CN1829713A - 3,5 disubstituted indazole compounds, pharmaceutical compositions, and methods for mediating or inhibiting cell proliferation - Google Patents

Abstract

3,5 disubstituted indazole compounds that modulate and/or inhibit cell proliferation, such as the activity of protein kinases are described. These compounds and pharmaceutical compositions containing them are capable of mediating CDK dependent diseases to modulate and/or inhibit unwanted cell proliferation. The invention is also directed to the therapeutic or prophylactic use of pharmaceutical compositions containing such compounds, and to methods of treating cancer as well as other disease states associated with unwanted angiogenesis and/or cellular proliferation, such as diabetic retinopathy, neovascular glaucoma, rheumatoid arthritis, and psoriasis, by administering effective amounts of such compounds.

Description

The method of 3,5 disubstituted indazole compounds, pharmaceutical composition and mediation or inhibition cell proliferation

Technical field

The present invention relates at 3 of 3 benzoglyoxalines with replacement, 5 two substituted indazoles, its activity by protein kinase mediates and/or suppresses cell proliferation, especially the kinases by cyclin dependent for example the mediation of CDK1, CDK2, CDK4 and CDK6 mediate and/or suppress cell proliferation.The invention further relates to the method for the pharmaceutical composition that contains this compound and composition and this compounds for treating tumour by giving significant quantity and other morbid state relevant with deleterious vasculogenesis and/or cell proliferation.

Background technology

Cell proliferation is for the middle appearance of replying of various stimulus, and can be lost regulation and control and caused by cell division cycle (or cell cycle), is multiplied and divides by this process cell.Height proliferative disease state comprises that tumour is characterised in that owing to for example damaged the gene of regulating cycle progress directly or indirectly, and makes cell have uncontrolled healthy and strong growing way, spread beyond control in the whole cell cycle.Like this, regulate the medicament of cell cycle and this height propagation, can be used for treating various and uncontrolled or deleterious cell proliferation disease states associated.

The mechanism of the cell proliferation on cell and molecular level is in the positive research: for signal pathway lose the forfeiture of regulation and control, cell cycle control, the research of cell levels has been carried out in the stimulation that does not add contained vasculogenesis or inflammatory approach, and on molecular level, these processes are regulated by various albumen, in these albumen, protein kinase is the doubtful thing of giving prominence to.The overall minimizing of propagation can also be produced by apoptosis or apoptosis, and simultaneously adjusted by means of multiple approach, some approach relate to the proteolysis zymoprotein.In candidate's modulin, protein kinase is the enzyme family of the hydroxyl phosphorylation of tyrosine, Serine or threonine residues specific in gang's catalytic protein.Typically, this phosphorylation is the function of interferencing protein significantly, and this protein kinase is critical in the regulation and control of various kinds of cell process.For example, do not wish to be fettered by particular theory, it is believed that as the protein kinase inhibitor of the kinases of cyclin dependent (" CDK ") for example, medicament of the present invention can be regulated the synthetic level of cell RNA and DNA, and therefore is expected to be effective to treat virus infection for example HIV, Human papilloma virus HPV, simplexvirus, Epstein-Barr virus, adenovirus, sindbis alphavirus, poxvirus or the like.(referring to Schang, waiting the people, J.Virol.74,2107-2120 (2000)).In addition, CDK5 has been contemplated as falling with in the phosphorylation of Protein tau, thinks that it is the potential method (Hosoi waits the people, J.Biochem. (Tokyo), 117,741-749 (1995)) of treatment or prevention Alzheimer.CDKs is the serine-threonine protein kinase enzyme that plays a crucial role in the transformation of regulating between the different steps of cell cycle, for example the resting stage from G1 (in the gap between mitotic division and the fissional dna replication dna of a new round initial) is to the S development in (active dna synthetic stage), or the development from G2 to the M stage, active mitotic division and cell fission appearred in this stage.Form the CDK mixture by control cyclin (cyclin) hypotype (for example, cyclin A, B1, B2, D1, D2, D3 and E) with the associating of catalysis kinases hypotype (for example, CDK1, CDK2, CDK4, CDK5 and CDK6).As its name suggests, for the target substrate of phosphorylation CDKs, CDKs has shown the absolute dependency for the cyclin hypotype, and different kinases/cyclins is to playing the effect of regulating progress in the specified phase that runs through the cell cycle.

Determined that a large amount of indazole derivativess have potential curative effect: GB 2345486 and disclose indazole derivatives as tyrosine kinase inhibitor, EP0518805 has determined to have the indazole that the piperidines of sigma receptor active replaces; WO 89/43969 discloses the indazole as the ring-type urea of HI V protein enzyme inhibitors; US 4,415, and 569 have determined to have the pyrazolo indazole derivatives of bronchiectatic activity; US 5,208, and 248 disclose the migrainous indazole of treatment.Other treatment of indazole derivatives is applied in people's such as WO 96/20192, EP 04994774, JP 60/004184, EP0023633, US 4051145, JP59/228248, GB 1/376600, US4978603, EP0904769 and De Lucca document Journal of Medicinal Chemistry, 42,135-52 has obtained discussion in (1999).The general building-up reactions route of preparation indazole derivatives by people such as Wentrup at Joumal Organic Chemistry, 43, people such as 2037-41 (1978) and Fugimura are at Chemical Abstracts, disclose in 1070,749 (1987).In more detail, determined that 3,5 indazoles that replace are kinases inhibitor: WIPOInternational Publication No.01/85726 discloses by 1, and the compound that the different thiazolidine of 1-dioxo replaces is as the CDK inhibitor; WO 02/10137 discloses 3,5 indazoles that replace as the inhibitor of the terminal kinase inhibitor of JunN-; With U.S. Patent No. 6,555,539 and WO 03/004488 disclose at 3 and had the indazoles that 3,5 of benzoglyoxaline replaces.

Yet also need more effective CDK inhibitor, specifically, the CDK inhibitor that need have high affinity and have highly selective with respect to other protein kinase for target CDK kinases.Compound of the present invention has more selectivity than the compound described in the previous publication usually for the CDK inhibitor.

Summary of the invention

The purpose of this invention is to provide effectively and CDK inhibitor selectively.Correspondingly, an object of the present invention is to obtain to suppress active compound of CDK and pharmaceutical composition, or their cyclin mixture.Further purpose provides the effective means that suppresses to treat the tumour indication by CDK.Another purpose is the pharmaceutical composition that obtains to contain compound, and this compound can effectively be blocked cancer cells and be converted to their proliferative phase.These and other purpose of the present invention and advantage will become clearly according to following detailed description, can realize these purposes and advantage by using cell cycle control agent of the present invention as described below.

According to these purposes, according to the compound that the invention provides formula I or pharmacologically acceptable salts or solvate:

R wherein ¹, R ², R ³And R ⁴Be selected from H, halogen, cyano group, nitro, trifluoromethoxy, trifluoromethyl, azido-, hydroxyl, C ₁-C ₆Alkoxyl group, C ₁-C ₁₀Alkyl, C ₂-C ₆Thiazolinyl, C ₂-C ₆Alkynyl ,-C (O) R ⁵,-C (O) OR ⁵,-OC (O) R ⁵,-NR ⁵C (O) R ⁶,-C (O) NR ⁵R ⁶,-(CR ⁵R ⁶) NR ⁷R ⁸,-CR ⁵R ⁶NR ⁷R ⁸,-NR ⁵OR ⁶,-SO ₂NR ⁵R ⁶,-S (O) _j(C ₁-C ₆Alkyl), wherein j is 0 to 2 integer ,-(CR ⁵R ⁶) _t(C ₆-C ₁₀Aryl) ,-(CR ⁵R ⁶) _t(C ₃-C ₁₀Cycloalkyl) ,-(CR ⁵R ⁶) _t(4-10 unit heterocycle) ,-(CR ⁵R ⁶) _qC (O) (CR ⁷R ⁸) _t(C ₆-C ₁₀Aryl) ,-(CR ⁵R ⁶) _qC (O) (CR ⁷R ⁸) _t(C ₃-C ₁₀Cycloalkyl) ,-(CR ⁵R ⁶) _qC (O) (CR ⁷R ⁸) _t(4-10 unit heterocycle) ,-(CR ⁵R ⁶) _tO (CR ⁷R ⁸) _q(C ₆-C ₁₀Aryl) ,-(CR ⁵R ⁶) _tO (CR ⁷R ⁸) _q(C ₃-C ₁₀Cycloalkyl) ,-(CR ⁵R ⁶) _tO (CR ⁷R ^g) _q(4-10 unit heterocycle) ,-(CR ⁵R ⁶) _qSO ₂(CR ⁷R ⁸) _t(C ₆-C ₁₀Aryl) ,-(CR ⁵R ⁶) _qSO ₂(CR ⁷R ⁸) _t(C ₃-C ₁₀Cycloalkyl) and-(CR ⁵R ⁶) _qSO ₂(CR ⁷R ⁸) _t(4-10 unit heterocycle), wherein q and t each be from 0 to 5 integer independently, above-mentioned R ¹, R ², R ³Or R ⁴1 or 2 ring carbon atom of the cycloalkyl of group or heterocyclic moiety is optional by an oxo (=O) group replacement, each R ⁵, R ⁶, R ⁷And R ⁸Be independently selected from H, C ₁-C ₆Alkyl; And R wherein ¹, R ², R ³And R ⁴Not H simultaneously.

In one embodiment, R ¹Be-(CR ⁵R ⁶) NR ⁷R ⁸, R ², R ³And R ⁴Be independently selected from H or F.

In one embodiment, R ¹Be the ethylamino methyl, R ³Be H, R ²And R ⁴Be F.

In another embodiment, R ¹Be the ethylamino methyl, R ²And R ⁴Be H, R ³Be F.

In further embodiment, the present invention relates to be selected from following compound

Or

The present invention also provides the method that is used for preparation I compound.

Be used for the treatment of by the compound that suppresses the disease that kinases mediates or the method for obstacle, comprise to need their patient formula I or the pharmacologically acceptable salts or the solvate of formula I compound according to having the present invention further provides the compound that uses as the cell cycle control agent.

The present invention further provides the method for treatment fungi infestation, cancer or tumour and other and deleterious vasculogenesis and/or cell proliferation disease states associated, comprised the compound of the formula I that needs the patient of this treatment significant quantity or the pharmacologically acceptable salts or the solvate of formula I compound.

The present invention also provides by the compound of the patient's formula I that needs or the pharmacologically acceptable salts or the solvate of formula I compound, suppresses the method for CDK kinase activity selectively.

According to the present invention also provide the pharmaceutical composition of the pharmacologically acceptable salts that contains formula I compound or formula I compound or solvate and said composition in treatment by the disease of kinase activity mediation tumour and other and deleterious vasculogenesis and/or the cell proliferation disease states associated therepic use in diabetic retinopathy, neovascular glaucoma, rheumatoid arthritis and the psoriasis for example for example.

