Pyrimidine-substituted purine compounds as kinase(s) inhibitors

FIELD: chemistry.

SUBSTANCE: claimed invention relates to novel pyrimidine-substituted purine compound of formula (I) or pharmaceutically acceptable salt of said compound, which possess inhibiting action with respect to mTOR and P13 kinases and can be applied in cancer treatment. Formula (I) compound corresponds to structural formula Formula (I)

EFFECT: invention relates to pharmaceutical composition which contains therapeutically effective quantity of said compound and pharmaceutically acceptable diluents, auxiliary substance or carrier, to method of inhibition of proteinkinase, selected from mTOR or P13 kinase, and to method of treatment or prevention of state in mammal, associated with inhibition of mTOR and/or P13 kinase activity.

11 cl, 1 tbl, 3 ex

 

The present invention relates to 5-(9-isopropyl-8-methyl-2-morpholine-4-yl-9-hydro-purine-6-yl)-pyrimidine-2-ylamino, the methods of its production, pharmaceutical compositions containing this compound and to the use of this compound for the treatment of certain disorders or conditions associated with kinases.

The search for inhibitors of kinases is a fertile ground for the creation of the substances used in the pharmaceutical industry. Kinase, also known as phosphotransferase are enzymes transferring phosphate groups from molecules makroergov (e.g., ATP) to specific molecules target (usually referred to as substrates) in a process called phosphorylation. One of the most numerous groups of kinases are protein kinases that influence and modify the activity of specific proteins.

Because of the ability of inhibitors of the kinase to act as a pharmaceutically active compounds was carried out substantial amount of research to identify compounds with the highest activity against these targets. In the field of Oncology, the two most important targets for therapeutic compounds are mTOR kinase and PI3. As an example of research in this area can result in the application PCT/SG 2008/000379 in which the disclosed compounds with activity in the RH is to kinases of both types: mTOR and PI3.

Compounds that inhibit both type kinase (mTOR and PI3), can have a strong anti-proliferative, anti-angiogenic and anti-cancer activity, as affect the way PDK/Akt/mTOR in several places. Now for the first time to conduct research of a number of inhibitors of this type in the clinical setting (for example, BEZ235, XL765, GDC0941, RH, SF1126).

In the process of finding the most suitable as a potential drug in order to make a final conclusion about whether the connection is a suitable potential drug take into account several factors. Accordingly, in the study of potential compounds for further development in addition to its inhibitory activity include a number of properties of the connection. When evaluating a specialist in the field of medicinal chemistry considers pharmacological ("lekarstovedenie properties of molecules and includes consideration of such factors as, for example, the activity of the compounds against the target, the solubility of the compounds (if they are not soluble, typically such compounds are potential bad drugs), metabolic activity of the compounds in vitro and in vivo, as well as possible side effects that this compound can cause in the body, etc., the Authors present invention IDA who was tapicerowane connection, with significantly improved pharmacological properties compared with other compounds in this field.

SHORT DESCRIPTION

According to the present invention proposed a compound of the formula I

or pharmaceutically acceptable salt of the compounds.

In addition to the compound of the formula I are also described embodiments, referring to pharmaceutically acceptable salts of the compounds, pharmaceutically acceptable N-oxides, pharmaceutically acceptable prodrugs, pharmaceutically acceptable metabolites of the compounds and pharmaceutically acceptable salts of these metabolites.

The present invention also relates to pharmaceutical compositions comprising the compound according to the present invention with a pharmaceutically acceptable carrier, diluent, or auxiliary agent.

Another aspect of the present invention relates to a method of inhibiting protein kinase selected from the group consisting of serine/threonine-protein kinase, fragment, or complex, a functional equivalent of the serine/threonine-protein kinase, and PI3 kinase, fragment or functional equivalent of PI3 kinase, including the effects of the protein kinase or a fragment, or functional equivalent, or to the fact that the R protein kinase effective amount of compound according to the present invention.

The proposed connection can influence directly and exclusively on the molecule kinase, or a fragment kinase and inhibit their biological activity. However, it is known that this compound may be at least partially influence of co-factors involved in the phosphorylation process. Known co-factors kinases include ionic groups (such as zinc and calcium), fats (such as phosphatidylserine) and diacylglycerol.

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

<> In some embodiments of the protein kinase is a PI3 kinase or a fragment complex, or a functional equivalent of PI3 kinase. In some embodiments of the PI3 kinase, fragment complex, or a functional equivalent of PI3 kinase is a PI3 class 1 or a fragment, or functional component of the PI3 class 1.

In one embodiment of the method for processing one or more protein kinases connection includes the introduction of the specified connection to the animal, the body which contains the one or more protein kinases.

The following aspect of the present invention proposed the use of the compounds for inhibiting one or more kinases selected from the group consisting of serine/threonine-protein kinase, fragment or functional equivalent of the serine/threonine-protein kinase, PI3 kinase, fragment or functional equivalent of PI3 kinase.

In some embodiments of the protein kinase is a serine/threonine-protein kinase or a fragment complex, or a functional equivalent of the serine/threonine-protein kinase. In some embodiments of the serine/threonine-protein kinase, fragment, or complex is a protein kinase mTOR, fragment or functional equivalent of the protein kinase mTOR. In some embodiments, the realization of the purpose serine/threonine-protein kinase, a fragment or a complex of serine/threonine-protein kinase is a protein kinase mTORC1 or fragment complex, or a functional equivalent protein kinase mTORC1. In some embodiments of the serine/threonine-protein kinase, a fragment or a complex of serine/threonine-protein kinase is a protein kinase mTORC2, fragment complex, or a functional equivalent protein kinase mTORC2.

In some embodiments of the protein kinase is a PI3 kinase or a fragment complex, or a functional equivalent of PI3 kinase. In some embodiments of the PI3 kinase, fragment complex, or a functional equivalent of PI3 kinase is a PI3 class 1 or a fragment, or functional component of the PI3 class 1.

The following aspect of the present invention, a method for treatment or prevention in a mammal of a condition in which inhibition of one or more protein kinases selected from the group consisting of serine/threonine-protein kinase, fragment or functional equivalent of the serine/threonine-protein kinase, PI3 kinase, fragment or functional equivalent of PI3 kinase prevents, reduces or alleviates pathology or condition, and this method includes the introduction of therapeutically effective amounts of compounds according to the present is obreteniyu.

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

In some embodiments of the protein kinase is a PI3 kinase or a fragment complex, or a functional equivalent of PI3 kinase. In some embodiments of the PI3 kinase, fragment complex, or a functional equivalent of PI3 kinase is a PI3 class 1 or a fragment, or functional component of the PI3 class 1.

In some embodiments of the condition is a cancer. In some embodiments of cancer selected from the group consisting of cancer shelter is, such as myeloproliferative disorders (idiopathic myelofibrosis, polycythemia Vera, essential thrombocytosis, chronic myeloid leukemia), myeloid metaplasia, myelomonocytic leukemia, acute lymphocytic leukemia, acute erythroblastosis leukemia, Hodgkin's disease and non-Jackinsky lymphoma, acute T-cell leukemia, myelodyplastic syndromes, diseases of the plasma cells, hairy cell leukemia, Kaposi's sarcoma, lymphoma, gynecologic cancers, such as breast carcinoma, ovarian cancer, cervical cancer, cancer of the vagina and labia, hyperplasia of the endometrium, cancer of the gastrointestinal tract, such as carcinoma Tolstoy intestine and rectum polyps, liver cancer, stomach cancer, pancreatic cancer, gallbladder cancer; cancer of the urinary tract, such as cancer of the prostate, kidney, bladder, urethral cancer, penile cancer; skin cancer such as melanoma, brain tumors, such as glioblastoma, neuroblastoma, astrocytoma, oligodendroglioma, ependymoma, stem glioma brain, medulloblastoma, meningioma; head and neck cancer, such as carcinoma of the nasopharynx carcinoma of the larynx; cancer of the respiratory tract, such as carcinoma of the lung (non-small cell lung cancer and small cell lung cancer), mesothelioma; eye diseases such as retinoblastoma; sabol the requirements of musculoskeletal apparatus, such as osteosarcoma, neoplasia musculoskeletal, squamous cell carcinoma and fibroma. In other embodiments of the compound according to the present invention can be applied for the treatment of precancerous lesions or hyperplasia, including familial adenomatous polyposis, adenomatous, Polipo colon cancer, myeloid dysplasia, endometrial dysplasia, atypical hyperplasia of the endometrium, cervical dysplasia, intraepithelial the neoplasia of the vagina, benign prostatic hyperplasia, papillomas of the larynx, and the old solar keratosis, seborrheic keratosis and keratoakantoma.

