpasture's syndrome, graft-versus-host disease, graves disease, guillain-barre syndrome, hashimoto's disease, hemolytic uremic syndrome, herpetic keratitis, ichthyosis vulgaris, idiopathic interstitial pneumonia, idiopathic thrombocytopenic purpura, inflammatory bowel disease, catarrhal diseases, keratitis, keratoconjunctivitis, lambert-yie syndrome, leukoplakia vulgaris, lichen planus, lichen sclerosus, lyme disease, linear IgA bullous disease, macular degeneration, megalarval anemia, acute juvenile anemia, chronic glomerulonephritis, chronic myelopathy, chronic glomerulonephritis, chronic, Meniere's disease, morn's ulcer, moha-habermann's disease, polymyositis, multiple sclerosis, myasthenia gravis, necrotizing enterocolitis, ocular encephalomyelopathy, ocular pemphigus, ocular myoclonus syndrome, alder's thyroiditis, paroxysmal nocturnal hemoglobinuria, bardt's syndrome, pemphigus, periodontitis, pernicious anemia, pollen allergy, polyaphrenic autoimmune syndrome, posterior uveitis, monolobular cirrhosis, proctitis, pseudomembranous colitis, psoriasis, emphysema, pyoderma, reiter's syndrome, reversible obstructive airway disease, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleritis, sezary syndrome, sjogren's syndrome, subacute bacterial endocarditis, systemic lupus erythematosus, macroarteritis, temporal arteritis, tuer-henry syndrome, tuscher-henschel's syndrome, chronic bronchitis, chronic obstructive pulmonary disease, chronic obstructive pulmonary, Type I diabetes, ulcerative colitis, urticaria, spring conjunctivitis, vitiligo, Waji-salix minor-Protozoa syndrome and Wechsler's granulomatosis. In some embodiments, the disorder is endometriosis.

The compounds of the invention are also useful for the preparation of medicaments for the treatment of disorders in animals, wherein inhibition of a protein kinase prevents, inhibits or ameliorates the symptoms or symptoms of the disease. The compounds of the invention can also be used for the preparation of medicaments for the treatment or prevention of kinase-associated diseases.

Administration of a compound of formula (I) to a human may be by any acceptable mode for enteral administration, such as oral or rectal, or by parenteral administration, such as subcutaneous, intramuscular, intravenous and intradermal routes. The injection may be a bolus injection or via continuous or intermittent infusion. The active compound is typically contained in a pharmaceutically acceptable carrier or diluent in an amount sufficient to deliver a therapeutically effective dose to the patient. In various embodiments, the inhibitor compound may be selectively toxic or more toxic to rapidly proliferating cells, such as cancerous tumors, as compared to normal cells.

The compounds of the invention, when used, can be administered in any bioavailable form or mode of administration of the compounds. One skilled in the art of preparing formulations can readily select the appropriate form and mode of administration depending on the particular nature of the compound selected, the condition to be treated, the stage of the condition to be treated and other relevant conditions. We recommend the reader to refer to the Remington pharmaceutical sciences, 19 th edition, Mark Press (1995) for additional information.

The compounds of the present invention may be administered alone or in combination with pharmaceutically acceptable carriers, diluents or excipients in the form of a pharmaceutical composition. While the compounds of the present invention are effective per se, they are generally formulated and administered as pharmaceutically acceptable salts, since these forms are generally more stable, more readily crystallized, and have higher solubility.

However, these compounds are generally used in the form of pharmaceutical compositions formulated according to the desired mode of administration. Thus, in another embodiment, the present invention provides a pharmaceutical composition comprising a compound of formula (I) and a pharmaceutically acceptable carrier, diluent or excipient. These compositions can be prepared using techniques well known in the art.

In other embodiments, the present invention provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the present invention. In such a package or kit, a container containing a unit dose of the drug may be provided. These kits may contain compositions containing the active agent, which may be concentrates (including lyophilized compositions) that may be further diluted for use or may provide compositions at use concentrations, wherein the vials may contain one or more doses. Conveniently, the kit may be provided in single doses in sterile vials containing the required amounts and concentrations of the drug, so that the vials may be directly used by the physician. The containers may also be accompanied by written material such as instructions for use or in the form of government agencies regulating the manufacture, use or sale of pharmaceuticals or biological products, which instructions reflect approval by the agency of manufacture, use or sale for human administration.

