CN105218548A - A kind of novel heterocyclic compounds and preparation method thereof and the purposes as kinase inhibitor - Google Patents

A kind of novel heterocyclic compounds and preparation method thereof and the purposes as kinase inhibitor Download PDF

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Publication number
CN105218548A
CN105218548A CN201410250746.8A CN201410250746A CN105218548A CN 105218548 A CN105218548 A CN 105218548A CN 201410250746 A CN201410250746 A CN 201410250746A CN 105218548 A CN105218548 A CN 105218548A
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compound
preparation
paraffin
reaction
purposes
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Inventor
江磊
耿美玉
丁健
刘磊
黄敏
查传涛
艾菁
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Shanghai Institute of Materia Medica of CAS
Shanghai Haihe Pharmaceutical Co Ltd
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Shanghai Institute of Materia Medica of CAS
Shanghai Haihe Pharmaceutical Co Ltd
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Priority to CN201410250746.8A priority Critical patent/CN105218548A/en
Priority to PCT/CN2015/079384 priority patent/WO2015188681A1/en
Publication of CN105218548A publication Critical patent/CN105218548A/en
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/04—Ortho-condensed systems

Abstract

The present invention relates to a kind of novel heterocyclic compounds and preparation method thereof and the purposes as kinase inhibitor.Particularly, the invention discloses a kind of structure compound as shown in Equation 1 and preparation method thereof.This compound is a kind of effective kinase inhibitor and has very high bioavailability.