In some embodiments of the condition is an autoimmune or inflammatory disease or illness, supported by increased neovascularization. The disease, which to some extent attributed to diseases autoimmune etiologies include the following: acute disseminated encephalomyelitis, Addison disease, agammaglobulinemia, agranulocytosis, allergic asthma, allergic encephalomyelitis, allergic rhinitis, alopecia areata, senile alopecia, premature aging, americaplay, ankylosing spondylitis, antiphospholipid syndrome antibodies, aortic, aplastic anemia, atypical dermatitis, autoimmune hemolytic anemia, automanagement, autoimmune oophoritis, Balo disease, graves ' disease, Behcet's disease, bronchial asthma, symptoms of Castellana, celiac disease, Chagas disease, chronic inflammatory demyelinizing polyneuropathy syndrome Churg-Strauss syndrome Kogan, myocarditis Coxsackie, Crohn's disease, cutaneous eosinophilia, cutaneous T-cell lymphoma, poly-form dermatitic erythema, dermatomyositis, diabetic retinopathy, Dressler syndrome, epithelial corneal dystrophy, exanthematous dermatitis, endometriosis, eosinophilic fasciitis, eosinophilic gastroenteritis, bullous bullosa, gestational pemphigoid, Evans syndrome, fibrosing alveolitis, gestational pemphigoid, glomerulonephritis syndrome?, the disease "graft vs. host disease greivsa syndrome Guillain-Barre, Hashimoto's disease, hemolytic natriuretic syndrome, herpes keratitis, ichthyosis vulgaris, idiopathic intestinally pneumonia, idiopathic thrombocytopenic of pupura, inflammatory bowel disease, Kawasaki disease, keratitis, keratoconjunctivitis syndrome of Lambert-Eaton vulgar Leucoderma lichen planus, a shingles seal, Lyme disease, linear IgA disease, macular degeneration, megaloblastic anemia, Meniere's disease, ulcer Moray, illness Fly - Haberman, multiple myositis, multiple with Leros, malignant male, necrotorous enterocolitis, neuromyelitis optic nerve, pemphigus eyes, needs to be myoclonus syndrome, thyroiditis Horde, paroxysmal nocturnal hemoglobinuria syndrome Parsonage-Turner, pemphigus, periodontal disease, pernicious anemia, allergies to pollen, autoimmune polyglandular syndrome, posterior uveitis, primary biliary cirrhosis, proctitis, pseudomembranous colitis, psoriasis, emphysema, pyoderma, Reiter syndrome, reversible obstructive Airways disease, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleritis, syndrome Cesari, Sjogren syndrome, systemic bacterial endocarditis, systemic lupus erythematosus, Takayasu's arteritis, temporal Takayasu syndrome Tolosa-Hunt, sugar diabetes, ulcerative colitis, spring Qatar eyes, vitiligo syndrome Vogt-Koyanagi-Harada and granulomatous of Wegner. In some embodiments of the condition represents endometriosis.

The following aspect of the invention the application of the compounds according to the invention for the manufacture of a medicinal product for the treatment of the animal condition in which inhibition of one or more kinases selected from the group consisting of serine/threonine-protein kinase, fragment or functional equivalent of the serine/Trets the NIN-protein kinase, the PI3 kinase or a fragment or functional equivalent of PI3 kinase, prevents, inhibits or facilitates the pathology and symptoms of this condition.

In another aspect of the present invention proposed the use of compounds according to the present invention or a pharmaceutically acceptable salt, N-oxide or prodrug of the compounds in the treatment condition in which one or more kinases selected from the group consisting of serine/threonine-protein kinase, fragment or functional equivalent of the serine/threonine-protein kinase, PI3 kinase, or a fragment or functional equivalent of PI3 kinase, prevents, inhibits or facilitates the pathology and symptoms of this condition.

In some embodiments of the protein kinase is a serine/threonine-protein kinase or a fragment complex, or a functional equivalent of the serine/threonine-protein kinase. In some embodiments of the serine/threonine-protein kinase, fragment, or complex is a protein kinase mTOR, fragment or functional equivalent of the protein kinase mTOR. In some embodiments of the serine/threonine-protein kinase, a fragment or a complex of serine/threonine-protein kinase is a protein kinase mTORC1 or fragment complex, or a functional equivalent about sincinaty mTORC1. In some embodiments of the serine/threonine-protein kinase, a fragment or a complex of serine/threonine-protein kinase is a protein kinase mTORC2, fragment complex, or a functional equivalent protein kinase mTORC2.

In some embodiments of the protein kinase is a PI3 kinase or a fragment complex, or a functional equivalent of PI3 kinase. In some embodiments of the PI3 kinase, fragment complex, or a functional equivalent of PI3 kinase is a PI3 class 1 or a fragment, or functional component of the PI3 class 1.

In another aspect of the present invention, a method for preventing or treating proliferative pathologies in General.

In some embodiments of the condition is a cancer. In some embodiments of cancer selected from the group consisting of blood cancer, such as myeloproliferative disorders (idiopathic myelofibrosis, polycythemia Vera, essential thrombocytosis, chronic myeloid leukemia), myeloid metaplasia, myelomonocytic leukemia, acute lymphocytic leukemia, acute erythroblastosis leukemia, Hodgkin's disease and non-Jackinsky lymphoma, acute T-cell leukemia, myelodyplastic syndromes, diseases of the plasma cells, hairy cell leukemia, Kaposi's sarcoma, lymphoma; guinet the ideological types of cancer, such as breast carcinoma, ovarian cancer, cervical cancer, cancer of the vagina and labia, hyperplasia of the endometrium, cancer of the gastrointestinal tract, such as carcinoma of the colon and rectum polyps, liver cancer, stomach cancer, pancreatic cancer, gallbladder cancer; cancer of the urinary tract, such as cancer of the prostate, kidney, bladder, urethral cancer, penile cancer; skin cancer such as melanoma, brain tumors, such as glioblastoma, neuroblastoma, astrocytoma, oligodendroglioma, ependymoma, stem glioma brain, medulloblastoma the meningioma; head and neck cancer, such as carcinoma of the nasopharynx carcinoma of the larynx; cancer of the respiratory tract, such as carcinoma of the lung (non-small cell lung cancer and small cell lung cancer), mesothelioma; eye diseases such as retinoblastoma; diseases of the musculoskeletal system, such as osteosarcoma, neoplasia musculoskeletal, squamous cell carcinoma and fibroma. In some embodiments of the condition represents endometriosis.

It is additionally assumed that the formula (I) comprises soluble and insoluble forms of connections, where applicable. Thus, each formula includes a connection with the specified structure, including hydrated and UN-hydrated form.

These and the other the e sections of the present invention is presented below.

DETAILED DESCRIPTION

In this description uses a number of terms that are well known to the specialist. For clarity, you will define a number of terms.

The term "pharmaceutically acceptable salt" refers to salts that retain the essential biological activity of certain of the above compounds, and include pharmaceutically acceptable salts of accession of the acid and the salt of the attaching base. Suitable pharmaceutically acceptable salts of accession of the acid compounds of formula I can be obtained from both inorganic and organic acids. Examples of such inorganic acids are hydrochloric, sulfuric, phosphoric acid. Preferred organic acids are aliphatic, cycloaliphatic, aromatic, heterocyclic carboxylic and sulfonic classes of organic acids, such as formic, acetic, propionic, succinic, glycolic, gluconic, lactic, malic, tartaric, citric, fumaric, maleic, alkylsulfonate and arylsulfonate. For more information about the pharmaceutically acceptable salts may be found in Remington's Pharmaceutical Sciences, 19thEdition, Mack Publishing Co., Easton, PA 1995. In that case, if agents are solids for professionals to be clear, the connection of the invention, agents, salts can be is redstavleny in different crystal or polymorphic forms, each of which can be applied in the framework of the present invention and encompassed by the above formula.

