CN105037364B - The purine compound that pyrimidine as kinase inhibitor substitutes - Google Patents

The purine compound that pyrimidine as kinase inhibitor substitutes Download PDF

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CN105037364B
CN105037364B CN201510344718.7A CN201510344718A CN105037364B CN 105037364 B CN105037364 B CN 105037364B CN 201510344718 A CN201510344718 A CN 201510344718A CN 105037364 B CN105037364 B CN 105037364B
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disease
compound
syndrome
cancer
fragment
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CN105037364A (en
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陈迪忠
M·威廉姆斯
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SBio Pte Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D473/00—Heterocyclic compounds containing purine ring systems
    • C07D473/26—Heterocyclic compounds containing purine ring systems with an oxygen, sulphur, or nitrogen atom directly attached in position 2 or 6, but not in both
    • C07D473/32—Nitrogen atom
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D473/00—Heterocyclic compounds containing purine ring systems
    • C07D473/40—Heterocyclic compounds containing purine ring systems with halogen atoms or perhalogeno-alkyl radicals directly attached in position 2 or 6
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F5/00—Compounds containing elements of Groups 3 or 13 of the Periodic Table
    • C07F5/02—Boron compounds
    • C07F5/025—Boronic and borinic acid compounds

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

The present invention relates to the purine compound that can be used as kinase inhibitor.The compound has structure：

Description

The purine compound that pyrimidine as kinase inhibitor substitutes
Present patent application is that international application no is PCT/SG2009/000124, and international filing date is April 3 in 2009 Day, into the Application No. 200980159441.3 of National Phase in China, entitled " pyrimidine as kinase inhibitor takes The divisional application of the application for a patent for invention of the purine compound in generation ".
Technical field
The present invention relates to 5- (9- isopropyl -8- methyl -2- morpholine -4- base -9H- purine -6- bases)-pyrimidine -2-base amine, its Preparation method, comprising the pharmaceutical composition of the compound and the compound in some kinase-associated conditions/illnesss are treated Using.
Background technology
It is to develop the useful fruitful field of pharmaceutically active substances that research to kinase inhibitor, which has confirmed,.Kinases Or referred to as phosphotransferase, be can in referred to as Phosphorylation events by phosphate group from high energy donor molecule (such as ATP) It is transferred to the enzyme of certain target molecules (commonly referred to as substrate).Maximum one kind is protein kinase in kinases, they act on specific Protein simultaneously adjusts its activity.
Since kinase inhibitor can act as pharmaceutical active compounds, numerous studies are carried out and have had to develop to these target spots There is the compound of suitable activity.In cancer field, what is aroused attention can serve as two kinds of kinases of the potential target spot of therapeutic compounds Including mTOR and PI3.A research example in the field is shown in PCT/SG2008/000379, the application disclose many to mTOR All there is the compound of kinase activity with PI3.
It is expected that potent antiproliferative, anti-angiogenesis and antitumor work can be provided by suppressing the compound of mTOR and PI3K at the same time Property, because these compounds can act on multiple points in PI3K/Akt/mTOR paths.At present, many such inhibitor are being just Carry out clinical research first (e.g., BEZ235, XL765, GDC0941, PX866, SF1126).
During suitable drug candidate is found, finally determine whether a kind of compound is that suitable drug candidate needs Consider many factors.Therefore, during evaluating and being used for the potential compound further developed, except compound in itself Outside main inhibitory activity, it is also necessary to consider many other factors.In evaluation procedure, those skilled in the art are conceived to molecule " drug-like properties ", including evaluation such as its activity to target spot interested, the solubility of compound of interest (if they Be it is insoluble, they usually it is unsuitable as Subsequent pharmacological), compound in vivo with external metabolic stability, chemical combination Thing is to factors such as the issuable side effects of human body.The application identifies other compound phase ratios with the field, medicine sample The compound that matter significantly improves.
Summary of the invention
The present invention provides the compound of formula (I)：
Or its pharmaceutically acceptable salt.
Except the compound of Formulas I, revealed embodiment further relates to the compound pharmaceutically acceptable salt, pharmaceutically Acceptable N- oxides, pharmaceutically acceptable prodrug and drug active metabolite, and the metabolin can pharmaceutically connect The salt received.
The invention further relates to pharmaceutical composition, includes the compounds of this invention and pharmaceutically acceptable carrier, diluent Or excipient.
Another aspect of the present invention provides a kind of method for suppressing protein kinase, and the protein kinase is selected from the group：Silk Propylhomoserin/Serineprotein kinase either its fragment or compound or its functional equivalent and PI3 kinases or its fragment or Either its functional equivalent this method includes making protein kinase or its fragment or compound or its functional equivalent compound And/or its co-factor is contacted with a effective amount of the compounds of this invention.
The compound disclosed herein directly and can act solely on kinase molecule or its compound or fragment, to suppress biology Activity.However, it is understood that the compound can also at least partly act on the co-factor participated in Phosphorylation events. The kinases co-factor known includes ionic species (such as zinc and calcium), lipid (such as phosphatidy serine) and diacylglycerol.
In some embodiments, protein kinase be serine/threonine protein kitase or its fragment or compound or Its functional equivalent of person.In some embodiments, the serine/threonine protein kitase or its fragment or compound are MTOR protein kinases or its fragment or its compound or its functional equivalent.In some embodiments, the serine/ Serineprotein kinase is mTORC1 either its fragment or compound or its functional equivalent.In some embodiments, institute It is mTORC2 either its fragment or compound or its functional equivalent to state serine/threonine protein kitase.
In some embodiments, protein kinase is PI3 kinases either its fragment or its compound or its function equivalent Thing.In some embodiments, either its fragment or its compound or its functional equivalent are I classes PI3K to the PI3 kinases Either its fragment or its compound or its functional equivalent.
In a kind of embodiment for making method that one or more protein kinases contact with the compound, including by institute State compound and give the mammal containing one or more protein kinases.
In another aspect of the present invention, there is provided the compounds of this invention suppresses the application of one or more protein kinases, institute Protein kinase is stated to be selected from the group：Serine/threonine protein kitase either its fragment or compound or its functional equivalent, with And PI3 kinases either its fragment or compound or its functional equivalent.
In some embodiments, protein kinase be serine/threonine protein kitase or its fragment or compound or Its functional equivalent of person.In some embodiments, the serine/threonine protein kitase or its fragment or compound are MTOR protein kinases or its fragment or its compound or its functional equivalent.In some embodiments, the serine/ Serineprotein kinase is mTORC1 either its fragment or compound or its functional equivalent.In some embodiments, institute It is mTORC2 either its fragment or compound or its functional equivalent to state serine/threonine protein kitase.
In some embodiments, protein kinase is PI3 kinases either its fragment or its compound or its function equivalent Thing.In some embodiments, either its fragment or its compound or its functional equivalent are I classes PI3K to the PI3 kinases Either its fragment or its compound or its functional equivalent.
In another aspect of the present invention, there is provided a kind of method for treating or preventing mammalian diseases, wherein, suppress one Kind or a variety of either its fragment or compound or its functional equivalent and PI3 swash selected from serine/threonine protein kitase Either its fragment or compound or the protein kinase of its functional equivalent can prevent, suppress or improve the symptom of the disease to enzyme Or symptom, the described method includes the compound of the invention for giving therapeutically effective amount.
In some embodiments, protein kinase be serine/threonine protein kitase or its fragment or compound or Its functional equivalent of person.In some embodiments, the serine/threonine protein kitase or its fragment or compound are MTOR protein kinases or its fragment or its compound or its functional equivalent.In some embodiments, the serine/ Serineprotein kinase is mTORC1 either its fragment or compound or its functional equivalent.In some embodiments, institute It is mTORC2 either its fragment or compound or its functional equivalent to state serine/threonine protein kitase.
In some embodiments, protein kinase is PI3 kinases either its fragment or its compound or its function equivalent Thing.In some embodiments, either its fragment or its compound or its functional equivalent are I classes PI3K to the PI3 kinases Either its fragment or its compound or its functional equivalent.