Medicament of the present invention can be used for the treatment of various obstacles or the morbid state relevant with uncontrolled or deleterious cell proliferation, for example tumour, autoimmunization obstacle, virus disease, fungal disease, neurodegeneration obstacle and cardiovascular disorder with the composition that contains this medicament.Therefore, the invention still further relates to the method for the treatment of this disease by the medicament of the present invention that gives significant quantity.

Others of the present invention, advantage and feature will become apparent from following detailed description.

Compound of the present invention and composition are as the antiproliferative medicament with as the inhibitor of mammiferous kinase complex, insect kinases or fungi kinase complex.For example, can suppress the CDK mixture.This compound and composition also can be used for controlling propagation, differentiation and/or apoptosis.

Unless otherwise stated, term used herein " halogen " is meant fluorine, chlorine, bromine or iodine.Preferred halogen group is fluorine, chlorine and bromine.

Unless otherwise stated, term used herein " alkyl " comprises the saturated monovalence alkyl with straight or branched part." C ₁-C ₆Alkyl " show straight or branched moieties with 1 to 6 carbon atom, or the like.

Term " thiazolinyl " is meant the straight or branched thiazolinyl that has 2 to 12 carbon atoms in the chain.Illustrative thiazolinyl comprises third-2-thiazolinyl, the but-2-ene base, and fourth-3-thiazolinyl, 2-methyl-prop-2-thiazolinyl, oneself-the 2-thiazolinyl, vinyl, pentenyl, or the like.

Term " alkynyl " is meant the straight or branched alkynyl that has 2 to 12 carbon atoms in the chain.Illustrative alkynyl comprises Propargyl, fourth-2-alkynyl, and fourth-3-alkynyl, 2-methyl fourth-2-alkynyl, oneself-the 2-alkynyl, ethynyl, proyl, pentynyl or the like.

Term " cycloalkyl " is meant that each ring has saturated or fractional saturation, the monocycle of 3 to 12 annular atomses or condenses or spiral shell polycyclic carbocyclic ring.The example of cycloalkyl explanation comprises following part:

, or the like.

Unless otherwise stated, term used herein " aryl " comprises by removing a hydrogen derived from the organic group of aromatic hydrocarbons, for example phenyl or naphthyl.

Unless otherwise stated, term used herein " 4-10 unit heterocycle ", comprise the heterocyclic group that contains one to four heteroatomic aromatic hydrocarbons and non-aromatics, each heteroatoms is selected from O, S and N, wherein each heterocyclic group has 4-10 atom in its ring system, and condition is O or S atom that the ring of described group does not contain two adjacency.The non-aromatics heterocyclic group is included in the group that only has 4 atoms in its ring system, but aromatic heterocyclic group must have at least 5 atoms in its ring system.Heterocyclic group comprises the fused benzo ring system.The example of 4 yuan of heterocyclic groups is azetidinyl (derived from azetidines).The example of 5 yuan of heterocyclic groups is that the example of thiazolyl, 10 yuan of heterocyclic groups is quinolyls.The example of non-aromatic heterocyclic group is a pyrrolidyl, tetrahydrofuran base, the dihydrofuran base, tetrahydro-thienyl, THP trtrahydropyranyl, dihydro pyranyl, tetrahydrochysene sulfo-pyranyl, piperidyl, morpholino, thiomorpholine is for ， thioxane base, piperazinyl, azetidinyl, oxetanyl, the Thietane base, homopiperidinyl, oxepane alkyl (oxepanyl), thia suberane base (thiepanyl), oxa-azatropylidene base, diaza base, thia azatropylidene base, 1,2,3,6-tetrahydro pyridyl, 2-pyrrolinyl, the 3-pyrrolinyl, indolinyl, 2H-pyranyl, 4H-pyranyl ， alkyl dioxin, 1, the 3-dioxolanyl, pyrazolinyl, dithiane base, the dithiolane base, dihydro pyranyl, dihydro-thiophene base, dihydrofuran base, pyrazolidyl, imidazolinyl, imidazolidyl, 3-azabicyclo [3.1.0] hexyl, 3-azabicyclo [4.1.0] heptane base, 3H-indyl and quinolizinyl.The example of aromatic heterocycle group is a pyridyl, imidazolyl, pyrimidyl, pyrazolyl, triazolyl, pyrazinyl, tetrazyl, furyl, thienyl ， isoxazolyl, thiazolyl ， oxazolyl, isothiazolyl, pyrryl, quinolyl, isoquinolyl, indyl, benzimidazolyl-, benzofuryl, cinnolines base, indazolyl, indenes piperazine base, phthalazinyl, pyridazinyl, triazinyl, isoindolyl, pteridine radicals, purine radicals oxadiazole base, thiadiazolyl group, furazan base, benzo furazan base, benzothienyl, benzothiazolyl, benzoxazolyl, quinazolyl, quinoxalinyl, naphthyridinyl and furo pyridyl.Above-mentioned group when derived from top listed group, can be that C connects or N connects, under such connection is possible situation.For example, the group derived from the pyrroles can be pyrroles-1-base (N connection) or pyridin-3-yl (C connection).In addition, the group derived from imidazoles can be imidazoles-1-base (N connection) or imidazo-3-yl (C connection).4-10 unit heterocycle can be to choose wantonly on any ring carbon, sulphur or the nitrogen-atoms of each ring to be replaced by one or two oxo.Wherein the example of the heterocyclic group that partly replaced by oxo of 2 ring carbon atoms is 1,1-dioxo-thio-morpholinyl.4-10 unit other illustrative examples of heterocyclic derived from but be not limited to following group:

With

Unless otherwise stated, term " oxo " is meant=O.

Term " acid amides " be meant group-C (O) N (R ') (R "), wherein R ' and R " each is independently selected from hydrogen, alkyl, thiazolinyl, alkynyl ,-OH, alkoxyl group, cycloalkyl, Heterocyclylalkyl, heteroaryl, aryl, as mentioned above; Or R ' and R " form aforesaid Heterocyclylalkyl or heteroaryl with the nitrogen cyclisation.

Term " replacement " is meant and has one or more substituent specified group or part.Term " unsubstituted " is meant and does not have substituent specified group.

In the present invention, know that very the compound of formula I can demonstrate tautomeric phenomenon, and the molecular formula figure in this specification sheets has only represented a kind of in the possible tautomeric form.Should understand the tautomeric form that the present invention includes any adjusting and/or suppress kinase activity, and only be not limited to any tautomeric form among the employed molecular formula figure.

Compounds more of the present invention can exist with the form of the mixture of single stereoisomers (that is, being substantially free of other steric isomer), raceme and/or enantiomorph and/or diastereomer.All this single stereoisomers, raceme and their mixture will be defined as within the scope of the invention.Preferably, use optically active compound of the present invention with optically pure form.

As those skilled in the art common understand, has a chiral centre (promptly, a unsymmetrical carbon) optically pure compound is formed (promptly by one in two possible enantiomorphs basically, enantiomer-pure), and the optically pure compound with an above chiral centre be diastereomer pure be again enantiomer-pure.Preferably, use compound of the present invention with at least 90% optically pure form, promptly, contain at least 90% individual isomer (80% enantiomeric excess (" e.e. ") or diastereomeric excess (" d.e. ")), more preferably at least 95% (90%e.e. or d.e.), be more preferably at least 97.5% (95%e.e. or d.e.), most preferably the form of at least 99% (98%e.e. or d.e.).

In addition, general formula I and II be intended to comprise solvate and the form of not solvation of definite structure.For example, general formula I and II comprise the compound with structure shown in hydration and the non-hydrated form.Other example of solvate comprises and Virahol, ethanol, methyl alcohol, DMSO, ethyl acetate, acetate or thanomin bonded structure.

Term " pharmacologically acceptable salts " is meant the salt of the biological effectiveness of the free bronsted lowry acids and bases bronsted lowry that keeps particular compound, and is not undesirable or other the undesirable salt of biology.Compound of the present invention can possess enough acidity, enough groups of alkalescence, or these two kinds of functional groups, and correspondingly with many inorganic or organic basess, inorganic and any reaction of organic acid, form pharmacologically acceptable salts.Typical pharmacologically acceptable salts comprises compound of the present invention and salt inorganic or organic acid or mineral alkali prepared in reaction, and for example salt comprises, vitriol, pyrosulphate, hydrosulfate, sulphite, hydrosulphite, phosphoric acid salt, monohydric phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate salt, muriate, bromide, iodide, acetate, propionic salt, caprate, octylate, acrylate, formate, isobutyrate, hexanoate, enanthate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butine-1, the 4-diacid salt, hexin-1,6-diacid salt, benzoate, chloro benzoate, tolyl acid salt, dinitro-benzoate, hydroxy benzoate, methoxybenzoic acid salt, phthalate, sulfonate, xylenesulfonate, phenylacetate, phenpropionate, phenylbutyric acid salt, Citrate trianion, lactic acid salt, gamma hydroxybutyrate, glycollate, tartrate, mesylate, propanesulfonic acid salt, naphthalene-1-sulfonate, naphthalene-2-sulfonic acid salt, and mandelate.

If compound of the present invention is a kind of alkali, needed pharmacologically acceptable salts can prepare by the existing appropriate method in any this area, for example uses the mineral acid treatment free alkali, mineral acid is hydrochloric acid for example, Hydrogen bromide, sulfuric acid, nitric acid, phosphoric acid or the like, or handle with organic acid, acetate for example, toxilic acid, succsinic acid, amygdalic acid, fumaric acid, propanedioic acid, pyrovicacid, oxalic acid, oxyacetic acid, Whitfield's ointment, the pyranose thuja acid is glucuronic acid or galacturonic acid for example, alpha hydroxy acid is citric acid or tartrate for example, and amino acid is aspartic acid or L-glutamic acid for example, and aromatic acid is phenylformic acid or styracin for example, sulfonic acid is tosic acid or ethyl sulfonic acid for example, or the like.

If compound of the present invention is acid, needed pharmacologically acceptable salts can prepare by any suitable method, for example, for example amine (the primary, the second month in a season or uncle), alkali metal hydroxide or alkaline earth metal hydroxides or the like are handled free acid with inorganic or organic bases.The case illustrated of suitable salt comprises: derived from amino acid for example glycine and arginine, ammonia, primary, the second month in a season and tertiary amine and cyclammonium such as the organic salt of piperidines, morpholine and piperazine with derived from the inorganic salt of sodium, calcium, potassium, magnesium, manganese, iron, copper, zinc, aluminium and lithium.