The term "therapeutically effective amount" or "effective amount" means a quantity sufficient to provide a favorable or desired clinical results. An effective amount can be entered one or more times. An effective amount is usually sufficient to alleviate, improve, stabilize, reversing, slowing or eliminating the progression of the disease condition.

It is assumed that the term "functional equivalent" includes variants described herein of specific protein kinases. It is clear that the kinase may be isoforms, and primary, secondary, tertiary and Quaternary structure of individual isoforms of the kinase will be different from the original kinase, however, possess the same biological activity as the protein kinase. Isoforms can occur as a result of normal allelic differences in population and contain mutations such as substitution, deletion, joining, cutting, duplication of amino acids. Also the concept of "functional equivalent" is obtained at the level of transcription. Other functional equivalents include kinases, with alternative post-translational modifications, such as glycolysis the interview.

The connection according to the present invention demonstrates improved pharmacological properties, described in detail below in comparison with the structural blizzie connections in this area.

Such improved properties suggest that the compound according to the present invention can be a suitable choice for pharmaceutical development in this area. First, we study the connection according to the present invention exhibits a comparable, if not superior, activity in the inhibition of two interest kinase-mTOR and PI3. The active compounds according to the present invention in respect of PI3 higher than the activity of the other investigated compounds, and demonstrates activity against mTOR which is comparable with the activity of the other investigated compounds, and is characterized suitable for therapeutic applications.

Despite the fact that the tests for determination of enzyme activity show that almost all connections, checked out, had an acceptable level of activity, further tests revealed a number of compounds that have been excluded as unsuitable for further pharmaceutical development or for other reasons. For example, the connection according to the present invention have a moderate solubility in water (178 μm)defining that Saedinenie according to the present invention can be prepared pharmaceutical composition, absorbed when administered orally, while the number of Comparators compounds did not demonstrate acceptable solubility. Thus, the connection according to the present invention demonstrates the combination of high activity and good solubility characteristics.

Of the compounds that showed the combination of activity and solubility, the compound according to the present invention was the best indicators of metabolic stability. The compound showed high stability studies with human liver microsomes, it indicates a sufficient resistance to degradation in the physiological environment. In contrast, other compounds possessing activity and solubility, is not demonstrated even close to stability. Because the connection according to the present invention demonstrates a unique combination of activity, solubility and stability, this makes it an excellent candidate drug in comparison with other Comparators known compounds despite the apparent structural similarity of these compounds.

The connection according to the present invention has the ability to inhibit the activity of certain protein kinases. The ability to inhibit the activity of kinases may be due to the fact that sedimentogene the present invention acts directly and directed molecule kinase and inhibits its activity. However, it should be understood that the connection may also be partly co-factors kinases in the process of their participation in the phosphorylation. The connection may have activity against PI3 kinases or fragments, complexes or functional equivalents of PI3 kinases. This connection may have activity against serine/threonine protein kinases such as mTOR or their fragments, complexes or functional equivalents.

Inhibition of protein kinases can be carried out by any known in this field means. For example, if you want to carry out the inhibition of protein kinases in vitro in a solution containing the purified enzyme is a kinase, add appropriate amount of compound according to the present invention. In conditions when it is necessary to carry out the inhibition of the activity of protein kinases in a mammal, the inhibition of kinases usually includes the introduction of compounds according to the present invention to a mammal having this kinase.

Accordingly, the connection according to the present invention can find a wide range of applications, which can be used by its ability to inhibit protein kinases of the above. For example, the compounds can be used for inhibition of serine/threonine protein kinases. The compounds can be used to treat or is the avoiding of mammals States, in which inhibition of protein kinases and/or their co-factors prevents, reduces or simplifies the pathology or symptoms of pathology.

The proposed connection can be used to treat proliferative diseases. An example of such diseases is cancer. It is expected that the compound will have the ability to treat both solid and liquid tumors. In some embodiments of the cancers that can be treated with a compound according to the present invention, solid tumors and cancers of the blood.

In the present description, the term "cancer" is a General term that covers a huge number of States characterized by uncontrolled abnormal cell growth. It is expected that the compound according to the present invention can be applied to treat various types of cancer, vkluchaysya, as non-limiting examples, bone cancer, brain tumors and Central nervous system, breast cancer, colon cancer, cancer of the endocrine glands, including the adrenal cortical carcinoma, pancreatic cancer, cancer of the pituitary, thyroid cancer, parathyroid cancer, cancer of the thymus gland, cancer of the gastrointestinal tract, liver cancer, common hepatic duct, carcinoid tumor of the gastrointestinal tract, gallbladder cancer, cancer of the urinary system, cancer of the reproductive system, cancer of the head and neck, is Akamai, myeloma, blood disease, lung cancer, lymphoma, cancer of the eye, skin cancer, soft tissue sarcoma, Mature soft tissue sarcoma, Kaposi's sarcoma, cancer of the urinary system.

Examples of cancers that can be treated with a compound according to the present invention includes a blood cancer, such as myeloproliferative disorders (idiopathic myelofibrosis, polycythemia Vera, essential thrombocytosis, chronic myeloid leukemia), myeloid metaplasia, myelomonocytic leukemia, acute lymphocytic leukemia, acute erythroblastosis leukemia, Hodgkin's disease and non-Jackinsky lymphoma, acute T-cell leukemia, myelodyplastic syndromes, diseases of the plasma cells, hairy cell leukemia, Kaposi's sarcoma, lymphoma, gynecologic cancers, such as breast carcinoma, ovarian cancer, cervical cancer, cancer of the vagina and labia, hyperplasia of the endometrium, cancer of the gastrointestinal tract, such as carcinoma of the colon and rectum polyps, liver cancer, stomach cancer, pancreatic cancer, gallbladder cancer; cancer of the urinary tract, such as cancer of the prostate, kidney, bladder, urethral cancer, penile cancer; skin cancer such as melanoma, brain tumors, such as glioblastoma, neuroblastoma, astrocytoma, oligodendroglioma, ependymoma, stem glioma brain, medulloblastoma, men who Ngoma; head and neck cancer, such as carcinoma of the nasopharynx carcinoma of the larynx; cancer of the respiratory tract, such as carcinoma of the lung (non-small cell lung cancer and small cell lung cancer), mesothelioma; eye diseases such as retinoblastoma; diseases of the musculoskeletal system, such as osteosarcoma, neoplasia musculoskeletal, squamous cell carcinoma and fibroma. The connection according to the present invention can also be used to treat precancerous conditions or hyperplasia, including familial adenomatous polyposis, adenomatous polyposis of the colon, myeloid dysplasia, endometrial dysplasia, atypical hyperplasia of the endometrium, cervical dysplasia, intraepithelial the neoplasia of the vagina, benign prostatic hyperplasia, papillomas of the larynx, and the old solar keratosis, seborrheic keratosis and keratoacanthoma.

It is also expected that the compound according to the present invention will be applicable in the treatment of autoimmune or inflammatory diseases, and diseases, supported by increased neovascularization. The disease, which to some extent attributed to diseases autoimmune etiologies include the following: acute disseminated encephalomyelitis, Addison disease, agammaglobulinemia, agranulocytosis, allergic asthma, allergic EN is elomari, allergic rhinitis, alopecia areata, senile alopecia, premature aging, americaplay, ankylosing spondylitis, antiphospholipid syndrome antibodies, aortic, aplastic anemia, atypical dermatitis, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune oophoritis, Balo disease, graves ' disease, Behcet's disease, bronchial asthma, symptoms of Castellana, celiac disease, Chagas disease, chronic inflammatory demyelinizing polyneuropathy syndrome Churg-Strauss syndrome Kogan, myocarditis Coxsackie, Crohn's disease, cutaneous eosinophilia, cutaneous T-cell lymphoma, poly-form dermatitic erythema, dermatomyositis, diabetic retinopathy, Dressler syndrome, epithelial dystrophy of the cornea, exanthematous dermatitis, endometriosis, eosinophilic fasciitis, eosinophilic gastroenteritis, bullous bullosa, gestational pemphigoid, Evans syndrome, fibrosing alveolitis, gestational pemphigoid, glomerulonephritis syndrome?, disease, graft vs. host disease greivsa syndrome Guillain-Barre, Hashimoto's disease, hemolytic natriuretic syndrome, herpes keratitis, ichthyosis vulgaris, idiopathic intestinally pneumonia, idiopathic thrombocytopenic of pupura, inflammatory bowel disease, Kawasaki disease, keratitis, kerato conjuctiva, the syndrome of Lambert-Eaton vulgar Leucoderma lichen planus, a shingles seal, Lyme disease, linear IgA disease, macular degeneration, megaloblastic anemia, Meniere's disease, ulcer Moray, illness Fly-Haberman, multiple myositis, multiple sclerosis, malignant male, necrotorous enterocolitis, neuromyelitis optic nerve, pemphigus eyes, needs to be myoclonus syndrome, thyroiditis Horde, paroxysmal nocturnal hemoglobinuria syndrome Parsonage-Turner, pemphigus, periodontal disease, pernicious anemia, allergies to pollen, autoimmune polyglandular syndrome, posterior uveitis, primary biliary cirrhosis, proctitis, pseudomembrane colitis, psoriasis, emphysema, pyoderma, Reiter syndrome, reversible obstructive Airways disease, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleritis, syndrome Cesari, Sjogren syndrome, systemic bacterial endocarditis, systemic lupus erythematosus, Takayasu's arteritis, temporal Takayasu syndrome Tolosa-Hunt, sugar diabetes, ulcerative colitis, spring Qatar eyes, vitiligo syndrome Vogt-Koyanagi-Harada and granulomatous of Wegner. In some embodiments of pathology is endometriosis.