In some embodiments, the illness is cancer.In some embodiments, cancer is selected from the group：Hematology Cancer, such as myeloproliferative disease (idiopathic myelofibrosis, polycythemia vera, essential thrombocythemia, chronic marrow Cell sample leukaemia), myeloid metaplasia, chronic myelomonocytic leukemia, acute lymphatic leukemia, acute Cheng Hong Cell leukemia, Huo Qijin and non-Hodgkin lymphoma, B cell lymphoma, acute T-cell leukemia, myeloproliferative disorder are comprehensive Simulator sickness, thick liquid cell obstacle, hairy cell, Kaposi sarcoma, lymthoma and hyperproliferation illness such as psoriasis and narrow again It is narrow；Gynecologic cancer, such as breast cancer, oophoroma, cervix cancer, vagina and vulva cancer, endometrial hyperplasia；Gastrointestinal cancer, such as colon The carcinoma of the rectum, polyp, liver cancer, stomach cancer, cancer of pancreas, gallbladder cancer；The urinary tract cancer, such as prostate cancer, kidney and kidney portion cancer；Carcinoma of urinary bladder, Carcinoma of urethra, carcinoma of penis；Cutaneum carcinoma, such as melanoma；Brain tumor, such as glioblastoma, neuroblastoma, astrocytoma, room Ependymal Cell knurl (ependynoma), brain stem glioma, medulloblastoma, meningioma (menigiomas), star Cytoma, oligodendroglioma；Incidence cancer, such as nasopharyngeal carcinoma, laryngocarcinoma；Respiratory cancer, such as lung cancer (NSCLC and SCLC), celiothelioma； Illness in eye, such as retinoblastoma；Musculoskeletal disease, such as osteosarcoma, flesh and bone tumor；Dermoid cancer and fibre Tie up knurl.In other embodiments, compound of the invention can be used for treatment precancerosis disease (pre-cancer condition) Or hyperplasia, including：Familial adenomatous polyposis, adenomatous polyposis coli, myelodysplasia, Endometrium development are different Often, endometrial hyperplasia with atypia, cervical atypical hyperplasia, knurl formation, benign prostatic hyperplasis, larynx fine hair in vagina epithelium Shape knurl, photochemical and solar keratosis, seborrheic keratosis and keratoacanthoma.
In some embodiments, the illness is autoimmunity or inflammatory disease or the disease as caused by excessive neovascularization Disease.The autoimmunity cause of disease is attributed to a certain extent or is related to the disease of pathologic inflammatory and new vessels formation reaction Including following disease：Acute diseminated encephalomyelitis, Addison's disease, without gamma-globulin mass formed by blood stasis (agammaglobulinermia), agranulocytosis, allergic asthma, allergic encephalitis, allergic rhinitis, alopecia areata, Alopecia senilis, red blood cell occur cannot, ankylosing spondylitis, antiphospholipid antibody syndrome, aortitis syndrome, regeneration barrier Impenetrability anaemia, atopic dermatitis, autoimmune hemolytic anemia, oneself immunity hepatitis, autoimmunity oaritis, Ba Luo Disease, Basedow disease (Basedow's disease), behcet disease/syndrome, bronchial asthma, Ka Siman synthesis Sign (Castleman ' s syndrome), chylous diarrhea, chagas disease, chronic inflammatory demyelinating polyneuropathy, Qiu-apply Er Shi are comprehensive Simulator sickness (Chrug-Strauss syndrome), Ke's radicular syndrome (Cogans syndrome), keratoconus (cornical Cornea), keratoconus hickie (cornical leukoma), coxsackie myocarditis, CREST diseases, Crohn disease, Epidermis eosinophilia, cutaneous T-cell lymphoma, diversity dermatitis erythremia, dermatomyositis, glycosuria It is characteristic of disease retinopathy, postmyocardial infarction syndrome, dystrophia epithelialis corneae, eczematous dermatitis, eosinophilic fasciitis, thermophilic Sour cellularity gastroenteritis, epidermolysis bullosa, Evans syndrome (Evans syndrome), fibrosing alveolitis, gestation Pemphigoid, glomerulonephritis, Goodpasture's syndrome, graft versus host disease(GVH disease), Graves disease, Ge-bar Er Shi synthesis Levy (Guillain-Barre syndrome), chronic lymphocytic thyroiditis, hemolytic uremic syndrome, herpetic keratitis, ichthyosis vulgaris, spy Hair property interstitial pneumonia, Idiopathic Thrombocytopenic Purpura, inflammatory bowel disease, kawasaki disease, keratitis, keratoconjunctivitis sicca, Lambert- Eton syndrome, common Leucoplakia, lichen planus, lichen sclerosus, Lyme disease, linear IgA bullous disease (linear IgA Disease), macular degeneration, megaloblastic anemia, Meniere disease (Meniere ' sdisease), Mooren's ulcer (Mooren ' s ulcer), mucha-Habermann disease, polymyositis, multiple sclerosis, myasthenia gravis, gangrenosum acne Enterocolitis, neuromyelitis optica, ocular pemphigus, ocular myoclonus syndrome, Ao Deshi thyroiditis (Ord ' s Thyroiditis), paroxysmal nocturnal hemoglobinuria, two Cotard (Parsonnage-Turner of Ba-spy Syndrome), pemphigus, periodontitis, pernicious anaemia, pollen allergy, polyadenous autoimmune syndromes, posterior uveitis, Monolobular cirrhosis, rectitis, pseudomembranous colitis, psoriasis, pulmonary emphysema, pyoderma, Reiter syndrome, reversible obstructive Airway disorders, rheumatic fever, rheumatoid arthritis, sarcoidosis, sclerotitis, Sezary syndrome, Sjogren syndrome, Asia are anxious Property bacterium endocarditis, systemic loupus erythematosus, aorto-arteritis, temporal arteritis, painful ophthalmoplegia, type i diabetes, burst Ulcer colitis, nettle rash, spring conjunctivitis, Leucoplakia, Wo Ji-Vogt-Koyanagi-Harada syndrome (Vogy-Koyanagi-Harada ) and Wegener granulomatosis syndrome.In some embodiments, the illness is mullerianosis.
In another aspect of the present invention, there is provided the compounds of this invention is preparing answering for the medicine for treating Animal diseases With, wherein, suppress one or more and be selected from serine/threonine protein kitase either its fragment or compound or its function etc. Either its fragment or compound or the protein kinase of its functional equivalent can be prevented, suppress or improved for effect thing and PI3 kinases The symptom or symptom of the disease.
In another aspect of the present invention, there is provided the compounds of this invention or its pharmaceutically acceptable salt, N- oxides or Application of its prodrug in disease is treated, wherein, suppress one or more and be selected from serine/threonine protein kitase or its piece Section or compound either its functional equivalent and PI3 kinases or its fragment or compound or the albumen of its functional equivalent Kinases can prevent, suppress or improve the symptom or symptom of the disease.
In some embodiments, protein kinase be serine/threonine protein kitase or its fragment or compound or Its functional equivalent of person.In some embodiments, the serine/threonine protein kitase or its fragment or compound are MTOR protein kinases or its fragment or its compound or its functional equivalent.In some embodiments, the serine/ Serineprotein kinase is mTORC1 either its fragment or compound or its functional equivalent.In some embodiments, institute It is mTORC2 either its fragment or compound or its functional equivalent to state serine/threonine protein kitase.
In some embodiments, protein kinase is PI3 kinases either its fragment or its compound or its function equivalent Thing.In some embodiments, either its fragment or its compound or its functional equivalent are I classes PI3K to the PI3 kinases Either its fragment or its compound or its functional equivalent.
In another aspect of the present invention, there is provided a kind of method of the proliferative diseases of prevention or treatment target, this method Compound of the invention including giving therapeutically effective amount.
In another aspect of the present invention, there is provided the compounds of this invention is preparing the medicine for treatment target proliferative diseases The application of thing.