The compounds of the present invention may be combined or administered with one or more other drugs to treat the condition/disease. The components may be administered in the same formulation or in different formulations. If administered in different formulations, the compounds of the invention may be administered sequentially or simultaneously with other drugs.

In addition to being administered in combination with one or more other drugs, the compounds of the present invention may be used in combination therapy. At this time, these compounds are usually administered in combination with each other. Thus, one or more compounds of the invention may be administered simultaneously (as a combined preparation) or sequentially to achieve the desired effect. This is particularly desirable if the therapeutic properties of each compound differ so that the combined effect of the two drugs improves the therapeutic outcome.

The pharmaceutical composition of the present invention for parenteral injection comprises pharmaceutically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions and sterile powders, so that it can be reconstituted into a sterile injectable solution or dispersion before use. Examples of suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, and the like) and suitable mixtures thereof, vegetable oils (e.g., olive oil), and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of a coating material, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

These compositions may also contain adjuvants such as preserving, wetting, emulsifying, and dispersing agents. The inclusion of various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like, ensures prevention of microbial action. It may also be desirable to include isotonic agents, for example, sugars, sodium chloride, and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.

If more efficient distribution is desired, the compounds can be incorporated into slow release or targeted delivery systems, such as polymer matrices, liposomes and microspheres.

Injectable formulations can be sterilized, for example, by filtration through a bacteria-retaining filter or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium just prior to use.

Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In these solid dosage forms, the active compound is mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and/or: a) fillers or extenders, such as starch, lactose, sucrose, glucose, mannitol, and silicic acid; b) binders such as carboxymethyl cellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and acacia; c) humectants, such as glycerol; d) disintegrants, for example agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates and sodium carbonate; e) solution retarding agents, such as paraffin; f) absorption accelerators, such as quaternary ammonium compounds; g) wetting agents, such as cetyl alcohol and glyceryl monostearate; h) adsorbents such as kaolin and bentonite; and i) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate and mixtures thereof. In the case of capsules, tablets and pills, the dosage forms may also contain buffering agents.

Solid compositions of a similar type may also be filled into soft and hard-filled gelatin capsules using polymers such as lactose and high molecular weight polyethylene glycols as excipients.

Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings or other coatings well known in the pharmaceutical formulating art. They may optionally contain opacifying agents and may also be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymers and waxes.

If applicable, these active compounds with one or more of the above-mentioned excipients may also be employed in microencapsulated form.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixirs. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1, 3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.

In addition to inert diluents, these oral compositions may also contain auxiliary agents such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

Suspensions, in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.

Compositions for rectal or vaginal administration are preferably suppositories which can be prepared by mixing the compounds of the invention with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax which are solid at room temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active compound.

Dosage forms for topical administration of the compounds of the present invention include powders, patches, sprays, ointments and inhalants. The active compound is mixed under sterile conditions with a pharmaceutically acceptable carrier and any required preservatives, buffers, or propellants which may be required.

The compound is preferably administered in an amount that treats and reduces or alleviates the condition. The therapeutically effective amount can be readily determined by the attending physician using conventional techniques and by observing the results obtained under similar conditions. In determining the therapeutically effective amount, several factors should be considered, including but not limited to: animal species, size, age, and general health, the particular condition involved, severity of the condition, patient response to treatment, the particular compound administered, mode of administration, bioavailability of the formulation administered, the selected dosage therapy, use of other agents, and other related conditions.

The preferred dosage is in the range of about 0.01 to 300mg per kg body weight per day. More preferred doses are in the range of 0.1 to 100mg per kg body weight per day, more preferably 0.2 to 80mg per kg body weight per day, more preferably 0.2 to 50mg per kg body weight per day. The appropriate dose may be administered in multiple sub-doses per day.

Synthesis of Compounds of the invention

Using the 5-step procedure outlined in scheme 1, 5- (9-isopropyl-8-methyl-2-morpholin-4-yl-9H-purin-6-yl) -pyrimidin-2-ylamine was prepared from dichloropurine.