At medicament is under the solid situation, and those skilled in the art can know that compound of the present invention can exist with different crystallizations or polymorphic form with salt, and all these is defined as in the present invention and the concrete formula scope.

As treating the particularly medicine of human proliferative disorders of Mammals, this obstacle is a sign with originality hyperblastosis in deleterious according to cell cycle control agent of the present invention.The compound of formula I can be used for the treatment of the patient who suffers from the illness relevant with excessive cell proliferation, these illnesss for for example tumour, psoriasis, relate to dysimmunity and restenosis and other smooth muscle disorders of undesirable white corpuscle propagation.In addition, this compound can be used for preventing the releasing differentiation of tissue after the mitotic division and/or cell.

With uncontrolled or unusual cell proliferation diseases associated or obstacle including, but not limited to following:

-various tumours include but not limited to that green blood tumour, a matter source tumour, maincenter and peripheral nervous system tumour and other tumour of cancer, lymphoid green blood tumour, marrow system comprise melanoma, spermocytoma and Kaposi's sarcoma or the like.

-be the disease pathology of feature with the abnormal cell proliferation, for example the restenosis after benign prostatauxe, familial adenomatous polyposis disease, neurofibromatosis, atherosclerosis, pulmonary fibrosis, sacroiliitis, psoriasis, glomerulonephritis, angioplasty or the vascular surgery, hypertrophic cicatrix form thing, inflammatory bowel, transplant rejection, endotoxin shock and fungi infestation.

-the illness related, for example tumour (including, but not limited to those types mentioned above) with defective apoptosis, virus infection is (including, but not limited to simplexvirus, poxvirus, Epstein-Barr virus, sindbis alphavirus and adenovirus), the prevention of the AIDS development in the HI V infected individuals, autoimmune disease is (including, but not limited to systemic lupus erythematous, rheumatoid arthritis, psoriasis, the glomerulonephritis of autoimmunization mediation, inflammatory bowel and autoimmune diabetes), neurodegenerative obstacle is (including, but not limited to Alzheimer, amyotrophic lateral sclerosis, retinitis pigmentosa, Parkinson's disease, the dementia that AIDS-is relevant, Duchenne-Arandisease and cerebellum degeneration), myelodysplastic syndrome, aplastic anemia, the ischemia injury relevant with myocardial infarction, apoplexy and reperfusion injury, irregular pulse, atherosclerosis, the hepatic diseases that toxin causes or alcohol is relevant.

Degenerative disease (including, but not limited to osteoporosis and sacroiliitis), Asprin susceptibility sinusitis paranasal sinusitis, cystic fibrosis, multiple sclerosis, kidney disease and the relaxing tumor pain of-hematological disease (including, but not limited to long-term anaemia and aplastic anemia), flesh osseous system.

Promoting agent of the present invention can also be effective to suppress to invade development, tumor-blood-vessel growth and the transfer of tumour.

In addition, promoting agent of the present invention is for example as the inhibitor of CDKs, can regulate cell RNA and DNA synthetic level, and therefore estimate that it can be effective to treat virus infection for example HIV, Human papilloma virus HPV, simplexvirus, Epstein-Barr virus, adenovirus, sindbis alphavirus, poxvirus or the like.

Compound of the present invention and composition suppress for example kinase activity of CDK/ cyclin mixture, this mixture be for example those at the G0 or the actives in G1 stage of cell cycle, for example CDK2, CDK4 and/or CDK6 mixture.

In order to obtain treatment or to suppress effect, the concrete dosage of the cell cycle control agent that is given can be with manner known in the art, according to the concrete medicament, route of administration, the illness of being treated and patient who is treated or the host that determine around the specific environment of case, for example comprise to be given.Can give exemplary total per daily dose of cell cycle control agent with single or multiple doses, contain dosage level from about 0.01mg/kg body weight to about 50mg/kg body weight.

Cell cycle control agent of the present invention can give by any of multiple suitable pathways, gives in for example oral, rectum, transdermal, subcutaneous, intravenously, intramuscular or the nose.Preferably, before giving, with cell cycle control agent preparation the becoming composition that is suitable for the approach that requires.

Comprise the cell cycle control agent of significant quantity, optional one or more other promoting agent and pharmaceutically acceptable carrier according to pharmaceutical composition of the present invention or preparation, for example be used for the thinner or the vehicle of medicament; When carrier during as thinner, it can be the medium of solid, semisolid or liquid substance, vehicle or the active ingredient that plays the vehicle effect.Can be by active ingredient be mixed with carrier according to composition of the present invention, or it is diluted with carrier, or its is sealed or be encapsulated within the carrier prepare, it can exist with the form of capsule, pouch, paper container or the like.Typical component except that one or more cell cycle control agent and any other active ingredient, comprises Microcrystalline Cellulose (Microcrystalline Cellulose), starch, lactose, calcium sulfate dihydrate, terra alba, sucrose, talcum powder, gelatin, agar, pectin, gum arabic, Magnesium Stearate, stearic acid, peanut oil, sweet oil, glyceryl monostearate, Tween 80 (polysorbate 80), 1, the 3-butyleneglycol, theobroma oil, beeswax, polyoxyethylene glycol, propylene glycol, Stearinsaeure sorbitan ester, polysorbate 60,2-Standamul G, phenylcarbinol, glycine, Sorbic Acid, potassium sorbate, Sodium phosphate dibasic, sodium-chlor, and water.

Composition can be suitable in the needed form that gives mode any and prepares according to various.For example, pharmaceutical composition can prepare with following form: tablet, pill, powder, lozenge, pouch, cachet, elixir, suspension, milk sap, solution, syrup, aerosol (with solid or liquid as medium), ointment is (for example, calculate by weight, contain 10% cell cycle control agent at the most), soft gel and hard gel capsule, suppository, aseptic injectable solution, aseptic packaging powder or the like.

Similarly, carrier or thinner can comprise time-delay known in the art or the material that on time discharges, for example glyceryl monostearate or distearin, it can use separately or mix with paraffin, ethyl cellulose, Vltra tears, methyl methacrylate or the like.

Can use various medicament forms.Therefore, if use solid carrier, preparation can be a tablet, be placed in the hard gelatin capsule with the form of powder or piller, or the form of lozenge or lozenge.The amount of solid carrier can change, but usually from about 25mg to about 1g.If use liquid vehicle, preparation can be syrup, milk sap, soft gelatin capsule, be placed on aseptic injectable solution or suspension or on-aqueous liquid suspension in ampoule or the bottle.

In order to obtain stable water-soluble dosage form, the pharmacologically acceptable salts of invention medicament is dissolved in the organic or inorganic aqueous acid, for example the solution of 0.3M succsinic acid or citric acid.If the form of soluble salt is not suitable for, medicament can be dissolved in the combination of suitable cosolvent or cosolvent.The example of suitable cosolvent includes, but are not limited to, and concentration range is from the ethanol of the 0-60% of cumulative volume, propylene glycol, Liquid Macrogol, polysorbate 80, glycerine or the like.The compound of formula I can be dissolved among the DMSO and dilute with water.Composition can also be in the suitable aqueous excipient solution form of the salt that forms of the active ingredient in water or isotonic saline solution or the glucose solution for example.

Composition of the present invention can prepare with the mode of common known pharmaceutical compositions, for example, uses ordinary method for example to mix, dissolving, and drageeing is produced in granulation, grinds, emulsification, encapsulation is collected or lyophilize.Pharmaceutical composition can be with the mode of routine, use one or more physiology acceptable carrier to prepare, and this carrier can be selected from those and be convenient to process vehicle and the auxiliary agent that active compound becomes the spendable preparation of pharmacy.

Appropriate formulations depends on selected route of administration.For injection, medicament preparation of the present invention can be become the aqueous solution, preferably use for example Hanks ' s solution of physiology compatible buffers, Ringer's solution, or normal saline buffer solution.For transmucosal administration, in preparation, use for the suitable permeate agent of the barrier that is permeated.Such permeate agent is known in this area usually.

For oral administration, can easily, prepare active compound by being mixed with pharmaceutically acceptable carrier known in the art.Such carrier can be formulated as tablet with compound of the present invention, pill, and drageeing, capsule, liquid, gelifying agent, syrup, slurries, suspension or the like, the patient who passes through to be treated carries out orally ingestible.The pharmaceutical preparation that orally uses can obtain according to following manner: the mixture that uses solid excipient and active ingredient (medicament), the resulting mixture of optional grinding, add the mixture of proper auxiliary agent processing granular agent afterwards, if necessary, obtain tablet or drageeing core.Suitable vehicle comprises: filler is sugar for example, comprises lactose, sucrose, mannitol or Sorbitol Powder; Cellulose preparation for example, W-Gum, wheat starch, W-Gum, yam starch, gelatin, natural gum, hydroxypropylmethyl-Mierocrystalline cellulose, Xylo-Mucine, methylcellulose gum or polyvinylpyrrolidone (PVP).If necessary, can add disintegrating agent, for example crosslinked Polyvinylpyrolidone (PVP), agar or alginic acid or its salt, for example sodiun alginate.

The drageeing core has suitable dressing.For this purpose, can use concentrated sugar solution, it can randomly contain Sudan Gum-arabic, Polyvinylpyrolidone (PVP), carboxyvinyl polymer gel, polyoxyethylene glycol and/or titanium dioxide, lacquer solution and appropriate organic solvent or solvent mixture.In order to differentiate or, dyestuff or pigment can be joined in tablet or the drageeing dressing in order to characterize the combination of different promoting agents.

Can be used for oral pharmaceutical preparation and comprise leak proof fit (push-fit) capsule of making by gelatin, and by gelatin and the softening agent soft seal capsule made of glycerine or Sorbitol Powder for example.The leak proof fit capsule can contain for example lactose, tackiness agent starch and/or the lubricant mixture of talcum powder or Magnesium Stearate and optional stablizer for example for example of active ingredient and filler.In soft capsule, promoting agent can be dissolved or suspended in for example aliphatic oils of suitable liquid, whiteruss or the liquid macrogol.In addition, can add stablizer.The oral preparation that is useful on should be by the dosage that is suitable for this administration.For the buccal administration, composition can be taked the tablet prepared in a usual manner or the form of lozenge.

For intranasal administration or pass through inhalation, compound can be given with the form that aerosol injection gives by pressurized package or atomizer easily by means of suitable propelling agent for example Refrigerant 12, trichlorofluoromethane, dichloro tetrafluoro ethane, carbonic acid gas or other suitable gas used according to the present invention.Under the situation of pressurised aerosol, can determine dose unit by a kind of valve of sending metered amount is provided.Being used for the capsule of sucker or insufflator or the like and gelatin cylinder can be formulated into and contain for example powdered mixture of lactose or starch of compound and suitable powder matrix.