In some embodiments, the illness is cancer.In some embodiments, cancer is selected from the group：Hematology Cancer, such as myeloproliferative disease (idiopathic myelofibrosis, polycythemia vera, essential thrombocythemia, chronic marrow Cell sample leukaemia), myeloid metaplasia, chronic myelomonocytic leukemia, acute lymphatic leukemia, acute Cheng Hong Cell leukemia, Huo Qijin and non-Hodgkin lymphoma, B cell lymphoma, acute T-cell leukemia, myeloproliferative disorder are comprehensive Simulator sickness, thick liquid cell obstacle, hairy cell, Kaposi sarcoma, lymthoma；Gynecologic cancer, such as breast cancer, oophoroma, uterus Neck cancer, vagina and vulva cancer, endometrial hyperplasia；Gastrointestinal cancer, such as colorectal cancer, polyp, liver cancer, stomach cancer, cancer of pancreas, courage Capsule cancer；The urinary tract cancer, such as prostate cancer, kidney and kidney portion cancer；Carcinoma of urinary bladder, carcinoma of urethra, carcinoma of penis；Cutaneum carcinoma, such as melanoma；Brain Tumour, as glioblastoma, neuroblastoma, astrocytoma, ependymocytoma, brain stem glioma, into nerve Solencyte knurl, meningioma, astrocytoma, oligodendroglioma；Incidence cancer, such as nasopharyngeal carcinoma, laryngocarcinoma；Respiratory cancer, such as lung cancer (NSCLC and SCLC), celiothelioma；Illness in eye, such as retinoblastoma；Musculoskeletal disease, such as osteosarcoma, flesh and bone tumor； Dermoid cancer and fibroma.In some embodiments, the illness is mullerianosis.
In addition, if if being applicable in, formula (I) should cover the solvated and unsolvated form of compound.Therefore, various bag Include the compound with specified structure, including hydration and nonhydrated form.
These features of the invention and other features are as follows.
Detailed description of the invention
Term known to multiple technical staff is used in this description.But multiple terms will be defined for clarity.
Term " pharmaceutically acceptable salt " refers to the salt for retaining the required bioactivity of the compound of identification above, including medicine Acceptable acid-addition salts and base addition salts on.The suitable pharmaceutically acceptable acid-addition salts of compound shown in formula (I) can Prepared from inorganic acid or organic acid.The example of these inorganic acids includes hydrochloric acid, sulfuric acid and phosphoric acid.Appropriate organic acid may be selected from organic Acid aliphatic series, cyclic aliphatic, aromatics, heterocyclic carboxylic acid and sulphonic acids, the example are formic acid, acetic acid, propionic acid, butanedioic acid, glycolic, Portugal Saccharic acid, lactic acid, malic acid, tartaric acid, citric acid, fumaric acid, maleic acid, alkyl sulfonic acid, aryl sulfonic acid.On medicine The other information of acceptable salt is shown on《Remington pharmaceutical science》(Remington's Pharmaceutical Sciences), the 19th edition, Mack Publishing Company (Mack Publishing Co.), Easton, Pennsylvania, 1995. In the case of medicine is solid, technical staff should be recognized that the compounds of this invention, medicament and salt may have the not syncrystallization or more Crystalline forms, all these materials should all belong in the range of of the invention and shown structural formula.
Term " therapeutically effective amount " or " effective dose " are the dosage for being enough to realize beneficial or required clinical effectiveness.Effective dose can Once or several times to give.Effective dose is typically enough to make the process of morbid state to mitigate, improve, stablize, reverse, slow down or prolong Late.
Term " function equivalent " should include the variation of specific protein kinase species described herein.It should be recognized that kinases can have Various isotypes so that although level-one, two level, the three or four structure of given kinase isoform are different from prototype kinases, The molecule remains the bioactivity of protein kinase.Isotype can produce in normal allele mutation out of crowd, including such as The mutation such as 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor, deletion, insertion, truncation or repetition.Term " function equivalent " further includes the change of transcriptional level generation Body.Other function equivalents include the kinases that posttranslational modification (such as glycosylation) is changed.
The compound phase ratio similar with ability domain structure, the compounds of this invention have excellent drug-like properties, below will It is more fully described.These excellent property promptings, the compounds of this invention may be suitable as the candidate of this area drug development Thing.Observed as first time, the compounds of this invention is shown quite (i.e. in terms of two kinds of kinases, that is, mTOR and PI3 interested are suppressed Make not to be more excellent) activity.The activity of the compounds of this invention is more stronger than all comparative compounds of test, it resists the work of mTOR Property is suitable with comparative compound and in the acceptable level for the treatment of use.
Although enzymatic activity test shows that almost all of comparative compound has acceptable activity level, compound The further many compounds of test prompts cannot act as drug development based on other reasons.Closed for example, the compounds of this invention has Suitable water solubility is horizontal (178 μM), it is thus possible to the pharmaceutical preparation of oral absorption is configured to, and many comparative compounds do not have Acceptable solubility.Therefore, the compounds of this invention has excellent active and acceptable solubility characteristics at the same time.
In the active at the same time and compound of solubility, the metabolic stability of the compounds of this invention is more excellent.This hair Bright compound has excellent stability in the research of human liver's microsome, shows that it is sane and opposite in physiological environment Tolerance degraded.On the contrary, active and solubility other compounds are at all unstable in these researchs.Therefore, it is of the invention Compound is active, the unique combination of solubility and stability, make it compared to the related compound of this area be excellent Drug candidate, although some in these compounds have obviously structural similarity.
The compound of the present invention can suppress the activity of some protein kinases.The ability for suppressing kinase activity is of the present inventionization Compound directly acts solely on kinase molecule to suppress the result of bioactivity.However, it is understood that the compound can also be extremely Small part acts on the co-factor for participating in the kinases in Phosphorylation events.Compound is to PI3 protein kinases or its fragment Or compound or its functional equivalent it is active.Compound can to some serine/threonine kinases such as mTOR or Its fragment or compound or its functional equivalent are active.
Can by it is known in the art it is many in a manner of carry out protein kinase suppression.For example, suppress in vitro if necessary to protein kinase, The compounds of this invention of appropriate amount can be added in the solution of the kinases containing purifying.The kinase activity in suppression mammal is required In situation, suppression kinases, which is usually directed to, gives the compound to the mammal containing the kinases.
Therefore, the compounds of this invention can have various applications, suppress the ability of the above-mentioned type protein kinase using it.Example Such as, the compound can be used for suppressing serine/threonine protein kitase.It can be additionally used in the disease for treating or preventing mammal Disease, wherein the symptom or symptom of the disease can be prevented, suppresses or improve by suppressing its protein kinase and/or co-factor.
The compound of announcement has the ability that can be used in treating proliferative diseases.The example of the disease is cancer.In advance Compound described in phase can treat solid tumor and liquid tumors.In some embodiments, can be treated by the compounds of this invention The example of cancer includes solid tumor and blood cancer.
As used herein, term " cancer " is intended to include many diseases characterized by uncontrolled abnormal growth of cells The generic term of disease.It is expected that the compound of the present invention can be used to treat various cancers, include but not limited to：Osteocarcinoma, brain and CNS swell Knurl, breast cancer, colorectal cancer, endocrine gland cancer include adrenocortical carcinoma, cancer of pancreas, pituitary gland cancer, thyroid cancer, pair Thyroid cancer, thymic carcinoma, human primary gastrointestinal cancers, liver cancer, hepatic duct outer cancer (extra hepatic bile duct cancer), stomach and intestine Carcinoid tumor, carcinoma of urinary bladder, apparatus urogenitalis cancer, gynecologic cancer, incidence cancer, leukaemia, myeloma, blood disorder, lung cancer, leaching Bar knurl, cancer eye, cutaneum carcinoma, soft tissue sarcoma, adult soft tissue sarcoma, Kaposi sarcoma, urinary system cancer.