Scheme 1

Examples

In the examples described below, all temperatures are in degrees Celsius and all parts and percentages are by weight unless otherwise indicated.

Various starting materials and other reagents were purchased from chemical suppliers such as Aldrich chemical company or lanchester Synthesis Ltd and used without further purification unless otherwise indicated. Tetrahydrofuran (THF) and N, N Dimethylformamide (DMF) were purchased from Aldrich (Aldrich) in a SureSeal bottle and used as received. All solvents were purified using standard methods in the art, unless otherwise indicated.

The following reactions were carried out under positive pressure of nitrogen, argon or using a dry tube at room temperature (unless otherwise indicated) in anhydrous solvents, and the reaction flask was equipped with a rubber septum for introducing the materials and reagents via syringe.

Glassware was oven dried and/or heat dried. Analytical thin layer chromatography was performed on silica gel 60F 254 glass plates (E Merck (0.25mm)) eluting with the appropriate solvent ratio (v/v). The reaction was analyzed by TLC and the reaction was terminated as judged by the consumption of starting material.

TLC plates were visualized via UV absorption or using p-anisaldehyde spray reagent or phosphomolybdic acid reagent (aldrich chemical, 20 wt% in ethanol) (heat activated) or staining in an iodine chamber.

The work-up is usually carried out by doubling the reaction volume with the reaction solvent or extraction solvent, followed by washing with the indicated aqueous solution at 25% of the extraction volume by volume (unless otherwise stated). The product solution was dried over anhydrous sodium sulfate and filtered, the solvent was evaporated under reduced pressure using a rotary evaporator, and attention was paid to the removal of the solvent in vacuo.

Unless otherwise indicated, flash column chromatography [ Still et al, J.org.chem.,43,2923(1978) ] uses E Merck (Merck) grade flash silica gel (47-61mm) with a silica gel to crude material ratio of about 20:1 to 50: 1. Hydrogenolysis was carried out at the indicated pressure or at ambient pressure.

Bruker instruments were operated at 400MHz for recording¹H NMR spectra, recorded at 100MHz operation¹³C-NMR spectrum. Chloroform was used as reference standard (7.27ppm and 77.00ppm) or CD was used₃OD (3.4 and 4.8ppm and 49.3ppm) as reference standard or, where appropriate, with tetramethylsilane internal standard (0.00ppm), CDCl was obtained₃NMR spectrum of the solution (reported in ppm). Other NMR solvents may be used if desired. When peak diversity is reported, the following abbreviations are used: s is singlet, d is doublet, t is triplet, m is multiplet, br is broadened, dd is doublet, dt is doublet. When given the coupling constant, it is reported in hertz. Mass spectra were obtained using LC/MS with ESI or APCI. All melting points are uncorrected. All final products were more than 90% pure (via HPLC, 220nm and 254nm wavelength).

The following synthesis examples are intended to illustrate one method of synthesizing the compounds of the present invention and should not be construed as being limited to these examples.

Example 1: synthesis of Compounds of the invention

Synthesis of 2, 6-dichloro-9-isopropyl-9H-purine

2, 6-dichloropurine (2 mmol), isopropanol (8 mmol) and triphenylphosphine (4 mmol) were taken up in 40 ml of dry tetrahydrofuran at room temperature over 30 minutes and diisopropyl diazodicarboxylate (4 mmol) was added dropwise. The reaction mixture was stirred at room temperature for 24 hours. The reaction was monitored periodically by TLC or LC/MS. The reaction mixture was poured into a beaker containing ice-cooled water. The aqueous layer was extracted with 3 × 100 ml of ethyl acetate to obtain the crude product. The crude product was purified by column chromatography on silica gel (10-80% ethyl acetate in petroleum ether, gradient elution) to give 2, 6-dichloro-9-isopropyl-9H-purine in 77% yield.