Compound can be mixed with form by the injection administered parenterally, for example, by bolus injection or transfusion continuously.The preparation that is used for injecting may reside in unit dosage, for example in the ampoule or multi-dose container that add sanitas.Composition can adopt the form of suspension, solution or milk sap in oily or aqueous excipient, and can contain for example suspension of allotment reagent, stabilization and/or dispersion agent.

The pharmaceutical formulations that is used for administered parenterally comprises the aqueous solution of the active compound of water-soluble form.In addition, the suspension preparation of promoting agent can be become suitable oily injectable suspensions.Suitable lipophilic solvent or vehicle comprise for example sesame oil of aliphatic oils, or Acrawax, for example ethyl oleate or triglyceride level, or liposome.Can contain the material that increases suspension viscosity in the moisture injectable suspensions, for example Xylo-Mucine, Sorbitol Powder or dextran.Randomly, suspension can also contain suitable stabilizers or increase the reagent of the solubleness of compound, to allow the preparation of highly concentrated solution.

For dosing eyes, promoting agent gives in vehicle at the acceptable eye of pharmacy, and compound can keep in touch one section adequate time with ocular surface like this, so that compound infiltrates the cornea and the interior region of eye, comprise, for example, the anterior chamber, back room, vitreum, aqueous humor, vitreous humor, cornea, iris/eyelash, lens, choroid/retina and sclera.The acceptable eye of pharmacy can be ointment, vegetables oil or sealing material with vehicle.Compound of the present invention directly can also be injected in vitreum and the aqueous humor.

Perhaps, before the use, active ingredient can be a powder type, is used for and for example aseptic, the pyrogen-free water combination of suitable vehicle.Can also for example, contain conventional suppository base for example theobroma oil or other glyceride with compound with rectal compositions suppository or be detained the form preparation of enema for example.

Compound can also be formulated as prolonged action preparation.This prolonged action preparation can give (for example, subcutaneous or intramuscular) by implantation or give by intramuscular injection.Therefore, for example, compound can be prepared with suitable polymerization or hydrophobic material (for example, the milk sap in acceptable oil) or ion exchange resin, or with sl. sol. derivative, for example the form of sl. sol. salt.

The pharmaceutical carriers that is used for hydrophobic compound is the organic polymer that comprises phenylcarbinol, non-polar surfactant, can dissolve each other with water and the cosolvent system of water.The cosolvent system can be a VPD cosolvent system.VPD is the non-polar surfactant's polysorbate 80 of phenylcarbinol, 8%w/v of 3%w/v and the solution of 65% w/v Liquid Macrogol, with the solution of straight alcohol compensation volume.VPD cosolvent system (VPD:5W) contains the VPD of the 1:1 of useful 5% D/W dilution.This cosolvent system is the solubilizing hydrophobic compound well, and when whole body gave, itself produced hypotoxicity.Naturally, under the condition of not destroying its solubleness and toxic characteristic, the ratio of cosolvent system can change significantly.In addition, the characteristic of cosolvent component can change: for example, can use other hypotoxic non-polar surfactant to replace polysorbate 80; The segment size of polyoxyethylene glycol can change; Other biocompatible polymkeric substance can substitute polyoxyethylene glycol, for example Polyvinylpyrolidone (PVP); Other sugar or polysaccharide can replace glucose.

Perhaps, can use other to be used for the delivery system of hydrophobicity pharmaceutical compound.Liposome and milk sap are the vehicle of hydrophobic drug delivery or the known example of carrier.Can also use for example methyl-sulphoxide of some organic solvent, although be cost with bigger toxicity usually.In addition, can use slow-released system to send compound, for example contain the semipermeability matrix of the solid hydrophobic polymkeric substance of therapeutical agent.Determined various slow-release materials, and understood by those skilled in the art.According to the chemical property of slow releasing capsule, slow releasing capsule can discharge several weeks of compound up to above 100 days.According to the chemical property and the biological stability of treatment reagent, can use other to make the strategy of protein stabilization.

Pharmaceutical composition can also comprise suitable solid or gel phase carrier or vehicle.The example of this carrier or vehicle comprises lime carbonate, calcium phosphate, and sugar, starch, derivatived cellulose, gelatin and polymkeric substance be polyoxyethylene glycol for example.

With the form of the salt that counter ion become compatible, can provide some compounds of the present invention with pharmacy.Can comprise that hydrochloric acid, sulfuric acid, acetate, lactic acid, tartrate, oxysuccinic acid, succsinic acid or the like form the compatible salt of pharmacy with many acid.Salt tends to more soluble in water than corresponding free alkali form or other protonic solvent.

Comprise cell cycle control agent and one or more optional other active ingredient according to pharmaceutical composition of the present invention, for example compatible and be suitable for the known antiproliferative medicament of the indication of being treated with the cell cycle control agent.

This compound is as the angiogenesis inhibitor medicament and as regulating and/or the medicament of arrestin kinase activity, thereby provides treatment for tumour or other other disease relevant with protein kinase mediated cell proliferation.

Can use the medicament of the present invention for the treatment of significant quantity to treat the disease that adjustment or adjusting mediated of protein kinase." significant quantity " be meant, when needing the Mammals of this treatment, is enough to implement to treat the amount of the disease that one or more kinase activity mediates.Therefore, for example, the treatment significant quantity of formula I compound, its salt, active metabolite or prodrug is enough adjustment, adjusting or the quantity that suppresses one or more kinase activity, can alleviate or alleviate the disease symptoms that is mediated by this activity like this.

" treatment " is meant that minimally alleviates by one or more kinase activity influence, the Mammals Human diseases situation for example of some effects at least, comprise: disease condition appears in the prevention Mammals, particularly when the discovery Mammals tends to suffer from this disease condition but also do not make diagnosis; Regulate and/or the inhibition disease condition; And/or the situation that palliates a disease.

In each specific embodiments of the inventive method described herein, unusual cell growth is a cancer, includes but not limited to lung cancer, osteocarcinoma, carcinoma of the pancreas, skin carcinoma, head or neck cancer, the melanoma of skin or intraocular, uterus carcinoma, ovarian cancer, the rectum cancer, cancer of the anal region, cancer of the stomach, colorectal carcinoma, mammary cancer, uterus carcinoma, carcinoma of fallopian tube, carcinoma of endometrium, cervical cancer, carcinoma of vagina, carcinoma vulvae, lymphogranulomatosis, esophagus cancer, carcinoma of small intestine, the endocrine system cancer, thyroid carcinoma, parathyroid carcinoma, adrenal carcinoma, soft tissue sarcoma, urethral carcinoma, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, bladder cancer, kidney or carcinoma of ureter, renal cell carcinoma, carcinoma of renal pelvis, central nervous system (CNS) tumour, primary CNS lymphoma, spine axle tumour, the brain stem neurospongioma, pituitary adenoma, or the combination of one or more above-mentioned tumour.In another embodiment of described method, described unusual cell growth is optimum hyperplasia, includes but not limited to psoriasis, benign prostatauxe or restinosis.

In the further specific embodiments of each the inventive method described herein, this method comprises that further giving a certain amount of one or more of Mammals is selected from the material of anti-tumor agents, angiogenesis inhibitor medicament, signal transduction inhibitor and antiproliferative medicament, and this quantity is effective simultaneously in the treatment of described abnormal cell growth.Compound of the present invention can combine with other anti-tumor agents, such method is disclosed among WO038716, WO038717, WO038715, WO038730, WO038718, WO038665, WO037107, WO038786, the WO038719, in they whole its content being incorporated herein as a reference.The example of anti-tumor agents comprises mitotic inhibitor, for example for example vincaleucoblastine, Vinorelbine, vindesine and vincristine(VCR) of vinca alkaloids derivative; Colchicum autumnale bases allochochine; halichondrins (halichondrine); N-benzoyl trimethylammonium-colchicinic acid methyl ether; dolastatin (dolastatin) 10; maytansine; rhizoxine, taxanes be taxol (Paclitaxel) for example; Docetaxel (taxotere); 2 '-N-[3-(dimethylamino) propyl group] glutarate (D51-7059); thio-colchicine; the trityl halfcystine; teniposide; methotrexate; azathioprine; the fluorine glycosides; cytosine arabinoside; 2 ' 2 '-difluoro Deoxyribose cytidine (gemcitabine); Zorubicin and mitomycin.Alkylating agent; cis-platinum for example; carboplatin oxiplatin; iproplatin; N-ethanoyl-DL-sarcosyl-L-leucinethylester (Asaley or Asalex); 1; 4-cyclohexadiene-1; the 4-diamino acid; 2; 5-two (1-'-aziridino)-3; 6-dioxo-diethyl ester (diaziquone); 1; 4-two (mesyloxy) butane (bisulfan or leucosulfan) chlorozotocin; clomesone (clomesone); cyano group morpholinyl Zorubicin; cyclodisone; dianhydrogalactitol (dianhydroglactitol); fluorodopan; hepsulfam; ametycin; the hycanthone ametycin; mitozolamide; 1-(2-chloroethyl)-4-(3-chloropropyl)-piperazine dihydrochloride; piperazinedione; pipobroman; porfiromycin; spiral shell is appropriate in mustard (spirohydantoin mustard); teroxirone; ormaplatin; thiotepa; triethylene melamine; uracil mustard; two (3-mesyloxy propyl group) amine hydrochlorate; mitomycin; the nitrosourea medicine is cyclohexyl-chlorethylnitrosourea for example; methylcyclohexyl-chlorethylnitrosourea 1-(2-chloroethyl)-3-(2,6-dioxo-3-piperidyl)-1-nitrosourea; two (2-chloroethyl) nitrosourea; procarbazine; dacarbazine; the compound that mustargen is correlated with is mustine hydrochlcride for example; endoxan; ifosfamide; melphalan; Chlorambucil; estramustine phosphate sodium; U-9889 and Temozolomide.The DNA metabolic antagonist, 5 FU 5 fluorouracil for example, cytosine arabinoside, hydroxyurea, 2-[(3-hydroxyl-2-pyridyl (pyrinodinyl)) methylene radical]-hydrazine carbonylsulfide acid amides, doxifluridine, 5-hydroxyl-2-formylpyridine thiosemicarbazone, α-2 '-deoxidation-6-thioguanosine, the aphidicolin glycinate, 5-azepine Deoxyribose cytidine, β-Tioguanine dezyribonucleoside, cyclotidine, guanazole, inosine ethylene glycol dialdehyde, macbecin II, pyrazolo imidazoles (pyrazolimidazole), CldAdo, pentostatin, Tioguanine, purinethol, bleomycin, 2-chlorodeoxyadenosine, thymidylate synthase inhibitor be Raltitrexed and the bent azoles disodium of Pei Mei for example, clofarabine, floxuridine and fludarabine.The DNA/RNA metabolic antagonist, for example: the L-alanosin, U-18496, U 42126, aminopterin and their derivative be N-[2-chloro-5-[[(2 for example, 4-diamino-5-methyl-6-quinazolyl) methyl] amino] benzoyl]-the L-aspartic acid, N-[4-[[(2,4-diamino-5-ethyl-6-quinazolyl) methyl] amino] benzoyl]-the L-aspartic acid, N-[2-chloro-4-[[(2,4-diamino pteridine radicals) methyl] amino] benzoyl]-the L-aspartic acid, the solubility triazinate, two chlorallyl lawsones, brequinar, Tegafur, dihydro-U-18496, methotrexate, N-(phosphono ethanoyl)-L-aspartic acid tetra-na salt, pyrazolo furans, trimetrexate, Plicamycin, dactinomycin, cryptophycin (a kind of depsipeptide class antitumour drug) and analogue for example cryptophycin-52 or, one of disclosed preferred metabolic antagonist N-(5-[N-(3,4-dihydro-2-methyl-4-oxo quinazoline-6-ylmethyl)-N-methylamino)-2-thenoyl for example in european patent application No.239362 for example)-L-L-glutamic acid; Growth factor receptor inhibitors; Cell cycle inhibitor; Embed microbiotic (intercalating antibiotics), for example Zorubicin and bleomycin; Albumen, for example Interferon, rabbit; And hormone antagonist, for example estrogen antagonist is such as Nolvadex ^TM(tamoxifen) or, for example androgen antagonist is such as Casodex ^TM(4 '-cyano group-3-(4-fluorophenyl alkylsulfonyl)-2-hydroxy-2-methyl-3 '-(trifluoromethyl) propionanilide).The mode of the separate constituent that this combination therapy can be by simultaneously, order or separate doses are treated realizes.