The connection according to the present invention can also be applied to manufacture the Oia medicines for the treatment of a mammal status, thus the inhibition of protein kinases can prevent, reduce or alleviate the pathology and symptoms of the condition. The connection according to the present invention can also be applied to the manufacture of medicines for the treatment or prevention of diseases associated with protein kinases.

Introduction the compounds of formula I according to the present invention can be implemented using any acceptable enteral routes of administration, such as oral or rectal, or parenteral administration such as subcutaneous, intramuscular, intravenous and intradermal. The injection may be a bolus or by continuous or discrete infusion. Generally, the active compound include pharmaceutically acceptable carrier or solvent in an amount necessary to obtain the patient an effective therapeutic amount. In various embodiments of the inhibiting compound may be selectively toxic or more toxic to rapidly proliferating cells, such as compared to normal cells.

When using compounds according to the present invention it can be entered in any shapes and forms, ensuring the bioavailability of the compounds. Specialist in the field of obtaining compositions can easily choose the appropriate form and type of introduction, according to otopitelnyh characteristics of the selected connection, pathogenesis, stages of pathogenesis and any other relevant circumstances. Additional information can be found in "Remingtons Pharmaceutical Sciences, 19thedition, Mack Publishing Co. (1995).

The connection according to the present invention can be entered separately or in the form of pharmaceutical compositions in combination with pharmaceutically acceptable carrier, diluent, or auxiliary agent. The connection according to the present invention, effective by itself, usually in the form of pharmaceutically acceptable salts, because these forms are usually more stable, easier to crystallize and have a high solubility.

However, the present compound is usually used in the form of pharmaceutical compositions, which comprise, depending on the method of introduction. Therefore, in some embodiments of the present invention proposed a pharmaceutical composition comprising the compound according to the present invention and a pharmaceutically acceptable carrier, solvent or excipient. The composition is formed in accordance with methods known in this field.

In other embodiments of the proposed invention, a pharmaceutical package or kit containing one or more containers containing one or more components of the pharmaceutical compositions according to the present invention. In tako the packaging or the set of possible capacity is, containing a single dose of a substance (substances). The kit may include a composition comprising the active ingredient either as concentrates (including lyophilized concentrates that can be diluted before application, as well as ready-to-use concentrates, in this capacity include one or more doses. For convenience, the set of unit dose may be placed in sterile vials for immediate use by the physician, and the vials contain the required amount of substance (substances). Such container (containers) can be offered various written materials such as instructions for medical use or information about the use and distribution of pharmaceutical and biological products for use in humans.

The connection according to the present invention can be applied or enter appointed in combination with one or more drugs for treatment of disorders/diseases described above. Components can be entered in the same composition or different compositions. In the case of the introduction of different formulations of the compounds according to the present invention can be entered sequentially or simultaneously with other drugs (drugs).

In connection with the possibility of introducing a connection with one or more drugs, the connection according to the present izobreteny which can be used in combination therapy. Usually compounds administered in combination with each other. Thus, the connection according to the present invention can be typed in at the same time (as a combination drug), and consistently in order to provide enhanced therapeutic effect.

The pharmaceutical compositions according to the present invention for parenteral administration can be entered in the form of a pharmaceutically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, and sterile powders which, before the introduction diluted with sterile solutions or dispersions for injection. Examples of suitable aqueous and nonaqueous carriers, solvents, soluble substances or environments are water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol and the like), and suitable mixtures, vegetable oils (such as olive oil) and suitable for introduction of organic esters, such as etiloleat. The proper fluidity can be provided by use of coating materials, such as lecithin, by providing the required particle size in the case of dispersions and by the use of surfactants.

Such compositions can contain auxiliary substances such as preservatives, moisturizing agent, emulsifying agents, dispersing agents. For the of avoiding contamination by microorganisms can be used antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenolic carboxylic acid and the like. It may be possible and necessary inclusion isotonic agents such as sugars, sodium chloride or the like. Prolonged action of pharmaceutical forms can be achieved by the inclusion of agents which delay absorption such as aluminum monostearate or gelatin.

For more effective distribution, the compounds may be included in slowly released system or delivery systems such as polymer matrix, the liposomes, microspheres.

Sterility form for injection can be achieved by, for example, filtration through inhibiting bacteria filter, or by incorporating sterilizing agents in sterile solid composition, which before application can be dissolving or dispersing in sterile water or other sterile environments.

Solid dosage forms for oral administration include capsules, tablets, pills, powders, granules. In such solid forms of active substance is mixed with an inert pharmaceutically acceptable excipients or carriers, such as sodium citrate or caliceditore and/or a) fillers and diluents, such as starch, lactose, sucrose, glucose, mannitol, silicic acid, b) binders such as carbon metilzellulosa, alginate, gelatin, polyvinylpyrrolidone, sucrose, gum, C) humectants such as glycerol, d) dezinfeciruyuhimi substances such as agar-agar, calcium carbonate, potato or cassava starch, alginic acid, certain silicates, and sodium carbonate, e) delaying the dissolution of the reagents, such as paraffin, f) absorption accelerators such as Quaternary ammonium compounds, g) wetting agents, such cancerology alcohol and glycerylmonostearate,) absorbents such as kaolin and bentonite, j) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, and mixtures thereof. In capsules, tablets and pills can be enabled and buffer reagents.

Solid compositions of a similar type may also be employed as fillers in soft and hard gelatin capsules, excipients which may be lactose or milk sugar and high molecular weight glycols or similar substances.

Solid dosage forms of tablets, capsules, pills and granules can be produced with coatings and shells, such as coatings for enteral administration, and other coatings well known in the manufacture of pharmaceuticals. Forms can contain a reagent that renders them opaque, or is there to be with such a composition, which is released only active substance (substances), or, preferably, it is delayed for release in a certain part of the gastrointestinal tract. Examples included such compositions can be incorporated polymeric substances and waxes.

The active compound can be in the form of microcapsules, preferably with one or more auxiliary substance mentioned earlier.

Liquid forms for oral administration are pharmaceutically acceptable emulsions, suspensions, syrups and tinctures. In addition to the active compounds, the liquid form may contain solvents usually used in this field, for example, water or other solvents, dissolving agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethylcarbonate, ethyl acetate, benzyl alcohol, propylene glycol, 1,3-butyleneglycol, dimethylformamide, oils (in particular, cottonseed oil, groundnut, corn, germ oil, olive, castor, sesame) glycerol, tetrahexahedron alcohol, polyethylene glycol, fatty acid esters of sorbitane mixtures thereof.

In addition to the inert solvent, oral compositions can include excipients, such as moistening agents, emulsifiers and suspendiruemye agents, sweeteners, flavorings and fragrances.

In suspensions chrome is active substances may be contained suspendresume reagents, such as, for example, ethoxycarbonylmethylene alcohols, politician sorbitol and esters sorbitan, microcrystalline cellulose, bentonite, agar-agar, tragakant, or mixtures thereof.