Synthesis of 5- (2-chloro-9-isopropyl-9H-purin-6-yl) -pyrimidin-2-ylamine

To 2, 6-dichloro-9-isopropyl-9H-purine (5.21 mmol), 5- (4,4,5, 5-tetramethyl- [1,3, 2-d-etramethyl-)]Peroxide-free bis (diphenylphosphino) ferrocene palladium (II) chloride complexed with Dichloromethane and Dioxolane-2-ylamine (5.21 mmol)To the alkane (40 ml) solution was added 2M aqueous sodium carbonate (15.6 mmol). The reaction mixture was degassed and purged with nitrogen. The reaction mixture was then heated and stirred in an oil bath maintained at 80 ℃ for 3 hours. LC/MS monitored the consumption of the starting purine in the reaction.

The reaction mixture was cooled to room temperature and the solvent was removed under reduced pressure. The residue was taken up in a mixture of ethyl acetate and water. The organic phase was separated and the aqueous layer was further extracted with 3 × 100 ml of ethyl acetate. The organic phase was dried over sodium sulfate and the solvent was removed in vacuo to give 5- (2-chloro-9-isopropyl-9H-purin-6-yl) -pyrimidin-2-ylamine in 55% yield.

Synthesis of 5- (9-isopropyl-2-morpholin-4-yl-9H-purin-6-yl) -pyrimidin-2-ylamine

To a solution of 5- (2-chloro-9-isopropyl-9H-purin-6-yl) -pyrimidin-2-ylamine (2.84 mmol) in dimethylacetamide (18 ml) was added morpholine (2.84 mmol). The reaction mixture was heated and stirred in an oil bath maintained at 94 ℃ for 12 hours. Monitoring of the reverse by LC-MSWhether or not starting materials are present. The crude product was loaded directly onto a preparative HPLC column and chromatographed to give 5- (9-isopropyl-2-morpholin-4-yl-9H-purin-6-yl) -pyrimidin-2-ylamine in 58% yield.¹HNMR，DMSO-d6:9.53(s，2H)，8.32(s，1H)，7.30(bs，2H)，4.72(m，1H)，3.78(m，4H)，3.73(m，4H)，1.55(d，6H)。m/z:341.17[MH]⁺.

Synthesis of 5- (8-bromo-9-isopropyl-2-morpholin-4-yl-9H-purin-6-yl) -pyrimidin-2-ylamine

To a solution of 5- (9-isopropyl-2-morpholin-4-yl-9H-purin-6-yl) -pyrimidin-2-ylamine (1.03 g, 3.03 mmol) in 15 ml of chloroform at 5 ℃, NBS (594 mg, 3.34 mmol) was added slowly. The reaction was continued at this temperature for 2 hours. After a simple work-up, the product 5- (8-bromo-9-isopropyl-2-morpholin-4-yl-9H-purin-6-yl) -pyrimidin-2-ylamine was purified by flash column (solvent system: 50% ethyl acetate in hexanes) to give 5- (8-bromo-9-isopropyl-2-morpholin-4-yl-9H-purin-6-yl) -pyrimidin-2-ylamine in 52% yield (660 mg).¹H NMR，MeOD:9.67(s，2H)，4.90(m，1H)，3.89(m，4H)，3.82(s，4H)，1.72(d，6H)。m/z:419.31，421.07[MH]⁺。

Synthesis of 5- (9-isopropyl-8-methyl-2-morpholin-4-yl-9H-purin-6-yl) -pyrimidin-2-ylamine

To 5- (8-bromo-9-isopropyl-2-morpholin-4-yl-9H-purin-6-yl) -pyrimidin-2-ylamine (30 mg, 0.072 mmol) and Pd (dppf) Cl in a closed tube₂(3 mg, 5% mmol) of 3 ml of anhydrous bisIn solution of alkaneDimethylzinc (210. mu.l, 1.0M heptane) was added slowly. The mixture was heated to about 65 ℃. MeOH was added dropwise and the solvent was removed in vacuo. EtOAc was added to the residue and the resulting solution was washed with 1M HCl, water and brine, then Na₂SO₄And (5) drying. The solvent was removed and the crude mixture was flash chromatographed on silica gel to give 8 mg of 5- (9-isopropyl-8-methyl-2-morpholin-4-yl-9H-purin-6-yl) -pyrimidin-2-ylamine in 47% yield.¹H NMR，MeOD:9.40(s，2H)，4.81(m，1H)，3.89(m，4H)，3.82(s，4H)，3.71(s，3H)，1.73(d，6H)。m/z:355.16[MH]⁺。

Example 2: comparative biological test

The compounds of the invention were compared to a number of compounds synthesized and disclosed in PCT/SG2008/000379 for various biological parameters.