The angiogenesis inhibitor medicament comprises MMP-2 (matrix-metalloprotease 2) inhibitor, MMP-9 (matrix-metalloprotease 9) inhibitor and COX-II (cyclo-oxygenase II) inhibitor.Effectively the example of COX-II inhibitor comprises CELEBREX ^TM(alecoxib), valdecoxib and rofecoxib.Effectively the example of matrix metallo-proteinase inhibitor is described in the following document: WO 96/33172 (on October 24th, 1996 is open), WO 96/27583 (on March 7th, 1996 is open), european patent application No.97304971.1 (application on July 8th, 1997), european patent application No.99308617.2 (application on October 29th, 1999), WO 98/07697 (on February 26th, 1998 is open), WO 98/03516 (on January 29th, 1998 is open), WO98/34918 (on August 13rd, 1998 is open), WO 98/34915 (on August 13rd, 1998 is open), WO 98/33768 (on August 6th, 1998 is open), WO 98/30566 (on July 16th, 1998 is open), European patent publication EP 606,046 (on July 13rd, 1994 is open), European patent publication EP 931,788 (on July 28th, 1999 is open), WO90/05719 (May 31 nineteen ninety is open), WO 99/52910 (on October 21st, 1999 is open), WO 99/52889 (on October 21st, 1999 is open), WO 99/29667 (on June 17th, 1999 is open), PCT international application No.PCT/IB98/01113 (application on July 21st, 1998), european patent application No.99302232.1 (application on March 25th, 1999), Great Britain's patent application number 9912961.1 (application on June 3rd, 1999), U.S. Provisional Application No.60/148,464 (applications on August 12nd, 1999), U.S. Pat 5,863,949 (bulletins on January 26th, 1999), U.S. Pat 5,861,510 (bulletins on January 19th, 1999), with European patent publication EP 780,386 (on June 25th, 1997 open), during all these all are incorporated herein with it as a reference.Preferred L MP-2 and MMP-9 inhibitor are that those have seldom or do not have the active inhibitor of inhibition MMP-1.More more preferably those optionally suppress those of MMP-2 and/or MMP-9 with respect to other matrix-metalloprotein enzyme (being MMP-1, MMP-3, MMP-4, MMP-5, MMP-6, MMP-7, MMP-8, MMP-10, MMP-11, MMP-12 and MMP-13).

The example of signal transduction inhibitor comprises the medicament that can suppress EGFR (EGF-R ELISA) reaction, for example EGFR antibody, EGF antibody and EGFR inhibitor molecules; VEGF (vascular endothelial growth factor) inhibitor; With the erbB2 acceptor inhibitor, for example with the organic molecule or the antibody of erbB2 receptors bind, HERCEPTIN for example ^TM(Genentech, Inc.of SouthSan Francisco, California, USA).

The EGFR inhibitor is for example being made description: WO 95/19970 (July 27 nineteen ninety-five is open) in the following document, WO 98/14451 (on April 9th, 1998 is open), WO98/02434 (on January 22nd, 1998 is open) and U.S. Pat 5,747,498 (bulletins on May 5th, 1998).The EGFR inhibitor comprises but is not limited to monoclonal antibody C225 and anti-EGFR 22Mab (ImClone Systems incorporated of New York, New York, USA), compound ZD-1839 (AstraZeneca), BIBX-1382 (BoehringerIngelheim), MDX-447 (Medarex Inc.of Annandale, New Jersey, and OLX-103 (Merck﹠amp USA); Co.of Whitehouse Station, New Jersey, USA), VRCTC-310 (Ventech Research) and EGF fusion toxin (Seragen Inc.ofHopkinton, Massachusettes).The VEGF inhibitor, for example SU-5416 and SU-6668, (Sugen Inc.of South San Francisco, California USA), also can merge with the compound of formula I or give jointly.The VEGF inhibitor is described in for example following document: WO 99/24440 (on May 20th, 1999 is open), PCT International Application PCT/IB99/00797 (application on May 3rd, 1999), WO 95/21613 (August 17 nineteen ninety-five is open), WO 99/61422 (on December 2nd, 1999 is open), U.S. Pat 5,834,504 (bulletins on November 10th, 1998), WO98/50356 (on November 12nd, 1998 is open), U.S. Pat 5,883,113 (bulletins on March 16th, 1999), U.S. Pat 5,886,020 (bulletin on March 23rd, 1999), U.S. Pat 5,792,783 (bulletins on August 11st, 1998), WO 99/10349 (on March 4th, 1999 is open), WO 97/32856 (on September 12nd, 1997 is open), WO 97/22596 (on June 26th, 1997 is open), WO 98/54093 (on December 3rd, 1998 is open), WO98/02438 (on January 22nd, 1998 is open), WO 99/16755 (on April 8th, 1999 is open), with WO 98/02437 (on January 22nd, 1998 open), during all these all are incorporated herein with it as a reference.Other example of some concrete VEGF inhibitor be IM862 (CytranInc.of Kirkland, Washington, USA); Genentech, Inc.of SouthSan Francisco, the anti-VEGF monoclonal antibody of California; And angiozyme, a kind of come from ribozyme (Boulder, Colorado) and Chiron (Emeryville, synthetic kernel carbohydrase California).The ErbB2 acceptor inhibitor, GW-282974 (GlaxoWelcome pic) and monoclonal antibody AR-209 (Aronex Pharmaceuticals Inc.of The Woodlands, Texas for example, USA) and 2B-1 (Chiron), can give with formula I combination of compounds.This erbB2 inhibitor comprises those described in following document: WO 98/02434 (on January 22nd, 1998 is open), WO99/35146 (on July 15th, 1999 is open), WO 99/35132 (on July 15th, 1999 is open), WO 98/02437 (on January 22nd, 1998 is open), WO 97/13760 (on April 17th, 1997 is open), WO95/19970 (July 27 nineteen ninety-five is open), U.S. Pat 5,587,458 (bulletin on December 24th, 1996) and U.S. Pat 5,877,305 (bulletins on March 2nd, 1999), every piece is incorporated into herein as a reference with its integral body.Effectively the ErbB2 acceptor inhibitor also is described in the following document in the present invention: U.S. Provisional Application No.60/117,341, application on January 27th, 1999, U.S. Provisional Application No.60/117 with application on January 27th, 1999,346, all these two pieces all be incorporated herein with it in as a reference.

Operable other antiproliferative medicament comprises the enzyme inhibitors of farnesyl protein transferase and the inhibitor of receptor tyrosine kinase PDGFr, comprises the compound that following U.S. Patent application is disclosed and require: 09/221946 (application on December 28th, 1998); 09/454058 (application on December 2nd, 1999); 09/501163 (application on February 9th, 2000); 09/539930 (application on March 31st, 2000); 09/202796 (application on May 22nd, 1997); 09/384339 (application on August 26th, 1999); With 09/383755 (application on August 26th, 1999); Disclosed with following U.S. Provisional Patent Application and require compound: 60/168207 (application on November 30th, 1999); 60/170119 (application on December 10th, 1999); 60/177718 (application on January 21st, 2000); 60/168217 (application on November 30th, 1999) and 60/200834 (application on May 1st, 2000).During the patent application of front and each of temporary patent application piece of writing is incorporated herein with its integral body as a reference.

The compound of formula I also can use with other efficacious agents in treatment abnormal cell growth or tumour, this medicament includes but not limited to: the medicament that can improve the antitumor immune reaction, for example CTLA4 (cytotoxic lymphocyte (lymphocite) antigen 4) antibody reaches other and can block the medicament of CTLA4; With antiproliferative medicament other farnesyl protein transferase inhibitors for example.Can be used to concrete CTLA4 antibody of the present invention and comprise described in the U.S. Provisional Application 60/113,647 (application on December 23rd, 1998) those, during it all is incorporated herein as a reference.

Detailed description of the present invention and preferred embodiment

Medicament of the present invention can use reaction path as described below and synthetic route, adopts the obtainable technology in this area, use the starting raw material that obtains easily to prepare.

The following examples are described the preparation of the concrete preferred compound of the present invention in detail.The technician will recognize that described chemical reaction can be suitable for preparing many other kinase inhibitor of the present invention easily.For example; can successfully be undertaken according to the synthetic of non-illustrative compound of the present invention by the improvement that it will be apparent to those skilled in the art; for example; by protecting interfering group rightly; by becoming other suitable reagent known in the art, or improve by the routine of carrying out reaction conditions and to carry out.Perhaps, other reaction disclosed herein or known in the art will be had the practicality of preparation other compound of the present invention by approval.

In embodiment as described below, unless otherwise stated, all temperature show with centigradetemperature, and all umbers and percentage ratio show with weight.Reagent from goods providers for example Aldrich chemical company or Lancaster Synthesis Ltd. buy, and can use, unless otherwise stated without being further purified.With hydrolith distillatory tetrahydrofuran (THF) (THF) and N, dinethylformamide (DMF) is bought from Aldrich, in the bottle of positiver sealing, and the state use to receive.All solvents can use standard method purifying well known by persons skilled in the art, unless otherwise stated.