The example for the cancer that can be treated by the compounds of this invention includes：Hematologic cancer, such as myeloproliferative disease (idiopathic Myelofibrosis, polycythemia vera, essential thrombocythemia, chronic myeloid leukemia), myeloid metaplasia, Chronic myelomonocytic leukemia, acute lymphatic leukemia, acute erythroblastic leukemia, Huo Qijin and non- Hodgkin's disease, B cell lymphoma, acute T-cell leukemia, myelodysplastic syndrome, thick liquid cell obstacle, hairy cell Leukaemia, Kaposi sarcoma, lymthoma and hyperproliferation illness, such as psoriasis and restenosis；Gynecologic cancer, such as breast cancer, ovary Cancer, cervix cancer, vagina and vulva cancer, endometrial hyperplasia；Gastrointestinal cancer, such as colorectal cancer, polyp, liver cancer, stomach cancer, pancreas Gland cancer, gallbladder cancer；The urinary tract cancer, such as prostate cancer, kidney and kidney portion cancer；Carcinoma of urinary bladder, carcinoma of urethra, carcinoma of penis；Cutaneum carcinoma is for example black Plain knurl；Brain tumor, as glioblastoma, neuroblastoma, astrocytoma, ependymocytoma, brain stem glioma, Medulloblastoma, meningioma, astrocytoma, oligodendroglioma；Incidence cancer, such as nasopharyngeal carcinoma, laryngocarcinoma；Respiratory cancer, Such as lung cancer (NSCLC and SCLC), celiothelioma；Illness in eye, such as retinoblastoma；Musculoskeletal disease, such as osteosarcoma, flesh and bone Bone tumour；Dermoid cancer and fibroma.The compound of the present invention is additionally operable to treatment precancerosis disease or hyperplasia, including family Adenomatous Polyposis, adenomatous polyposis coli, myelodysplasia, Endometrium development are abnormal, endometrium atypia It is hyperplasia, cervical atypical hyperplasia, knurl formation in vagina epithelium, benign prostatic hyperplasis, larynx papilloma, photochemical and solar lentigines Keratosis, seborrheic keratosis and keratoacanthoma.
It is also contemplated that the compound of the present invention can be used for treatment autoimmunity or inflammatory disease or be caused by excessive neovascularization Disease.The disease of the autoimmunity cause of disease is attributed to a certain extent, or participates in Inflammation and neovascularization reaction Disease include but not limited to：Acute diseminated encephalomyelitis, Addison's disease, without gamma-globulin mass formed by blood stasis, agranulocytosis, Allergic asthma, allergic encephalitis, allergic rhinitis, alopecia areata, alopecia senilis, red blood cell occur cannot, tatanic ridge Column inflammation, antiphospholipid antibody syndrome, aortitis syndrome, alpastic anemia, atopic dermatitis, autoimmune haemolytic Anemia, autoimmune hepatitis, autoimmunity oaritis, Balo disease, Basedow disease, behcet disease/syndrome, Bronchial asthma, to block this Mann syndrome, chylous diarrhea, chagas disease, chronic inflammatory demyelinating polyneuropathy, Qiu-apply Er Shi comprehensive Simulator sickness, Ke's radicular syndrome, keratoconus, keratoconus hickie, coxsackie myocarditis, CREST diseases, Crow grace Disease, epidermis eosinophilia, cutaneous T-cell lymphoma, diversity dermatitis erythremia, dermatomyositis, Diabetic retinopathy, postmyocardial infarction syndrome, dystrophia epithelialis corneae, eczematous dermatitis, eosinophil driven's fascia Inflammation, eosinophilic gastroenteritis, epidermolysis bullosa, Evans syndrome, fibrosing alveolitis, pregnant pemphigoid, kidney It is bead ephritis, Goodpasture's syndrome, graft versus host disease(GVH disease), Graves disease, guillain-Barre syndrome, chronic lymphocytic thyroiditis, molten Courageous and upright uremic syndrome, herpetic keratitis, ichthyosis vulgaris, idiopathic interstitial pneumonia, Idiopathic Thrombocytopenic Purpura, Inflammatory bowel disease, kawasaki disease, keratitis, keratoconjunctivitis sicca, Lambert-Eaton syndrome, common Leucoplakia, lichen planus, hardening Property lichen, Lyme disease, linear IgA bullous disease, macular degeneration, megaloblastic anemia, Meniere disease, Mooren's ulcer, Mucha-Habermann disease, polymyositis, multiple sclerosis, myasthenia gravis, necrotizing enterocolitis, eye brain ridge Marrow disease, ocular pemphigus, ocular myoclonus syndrome, Order thyroiditis, paroxysmal nocturnal hemoglobinuria, Ba-spy Er Shi synthesis Sign, pemphigus, periodontitis, pernicious anaemia, pollen allergy, polyadenous autoimmune syndromes, posterior uveitis, list are lobular Cirrhosis, rectitis, pseudomembranous colitis, psoriasis, pulmonary emphysema, pyoderma, Reiter syndrome, reversible obstructive airway disease Disease, rheumatic fever, rheumatoid arthritis, sarcoidosis, sclerotitis, Sezary syndrome, Sjogren syndrome, subacute bacterium Endocarditis, systemic loupus erythematosus, aorto-arteritis, temporal arteritis, painful ophthalmoplegia, type i diabetes, exedens knot Enteritis, nettle rash, spring conjunctivitis, Leucoplakia, Wo Ji-Vogt-Koyanagi-Harada syndrome and Wegener granulomatosis.In some embodiment party In formula, the illness is mullerianosis.
The compound of the present invention can be additionally used in the medicine for preparing treatment animal disorders, wherein, suppressing protein kinase can be to prevent Only, suppress or improve the symptom or symptom of the disease.The compound of the present invention, which can also be used to prepare, treats or prevents kinases phase The medicine of related disorders.
Give the mankind belong to compound shown in formula (I) can by for through enteral administration any accepted pattern (such as Oral or per rectum), or by parenteral, such as subcutaneously, intramuscular, intravenous and intradermal route.Injection can be to push away Note or via lasting or intermittent infusion.The reactive compound is generally contained within pharmaceutically acceptable carrier or diluent, its Dosage, which is enough to deliver to patient, treats effective dosage.In various embodiments, compared with normal cell, the inhibitor chemical combination Thing can have selection toxicity or more toxic to the fast proliferating cells such as tumor.
The compounds of this invention when in use, can use any bioavailable form of the compound or pattern to give.System The specific natures of the visual selected compounds of technical staff of standby formulation art, illness to be treated, illness to be treated stage and Other conditions associated appropriate formats and pattern for being readily selected administration.We prefer that reader's reference《Remington pharmaceutical science》, 19th edition, Mack Publishing Company (1995) is to obtain other information.
The compounds of this invention can individually be given or with pharmaceutically acceptable carrier, diluent or excipient composition into medicine The form of composition is given.Although the compounds of this invention is effective in itself, pharmaceutically acceptable salt form is typically formulated as simultaneously Give, because these forms are usually more stable, are easier to crystallize and have the solubility of higher.
However, these compounds are usually used with the pharmaceutical compositions prepared depending on required mode of administration.Therefore, exist In another embodiment, the present invention provides and includes compound and pharmaceutically acceptable carrier, diluent or figuration shown in formula (I) The pharmaceutical composition of agent.These compositions can be prepared using technology well known in the art.
In other embodiments, the present invention provides a kind of medicine bag or kit, it is equipped with one or more filled with this The container of one or more compositions of invention pharmaceutical composition.It can be equipped with this medicine bag or kit containing unit dose The container of medicine.These kits can be equipped with the composition containing active drug, and the composition can be that concentrate is (including lyophilized Composition), it can first make the composition that further dilution reused or can provide concentration, and wherein these bottles can be equipped with one Part or more doses.For conventionally, single dose can be provided with sterile vials in kit, so that doctor can be directly sharp With these bottles, wherein medicine of these bottles equipped with institute's expense and concentration.These containers can also post various written materials, Such as the explanation of government organs' prescribed form of production, use or the sale of operation instructions or management medicine or biological product, The explanation reflects production, use or sale of the mechanism approval for human administration.