The parameters tested were:

activity of TOR assay;

activity of PI3K assay;

solubility test

Human microsome stability test

The method of each test will be described in detail below:

mTOR assay

Truncated mTOR kinase and His-tag was prepared internally of 4eBP 1. [ gamma ] gamma³³P]ATP was purchased from Amersham (GE Healthcare). All chemicals were obtained from Sigma Aldrich (Sigma-Aldrich) unless otherwise indicated.

Phosphorylation assays were initially performed on 384-well polypropylene plates (Greiner) with a final volume of 20 microliters. Compounds were tested in the range of 100 μ M to 0.006 μ M, diluted in 8 steps in duplicate. First, in pure DMSO, 10 microliters/well of test compound are placed on a sample plate containing 1 microliter/well of test compoundWell 2 Xenzyme-substrate solution (1.5. mu.g/ml mTOR, 40. mu.g/ml 4eBP1 in 1 Xtest buffer: 10mM Hepes pH 7.5, 50mM NaCl and 10mM MnCl₂). By adding 10. mu.l/well of 20. mu.M ATP solution (final assay concentration of 10. mu.M ATP and 0.4. mu. Ci/well [ gamma. ])³³P]-ATP) initiates the reaction. After incubation at room temperature for 1 hour, the reaction was stopped with 40. mu.l/well of 20mM EDTA/1mM MATP solution.

Then, 50. mu.l/well of the reaction-terminated mixture was transferred to a 384-well MultiScreen HTS-PH filter plate (Millipore) to which 50. mu.l/well of 1% phosphoric acid was previously added. By vacuum filtration, the plate was washed four times with 120. mu.l/well of 0.5% phosphoric acid. Finally, 10. mu.l/well of Optiphase was added^TMSupermix liquid scintillation cocktail (cocktail) (Perkin Elmer). After a minimum of 1 hour incubation, counts were performed in a Wallac MicroBeta TriLux scintillation counter using a cross-talk corrected coincidence counting mode. IC (integrated circuit)₅₀Defined as the concentration of compound required for 50% of the maximum possible inhibition of kinase activity.

PI3K test

Recombinant PI3K p110 α/p85 prepared internally phosphatidylinositol (PtdIns), phosphatidylserine (PtdSer), and all other chemicals not specified were purchased from Sigma-Alder³³P]ATP and Optiphase scintillating materials are available from Parkinelmer (Perkin Elmer).

The assay was performed on 384-well Maxisorp well plates (Nunc) at a final assay volume of 25 microliters. Compounds were tested at 8 concentrations, typically starting at 10 μ M, at 3-fold serial dilutions. A1: 1 mixture of 20. mu.l/well of PtdIns and PtdSer [ each dissolved at 0.1 mg/ml in chloroform: ethanol (3:7) ] was coated onto a Maxisorp well plate and dried overnight in a fume hood at Room Temperature (RT).

5. mu.l/well of compound (2.5% DMSO solution), 10. mu.l/well of enzyme (0.5. mu.g/ml p110 α + 1. mu.g/ml p85) and 10. mu.l/well of 5. mu.M ATP and 5. mu. Ci/ml [ gamma. ] by pipette³³P]ATP was added to the test buffer (final concentration: 0.2. mu.g/ml p110 α)2 μ M ATP,0.05 μ Ci/well [ γ ]³³P]ATP in 1X assay buffer: 100mM Tris-HCl pH 7.0,200mM NaCl, 8mM MgCl₂) The enzyme reaction is started. The reaction was incubated at room temperature for 1 hour and stopped with 30. mu.l/well of 50mM EDTA solution. The plates were then washed twice with TBS, dried and then 30. mu.l/well of scintillation material was added and counted on a Microbeta Trilux. IC (integrated circuit)₅₀Defined as the concentration of compound required for 50% of the maximum possible inhibition of kinase activity.