Compositions for rectal or vaginal use are mostly suppositories, which can be obtained by mixing the substances of the invention with suitable not cause irritation excipients or carriers, such as coconut oil, polyethylene glycol, or suppositories made of wax that is solid at room temperature, but liquid at body temperature, so when dissolved in rectal or vaginal channels allocated active substance.

Dosage forms for topical use the compounds according to the present invention are powders, patches, sprays, ointments and inhalers. The active substance is mixed under sterile conditions with a pharmaceutically acceptable carrier or needed preservatives, buffers or required spraying substances.

Enter the number of connections in the preferred case provides treatment, mitigation or relief pathology action. therapeutically effective amount can be determined by the diagnostician by using standard equipment and research results obtained analogues of the circumstances. When determining therapeutically effective amount there are a number of factors, including but not limited to the following factors: type of animal, size, age, General health status, a feature of the disease, extent of disease, the sensitivity of the patient to treatment, the feature assigned to the substance, method of appointment, the bioavailability of the assigned drug, the selected dosage regimen, use of other medications and other relevant circumstances.

The preferred dosage will be in the range from 0.01 to 300 mg per kilogram of body weight. The preferred dose is a daily dose of 0.1-100 mg per kilogram of body mass, as a daily dose of 0.2-80 mg per kilogram of body weight, and even more preferred is a daily dosage of 0.2-50 mg per kilogram of body weight. A suitable daily dose can be assigned to several sub-doses.

The SYNTHESIS of COMPOUNDS ACCORDING to the INVENTION

5-(9-isopropyl-8-methyl-2-morpholine-4-yl-9-hydro-purine-6-yl)-pyrimidine-2-ylamine was obtained from dichloropurine in 5 stages, shown in figure 1.

Scheme 1

EXAMPLES

In the following examples, unless otherwise indicated, all temperatures are given in degrees Celsius and all parts and percentages are calculated by weight.

Various starting materials and other reagents were purchased from commercial the ski suppliers such as Aldrich Chemical Company and Lancaster Synthesis Ltd., and used without further purification, unless otherwise stated. Tetrahydrofuran and N,N-dimethylformamidine from Aldrich in airtight bottles and used in the form in which it was received. All solvents were purified by standard methods used in this field, unless you specify otherwise.

The following reaction was carried out at a gauge pressure of nitrogen, argon or with a drying tube at ambient temperature (unless stated otherwise), in anhydrous solvents in flasks with a rubber septum for the introduction of substrates and reagents via syringe. Glassware was pre-dried in a drying Cabinet and/or progulivali. Analytical thin-layer chromatography was performed on Silicagel plates R on glass (Merck thickness 0.25 mm) and elution with a suitable ratio of solvent (volume/volume). Reactions were analyzed by thin layer chromatography and stopped when the magazine is empty, the source materials.

Plates for TLC were visualized by UV absorption using spraying p-anise aldehyde or phosphomolybdenum acid (manufacturer : Aldrich Chemical Company, with 20% ethanol content) with activation by heat or location in the iodine chamber.

The reaction product is made by doubling the volume of the reaction with the art by adding a reaction solvent or extraction of the solvent with subsequent washing specified water solvent in the amount of 25% of the extraction mixture (unless specified otherwise). The obtained solutions were dried anhydrous sodium sulfate prior to filtration, evaporation of solvents was carried out under reduced pressure in a rotary evaporator, in the description of this process is called removal of the solvents under vacuum.

Flash chromatography (Stillatal, J. Org. Chem., 43, 2923 (1978)) was carried out using silica gel brand EMerck (47-61 mm)with a ratio of the silica raw material is from 20:1 to 50:1, if not stated otherwise. Hydrogenolysis was carried out at the specified pressure or at atmospheric pressure.

1H NMR spectra were shot on the equipment Bruker 400 MHz,13C-NMR spectra were shot at 100 MHz. NMR spectra were obtained in the form of solutions in CdCl3(in ppm), using chloroform as the reference solution (7,27 ppm and of 77.0 ppm)or Cd3OD (of 3.4 and 4.8 ppm and 49.3 ppm), or internal standard tetramethylsilane (TMS) (0,00 ppm) if necessary. If necessary used the other solvents for NMR. When detecting multiple peaks use the following abbreviations: s - singlet, d - doublet, t - triplet, t - multiplet, br : broad, d - doublet of doublets, dt - doublet of triplets. Constant interaction, if any, given in Hz. Mass spectra were obtained by liquid chromatography (LC) - mass spectrometry (MS) detectors ESI or APCI, respectively. The melting temperature is not corrected. All final products were is istota more than 90% (according to the method of high-performance chromatography with wavelengths 220-240 nm).

The following examples illustrate the synthesis one way to obtain according to the present invention, but they should not be construed as a limitation of the synthesis methods.

Example 1 Synthesis of compounds according to the invention

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

2,6-dichloropurine(2 mmol), isopropanol (8 mmol) and triphenylphosphine (4 mmol) dissolved in 40 ml of anhydrous tetrahydrofuran, and to the mixture dropwise at room temperature within 30 minutes was added diisopropylethylamine (4 mmol). The reaction mixture was stirred at room temperature for days. Periodically watched the course of the reaction by TLC or LC-MS. The reaction mixture is poured into the beaker with ice water. As a result of extraction of the aqueous layer was extracted three times with ethyl acetate 100 ml) received intermediate. The intermediate was purified by chromatography on silikagelevye column (10-80% ethyl acetate in kerosene ether, gradient leaching), the result obtained 2,6-dichloro-9-isopropyl-9-hydro-purine with a yield of 77%.

Synthesis of 5-(2-chloro-9-isopropyl-9-hydro-purine-6-yl)-pyrimidine-2-ylamine

To a solution of 2,6-dichloro-9-isopropyl-9-hydro-purine (to 5.21 mmol), 5-(4,4,5,5-tetramethyl-[1,3,2] dioxaborolan-2-ylamine (to 5.21 mmol) and 1,1-bis-(diphenylphosphino)-ferrocin palladium (II) chloride complexes with dichloromethane (0.26 mmol) in peroxide-free dioxane (40 ml) was added 2 M aqueous solution of sodium carbonate (15.6 mmol). The resulting mixture was degirolami and purged with nitrogen. Then the reaction mixture was mixed and heated at an oil bath at a temperature of 80°C for 3 hours. The reaction mixture was checked by LC-MS in the absence of the source of purines.

The reaction mixture was cooled to room temperature, the solvents were removed under reduced pressure. Sediment was added to a mixture of ethyl acetate and water. The organic phase was separated, the aqueous phase was extracted with ethyl acetate in the ratio of 3:100. After removal of organic substances by vacuum received 5-(2-chloro-9-isopropyl-9-hydro-purine-6-yl)-pyrimidine-2-ylamine with the release of 55%.

Synthesis of 5-(9-isopropyl-2-morpholine-4-yl-9-hydro-purine-6-yl)-pyrimidine-2-ylamine

To a solution of 5-(2-chloro-9-isopropyl-9-hydro-purine-6-yl)-pyrimidine-2-ylamine (2,84 mmol) in dimethylacetamide(18 ml) was added morpholine (2,84 mmol). The reaction mixture was stirred under heating at an oil bath at a temperature of 94°C for 12 hours. The reaction was checked for the absence of starting materials using LC-MS. Intermediate immediately skipped through the HPLC column and purified, thus obtained 5-(9-isopropyl-2-morpholine-4-yl-9-hydro-purine-6-yl)-pyrimidine-2-ylamine with the release of 58%.1H NMR, DMSO-d6: at 9.53 (s, 2H), 8,32 (s, 1H), 7,30 (bs, 2H), 4.72 in (m, 1H), 3,78 (m, 4H), to 3.73 (m, 4H), of 1.55 (d, 6H). m/z: 341,17 [MH]+.

Synthesis of 5-(8-bromo-9-isopropyl-2-morpholine-4-yl-9-hydro-p is Rin-6-yl)-pyrimidine-2-ylamine.