The compounds of this invention can be combined or be administered with one or more other drugs, to treat the condition/disease.It can use Same preparation gives all components with different preparations.If with different preparations be administered, the compounds of this invention can with other medicines according to It is secondary or give at the same time.
In addition to it can be given in combination with one or more other medicines, the compounds of this invention can be used in combination treatment.At this time, Usually it is combined with each other and gives these compounds.Therefore one or more present invention can be given (as combination preparation) or successively at the same time Compound is to realize required effect.If the treatment characteristic of each compound is different, it is especially desirable to so so that two kinds of medicines Combined effect can improve treatment results.
Pharmaceutical composition of the present invention for parental injection includes pharmaceutically acceptable sterile aqueous or non-aqueous molten Liquid, dispersion liquid, supensoid agent and aseptic powdery, reuse so as to first be reconstructed into sterile injectable solution or dispersion liquid.Appropriate It is (such as glycerine, propane diols, poly- that water-based and non-aqueous carrier, diluent, the example of solvent or mediator include water, ethanol, polyalcohol Ethylene glycol etc.) and its injectable organic ester such as appropriate mixture, vegetable oil (such as olive oil) and ethyl oleate.It can lead to Cross using coating material such as lecithin, if by granularity needed for holding and using surfactant if dispersion, to tie up Hold suitable mobility.
These compositions can also include auxiliary material, such as preservative, wetting agent, emulsifying agent and dispersant.Include various antibacteriums Agent and antifungal agent, such as p-hydroxybenzoate, methaform, phenol, sorbic acid etc. ensure to prevent microbial action.Also can need To include isotonic agent, such as sugar, sodium chloride etc..Can be real by the material absorbed comprising the delay such as aluminum monostearate and gelatin The extension of existing injectable drug form absorbs.
If necessary to more effectively distribution, can by the compound mix sustained release or targeted delivery systems, as polymer substrate, In liposome and microsphere.
It can make by, for example, with bacteria retaining filter filtering or the bactericidal agent by mixing aseptic solid composite form Injectable formulation is sterile, the composition can be dissolved or dispersed in sterile water or other sterile injectables before by use In medium.
Solid dosage forms for oral administration includes capsule, tablet, pill, powders and granules.In these solid dosage forms In, reactive compound and at least one inertia such as sodium citrate or dicalcium phosphate, pharmaceutically acceptable excipient or Carrier and/or following material mixing：A) filler or enriching substance, such as starch, lactose, sucrose, glucose, mannitol and silicon Acid；B) adhesive, such as carboxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and Arabic gum；C) protect Humectant, such as glycerine；D) disintegrant, such as agar, calcium carbonate, potato or tapioca, alginic acid, some silicates and carbon Sour sodium；E) solution retarding agents, such as paraffin；F) sorbefacient, such as quaternary ammonium compound；G) wetting agent, for example, cetanol and Glycerin monostearate；H) adsorbent, such as kaolin and bentonite；And i) lubricant, such as it is talcum powder, calcium stearate, hard Fatty acid magnesium, solid polyethylene glycol, NaLS and their mixture.In the case of capsule, tablet and pill, formulation In can also contain buffer.
Also the polymer such as lactose and high molecular weight polyethylene glycol can be used as excipient, by the similar solid of type Composition is filled into soft hard-filled gelatin capsule.
Coating and capsid, such as casing known to pharmaceutical-formulating art or other coatings can be used to prepare tablet, lozenge, glue The solid dosage forms such as wafer, pill and granule.They can optionally contain opacifier, can also be only or preferentially enteron aisle certain Partly (optional) is with the composition of delayed mode release active ingredient.The example of workable embedding composition include polymer and Wax.
If being applicable in, these reactive compounds can also use micro-encapsulated form with one or more above-mentioned excipient.
The liquid dosage form of oral administration includes pharmaceutically acceptable emulsion, solution, supensoid agent, syrup and elixir.Remove Active ingredient beyond the region of objective existence, liquid dosage form contain inert diluent commonly used in the art, such as water or other solvents, solubilizer and breast Agent, such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzylalcohol, Ergol, propane diols, 1,3-BDO, diformazan Base formamide, oils (especially cottonseed oil, peanut oil, corn oil, embryo oil, olive oil, castor oil and sesame oil), glycerine, four Hydrogen furan alcohol, polyethylene glycol and sorbitan fatty ester and their mixture.
Besides inert diluents, these Orally administered compositions also can include such as wetting agent, emulsifying and suspending agent, sweetener, The auxiliary reagent such as flavor enhancement and aromatic.
In addition to the active compound, supensoid agent can contain such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitan and Sorbitan esters, microcrystalline cellulose, inclined aluminium hydroxide, bentonite, agar and bassora gum and their mixture etc. suspend Agent.
The preferred suppository of compositions for rectal or vaginal administration, it can be by mixing the compounds of this invention and such as cocoa (they are solid-state in room temperature but are liquid under body temperature, therefore melt in rectum or vaginal canal for fat, polyethylene glycol or suppository wax And release of active compounds) etc. appropriate non-stimulated excipient or carrier and prepare.
Formulation for the compounds of this invention local administration includes powder agent, patch, spray, ointment and inhalant. Reactive compound is aseptically with that pharmaceutically acceptable carrier and any desired preservative, buffer or may need Propellants.
The administered dose of compound preferably can be treated and reduced or slow down the illness.Attending doctor is using routine techniques and passes through The result obtained under similar state is observed to be not difficult to determine therapeutically effective amount.Determine therapeutically effective amount when, be considered as it is several because Element, includes but not limited to：Animal species, volume, age and general health, involved specific illness, disease serious degree, The reaction of patient for treatment, the particular compound given, mode of administration, the biological availability for giving preparation, selected dosage Therapy, the use of other medicaments and other conditions associated.
Preferable dosage is daily per kilogram of body weight about 0.01 to 300mg scopes.Preferred dosage is daily per kilogram Weight 0.1 is to 100mg scopes, more preferably daily per kilogram of body weight 0.2 to 80mg, more preferably daily per kilogram of body weight 0.2 to 50mg.Daily multiple sub-doses can be divided to give suitable dosage.
The synthesis of the compounds of this invention
Using 5 step processes shown in scheme 1, by dichloropurine prepare 5- (9- isopropyl -8- methyl -2- morpholine -4- bases - 9H- purine -6- bases)-pyrimidine -2-base amine.
Scheme 1
Embodiment
In the embodiment described below, unless otherwise noted, in being described below all temperature by degree Celsius, all parts and Percentage is parts by weight and percetage by weight.
Various starting materials and other reagents are from such as Aldrich chemical company (Aldrich Chemical Company) or Lancaster synthesizes the chemical supplier such as Co., Ltd (Lancaster Synthesis Ltd.) and buys, unless It is otherwise noted, otherwise can be used without further purification.Tetrahydrofuran (THF) and N, N-dimethylformamide (DMF) Zi Aer The strange company in Delhi (Aldrich) buys in SureSeal bottles, receives and can be used.Unless otherwise noted, otherwise all solvents Purified using the standard method of this area.
React below in nitrogen, the direct draught of argon or using drying tube, in room temperature (unless otherwise indicated) in anhydrous solvent Carry out, and reaction flask is used to introduce material and reagent via syringe equipped with rubber septum.
Glass wares is through oven drying and/or heat drying.At 254 glass plates of silica gel 60F (E Merck (0.25mm)) On carry out analysis thin-layer chromatography, with appropriate solvent ratio (v/v) elute.By TLC analyze react, by the consumption of starting material Lai Judge reaction terminating.
TLC plates are absorbed via UV or (Aldrich chemicals is public using p-anisaldehyde spraying reagent or Sonnenschein's reagent Department, ethanol prepare 20 weight %) (thermal activation) or dye in the iodine room and observe.
Usually double reaction volume with reaction dissolvent or extractant, then using by volume 25% extraction volume (unless otherwise stated) shown aqueous solution washing is post-processed.Reaction mixture filters after being dried over anhydrous sodium sulfate, and uses Solvent is evaporated under reduced pressure in Rotary Evaporators, should be noted that when solvent is removed in vacuum.