The reaction of below listing is normally carried out under following condition: at the direct draught of argon gas or have a drying tube, in room temperature (except as otherwise noted), in anhydrous solvent, and this reaction flask has rubber septum, to introduce substrate and reagent by syringe.Glassware be oven dry and/or heat drying.Analytical tlc (TLC) is carried out on the dull and stereotyped Analtech of silica gel 60F254 (0.25mm) of glass back plate, and shows with suitable solvent ratios (V/V) wash-out with in suitable place.By the TLC assaying reaction, and by consumption of raw materials amount judgement reaction terminating.

The colour developing of chromatographic sheet is carried out and heat-activated with aubepine streak reagent or Sonnenschein's reagent (Aldrich Chemical, 20wt% is in ethanol).Aftertreatment typically by with reaction solvent or extraction solvent reaction volume being doubled, is carried out with specified solution washing then, uses 25% extraction volume by volume, unless otherwise stated.Product solution was used anhydrous Na before filtering ₂SO ₄Or MgSO ₄Drying, and on rotatory evaporator this solvent of reduction vaporization, and indicated when desolvating when removing in a vacuum.Use quick silica gel of Baker level (47-61 μ m) and silica gel: the ratio of raw material is approximately 20: 1 to 50: 1 carries out flash column chromatography people such as (, J.Org.Chem., 43,2923 (1978)) Still, except as otherwise noted.Under pressure that embodiment shows or environmental stress, carry out hydrogenation.

¹The H-NMR spectrum is what to write down on the Bruker instrument that moves under 300MHz or the 500MHz, and ¹³C-NMR spectrum operating records under 75MHz.The NMR wave spectrum is with CDCl ₃Solution (with the ppm report) obtains, and uses chloroform as reference standard (7.25ppm and 77.00ppm) or CD ₃OD (3.4ppm and 4.8ppm and 49.3ppm), or in suitable time marquis uses, mark tetramethylsilane (0.00ppm).Can use other NMR solvent if desired.When report peak value multiplicity, use following abbreviation: s (unimodal), d (bimodal), t (triplet), m (multiplet), br (wide), dd (double doublet), dt (two triplet).When obtaining coupling constant, report with hertz (Hz).

Infrared (IR) spectrum be with pure oil or KBr compressing tablet form, on the Perkin-ElmerFT-IR spectrometer, write down, and when providing when spectrum, with wave number (cm ^-1) report.Mass spectrum is to use LSIMS or electron spray(ES) to obtain.All fusing points (mp) are uncorrected.

The starting raw material of Shi Yonging can commercially be bought in an embodiment, and/or can be by technology preparation known in the art.

Embodiment 1:{5-[3-(4,6-two fluoro-1H-benzimidazolyl-2 radicals-yl)-1H-indazole-5-yl]-4-methyl-pyridin-3-yl methyl }-ethyl-amine

(a) intermediate 1a-(5-bromo-4-methyl-pyridin-3-yl methyl)-ethyl-amine

Under nitrogen atmosphere, (6.74g 33.7mmol) [for this compound of preparation, sees Reich, S.R. with 5-bromo-4-methyl-pyridine-3-formaldehyde; Bleckman, T.M.; Kephart, S.E.; Romines, W.H.; Wallace, M.B., US patent6,555,539, April 29,2003.] be dissolved in the methyl alcohol (290mL).With the methanol solution that dripped ethamine in 30 minutes (2.0M, 90ml, 180mmol).At room temperature further continue to stir 30 minutes.

In the flask that separates, (2.33g 37.1mmol) is dissolved in the methyl alcohol (150mL) with sodium cyanoborohydride.(2.53g 18.5mmol) and at room temperature continues to stir 20 minutes to add Zinc Chloride Anhydrous.Then this solution (zinc/cyano group hydroborate) is joined in above-mentioned aldehyde/ethylamine solution at leisure.With 2.0M HCl/ methyl alcohol (120mL) reaction soln is acidified to pH value 4, at room temperature stirred then 18 hours.

Remove by rotary evaporation and to desolvate, and resistates is distributed between ethyl acetate and 10% aqueous sodium carbonate.With dried over mgso organic extraction and concentrated, obtain crude product amine 1a (7.36g, 95%) orange oil, it is not further purified and in next step, uses:

¹HNMR(CDCl ₃)δ8.53(s，1H)，8.31(s，1H)，3.77(s，2H)，2.67(q，J＝7.0Hz，2H)，2.42(s，3H)，1.11(t，J＝7.0Hz，3H).

(b) intermediate 1b-(5-bromo-4-methyl-pyridin-3-yl methyl)-ethyl-carboxylamine tertiary butyl ester

With two dimethyl dicarbonate butyl esters (10.43g, 47.8mmol) join crude product amine 1a (7.36g, in THF 32.1mmol) (400mL) solution, then add aqueous sodium hydroxide solution (1.0M, 101mL).At room temperature this two-phase solution was stirred 20 hours energetically.Solution is distributed between water and ethyl acetate; Use the dried over mgso organic extraction, filter and concentrate.The crude product yellow oil that so obtains by silica gel chromatography purifying (ethyl acetate/hexane with 10% to 30% is carried out gradient elution), is obtained bromopyridine 1b (5.37g, 51%) yellow oil:

¹H NMR(CDCl ₃)δ8.58(s，1H)，8.22(s，1H)，4.47(s，2H)，3.17(br s，2H)，2.37(s，3H)，1.45(s，9H)，1.03(t，J＝7.2Hz，3H).

(c) intermediate 1c-5-iodo-1-(tetrahydrochysene-pyrans-2-yl)-1H-indazole-3-carboxylic acid methoxyl group-methyl-acid amides

According to people such as Sun [Sun, J.-H.; Teleha, C.A.; Yan, J.-S.; Rogers, J.D.; And Nugiel, D.A., J.Org.Chem.1997,62,5627] method, with dihydropyrane 5-iodo-1H-indazole-3-carboxylic acid methoxyl group-methyl-acid amides [for the preparation of this compound, is seen Reich, S.R.; Bleckman, T.M.; Kephart, S.E.; Romines, W.H.; Wallace, M.B., US patent 6,555,539 B2, April29,2003.] alkylation, obtain acid amides 1c (typically＞90%) off-white powder:

¹HNMR(DMSO-d ₆)δ8.37(s，1H)，7.74(dd，J＝1.5，8.8Hz，1H)，7.68(d，J＝8.8Hz，1H)，5.97(dd，J＝2.3，9.0Hz，1H)，3.88(m，2H)，3.79(s，3H)，3.42(s，3H)，2.35(m，1H)，2.03(m，2H)，1.75(m，1H)，1.58(m，2H).

(d) intermediate 1d-5-iodo-1-(tetrahydrochysene-pyrans-2-yl)-1H-indazole-3-formaldehyde

Lithium aluminum hydride (1.2 equivalent) is added in batches in THF cold (＜5 ℃) solution of acid amides 1c (1.0 equivalent).Continue down to stir at＜5 ℃, till reaction is finished, typically stirred 30 minutes.Under＜5 ℃ condition,, whole mixtures are poured into 0.4N NaHSO by slow adding ethyl acetate quencher reaction ₄In.With salt water washing organic layer, use dried over mgso, concentrate, obtain aldehyde 1d (typically～70%) off-white powder with the silica gel chromatography purifying:

¹H NMR(CDCl ₃)δ10.15(s，1H)，8.47(s，1H)，7.82(dd，J＝1.5，8.7Hz，1H)，7.78(d，J＝8.5Hz，1H)，6.04(dd，J＝2.3，9.28Hz，1H)，3.85(m，2H)，2.35(m，1H)，2.05(m，2H)，1.76(m，1H)，1.60(m，2H).

(e) intermediate 1e-ethyl-{ 5-[3-formyl radical-1-(tetrahydrochysene-pyrans-2-yl)-1H-indazole-5-yl]-4-methyl-pyridin-3-yl methyl }-carboxylamine tertiary butyl ester

With iodo indazole 1d (3.56g, 10.0mmol), two (tetramethyl ethylene ketone acyls) (pinacolato) two boron (2.79g, 11mmol), potassium acetate (2.74g, 30mmol) with [1,1 '-two (diphenylphosphino)-ferrocene] dichloro palladium (II) and methylene dichloride (245mg, 0.3mmol) mixture be dissolved in the N,N-dimethylacetamide (60mL).By vacuumizing solution is outgased (till solvent begins bubbling), and purge, in 80 ℃ oil bath, heated 2 hours then with argon gas (3 circulations).Slightly after the cooling (to～50 ℃), add bromopyridine 1b (3.62g, N,N-dimethylacetamide 11mmol) (40mL) solution, then add deionized water (10mL) and potassiumphosphate (3.18g, 15mmol).With the solution degassing, (347mg 0.3mmol), outgases once more to add tetrakis triphenylphosphine palladium (O).Mixture was stirred 4.5 hours in 90 ℃ oil bath.Be cooled to after the room temperature, mixture with ethyl acetate (300mL) dilution, is washed with deionized water (150mL) and saturated sodium-chloride (100mL).Use the dried over mgso organic layer, filter and be concentrated into crude product reddish black oil (9.43g).By silica gel chromatography purifying (with 50-100% ethyl acetate/hexane wash-out), obtain coupling product 1e (2.9462g) orange oil.This product ¹H NMR shows that it is by～1 normal tetramethyl ethylene ketone pollution.Obtain the tiny yellow powder of pure 1e (2.0853g, 44%) with the hexane grinding:

¹H NMR(CDCl ₃)δ10.25(s，1H)，8.39(s，1H)，8.34(s，1H)，8.22(s，1H)，7.74(d，J＝8.7Hz，1H)，7.38(dd，J＝1.5，8.5Hz，1H)，5.88(dd，J＝2.8，9.2Hz，1H)，4.53(s，2H)，4.03(m，1H)，3.81(m，1H)，3.24(br s，2H)，2.60(m，1H)，2.18(s，3H)，2.15(m，2H)，1.77(m，1H)，1.65(m，2H)，1.47(s，9H)，1.09(t，J＝7.0Hz，1H).