To a solution of 5-(9-isopropyl-2-morpholine-4-yl-9-hydro-purine-6-yl)-pyrimidine-2-ylamine (1,03 g, 3.03 mmol) in 15 ml of chloroform is slowly added N-bromosuccinimide (594 mg, 3,34 mmol) at 5°C. At this temperature, the reaction was performed for 2 hours. The resulting product 5-(8-bromo-9-isopropyl-2-morpholine-4-yl-9-hydro-purine-6-yl)-pyrimidine-2-ylamine was purified on a flash column (solvent system: 50% ethyl acetate in hexane) and received 5-(8-bromo-9-isopropyl-2-morpholine-4-yl-9-hydro-purine-6-yl)-pyrimidine-2-ylamine with the release of 52% (660 mg).1H NMR, MeOD: 9,67 (s, 2H), 4,90 (m, 1H), with 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-morpholine-4-yl-9-hydro-purine-6-yl)-pyrimidine-2-ylamine

In a closed vessel to a solution of 5-(8-bromo-9-isopropyl-2-morpholine-4-yl-9-hydro-purine-6-yl)-pyrimidine-2-ylamine (30 mg, 0,072 mmol)and Pd(dppf)Cl2(3 mg, 5% mmol) in 3 ml of anhydrous dioxane was slowly added dimethylzinc (210 μl, 1.0 M in heptane solution). The mixture was heated to 65°C. Drip added methanol, and then the solvent was removed by vacuum. To the residue was added ethyl acetate, the resulting solution was washed 1M hydrochloric acid, water and brine, then dried Na2SO4After removal of the solvent the mixture pass through a flash column on silica gel and identified 5-(9-isopropyl-8-IU the Il-2-morpholine-4-yl-9-hydro-purine-6-yl)-pyrimidine-2-ylamine in the amount of 8 g with a yield of 47%. 1HNMR, MeOD: 9,40 (s, 2H), to 4.81 (m, 1H), with 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 tests

The connection according to the present invention compared with a number of substances obtained and described in PCT/SG 2008/000379 on some biological parameters. The test parameters were:

the activity of mTOR test

activity in PI3K test

- Solubility

stability in human microsomes.

The detailed methodology for each of the tests below.

mTOR test

Truncated mTOR kinase and His-tag4eBP1 were obtained independently. [γ33P]-ATO was purchased from Amersham (GEHealthcare). Other reagents, unless otherwise specified, purchased from Sigma-Aldrich.

Tests phosphorylation were initially carried out in a volume of 20 ál in 384-well polypropylene tablet (Greiner). Compounds are usually tested in the range from 100 μm to 0.006 μm, with eight breeding, in two repetitions. 10 ál/well of 2X solution (1.5 mcg/ml mTOR, 40 μg/ml EUR in 1X sample buffer consisting of 10 ml MN-2-hydroethyl-piperazine-N-2-econsultancy acid with pH 7.5, 50 mm sodium chloride and 10 μm manganese chloride (II) inflicted on the tablet with samples containing 1 μl/per well tested compound in pure DMSO. To start the reaction was added 10 μl of a 20 μm solution of ATP (final concentration 10 μm ATP and 0.4 µci/well [γ33P]-ATP). And the 1 hour incubation at room temperature the reaction was stopped by adding 40 μl/Lungu mm EDTA/1 mm solution of ATP.

50 μl of the resulting reaction mixture was transferred to a 384-well MultiscreenHTP-PH filter tablet (Millipore) pre-filled with 50 µl/well of a 1% solution of phosphoric acid. The tablet was washed 4 times with 120 μl/well of 0.5% solution of phosphoric acid by vacuum filtration. In the end added 10 μl/per well Optiphase™ Supermix liquid scintillation mixture (PerkinElmer). After at least 1 hour incubation assessed on WallacMicrobetaTrilux luminiferous counting device, in view of the amendment calculus. IC50identified as the desired concentration for 50%of goat maximum possible inhibition of kinase activity.

PI3 test

Recombinant PI3 Kp 110α/R received independently. Phosphatidylinositol (Ptdlns), phosphatidylserine (PtdSer), and all other unspecified readentirefile from Sigma-Aldrich. [γ33P]-ATP and Optiphasec scintillator purchased from PerkinElmer.

The tests were performed in a volume of 25 ál for 384-well the tablet Maxisorp. The substances investigated in 8 concentrations in threefold serial dilution, mainly since the concentration of 10 μm. The tablet is coated Maxisorp 20 μl/well of a mixture of Ptdlns and PtdSer in the ratio of 1:1 (0.1 mg/ml each in a mixture of chloroform and ethanol in the ratio 3:7) and left overnight in a fume hood at room temperature for drying.

For reaction with the enzyme was dosed out connection 5 µl/is as well (2.5% DMSO), 10 μl/well enzyme(0.5 μg/ml 110α + 1 mg/ml R) and 10 µl/Lungu μm ATP with µci/ml [γ33P]ATP in a test buffer (final concentration of 0.2 μg/ml p110α, 2 μm ATP, of 0.05 µci/ well [γ33P]ATP in 1X sample buffer: buffer consists of 100 mm Tris-hydrochloric acid to pH 7.0, 200 mm sodium chloride, 8 mm magnesium chloride (II)). The reaction was conducted at room temperature for 1 hour, then was interrupted by adding 30 ál/well of 50 mm EDTA. The tablet then washed twice in TBS, dried, was added 30 μl/well of scintillator before counting on MicrobetaTrilux. IC50identified as the desired concentration for 50%of the maximum inhibition of kinase activity.

Test microsomal stability

First evaluated the stability of the substance in vitro in high-performance format 96-hole tablet (Whatman). The test consisted of incubation with human liver microsomes (MPH). As the reference standard used verapamil purchased at Sigma-Aldrich. MPH acquired in Xenotech (20 mg/ml in 250 mm sucrose). Pre-prepared 100 mm reaction mixture potassium phosphate buffer by mixing 80 ml of 1M K2HPO4and 20 ml of 1M K2HPO4900 water (pH brought to 7.4 with hydrochloric acid), the buffer was stored at room temperature. K2HPO4·3H2O and K2HPO4were the scientists at Sigma Aldrich, NADP·H and restoring system solutions a and B were acquired in Gentest. Pre-prepared solution to stop reaction, consisting of acetonitrile and DMSO (80:20); the solution was stored at 4°C. All used solvents were of class "for HPLC", water to prepare a reaction solution for analysis by LC-MS was maionizirebeli on installing Milli-Q.

a 2.5 μl of 10 mm stock solution of test compound in DMSO was diluted 200 times by mixing with 500 ál of 50 mm potassium phosphate buffer (pH of 7.4, prepared by diluting 100 mm uterine buffer solution with water). 8 μl of a mixture containing compound, was added to 72 μl of pre-cooked incubation mixture (2250 μl water, 2900 μl of 100 mm potassium phosphate buffer, 290 ál of NADP·H healing system solutions a and B and 250 ál MPH). The resulting mixture (concentration 5 μm) were incubated at 37°C in C. incubator Braun Certomat H, after which each well on the tablet containing 100 μl of a solution to stop the reaction, was added to the aliquot volume of 50 µl. After centrifugation at 4°C for 15 minutes at 2000 rpm/min 100 μl of the obtained supernatant was transferred to a tablet for LC-MS for analysis. Each test compound was tested many times, and incubated for several time periods (5, 15, 30,45 and 60 minutes). The residual concentration of the compounds were determined by liquid chromatography and mass spectrometry (ABIQtrap 3200) and compared with a reference solution of known concentration. Stability was expressed in the form of half-life in minutes (t1/2).

Performance analysis of solubility

The solubility of the compounds were determined using high-performance method of kinetic analysis of solubility using a 96-well format. The solubility of the compounds was determined using a microplate spectrophotometer in the UV region (Molecula Devices Spectra Max Plus 384). As reference standards used Vorinostat (SAHA) and Nicardipine (purchased in Sigma).

Compounds in DMSO were dissolved in phosphate buffer (Sigma) to a final concentration of 250 μm (5 ál of 10 mm stock solution in 195 μl of phosphate buffer PH 7.0) and thoroughly mixed. Then the mixture was stirred at 600 rpm for 1.5 hours, then the mixture was left for sedimentation at room temperature for 2 hours. The tablet then centrifuged at 1500 g for 15 minutes. The obtained supernatant (80 μl) was transferred to the tablet for UV analysis and diluted DMSO (20 µl). Quantitative analysis of samples was performed using calibration solutions of the respective compounds prepared in a mixture of phosphate buffer/DMSO (8020).