Unless otherwise indicated, otherwise rapid column chromatography (flash column chromatography) [Still et al., J.Org.Chem., 43,2923 (1978)] E Merck (Merck) level Flash silica (flash silica) (47-61mm) is used, Silica gel:Roughage ratio is about 20:1 to 50:1.Carry out in shown pressure or under environmental pressure hydrogenesis.
Bruker instruments are operated in 400MHz to record1H H NMR spectroscopies, in 100MHz operation notes13C-NMR is composed.Use chlorine Replication is reference standard (7.27ppm and 77.00ppm) or uses CD3OD (3.4 and 4.8ppm and 49.3ppm) is used as reference Standard items or it is appropriate when utilize tetramethylsilane internal standard (0.00ppm), obtain CDCl3The H NMR spectroscopy of solution (is reported in units of ppm Lead).If desired other NMR solvents can be used.When reporting peak diversity, following abbreviation is used：S=is unimodal, and d=is bimodal, t= Triplet, m=multiplets, br=widen, dd=double doublets, the double triplets of dt=.When providing coupling constant, using hertz as Unit is reported.LC/MS is used to obtain mass spectrum with ESI or APCI.All fusing points are not calibrated.The purity of all end-products is more than 90% (via HPLC, 220nm and 254nm wavelength).
Following synthetic example is used to illustrate a kind of method for synthesizing the compounds of this invention, should not be construed as limited to these Embodiment.
Embodiment 1：Synthesize the compounds of this invention
Synthesize the chloro- 9- isopropyls -9H- purine of 2,6- bis-
At room temperature, in 30 minutes, by 2,6- dichloropurines (2 mMs), isopropanol (8 mMs) and triphenylphosphine (4 MM) 40 milliliters of anhydrous tetrahydro furans are absorbed in, diazonium dicaxboxylate (4 mMs) is added dropwise.The reaction mixes Compound be stirred at room temperature 24 it is small when.The reaction is periodically monitored by TLC or LC/MS.Reaction mixture is poured into containing ice cooling In the beaker of water.With 3x100 milliliters of ethyl acetate aqueous layer extracteds, crude product is obtained.Crude product chromatographic purifying on a silica gel column (petroleum ether solution of 10-80% ethyl acetate, gradient elution), obtains the chloro- 9- isopropyls -9H- purine of 2,6- bis-, yield 77%.
Synthesize 5- (the chloro- 9- isopropyls -9H- purine -6- bases of 2-)-pyrimidine -2-base amine
To the chloro- 9- isopropyls -9H- purine of 2,6- bis- (5.21 mMs), 5- (4,4,5,5- tetramethyls-[1,3,2] dioxy Heterocycle pentaborane -2- bases amine (5.21 mMs) and 1,1 '-bis- (diphenylphosphino) the ferrocene chlorinations with dichloromethane complexing Two without peroxide of palladium (II)2M aqueous sodium carbonates (15.6 mMs) are added in alkane (40 milliliters) solution.Will be anti- Answer mixture to deaerate and purged with nitrogen.Then heated in keeping 80 DEG C of oil bath and stir the reaction mixture 3 it is small when. The consumption of purine is originated in LC/MS monitoring reactions.
Reaction mixture is cooled to room temperature, depressurizes lower removing solvent.Residue is absorbed in the mixed of ethyl acetate and water In compound.Organic phase is separated, water layer is further extracted with 3x100 milliliters of ethyl acetate.Organic phase is dried with sodium sulphate, under vacuum Solvent is removed, obtains 5- (the chloro- 9- isopropyls -9H- purine -6- bases of 2-)-pyrimidine -2-base amine, yield 55%.
Synthesize 5- (9- isopropyl -2- morpholine -4- base -9H- purine -6- bases)-pyrimidine -2-base amine
To the dimethylacetamide of 5- (the chloro- 9- isopropyls -9H- purine -6- bases of 2-)-pyrimidine -2-base amine (2.84 mMs) Morpholine (2.84 mMs) is added in amine (18 milliliters) solution.Reaction mixture heats in 94 DEG C of oil bath is kept and stirs 12 Hour.By whether starting material being not present in LC-MS monitoring reactions.Crude product is loaded directly on preparation HPLC column, 5- (9- isopropyl -2- morpholine -4- base -9H- purine -6- bases)-pyrimidine -2-base amine, yield 58% are obtained after chromatogram purification.1H NMR, 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]+.
Synthesize 5- (the bromo- 9- isopropyls -2- morpholines -4- bases -9H- purine -6- bases of 8-)-pyrimidine -2-base amine
At 5 DEG C, to 5- (9- isopropyl -2- morpholine -4- base -9H- purine -6- bases)-pyrimidine -2-base amine (1.03 grams, 3.03 MM) 15 milliliters of chloroformic solutions in, be slowly added to NBS (594 milligrams, 3.34 mMs).The reaction was continued at this temperature 2 Hour.After simple post processing, product 5- (the bromo- 9- isopropyls -2- morpholines -4- bases -9H- purine -6- bases of 8-)-pyrimidine -2-base amine Pass through flash column (dicyandiamide solution：The hexane solution of 50% ethyl acetate), acquisition 5- (the bromo- 9- isopropyls -2- morpholines of 8- - 4- base -9H- purine -6- bases)-pyrimidine -2-base amine, yield 52% (660 milligrams).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]+。
Synthesize 5- (9- isopropyl -8- methyl -2- morpholine -4- base -9H- purine -6- bases)-pyrimidine -2-base amine
In sealed tube, to 5- (the bromo- 9- isopropyls -2- morpholines -4- bases -9H- purine -6- bases of 8-)-pyrimidine -2-base amine (30 milligrams, 0.072 mM) and Pd (dppf) Cl23 milliliters anhydrous two of (3 milligrams, 5% mM)In alkane solution, delay It is slow to add zinc methide (210 microlitres, 1.0M n-heptane solutions).The mixture is heated to about 65 DEG C.MeOH is added dropwise, is removed under vacuum Remove solvent.EtOAc is added in residue, solution 1M HCl, water and the salt water washing of generation, then use Na2SO4It is dry.Remove Solvent is removed, crude mixture carries out flash chromatography on silica gel, and obtaining 5-, (9- isopropyl -8- methyl -2- morpholine -4- bases -9H- is fast Purine -6- bases) 8 milligrams of-pyrimidine -2-base amine, yield 47%.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]+。
Embodiment 2：Compare biological test
By synthesis in the compounds of this invention and PCT/SG2008/000379 and the more various biologies of many compounds disclosed Learn parameter.
The parameter of test is：
The activity of TOR experiments；
The activity of PI3K experiments；
Solubility test
People's Microsomal Stability is tested
The method that will be detailed below each test：
MTOR is tested
Prepared inside the 4eBP1 of truncated mTOR kinases and His- labels.[γ33P]-ATP is from peace agate West Asia (Amersham) (GE Healthcare) is bought.Unless otherwise noted, all chemicals all derive from public in Sigma-Alder Take charge of (Sigma-Aldrich).
Phosphorylation test initially carries out on the 384 hole polypropylene boards (Greiner) that final volume is 20 microlitres.Compound Test scope be usually 100 μM to 0.006 μM, the dilution of point 8 steps, in duplicate.First, in pure DMSO, micro- containing 1 Liter/hole test compound sample panel on add 10 microlitres/hole 2X enzyme-substrates solution (1.5 mcg/ml mTOR, 40 micrograms/ Milliliter 4eBP1 is in 1X assay buffers：10mM Hepes pH 7.5,50mM NaCl and 10mM MnCl2).By adding 10 Microlitre/(final test concentration is 10 μM of ATP and 0.4 μ Ci/ hole [γ for 20 μM of ATP solution in hole33P]-ATP) start reaction. After when incubated at room temperature 1 is small, reaction is terminated with the 20mM EDTA/1mMATP solution in 40 microlitres/hole.