(f) intermediate 1f-{5-[3-(4,6-two fluoro-1H-benzimidazolyl-2 radicals-yl)-1-(tetrahydrochysene-pyrans-2-yl)-1H-indazole-5-yl]-4-methyl-pyridin-3-yl methyl-ethyl-carboxylamine tertiary butyl ester

With aldehyde 1e (2.05 grams, 4.28mmol), 1,2-diamino-3, the 5-phenyl-difluoride (617 milligrams, 4.28mmol) and sodium bisulfite (891 milligrams 8.57mmol) are dissolved in N, in the N-N,N-DIMETHYLACETAMIDE (43 milliliters), and in 120 ℃ oil bath, heated 21 hours.Be cooled to after the room temperature, mixture is diluted with ethyl acetate (100mL), and wash with semi-saturation sodium chloride aqueous solution (75mL, 1: 1 mixture of deionized water and saturated sodium-chloride water solution).(2 * 100ml) strip with ethyl acetate with water layer.Merge all organic extractions, use dried over mgso, be concentrated into brown tar (3.39g).Crude product with silica gel chromatography purifying (ethyl acetate/hexane with 70% to 100% is carried out gradient elution), is obtained benzoglyoxaline product 1f (2.11g, 81%) brown foam: ¹H NMR (CD ₃OD) δ 8.46 (s, 1H), 8.41 (s, 1H), 8.29 (s, 1H), 7.87 (d, J=8.6Hz, 1H), 7.46 (dd, J=1.3,8.6Hz, 1H), 7.13 (m, 1H), 6.84 (m, 1H), 5.99 (dd, J=2.3,9.9Hz, 1H), 4.60 (s, 2H), 4.01 (m, 1H), 3.86 (m, 1H), 3.32 (m, 2H is covered by solvent peak) 2.67 (m, 1H), 2.28 (s, 3H), 2.18 (m, 2H), 1.89 (m, 1H), 1.73 (m, 2H), 1.47 (s, 9H), 1.13 (t, J=7.1Hz, 1H). ultimate analysis: (C ₃₃H ₃₆F ₂N ₆O ₃0.4H ₂O) C, H, N, F.

(g) embodiment 1-(5-[3-(4,6-two fluoro-1H-benzimidazolyl-2 radicals-yl)-1H-indazole-5-yl]-4-methyl-pyridin-3-yl methyl-ethyl-amine

With triethyl silicane (976 milligrams, 8.40mmol) and trifluoroacetic acid (12.9 milliliters, 168mmol) join 1f (2.02 the gram, in methylene dichloride 3.36mmol) (12.9 milliliters) solution.Mixture was at room temperature stirred 3.5 hours.Remove volatile matter by rotary evaporation, with hexanaphthene (10mL) and ammonium hydroxide aqueous solution (2N, 20mL) processing resistates.After the violent stirring 15 minutes, form the pink precipitation, it is collected by suction filtration.Extract filtrate with ethyl acetate (3x 50mL).Merge organic extraction, use dried over mgso, filtration, be concentrated into orange solid (～0.4g).This solid is joined in the pink precipitation that obtains above, with column chromatography purification (with the mixture wash-out of 1% strong aqua-19% straight alcohol-80% methylene dichloride).The product (1.23g off-white color solid) that so obtains is further ground purifying with hexanaphthene, obtains pure 1 (1.09g, 74%) off-white color solid:

¹H NMR (DMSO-d ₆) δ 13.81 (s of non-constant width, 1H), 8.46 (s, 1H), 8.35 (s, 2H), 7.76 (d, J=8.6Hz, 1H), 7.44 (dd, J=1.3,8.6Hz, 1H), 7.17 (m, 1H), 7.07 (t of d, Jt=1.5Hz, Jd=10.6Hz, 1H) 3.78 (s, 2H), 2.63 (q, J=7.1Hz, 2H), 2.25 (s, 3H), 1.07 (t, J=7.1Hz, 3H) .HRMS[M+H] ⁺Calculated value: 419.1791; Measured value: 419.1811. ultimate analysis (C ₂₃H ₂₀F ₂N ₆1.1H ₂O) C, H, N, F.

Embodiment 2: ethyl-5-[3-(5-fluoro-1H-benzimidazolyl-2 radicals-yl)-1H-indazole-5-yl]-4-methyl-pyridin-3-yl methyl }-amine

(a) intermediate 2a-ethyl-5-[3-(5-fluoro-1H-benzimidazolyl-2 radicals-yl)-1-(tetrahydrochysene-pyrans-2-yl)-1H-indazole-5-yl]-4-methyl-pyridin-3-yl methyl-the carboxylamine tertiary butyl ester

Use the method identical with synthetic intermediate 1f, (894mg is under existence 8.59mmol) at sodium bisulfite, with aldehyde 1e (2.06g, 4.29mmol) with 4-fluoro-1,2-phenylenediamine (542mg, 4.29mmol) condensation, obtain the orange foam of benzoglyoxaline 2a (2.04g, 78%): ¹H NMR (DMSO-d ₆, because tautomerization, some peaks are dual)

δ 13.15 and 13.13 (2s, 1H are together), 8.42 and 8.41 (2s, 1H are together), 8.39 (s, 1H), 8.32 (s, 1H), 7.77 (dd, J=1.0,8.9Hz, 1H), (7.70 (dd, J=4.8 is 8.8Hz) with 7.27 (dd, J=2.5,9.1Hz) 1H is together], 7.52 (m, 2H), 7.07 (m, 1H), 6.07 (d, J=9.3Hz 1H), 4.53 (s, 2H), 3.96 (m, 1H), 3.86 (m, 1H), 3.32 (m, and 2H) 2.60 (m, 1H), 2.18 (s, 3H), 2.12 (m, 2H), 1.83 (m, 1H), 1.65 (m, 2H), 1.41 (s, 9H), 1.05 (t, J=7.0Hz, 3H). ultimate analysis: (C ₃₃H ₃₇FN ₆O ₃0.5H ₂The O0.2 hexane) C, H, N, F.

(b) embodiment 2-ethyl-5-[3-(5-fluoro-1H-benzimidazolyl-2 radicals-yl)-1H-indazole-5-yl]-4-methyl-pyridin-3-yl methyl-amine

With the method same with the deprotection of 1f, with intermediate 2a (1.95g, 3.34mmol) change title compound 2 (1.04g, 74%) pale solid into:

¹H NMR(DMSO-d ₆，

Because tautomerization, some peaks are dual) and δ 13.80 (very br s, 1H), 13.12 (s of non-constant width) 1H), 8.46 (s, 1H), 8.39 (s, 1H), 8.34 (s, 1H), 7.69 with 7.25 (2m, 1H are together), 7.48 (m, 1H), 7.44 (dd, J=1.5,8.6Hz, 1H), 7.05 (m, 1H), 3.77 (s, 2H), 2.62 (q, J=7.3Hz, 2H), 2.25 (s, 3H), 1.07 (t, J=7.1Hz, 3H). ultimate analysis: (C ₂₃H ₂₁FN ₆1.1H ₂O) C, H, N, F.

Biological chemistry and biological assessment

The kinase whose activity of cyclin dependent be by the radiophosphorus hydrochlorate from [ ³²P] ATP or [ ³³P] ATP is incorporated into that enzymatic, the time-varying combination of protein substrate quantizes to measure.Unless otherwise noted, test be in 98 hole culture dish, with the cumulative volume of 50 μ L, each be reflected at 10mM HEPES (the N-[2-hydroxyethyl] piperazine-N '-[2-ethanesulfonic acid] (pH 7.4), 10mM MgCl ₂, 25 μ M Triphosadens (ATP), 1mg/mL ovalbumin, 5 μ g/mL leupeptins, 1mM dithiothreitol (DTT), 10mM β-glycerophosphate, 0.1mM vanadic acid sodium, 1mM Sodium Fluoride, 2.5mM ethylene glycol-two (β-aminoethyl ether)-N, N, N ', N '-tetraacethyl (EGTA), 2% (v/v) methyl-sulphoxide and 0.03-0.4 μ Ci[32/33P] carry out under the existence of ATP.With the enzyme initiation reaction of 30 ℃ of cultivations, after 20 minutes, by adding ethylenediamine tetraacetic acid (EDTA) (EDTA) to 250mM and termination reaction.Then on soluble cotton or phosphorylated cotton film, use 96 hole porous filters to catch phosphorylation matrix, by removing unconjugated radioactivity with 0.85% phosphoric acid repeated washing.By desciccator diaphragm is exposed to phosphorescence image scan analyser radioactivity is quantized.

Apparent Ki value is measured by following method: the enzymic activity of measuring in the presence of the different concns inhibitor compound deducts the radioactive background of measuring when not having enzyme.Use Kaleidagraph (Synergy Software) will suppress data and insert the competitive inhibition equation, or use software KineTic (BioKin Ltd.) inserts it competitiveness inhibition equation of combining closely.

The inhibition of CDK4/Cyclin D retinoblastoma kinase activity

Use traditional biochemical chromatographic technique, the mixture of (1-264) cyclin D3 of the mixture of human CDK4 of purification and cyclin D3 or human CDK4 and gene brachymemma (is for example seen from the insect cell that infects corresponding rhabdovirus expression vector simultaneously, Meijerand Kim, " Chemical Inhibitors of Cyclin-Dependent Kinases; " Methods in Enzymol, .vol.283 (1997), pp.113-128.).Measure enzyme complex (5 or 50nM) with 0.3-0.5 microgram purification of Recombinant retinoblastoma protein fragment (Rb) as matrix.For the ease of purifying, the Rb fragment of design (the residue 386-928 of natural retinoblastoma protein; 62.3kDa) contain the most phosphorylation site of in natural 106-kDa protein, finding, and the mark of six histidine residues.Catch phosphorylation Rb matrix by on nitrocellulose membrane, carrying out micro-filtration, and use aforesaid phosphorescence image scan analyser to quantize.In order to measure the inhibitor of combining closely, enzyme complex concentration is lower than 5nM, and the mensuration time length is extended down to 60 minutes, and during this period, the time-dependent manner of the formation of product is linear.

The inhibition of CDK2/Cyclin A retinoblastoma kinase activity

Use disclosed method (people such as Rosenblatt, " Purification andCrystallization of Human Cyclin-dependent Kinase 2; " J.MoLBiol., vol.230,1993, pp.1317-1319), CDK2 purifies from the insect cell of infecing rhabdovirus expression vector.Purification cyclin A from the Bacillus coli cells of expressing total length reconstitution cell cyclin A, and produce truncate cyclin A by the limited proteolysis effect and constitute thing, and by the previously described mode (people such as Jeffrey that purifies, " Mechanism of CDK activation revealed by the structureof a cyclin A-CDK2 complex; " Nature, vol.376 (27July 1995), pp.313-320).The mixture of preparation CDK2 and proteoclastic (proteolyzed) cyclin A is also by the gel-filtration purifying.The matrix of this test is identical with the Rb matrix fragment that is used for the CDK4 test, and the method for CDK2/ cyclin A and CDK4/ cyclin D3 test is substantially the same, except CDK2 provides with 150nM or 5nM.By measuring the Ki value as mentioned above.

By for example stimulation of the cell proliferation carried out of VEGF and other material of somatomedin, depend on that their induction phases answer each the automatic phosphorylation in the Tyrosylprotein kinase of acceptor.Therefore, by the ability of these somatomedin institute inductive kinases inhibitor blocking-up cell proliferations, ability direct and the automatic phosphorylation of its blocking-up acceptor has relation.In order to measure the protein kinase inhibiting activity of compound, use array structure down.