The tested compounds are shown below:

Table 1 shows the results of biological tests

Table 1
ConnectionConnectionConnectionConnection DConnection EThe connection according to the present invention
mTOR21 nm16 nm27 nm40 nm122 nm36 nm
PI3K43 nm17 nm16 nm15 nm18 nm11 nm
Solubility44 microns18 microns>250 μm>250 μm 35 microns178 microns
The half-life>60 min>60 min36 min26 min>60 min>60 min

As can be seen from the table, even though all connections have some activity, the connection according to the present invention had activity against both interesting enzymes, comparable with all the other compounds, except compound E, which has a significantly lower activity against mTOR kinase. Thus, the compound E is somewhat less suitable for use as a medicine than other compounds used in the above analysis for comparison because it has the lowest activity of mTOR kinase.

Not all investigated compounds showed acceptable solubility characteristics. For example, low scores on the solubility of the compounds And, In E indicate that these compounds may not be good potential drugs. Their low solubility prevents effective formulation in a physiologically acceptable carriers, therefore, decreases the likelihood that they will have the good pharmacokinetic characteristics for oral administration.

As for metabolic stability, the results were significantly different. In these tests, the compounds a, b, E, and the connection according to the present invention have acceptable stability in human liver microsomes. This indicates that in case of their successful introduction of these compounds will be stable enough to achieve the desired physiological effect in the body of the patient. Moreover, among the compounds exhibiting good inhibitory activity against both types of kinases mTOR and PI3K and good solubility, (b, D, and the connection according to the present invention), only the connection according to the present invention has acceptable metabolic stability.

All compounds a-E and the connection according to the present invention exhibit good activity in these tests, however, the connection according to the present invention is one of the most effective thanks to the combination of high inhibitory activity, good solubility in water and good metabolic stability.

In General, the biological results showed that the investigated compounds in the above-described tests showed that, despite the similarity of the compounds with the compound according to the present invention, only the connection according to the present invention has demonstrated a need for the needful combination of activity, good water solubility and metabolic stability, which makes this compound is suitable for use as a drug. These studies demonstrated the superiority of the compounds according to the present invention as a potential drug.

Example 3 Tests on the effectiveness of using biomarkers on cells (pp70-S6KT389, pAktS473)

For demonstrating the activity of the compounds according to the present invention proveli test using biomarkers in the cell. The procedure is described below:

Set Alphascreen® Surefire p-Akt (Ser 473) 384 (TGP, catalog No..: TGRAS500)

Alphascreen® Surefirephospho-p70 S6 kinase (Thr 389) 384 kit (TGR, Cat. No.: TGR70S500) HProxiplate-384 Plus (Perkin Elmer, catalog No.: 6008280) acquired in a Perkin Elmer. Cells (PC-3) prostate cancer person has acquired in ATSS. All reagents, unless otherwise stated, were purchased at Sigma-Aldrich.

Day 1 200 ál of 2 X 105 cells/ml cell solution cells RS were seeded into each well of 96-well plate. Compounds were added after 24 hours after seeding and usually investigated at concentrations of 10 μm to 4.6 nm, with 8 dilutions, three times. The final concentration of DMSO after 4-hour incubation at 37°C was 0.1%. After incubation, the supernatant layer was removed, the cells liofilizirovanny in 1X lyse buffer (set Alpha Screen) and gently stirred for 10 minutes. 4 µl l is the ZAT and 5 µl of the Reaction mixture buffer Trigger buffer containing Alpha Screen Acceptor pellets were added to each of the 384 wells (the ratio of the Reaction buffer: Activating buffer: Acceptor pellets was 40:10:1) and gently stirred for 2 hours at room temperature in the dark. 2 μl of dilution buffer containing Donor granules Alpha Screen® (ratio of dilution buffer: Donor sphere is 20:1) was added to each of the wells of 384-hole tablet was placed on a flatbed shaker for 1-2 minutes, and incubated at room temperature overnight.

To read the results used the device BMG Pherostar with default settings Alpha Screen (mode humidity: Alpha Screen, readout mode: Endpoint, the optical mode: Alpha Screen 680 570; retention time: 0,30, excitation: 0,10, start integration: 0,34 with, the beginning of integration: 0,30 s, gain: 3000).

IC50defined as the molar concentration of a substance that provides 50% of the maximum possible inhibitory activity of the kinase. The measured IC505-(9-isopropyl-8-methyl-2-morpholine-4-yl-9-hydro-purine-6-yl)-pyrimidine-2-ylamine in relation to inhibition of phosphorylation of p70-S6KT389 and pAktS473 was 24 nm and 9 nm, respectively.

The results biomarker analysis demonstrated the activity of the compounds according to the invention in the inhibition of the enzymatic activity of the kinase.

p> 1. The compound of formula (I):

or pharmaceutically acceptable salt of the compounds.

2. Pharmaceutical composition having inhibitory activity against mTOR and P13 kinase, and this composition contains a therapeutically effective amount of a compound according to claim 1 and a pharmaceutically acceptable diluent, excipient or carrier.

3. Method of inhibiting protein kinase selected from mTOR or P13 kinase, including the impact on the indicated protein kinase effective amount of a compound according to claim 1.

4. The method according to claim 3, wherein the kinase is a mTOR.

5. The method according to claim 3, wherein the protein kinase is a kinase P13.

6. A method of treating or preventing a condition in a mammal, in which the inhibition of one or two protein kinases selected from mTOR or P13, inhibits or facilitates the pathology and symptoms of this condition, including the introduction of a therapeutically effective amount of a compound according to claim 1.

7. The method according to claim 6, wherein the kinase is a mTOR.

8. The method according to claim 6, wherein the kinase is a kinase P13.

9. The method according to any of PP-8, characterized in that the specified condition is a cancer.

10. Method for the treatment or prevention of the situation of the tion, associated with the inhibition of mTOR activity and/or P13 kinase in a subject, comprising the introduction of a specified subject a therapeutically effective amount of a compound according to claim 1.

11. The method according to claim 10, in which the specified condition is a cancer.



 

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Compound // 2505532

FIELD: chemistry.

SUBSTANCE: claimed is compound of formula (I) where A is selected from CH2 and C=O; B is selected from CH2, C=O and -S(=O)2-; R1 represents -OSO2NH2; R2 is selected from C1-C6-alkoxygroups; R3 represents where R4, R5, R6, R7 and R8 is independently selected from H, -OH, C1-C6-alkyl, -O-C1-C5-alkyl, -OPh, -O-CF3, acyl, -O-acyl, -NH-acetyl, -OSO2NH2, -NH2, -CN, -NO2 and halogens; where R24 and R25 are independently selected from H and -CH3; and each R27 and R28 represents H, pharmaceutical composition, containing compound of formula (I).

EFFECT: prevention and inhibition of tumour growth.

10 cl, 2 dwg, 1 tbl

FIELD: medicine.

SUBSTANCE: group concerns a composition for the target elimination of mutation escape from anti-cancer therapeutic agent; a kit for eliciting the immune response, wherein the kit comprises the above composition. The compositions containing yeast carriers are applied in a combination with the other anti-cancer therapeutic agents.

EFFECT: group of invention provides the minimisation and complete elimination of the stability to the agent or higher effectiveness of the agent, or the minimisation, reduction or complete elimination of some symptoms of the disease.

20 cl, 2 ex, 5 dwg

Antibodies to her // 2504553

FIELD: biotechnologies.

SUBSTANCE: invention describes versions of bispecific antibodies specifically bound to EGFR and HER3, which contain amino-acid sequences of variable regions of heavy and light chains respectively, SEQ ID NO: 30 and 29; or SEQ ID NO: 28 and 27; or SEQ ID NO: 28 and 29; or contain complementary regions CDR of heavy and light chains of the above sequences of variable regions. The invention describes nucleic acid coding a versions antibody, and a host cell containing the above nucleic acid and used for expression of the anitbody. Immunoconjugate containing antibody versions and cytotoxic agent used for treatment of cancer containing cells that express EGFR and HER3 are presented. A method for obtaining a bispecific antibody, which involves cultivation of a host cell so that an antibody is generated, is described. The invention describes a pharmaceutical composition for treatment of cancer containing EGFR- and HER3-expressing cells, which contains effective amount of bispecific antibody and pharmaceutically acceptable carrier. The invention proposes a treatment method of cancer containing EGFR- and HER3-expressing cells and an inhibition method of biological activity of EGFR and/or HER3 of a specimen, which involve introduction of effective amount of a bispecific antibody. Use of the above antibody in production of a remedy for treatment of cancer, the cells of which express EGFR and HER3, is described.