Then, the mixture for 50 microlitres/hole being terminated to reaction is transferred to 384- holes MultiScreenHTS-PH filter plates (Millipore) on, 1% phosphoric acid in 50 microlitres/hole is previously added on the filter plate.By vacuum filter, the plate is micro- with 120 0.5% phosphoric acid washing in liter/hole four times.Finally, the Optiphase in 10 microlitres/hole is addedTMSuperMix liquid scintillations are concocted Thing (cocktail) (Perkin Elmer).It is minimum 1 it is small when culture after, using the coincidence counting pattern of crosstalk correction, Counted in Wallac MicroBeta TriLux scintillation counters.IC50It is defined as the suppression of 50% maximum possible kinase activity Required compound concentration.
PI3K is tested
Recombinate and prepared inside PI3K p110 α/p85.Phosphatidylinositols (PtdIns), phosphatidylserine (PtdSer) and Every other unspecified chemicals is all purchased from company in Sigma-Alder.[γ33P] ATP and Optiphase flicker Material can be obtained from Pa Jin Elmer Co., Ltd (Perkin Elmer).
Tested with 25 microlitres of final test volumes on 384- holes Maxisorp orifice plates (Nunc).Compound is according to 3 Times serial dilution, is tested with 8 kinds of concentration, usually since 10 μM.20 microlitres/hole is coated with Maxisorp orifice plates The 1 of PtdIns and PtdSer:1 mixture [is each dissolved in chloroform with 0.1 mg/ml:Ethanol (3:7) in], room temperature (RT) Under be dried overnight in fume hood.
By pipette by 5 microlitres/hole compound (2.5%DMSO solution), 10 microlitres/hole enzyme (0.5 mcg/ml + 1 mcg/ml p85 of p110 α) and Ci/ milliliters of [γ of 5 μM of 10 microlitres/hole ATP and 5 μ33P] ATP additions assay buffer is (finally Concentration：0.2 mcg/ml p110 α, 2 μM of ATP, 0.05 μ Ci/ hole [γ33P] ATP is in 1X assay buffers：100mM Tris- HCl pH 7.0,200mM NaCl, 8mM MgCl2), start enzyme reaction.The reaction cultivate at room temperature 1 it is small when, with 30 microlitres/ The 50mM EDTA solution in hole terminates reaction.Then, orifice plate is washed twice with TBS, and the flicker material in 30 microlitres/hole is added after dry Material, then counts on MicroBeta Trilux.IC50It is defined as the compound that 50% maximum possible kinase activity suppresses required Concentration.
Microsomal Stability is tested
Using the stability of the outer preliminary assessment compound of high throughput template body in 96 orifice plates (Whatman), including with people Liver microsomes (HLM) are cultivated.Verapamil is purchased from company in Sigma-Alder, and reference standard is used as in this experiment. HLM is purchased from Ze Nuo scientific ＆ technical corporation (Xeno Tech) (20 mg/mls, in 250mM sucrose solutions).It is pre- by the following method First prepare the mother liquor of 100mM kaliumphosphate buffers：By 80 milliliters of 1M K2HPO4With 20 milliliters of 1M KH2PO4In 900 milliliters of water Mix (being adjusted pH to 7.4 with dilute hydrochloric acid), room temperature storage.K2HPO4.3H2O and KH2PO4Purchased from public in Sigma-Alder Department, NADPH regenerative systems solution A and B are purchased from genetic test company (Gentest).Stop bath for reaction to be quenched is pre- The acetonitrile and DMSO (80 first prepared:20) mixed liquor and in 4 DEG C of storages.All solvents used are HPLC grades, prepared by mother liquor And the water used during LC-MS analyses is through Milli-Q system deionizations.
It is mixed with 500 microlitres of 50mM kaliumphosphate buffers (pH 7.4, is diluted with water the dilution of 100mM stock buffers and prepares) Close, the DMSO storing solutions that 2.5 microlitres of 10mM are tested to compound dilute 200 times, obtain 500 microlitres of 50 μM of solution.Then it is micro- by 8 Rise compound cocktail and add 72 microlitres by water (2250 microlitres), 100mM kaliumphosphate buffers (2900 microlitres), NADPH regeneration The previously prepared training of (250 microlitres) System Solution B (58 microlitres), NADPH regenerative systems solution A (290 microlitres) and HLM compositions Support in mixed liquor.Then by obtained reaction mixture (final compound concentration be 5 μM) in 37 DEG C in B.Braun Incubated in Certomat H incubators, then 50 microlitres of aliquots are distributed to and are trained comprising 100 microlitres of the different of stop bath In the hole for supporting plate.Centrifuged 15 minutes with 2000rpm in 4 DEG C, the sample of supernatant obtained by 100 μ L is transferred to LC-MS plates carries out Analysis.Every kind of test compound sampling is multiple, incubates a series of time point (5,15,30,45 and 60 minutes).Pass through LC-MS (ABI Qtrap 3200) determines the concentration of remaining compound, compared with the reference solution of concentration known.Then, partly to decline Phase (t1/2, minute) represents stability.
High throughput solubility test
Using 96- casement plates, the solubility of definite compound in high throughput dynamic solubility assay method.With it is ultraviolet/ Visible ray microplate spectrophotometer (Molecular Devices SpectraMax Plus384) evaluation compound Solubility.It is used as reference standard purchased from the Vorinostat (Vorinostat) (SAHA) and nicardipine of Sigma.
The compound being diluted in phosphate buffer (Sigma) in DMSO, (the 5 microlitres of 10mM storages of 250 μM of ultimate density Standby liquid is in 195 microlitres of phosphate buffer pH 7) and be sufficiently mixed.Then by mixture with 600rpm shake 1.5 it is small when, room When temperature standing 2 is small.Then plate is centrifuged 15 minutes with 1500g.Gained supernatant (80 microlitres) is transferred to UV- analysis plates to be used in combination (20 microlitres) dilutions of DMSO.Used in phosphate buffer/DMSO (80:20) the calibration mother liquor of the respective compound prepared in quantifies Sample.
The compound of test is as follows：
Table 1 summarizes the result of biological test.
Table 1
It can be seen from the above that all compounds are respectively provided with a degree of activity, (compound E is to mTOR in addition to compound E It is active significantly lower), the compounds of this invention is suitable with the activity of comparative compound to the activity of two kinds of enzymes interested.Therefore, All compounds are potential drug candidates, although some are low for activity of the compound E to mTOR.
But and the compound of not all test show acceptable solubility characteristics.For example, compound A, B and E Low solubility is not the result shows that these compounds are preferable drug candidates.Their low solubility makes it be difficult in a physiologically Effectively prepared in acceptable carrier, thus reduce it there is the excellent dynamic (dynamical) possibility of oral drugs in human body. On the contrary, the solubility of compound C, D and the compounds of this invention is acceptable for drug candidate.
However, in vitro in terms of metabolic stability, it is as a result dramatically different.In these tests, compound A, B, E and Ben Fa Bright compound has acceptable stability in the research of people's liver microsomes.This is suggested that, if these compounds can Successfully give, then they are sufficiently stable to realize required physiological effect in patients.Moreover, have at the same time excellent In the compound of mTOR/PI3K inhibitory activity and excellent solubility (compound of C, D and the present invention), unique one kind has can The compound of the metabolic stability of receiving is the compound of the present invention.
The compound of all compound A-E and the present invention show excellent activity, but the present invention in these trials The activity of compound is most strong, because it combines potent target spot inhibitory activity, excellent water-soluble and excellent metabolic stability Property.
In short, the biological results prompting that the compound tested in the biological study of above-mentioned progress is realized, although this The compound of invention tests compound compared with many has the similitude being significantly closer to, but the compounds of this invention is unique one Kind meets active, abundant water-soluble and metabolic stability requirement at the same time, thus is suitable as the compound of drug molecule.Therefore, These researchs show that the compound is used as the superiority of drug candidate.