Microsomal stability test

The stability of compounds was initially assessed in vitro in 96-well plates (Whatman) using high-throughput templates, including culture with Human Liver Microsomes (HLM). Verapamil was purchased from sigma alder and used as a reference standard in this experiment. HLM was purchased from zeno technologies (Xeno Tech) (20 mg/ml in 250mM sucrose solution). A mother liquor of 100mM potassium phosphate buffer was prepared in advance by the following method: 80 ml of 1M K₂HPO₄And 20 ml of 1M KH₂PO₄Mixed in 900 ml of water (pH adjusted to 7.4 with dilute hydrochloric acid) and stored at room temperature. K₂HPO₄.3H₂O and KH₂PO₄From Sigma-Aldrich, NADPH regeneration system solutions A and B were obtained from Gene testing (Gentest). The stop solution for quenching reaction was a mixture of acetonitrile and DMSO (80:20) prepared beforehand and stored at 4 ℃. All solvents used were HPLC grade, and the water used during mother liquor preparation and LC-MS analysis was deionized by a Milli-Q system.

Mixed with 500. mu.l of 50mM potassium phosphate buffer (pH 7.4, prepared by diluting 100mM stock buffer with water), 2.5. mu.l of 10mM stock DMSO solution of the test compound was diluted 200-fold to give 500. mu.l of 50. mu.M solution. Then, 8. mu.l of the compound mixture was added to 72. mu.l of a previously prepared culture mixture composed of water (2250. mu.l), 100mM potassium phosphate buffer (2900. mu.l), NADPH regenerating system solution B (58. mu.l), NADPH regenerating system solution A (290. mu.l) and HLM (250. mu.l). The resulting reaction mixture (final compound concentration of 5 μ M) was then incubated at 37 ℃ in a b.braunctomat H incubator, and 50 μ l aliquots were then dispensed into wells of different plates containing 100 μ l stop solution. After centrifugation at 2000rpm for 15 minutes at 4 ℃ a sample of 100. mu.L of the resulting supernatant was transferred to an LC-MS plate for analysis. Each test compound was sampled multiple times and incubated for a series of time points (5, 15, 30, 45 and 60 minutes). The concentration of the remaining compound was determined by LC-MS (ABI Qtrap 3200) and compared to a reference solution of known concentration. Stability was then expressed as half-life (t1/2, min).

High throughput solubility test

The solubility of the compounds was determined in a high throughput dynamic solubility assay using 96-well templates. The solubility of the compounds was evaluated using a UV/visible microplate spectrophotometer (Molecular Devices SpectraMax Plus 384). Vorinostat (SAHA) and nicardipine from sigma were used as reference standards.

Compounds in DMSO were diluted with phosphate buffer (sigma) to a final concentration of 250 μ M (5 μ l of 10mM stock solution in 195 μ l of phosphate buffer pH 7) and mixed well. The mixture was then shaken at 600rpm for 1.5 hours and allowed to stand at room temperature for 2 hours. The plates were then centrifuged at 1500g for 15 minutes. The resulting supernatant (80 μ l) was transferred to a UV-assay plate and diluted with DMSO (20 μ l). Samples were quantitated using a calibrator stock of the corresponding compound prepared in phosphate buffer/DMSO (80: 20).

The compounds tested were as follows:

table 1 summarizes the results of the biological tests.

TABLE 1

It can be seen that all compounds have some degree of activity, except for compound E, which is significantly less active against mTOR, the activity of the compounds of the invention against the two enzymes of interest is comparable to that of the comparative compound. Therefore, all compounds are potential drug candidates, although compound E is somewhat less active against mTOR.

However, not all compounds tested showed acceptable solubility characteristics. For example, the low solubility results of compounds A, B and E indicate that these compounds are not ideal drug candidates. Their low solubility makes them difficult to formulate efficiently in physiologically acceptable carriers, thus reducing the possibility of having good oral pharmacokinetics in humans. In contrast, the solubility of compound C, D and the compounds of the invention is acceptable for drug candidates.

However, the results differ significantly in the metabolic stability in vitro. In these tests, compound A, B, E and the compounds of the invention had acceptable stability in human liver microsome studies. This suggests that if these compounds can be successfully administered, they are sufficiently stable to achieve the desired physiological effect in the patient. Moreover, of the compounds that have both good mTOR/PI3K inhibitory activity and good solubility (C, D and the compounds of the invention), the only compound with acceptable metabolic stability is the compound of the invention.