EFFECT: invention allows obtaining bispecific antibodies binding EGFR and HER3, which are not conjugates of two antibodies.

22 cl, 33 dwg, 4 tbl, 19 ex

FIELD: biotechnologies.

SUBSTANCE: invention proposes an antibody that specifically binds heparin-binding EGF-like growth factor (HB-EGF) and its antigen-binding fragment. Invention describes a nucleic acid molecule, an expressing vector, a host cell and a method for obtaining an antibody or its antigen-binding fragment, as well as use of antibody or its antigen-binding fragment for obtaining pharmaceutical composition for diagnostics, prevention or treatment of hyperproliferation disease, methods and sets for diagnostics and prevention or treatment of the state associated with HB-EGF expression. This invention can be further found in therapy of diseases determined with or related to HB-EGF expression.

EFFECT: improving efficiency of composition and treatment method.

34 cl, 43 dwg, 28 ex, 12 tbl

FIELD: chemistry.

SUBSTANCE: invention relates to a compound of formula (1) or a salt thereof, where D1 is a single bond, -N(R11)- or -O-, where R11 is a hydrogen atom or C1-C3 alkyl; A1 is C2-C4 alkylene, or any of divalent groups selected from the following formulae , and ,

where n1 equals 0 or 1; n2 equals 2 or 3; n3 equals 1 or 2; R12 and R13 are each independently a hydrogen atom or C1 -C3 alkyl; v is a bond with D1; and w is a bond with D2; D2 is a single bond, C1-C3 alkylene, -C(O)-, S(O)2-, -C(O)-N(R15)-, or -E-C(O)-, where E is C1-C3 alkylene, and R15 is a hydrogen atom; R1 is a hydrogen atom, C1-C6 alkyl, a saturated heterocyclic group which can be substituted with C1-C6 alkyl groups, an aromatic hydrocarbon ring which can be substituted with C1-C3 alkyl groups, C1-C4 alkoxy groups, halogen atoms, cyano groups, a monocyclic aromatic heterocyclic ring containing one or two heteroatoms selected from a group consisting of a nitrogen atom, a sulphur atom and an oxygen atom, or the following formula ,

where n1 equals 0, 1 or 2; m2 equals 1 or 2; D12 is a single bond, -C(O)- or -S(O)2-; R18 and R19 denote a hydrogen atom; R17 is a hydrogen atom or C1-C3 alkyl; and x is a bond with D2; under the condition that when R17 denotes a hydrogen atom, D12 denotes a single bond; under the condition that when D1 denotes a single bond, A1 denotes a divalent group of said formula (1a-5) or (1a-6); when D1 denotes -N(R11)-, -O-, or -S(O)2-, A1 denotes a single bond, C2-C4 alkylene, or any of divalent groups selected from formulae (1a-1)-(1a-3), where, when A1 denotes a single bond, D2 denotes -E-C(O)-; and D3 is a single bond, -N(R21)-, -N(R21)-C(O) - or -S-, where R21 is a hydrogen atom; and R2 denotes a group of formula ,

where Q denotes an aromatic hydrocarbon ring, a monocyclic aromatic heterocyclic ring containing one or two heteroatoms selected from a group consisting of a nitrogen atom, a sulphur atom and an oxygen atom, a condensed polycyclic aromatic ring containing one or two heteroatoms selected from a group consisting of a nitrogen atom, a sulphur atom and an oxygen atom, or a partially unsaturated monocyclic or a condensed bicyclic carbon ring and a heterocyclic ring; and y denotes a bond with D3; and R23, R24 and R25 each independently denotes a hydrogen atom, a halogen atom, a cyano group, C1-C3 alkyl, which can be substituted with hydroxyl groups, halogen atoms or cyano groups, C1-C4 alkoxy group, which can be substituted with halogen atoms, alkylamino group, dialkylamino group, acylamino group, or the formula ,

where D21 denotes a single bond or C1-C3 alkylene; D22 denotes a single bond or -C(O)-; R26 and R27 each independently denotes a hydrogen atom or C1-C3 alkyl; and z denotes a bond with Q; under the condition that when D22 denotes a single bond, R27 is a hydrogen atom. The invention also relates to specific compounds, a pharmaceutical composition based on the compound of formula , a IKKβ inhibitor, a method of inhibiting IKKβ, a method of preventing and/or treating an NF-kB-associated or IKKβ-associated disease, and intermediate compounds of formulae and .

EFFECT: obtaining novel isoquinoline derivatives, having useful biological properties.

46 cl, 3 dwg, 38 tbl, 89 ex

FIELD: biotechnologies.

SUBSTANCE: invention refers to cyclohexyl ammonium salt of 2-[3-methyl-7-(1,1-dioxotiethanyl-3)-1-ethyl xanthenyl-8-tio]acetic acid of the following formula: .

EFFECT: obtaining a new compound that shows antithromboembolic action and can be used in medicine.

2 cl, 2 tbl, 3 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to chemical-pharmaceutical industry, namely to using a purine derivatives for preparing drug preparations for treating chronic lymphocytic leukaemia and polycystic kidneys.

EFFECT: invention provides preparing the compounds possessing higher activity on lymphocytic leukaemia and polycystic kidneys.

8 cl, 3 dwg, 1 ex, 3 tbl

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to pharmaceutics and represents a combined analgesic and anti-spasmodic drug containing caffeine and drotaverine hydrochloride, characterised by the fact that as active substances it additionally contains ketorolac or a compound thereof, such as ketorolac tromethamine and diphenylhydramine in the amount of: ketorolac or a compound thereof, such as ketorolac tromethamine 5 - 40 mg, caffeine 50 - 100 mg, diphenylhydramine 20 - 50 mg, drotaverine hydrochloride 40 - 80 mg.

EFFECT: invention provides developing the drug possessing strong analgesic action in moderate or severe pain syndromes, various cramping pains.

2 cl, 3 ex

FIELD: chemistry.

SUBSTANCE: invention relates to anti-tumour purine derivatives of formula (A) and salts thereof, as well as pharmaceutical compositions based thereon, a method for production thereof where W is an alkyl-substituted amine, a pyrrolidine, piperidine, morpholine or piperazine residue, optionally substituted with C1-C6 alkyl or hydroxy; Y is H or a saccharide residue, Z is H; Q is an optionally substituted quinoline residue. The disclosed method involves reacting a corresponding purine protected at the 9th position with corresponding precursors of groups W and Q.

EFFECT: novel compounds with low toxicity, a wide anticancer range, high anticancer activity, high stability, suitable for producing anti-tumour drugs.

12 cl, 3 tbl, 31 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely resuscitation and intensive care, and may be used in treating a craniocerebral injury. That is ensured by administering dexamethasone 0.3 ml and trental 0.4 ml into an inferior external orbital angle between an eyeball and a bone orbit, external and inferior rectus muscle of eye with underlying standard drug-induced therapy. The introduction is once a day daily for 5-7 days.

EFFECT: method provides the highest drug concentrations in the cerebral tissues once administered into the retrobulbar space over a shorter period of time than if administered otherwise that in turn leads to faster cerebral decongestion, peripapillar optic nerve decongestion, and as a consequence ensures faster coming out of coma of the patient with the craniocerebral injury.

3 ex

FIELD: medicine.

SUBSTANCE: dermal flap is simulated in laboratory animals on the second day of experiment. Sildenafil 2.2 mg/kg is introduced intraperitoneally on the first, third and fifth day of the experiment.

EFFECT: dermal flap survival rate in reduced circulation with no side effects.

1 tbl, 1 ex

FIELD: medicine.

SUBSTANCE: calf muscle ischemia is simulated, including in a combination with additional simulation of nitrogen oxide deficiency by intraperitoneal introduction of the nitrogen oxide synthesis blocker N-nitro-L-arginine of methyl ester (L-NAME) 25 mg/kg daily for 7 days. In both cases, the ischemia is corrected by intragastric introduction of sildenafil 2.2 mg/kg on the first, third and fifth days of experiment.

EFFECT: significant improvement of microcirculation in the ischemic calf muscle of rats.

1 tbl, 1 ex

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