Embodiment 3：Efficiency biomarker experiment (pp70-S6KT389, pAktS473) based on cell
In order to further illustrate the efficiency of the compounds of this invention, two kinds of lifes based on cell are carried out to the compound of the present invention Substance markers thing is tested.Method is as follows：
SureFire p-Akt (Ser 473) 384 kit (TGR, catalog number (Cat.No.)：TGRAS500),
SureFire phospho-p70S6 kinases (Thr 389) 384 kit (TGR, catalogue Number:) and Proxiplate-384Plus (Perkin Elmer, catalog number (Cat.No.) TGR70S500:6008280) it is purchased from Pa Jinaiermo Company (Perkin Elmer).Human prostate cancer cell line (PC-3) is purchased from ATCC.Unless otherwise indicated, all compounds are purchased From company in Sigma-Alder.
First day, 200 microlitres of PC3 cell solutions of 105 cells/mls of 2X are inoculated into each hole of 96- orifice plates.Connect Add compound when 24 is small after kind, test scope be usually 10 μM to 4.6nM, point 8 steps dilution, in triplicate.37 DEG C, 4 it is small when The ultimate density of DMSO is 0.1% during incubation step.After incubation step, supernatant is removed, with 1X lysis buffers (offer of AlphaScreen kits) cell lysis simultaneously gently shakes 10 minutes.4 microlitres of lysates and 5 microlitres are contained The reaction buffer of AlphaScreen acceptor globules adds activation buffer solution mixture to add each 384- holes (reaction buffer: Activate buffer solution:Acceptor globule is 40:10:1), gently shake 2 it is small when (room temperature, dark place).2 microlitres are containedDilution buffer (the dilution buffer of donor globule:Donor globule is 20:1) each of 384- plates is added Kong Zhong, is placed in plate and shakes and shaked on device 1-2 minutes, and is incubated overnight at room temperature.
(measurement pattern is configured using standard AlphaScreen:Alphascreen；Reading mode:Endpoint；Optical mode Formula:AlphaScreen 680570；Position postpones:0.10 second；Firing time:0.30 second；Integration starts:0.34 second；During integration Between:0.30 second；Gain:3000) 384- orifice plates, are read with BMG Pherostar plates readout instrument.
IC50The kinase activity for being defined as 50% maximum possible suppresses the molar concentration of required compound.5- (9- isopropyls Base -8- methyl -2- morpholine -4- base -9H- purine -6- bases) suppression of-pyrimidine -2-base amineP70-S6KT389 and pAktS473Phosphoric acid ChangeIC50Respectively 24nM and 9nM.
Biomarker the result shows that the compounds of this invention suppress in kinase activity the effect of.

Claims (16)

1. a kind of method of the compound of manufacture formula (I), this method include：
The compound and zinc methide for making formula (II) are reacted,
So as to provide the compound of formula (I).
2. the method as described in claim 1, it is characterised in that the reaction carries out in the presence of palladium compound, described Palladium compound is Pd (dppf) Cl2。
3. the method as described in claim 1, it is characterised in that the reaction carries out in the presence of solvent.
4. method as claimed in claim 3, it is characterised in that the solvent Shi dioxanes.
5. the method as described in claim 1, it is characterised in that the reaction carries out in seal pipe.
6. the method as described in claim 1, it is characterised in that the reaction is in about 65 DEG C of progress.
7. a kind of method of the compound of the formula (II) limited in manufacturing claims 1, this method include：
The compound and N-bromosuccinimide for making formula (III) are reacted,
So as to provide the compound of formula (II).
8. the method for claim 7, it is characterised in that the reaction carries out in the presence of solvent.
9. method as claimed in claim 8, it is characterised in that the solvent is chloroform.
10. the method for claim 7, it is characterised in that the reaction is in about 5 DEG C of progress.
11. the compound of the formula (I) of claim 1 is preparing the application in being used to treat the medicine of Animal diseases, wherein, suppress One or more are selected from serine/threonine protein kitase either its fragment or compound or PI3 kinases or its fragment or multiple The protein kinase of compound prevents, suppresses or improves the symptom or symptom of the disease, wherein, the disease is cancer, precancerosis Disease, hyperplasia, autoimmunity or inflammatory disease or the disease as caused by excessive neovascularization.
12. application as described in claim 11, it is characterised in that the illness is cancer.
13. application as claimed in claim 11, it is characterised in that the illness is precancerosis disease or hyperplasia.
14. application as claimed in claim 13, it is characterised in that the illness is selected from：Familial adenomatous polyposis, colon Polyposis adenomatous, myelodysplasia, Endometrium development exception, endometrial hyperplasia with atypia, cervical atypical hyperplasia, Knurl formation, benign prostatic hyperplasis, larynx papilloma, photochemical and solar keratosis, seborrheic keratosis in vagina epithelium And keratoacanthoma.
15. application as claimed in claim 13, it is characterised in that the illness is autoimmunity or inflammatory disease or by excessive Disease caused by neovascularization.
16. application as claimed in claim 15, it is characterised in that the illness is selected from：Acute diseminated encephalomyelitis, Ai Di Sick family name, without gamma-globulin mass formed by blood stasis, agranulocytosis, allergic asthma, allergic encephalitis, allergic rhinitis, alopecia areata, Alopecia senilis, red blood cell occur cannot, ankylosing spondylitis, antiphospholipid antibody syndrome, aortitis syndrome, regeneration barrier Impenetrability anaemia, atopic dermatitis, autoimmune hemolytic anemia, oneself immunity hepatitis, autoimmunity oaritis, Ba Luo Disease, Basedow disease, behcet disease/syndrome, bronchial asthma, block this Mann syndrome, chylous diarrhea, chagas disease, Chronic inflammatory demyelinating polyneuropathy, churg-Strauss syndrome, Ke's radicular syndrome, keratoconus, keratoconus are white It is spot, coxsackie myocarditis, CREST diseases, Crohn disease, epidermis eosinophilia, cutaneous T-cell lymphoma, more Sample dermatitis erythremia, dermatomyositis, diabetic retinopathy, postmyocardial infarction syndrome, on cornea Skin malnutrition, eczematous dermatitis, eosinophilic fasciitis, eosinophilic gastroenteritis, epidermolysis bullosa, ivens Syndrome, fibrosing alveolitis, pregnant pemphigoid, glomerulonephritis, Goodpasture's syndrome, graft versus host disease(GVH disease), Graves disease, guillain-Barre syndrome, chronic lymphocytic thyroiditis, hemolytic uremic syndrome, herpetic keratitis, ichthyosis vulgaris, special hair Property interstitial pneumonia, Idiopathic Thrombocytopenic Purpura, inflammatory bowel disease, kawasaki disease, keratitis, keratoconjunctivitis sicca, Lambert-she Syndrome, common Leucoplakia, lichen planus, lichen sclerosus, Lyme disease, linear IgA bullous disease, macular degeneration, huge children are red Cellulous anemia, Meniere disease, Mooren's ulcer, mucha-Habermann disease, polymyositis, multiple sclerosis, again Disease myasthenia, necrotizing enterocolitis, neuromyelitis optica, ocular pemphigus, ocular myoclonus syndrome, Ao Deshi thyroiditis, Paroxysmal nocturnal hemoglobinuria, two Cotard of Ba-spy, pemphigus, periodontitis, pernicious anaemia, pollen allergy, polyadenous Autoimmune syndromes, posterior uveitis, monolobular cirrhosis, rectitis, pseudomembranous colitis, psoriasis, pulmonary emphysema, purulence Skin disease, Reiter syndrome, reversible obstructive airway disease, rheumatic fever, rheumatoid arthritis, sarcoidosis, sclerotitis, Sai Zha In syndrome, Sjogren syndrome, subacute bacterium endocarditis, systemic loupus erythematosus, aorto-arteritis, temporal arteritis, support- Prosperous two Cotards, type i diabetes, ulcerative colitis, nettle rash, spring conjunctivitis, Leucoplakia, Wo Ji- little Liu-farmland on a plateau are comprehensive Simulator sickness and Wegener granulomatosis.
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GB2431156A (en) * 2005-10-11 2007-04-18 Piramed Ltd 1-cyclyl-3-substituted- -benzenes and -azines as inhibitors of phosphatidylinositol 3-kinase
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