The inhibition of cell growth: Cytotoxic evaluation

Use tetrazolium salts to test to measure the inhibition of cell growth, its based on viable cell with bromination 3-(4,5-dimethylthiazole-2-yl)-2,5-[2H]-phenylbenzene tetrazolium (MTT) is reduced to the ability of first (formazan).(Mossman，Journal of Immunological Methods，vol.65(1983)，pp.55-58)。Detect water-fast purple first (forrmazan) product with spectrophotometry then.HCT 116 cell line growths are in 96 hole culture dish.In McCoy ' s 5A medium, the volume of cell with 135 μ l/ holes applied in suitable medium.Before adding inhibitor compound, plate was cultivated four hours.The inhibitor compound that in 0.5% (v/v) methyl-sulphoxide (15 μ L/ hole), adds different concns, and with cell at 37 ℃ of (5%CO ₂) four to six days (according to cell type) of following cultivation.When cultivating end, add the final concn of MTT to 0.2mg/mL, and cell is cultivated above 4 hours down at 37 ℃.Centrifugal plate and remove medium after, the absorbancy of first (formazan) (being dissolved in the methyl-sulphoxide) can be measured in 540 nanometers.The concentration that causes 50% growth inhibiting inhibitor compound is by inhibitor concentration the linear portion of the semilog plot of inhibition percentage ratio to be assigned to determine.All results compare with the control cells of only handling with 0.5% (v/v) methyl-sulphoxide.

The protein kinase group

In order to measure the inhibition activity of the CDK that contrasts with the range protein kinases, compound of the present invention screens at many histone kinases.Use Davies, people such as S are at Specificity and Mechanism of Action of Some Commonly UsedProtein Kinase Inhibitors, Biochem J.351, method and the material described among the 95-105 (2000) are tested, and are incorporated herein by reference in their incorporated herein by.At a histone matter kinases SCREENED COMPOUND, protein kinase comprises: AMP-activated protein kinase (AMPK), checkpoint kinase (CHK1), casein kinase 1 and 2 (CK1 and CK2), cytoplasmic tyrosine kinase (CSK), dual serine/threonine/Tyrosylprotein kinase (DYRK1A), Glycogen Synthase kinase 3 (GSK3B), the terminal kinases (JNK) of c-Jun N-, lymphocyte kinases (LCK), mitogen-activated protein kinase 2, K-1a, K2 (MAPK2, MAPKAP-K1a and MAPKAP-K2), mapk kinase (MKK1, be also referred to as MEK), mitogen and stress-activated protein kinase 1 (MSK1), never-in-mitotic kinase 6 (NEK6), p70 ribosomal protein S6 kinases (P70s6K1), 3-phosphoinositide-deopendent protein kinase 1 (PDK1), phosphorylase kinase (PHK), protein kinase A (PKA), protein kinase B (PKB is also referred to as Akt), protein kinase C (PKCa), p38-adjusting/activated protein kinase (PRAK), Rho-deopendent protein kinase (ROCK-II), stress-activated protein kinase 2a, 2b, 3 and 4 (SAPK-2A or p38, SAPK-2b or p38 β 2, SAPK3 or p38 γ, SAPK4 or p38 δ, respectively), and serum-and glucocorticosteroid-inducible kinase (SGK).Compound activity is divided into weak type (＜50%), moderate type (50-75%) and strongly inhibited type (＞75%).Illustrated as following table 1, compound of the present invention is effective CDK inhibitor, and with select from U.S. Pat No.6,555,539 3,5 indazoles that replace are compared, and have beyond thought better CDK inhibitor selectivity.

The kinase activity of 3,5 indazole compounds

Table 1

	A *	B *	C *	D *	1	2
CDK2/A K 1 (nM)	0.52	0.25	0.80	2.40	0.78	0.47
							HCT-116IC 50 (nM)	70	90	86	＞500	120	22
Kinase
							AMPK	++	++	++	++	++	++
CDK2/A	++	++	++	++	++	++
							CHK1	+	+	-	-	-	-
CK1	-	-	-	-	-	-
							CK2	-	-	-	-	-	-
CSK	-	-	-	-	-	-
							DYRK1A	++	++	++	++	++	++
GSK3B	++	++	++	++	++	++
							JNK	-	-	-	-	-	-
LCK	++	++	+	-	+	+
							MAPK2	-	-	-	-	-	-
MAPKAP-K1a	++	++	++	+	++	-
							MAPKAP-K2	-	-	-	-	-	-
MKK1	++	+	+	+	-	-
							MSK1	-	-	-	-	-	-
NEK6	-	-	-	-	-	-
							P70s6K1	+	+	-	-	-	-
PDK1	+	-	-	-	-	-
							PHK	++	+	+	-	+	++
PKA	++	++	+	-	-	-
							PKB	-	-	-	-	-	-
PKCa	+	++	+	-	-	-
							PRAK	++	+	++	+	-	-
ROCK-II	++	+	+	-	-	+
							SAPK-2A	-	-	-	-	-	-
SAPK-2b	-	-	-	-	-	-
							SAPK3	-	-	-	-	-	-
SAPK4	-	-	-	-	-	-
							SGK	+	+	-	-	-	-

++ strongly inhibited＞75%

+ moderate suppresses 50-75%

-weak inhibition＜50%

* compd A, B, C and D be at U.S. Pat No.6, describes in 555,539.

Is VEGF or the kinase whose CDK selectivity of FGFR (fibroblast growth factor acceptor) in order to measure formula I compound to other kinases, can easily carry out extra test.Such test is at U.S. Pat No.6,555,539 and WO 03/004488 in describe, and be known in this area.

Can be with aforesaid exemplary compound, according to following common embodiment preparation becoming pharmaceutical composition.

Parenteral composition

In order to prepare the parenteral pharmaceutical composition that is suitable for drug administration by injection, the water-soluble salt of the formula I compound of 100mg can be dissolved among the DMSO, mix with 0.9% stroke-physiological saline solution of 10mL then.Mixture is incorporated in the dosage device formulation that is suitable for drug administration by injection.

Oral compositions

In order to prepare the pharmaceutical composition that is used for oral delivery, the formula I compound of 100mg can be mixed with the 750mg lactose.Can incorporate mixture into oral dosage units, for example be suitable for oral hard capsule.

The present invention has simultaneously quoted as proof concrete and embodiment preferred illustrates, and it will be recognized by those skilled in the art, by normal experiment and practice of the present invention, can carry out changes and improvements.Therefore, the present invention does not want to be limited by above-mentioned specification sheets, but is limited by additional claims and their equal mode.

Claims (9)

1. the compound of formula I or pharmacologically acceptable salts or solvate:

R wherein ¹, R ², R ³And R ⁴Be selected from H, halogen, cyano group, nitro, trifluoromethoxy, trifluoromethyl, azido-, hydroxyl, C ₁-C ₆Alkoxyl group, C ₁-C ₁₀Alkyl, C ₂-C ₆Thiazolinyl, C ₂-C ₆Alkynyl ,-C (O) R ⁵,-C (O) OR ⁵,-OC (O) R ⁵,-NR ⁵C (O) R ⁶,-C (O) NR ⁵R ⁶,-(CR ⁵R ⁶) NR ⁷R ⁸,-CR ⁵R ⁶NR ⁷R ⁸,-NR ⁵OR ⁶,-SO ₂NR ⁵R ⁶,-S (O) _j(C ₁-C ₆Alkyl), wherein j is 0 to 2 integer ,-(CR ⁵R ⁶) _t(C ₆-C ₁₀Aryl) ,-(CR ⁵R ⁶) _t(C ₃-C ₁₀Cycloalkyl) ,-(CR ⁵R ⁶) _t(4-10 unit heterocycle) ,-(CR ⁵R ⁶) _qC (O) (CR ⁷R ⁸) _t(C ₆-C ₁₀Aryl) ,-(CR ⁵R ⁶) _qC (O) (CR ⁷R ⁸) _t(C ₃-C ₁₀Cycloalkyl) ,-(CR ⁵R ⁶) _qC (O) (CR ⁷R ⁸) _t(4-10 unit heterocycle) ,-(CR ⁵R ⁶) _tO (CR ⁷R ⁸) _q(C ₆-C ₁₀Aryl) ,-(CR ⁵R ⁶) _tO (CR ⁷R ⁸) _q(C ₃-C ₁₀Cycloalkyl) ,-(CR ⁵R ⁶) _tO (CR ⁷R ⁸) _q(4-10 unit heterocycle) ,-(CR ⁵R ⁶) _qSO ₂(CR ⁷R ⁸) _t(C ₆-C ₁₀Aryl) ,-(CR ⁵R ⁶) _qSO ₂(CR ⁷R ⁸) _t(C ₃-C ₁₀Cycloalkyl) and-(CR ⁵R ⁶) _qSO ₂(CR ⁷R ⁸) _t(4-10 unit heterocycle), wherein q and t each be from 0 to 5 integer independently, above-mentioned R ¹, R ², R ³Or R ⁴1 or 2 ring carbon atom of the cycloalkyl of group or heterocyclic moiety is optional by an oxo (=O) group replacement, each R ⁵, R ⁶, R ⁷And R ⁸Be independently selected from H, C ₁-C ₆Alkyl; And R wherein ¹, R ², R ³And R ⁴Not H simultaneously.

2. according to compound or pharmacologically acceptable salts or solvate, the wherein R of claim 1 ¹Be-(CR ⁵R ⁶) NR ⁷R ⁸, R ², R ³And R ⁴Be independently selected from H or F.

3. according to compound or pharmacologically acceptable salts or solvate, the wherein R of claim 1 ¹Be the ethylamino methyl, R ³Be H, R ²And R ⁴Be F.

4. according to compound or pharmacologically acceptable salts or solvate, the wherein R of claim 1 ¹Be the ethylamino methyl, R ²And R ⁴Be H, R ³Be F.

5. be selected from following compound or pharmacologically acceptable salts or solvate:

Or

6. treatment is by the disease of kinase whose inhibition mediation or the method for obstacle, comprises patient that these needs are arranged compound or pharmacologically acceptable salts or the solvate according to claim 5.

7. a method for the treatment of fungi infestation, cancer or tumour and other and undesirable vasculogenesis and/or cell proliferation disease states associated comprises compound or pharmacologically acceptable salts or the solvate according to the claim 5 that give significant quantity.

8. by giving compound or pharmacologically acceptable salts or the solvate method that optionally suppress CDK kinase activity of son according to claim 5.

9. one kind contains according to the compound of claim 5 or the pharmaceutical composition of pharmacologically acceptable salts or solvate.