All compounds a-E and the compounds of the invention showed excellent activity in these assays, but the compounds of the invention were the most active because of their combination of potent target inhibition activity, excellent water solubility and excellent metabolic stability.

In summary, the biological results achieved by the compounds tested in the biological studies conducted above suggest that, although the compounds of the present invention have a significantly close similarity to many of the comparative test compounds, the compound of the present invention is the only one that simultaneously meets the requirements of activity, sufficient water solubility and metabolic stability and is therefore suitable for use as a pharmaceutical molecule. Thus, these studies indicate the superiority of this compound as a drug candidate.

Example 3: cell-based potency biomarker assay (pp70-S6KT389, pAktS473)

To further illustrate the potency of the compounds of the invention, two cell-based biomarker assays were performed on the compounds of the invention. The method comprises the following steps:

SureFire p-Akt (Ser 473)384 kit (TGR, Cat: TGRAS500),

SureFire phosphor-p 70S6 kinase (Thr 389)384 kit (TGR, Cat.: TGR70S500) and Proxiplate-384Plus (Perkin Elmer, Cat.: 6008280) were purchased from Parkinelmer (Perkin Elmer). Human prostate cancer cell line (PC-3) was purchased from ATCC. All compounds were purchased from sigma alder, unless otherwise noted.

The first day, 2X 105 cells/ml of 200 μ l PC3 cell solution was seeded into each well of a 96-well plate. Compounds were added 24 hours after inoculation, ranging typically from 10 μ M to 4.6nM, and diluted in triplicate in 8 steps. The final concentration of DMSO during the 4 hour incubation step at 37 ℃ was 0.1%. After the incubation step, the supernatant was removed, the cells were lysed with 1 × lysis buffer (provided by AlphaScreen kit) and gently shaken for 10 min. 4 microliters of lysate and 5 microliters of reaction buffer plus activation buffer mixture containing AlphaScreen acceptor beads were added to each 384-well (reaction buffer: activation buffer: acceptor beads 40:10:1) and gently shaken for 2 hours (room temperature, dark). 2 microliter ofDilution buffer for donor beads (dilution buffer: 20:1 for donor beads) was added to each well of the 384-plate, placed on a plate shaker, shaken for 1-2 minutes, and incubated overnight at room temperature.

The 384-well plate was read with a BMG Photometer plate reader using a standard AlphaScreen configuration (measurement mode: AlphaScreen; reading mode: Endpoint; optical mode: AlphaScreen 680570; position delay: 0.10 seconds; excitation time: 0.30 seconds; start of integration: 0.34 seconds; integration time: 0.30 seconds; gain: 3000).

IC₅₀Defined as the molar concentration of compound required for 50% of the maximum possible inhibition of kinase activity. 5- (9-isopropyl-8-methyl-2-morpholin-4-yl-9H-purin-6-yl) -pyrimidin-2-ylamine inhibitionp70-S6KT389 and pAktS473Phosphorylated ₅₀The IC was 24nM and 9nM, respectively.

Biomarker results indicate the efficacy of the compounds of the invention in inhibiting kinase activity.

Claims (16)

1. A method of making a compound of formula (I), the method comprising:

reacting a compound of formula (II) with dimethylzinc,

thereby providing a compound of formula (I).

2. The method of claim 1, wherein the reaction is carried out in the presence of a palladium complex that is pd (dppf) Cl₂。

3. The method of claim 1, wherein the reaction is carried out in the presence of a solvent.

4. The method of claim 3, wherein the solvent is dioxane.

5. The method of claim 1, wherein the reaction is carried out in a sealed tube.

6. The method of claim 1, wherein the reaction is carried out at about 65 ℃.

7. A process for the manufacture of a compound of formula (II) as defined in claim 1, which process comprises:

reacting a compound of formula (III) with N-bromosuccinimide,

thereby providing a compound of formula (II).

8. The method of claim 7, wherein the reaction is carried out in the presence of a solvent.

9. The method of claim 8, wherein the solvent is chloroform.

10. The method of claim 7, wherein the reaction is carried out at about 5 ℃.

11. Use of a compound of formula (I) according to claim 1 for the preparation of a medicament for the treatment of a disease in an animal, wherein inhibition of one or more protein kinases selected from the group consisting of serine/threonine protein kinases or fragments or complexes thereof or PI3 kinase or fragments or complexes thereof prevents, inhibits or ameliorates the pathology or symptomology of the disease, wherein the disease is cancer, a precancerous condition, a proliferative, autoimmune or inflammatory disease or a disease caused by excessive neovascularization.

12. The use as claimed in claim 11 wherein the condition is cancer.

13. The use of claim 11, wherein the condition is a precancerous condition or hyperplasia.

14. The use of claim 13, wherein the condition is selected from the group consisting of: familial adenomatous polyposis, adenomatous polyposis of the colon, myelodysplasia, endometriosis, endometrial dysplasia, cervical dysplasia, vaginal intraepithelial neoplasia, benign prostatic hyperplasia, laryngeal chorioma, actinic and solar keratoses, seborrheic keratoses and keratoacanthoma.

15. The use according to claim 13, wherein the condition is an autoimmune or inflammatory disease or a disease caused by excessive neovascularization.

16. The use of claim 15, wherein the condition is selected from the group consisting of: acute disseminated encephalomyelitis, Addison's disease, agammaglobulinemia, agranulocytosis, allergic asthma, allergic encephalomyelitis, allergic rhinitis, alopecia areata, senile alopecia, erythropoiesis inability, ankylosing spondylitis, antiphospholipid antibody syndrome, aortic inflammation syndrome, aplastic anemia, atopic dermatitis, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune oophoritis, Barlow's disease, Palygod's disease, Ocular-oral-genital triple syndrome, bronchial asthma, Cassman syndrome, celiac disease, Chagas' disease, chronic inflammatory demyelinating polyneuropathy, Church-Shidi's syndrome, Cotogen syndrome, keratoconus keratoderma, Coxsackie viral myocarditis, CREST disease, Crohn's disease, epidermal eosinophilia, epidermal T-cell lymphoma, Dermatitis diversities polycythemia myelomatosis, dermatomyositis, diabetic retinopathy, post-myocardial infarction syndrome, corneal epithelial dystrophy, eczematous dermatitis, eosinophilic fasciitis, eosinophilic gastroenteritis, epidermolysis bullosa, evans syndrome, fibrosing alveolitis, pemphigoid gestationis, glomerulonephritis, goodpasture's syndrome, graft-versus-host disease, graves ' disease, guillain-barre syndrome, hashimoto's disease, hemolytic uremic syndrome, herpetic keratitis, ichthyosis vulgaris, idiopathic interstitial pneumonia, idiopathic thrombocytopenic purpura, inflammatory bowel disease, catarrhal diseases, keratitis, keratoconjunctivitis, lambert-eaton syndrome, leukoplakia vulgaris, lichen planus, lichen sclerosus, lyme disease, linear bullous disease, macular degeneration, megaloblastic anemia, chronic glomerulonephritis, eosinophilic anemia, and glomerulonephritis, Meniere's disease, Moren's ulcer, Muha-Hubbmann's disease, polymyositis, multiple sclerosis, myasthenia gravis, necrotizing enterocolitis, eye encephalomyelopathy, ocular pemphigus, ocular myoclonus syndrome, Alder's thyroiditis, paroxysmal nocturnal hemoglobinuria, Bart's syndrome, pemphigus, periodontitis, pernicious anemia, pollen allergies, polyaphrenic autoimmune syndrome, posterior uveitis, monolobular cirrhosis, proctitis, pseudomembranous colitis, psoriasis, emphysema, pyoderma, Reiter's syndrome, reversible obstructive airway disease, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleritis, Saxieli syndrome, Sjogren's syndrome, subacute bacterial endocarditis, systemic lupus erythematosus, Takayasu's arteritis, temporal arteritis, Toho-Henbie's syndrome, Toyohimbe's syndrome, multiple sclerosis, necrotizing enterocolitis, chronic myelomato, Type I diabetes, ulcerative colitis, urticaria, spring conjunctivitis, vitiligo, Waji-salix minor-Protozoa syndrome and Wechsler's granulomatosis.