Description

A kind of novel heterocyclic compounds and preparation method thereof and the purposes as kinase inhibitor
Technical field
The invention belongs to biomedicine field.Particularly, the present invention relates to a kind of novel heterocyclic compounds and preparation method thereof and the purposes as kinase inhibitor.
Background technology
Tyrosylprotein kinase plays very important effect in the generation, evolution of tumour, and a wherein important class is non-receptor tyrosine kinase JAK.There are JAK1, JAK2, JAK3, TYK2 tetra-members in JAK family.At present, the dependency of each member of JAK family and tumour is the most full and accurate, clear and definite with the research of JAK2.The relation of the abnormal activation of JAK2 signal path and the generation of tumour, development is the closest, and early stage research is main pays close attention to the effects of multiple abnormal activation form in blood type tumour such as JAK2 gene fusion, amplification and sudden change.At present, studying more is JAK2V617F (the 617th valine mutation on false kinase domain JH2 the is phenylalanine) effect in myeloproliferative tumour, this sudden change makes JAK2 lose from inhibit feature, causes downstream signal excessive activation, finally causes malignant change of cell.Clinical data shows that the incidence of this sudden change in polycythemia vera, thrombocytosis, PMF is 81% ~ 99%, 41% ~ 72%, 39% ~ 57% (SzpurkaH etc. respectively, Refractoryanemiawithringedsideroblastsassociatedwithmark edthrombocytosis (RARS-T), anothermyeloproliferativeconditioncharacterizedbyJAK2V61 7Fmutation [J] .Blood, 2006,108 (7): 2173-2181).In recent years, increasing research shows that JAK2/STAT path may also play vital role in solid tumor, as Pro-inflammatory mediator IL-6, simultaneously also as the independent risk factor in liver cancer, the STAT of mediation activates this signal path and has very important effect, IL-6 only has by just with its acceptor formation IL-6/IL-6R/gp130 complex body carrying out signal transduction thus play a role, and one of main downstream of gp130 is exactly JAK2/STAT signal transduction pathway (YLiu etc., CelecoxibInhibitsIL-6/IL-6R-InducedJAK2/STAT3Phosphoryla tioninHumanHepatocellularCarcinomaCells.CancerPrev.Res.A ugust20114, 1296).Therefore, the research of target JAK2 micromolecular inhibitor is one of focus of current antineoplastic medicine research and development.
At present, the target JAK micromolecular inhibitor being in clinical study different steps has more than 10 to plant, and is mostly the selective depressant of JAK1, JAK2, and indication is blood system related neoplasms and small part solid tumor mainly.
At present, in the micromolecular inhibitor of target JAK2, only a medicine Ruxolitinib is gone on the market by U.S. FDA approval, be mainly used in treatment myelofibrosis (SaraC.MeyerandRossL.LevineMolecularPathways:MolecularBas isforSensitivityandResistancetoJAKKinaseInhibitors, Clin.CancerRes.2014; 20:2051-2059).
After Ruxolitinib listing, research finds that this pharmacological agent can improve the constitutional symptoms such as the relevant splenomegaly of myeloproliferative tumour, but does not does not significantly reduce or eliminate the tumor colonies in Most patients body.But the feature that this medicine has quick absorption in metabolism, high-efficient cleaning removes, cause its bioavailability in patient body low.
Therefore the scheme of daily twice must be taked in actual clinical operation to ensure the result for the treatment of that the concentration of Ruxolitinib in patient body reaches necessary.And in actually operating, patient often can not take medicine on time, thus cause patient's vivo medicine concentration to fluctuate excessive, affect medication effect.
Therefore, need the medicine developing a kind of metabolic stability badly, to reduce the frequency that patient takes medicine, and provide better result for the treatment of for patient.
Summary of the invention
The object of this invention is to provide a kind of efficient, small molecule kinase inhibitors that bioavailability is high.
In a first aspect of the present invention, provide structure compound as shown in Equation 1 or its steric isomer, or its pharmacy acceptable salt,
Wherein, R is the straight-chain paraffin of C1-C6 or branched paraffin, the straight-chain paraffin of C1-C6 of halo or branched paraffin or halogen;
A is H or CH 3;
X and Y is selected from independently of one another: N, CH or C-CH 3;
Z is N, CH or CX, and wherein, X is halogen.
In another preference, R is the straight-chain paraffin of C1-C4 or branched paraffin, the halogen of the branched paraffin of C1-C6, the straight-chain paraffin of the C1-C4 of halo or the C1-C6 of halo.
In another preference, the steric isomer of described compound such as formula shown in 1a,
Wherein, R is the straight-chain paraffin of C1-C4 or branched paraffin, the halogen of the branched paraffin of C1-C6, the straight-chain paraffin of the C1-C4 of halo or the C1-C6 of halo;
A is H or CH 3;
X and Y is selected from independently of one another: N, CH or C-CH 3;
Z is N, CH or CX, and wherein, X is halogen.
In second aspect present invention, provide the preparation method of compound described in first aspect present invention or its steric isomer, comprise step:
(1) in inert solvent, in the presence of base, compound 2 and compound 3 are carried out addition reaction, obtains compound 4;
(2) in inert solvent, under the existence of catalyzer and alkali, compound 4 and compound 5 are carried out linked reaction, obtains compound 6;
(3) in inert solvent, in the presence of base, compound 6 is carried out deprotection reaction, obtains compound 1;
Above-mentioned various in, R, A, X, Y, Z definition is as front.
In another preference, also comprise chiral separation step:
In formula, R, A, X, Y, Z definition is as front.
In third aspect present invention, provide a kind of pharmaceutical composition, comprise (a) activeconstituents: compound as described in the first aspect of the invention or its steric isomer, or its pharmacy acceptable salt; And (b) pharmaceutically acceptable carrier.
In fourth aspect present invention, provide the compound described in first aspect present invention or its steric isomer, or the purposes of its pharmacy acceptable salt or the pharmaceutical composition as described in third aspect present invention, for the preparation of kinase inhibitor or for the preparation of antitumor drug.
In another preference, described kinases is Tyrosylprotein kinase.
In another preference, described kinases is JAK2.
Should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the present invention and can combining mutually between specifically described each technical characteristic in below (eg embodiment), thus form new or preferred technical scheme.As space is limited, tiredly no longer one by one to state at this.
Embodiment
Contriver, through extensive and deep research, has surprisingly found the small molecule kinase inhibitors of a class formation novelty.And with existing Compound Phase ratio, compound of the present invention has higher bioavailability.On this basis, contriver completes the present invention.
Term
Term " straight-chain paraffin of C1-C6 or branched paraffin " refers to the straight chain with 1-6 carbon atom or the alkane with side chain, such as methyl, ethyl, propyl group, sec.-propyl, butyl, isobutyl-, the tertiary butyl, amyl group etc.
Term " halo " refer to fluoro, chloro, bromo, iodo.
Term " halogen " refers to fluorine, chlorine, bromine, iodine.
Activeconstituents
As used herein, term " the compounds of this invention " refers to the compound shown in formula 1.This term also comprises and the various steric isomers of formula 1 compound, crystalline forms, pharmacy acceptable salt, hydrate or solvate.
As used herein, term " pharmacy acceptable salt " refers to the salt being suitable as medicine that the compounds of this invention is formed with acid or alkali.Pharmacy acceptable salt comprises inorganic salt and organic salt.The preferred salt of one class is the salt that the compounds of this invention is formed with acid.The acid being applicable to being formed salt includes, but are not limited to: the mineral acids such as hydrochloric acid, Hydrogen bromide, hydrofluoric acid, sulfuric acid, nitric acid, phosphoric acid, formic acid, acetic acid, propionic acid, oxalic acid, propanedioic acid, succsinic acid, fumaric acid, toxilic acid, lactic acid, oxysuccinic acid, tartrate, citric acid, picric acid, methylsulfonic acid, benzene methanesulfonic acid, the organic acids such as Phenylsulfonic acid; And the acidic amino acid such as aspartic acid, L-glutamic acid.
Preparation method
More specifically describe the preparation method of formula 1 structural compounds below, but these concrete grammars do not form any restriction to the present invention.Various synthetic method that describe in this manual or known in the art can also optionally combine and obtain easily by the compounds of this invention, and such combination can be easy to carry out by those skilled in the art in the invention.
A kind of particularly preferred preparation flow is as follows:
(1) under certain temperature (as 70-100 DEG C), in inert solvent (as acetonitrile, DMF, DMSO, THF etc.), at alkali (as DBU, triethylamine, diisopropylethylamine, pyridine, K 2cO 3, Na 2cO 3, NaOH, KOH etc.) existence under, compound 2 and compound 3 are carried out Michael addition reaction for some time (as 4-20 hour), obtain compound 4;
(2) under certain temperature (as 70-100 DEG C), in inert solvent (as dioxane and water, toluene and water, DMSO, THF, DMF etc.), at catalyzer (as tetrakis triphenylphosphine palladium, three (dibenzalacetone) two palladium (Pd 2(dba) 3), two (dibenzalacetone) palladium, dichloro two (triphenylphosphine) palladium, triphenylphosphine palladium acetate, two (three adjacent phenmethyl phosphines) palladium chloride, 1, 2-bis-(diphenylphosphino) ethane palladium chloride etc.) and alkali (as salt of wormwood, Potassium monofluoride, cesium fluoride, Sodium Fluoride, potassiumphosphate, hypophosphite monohydrate potassium, sodium carbonate, sodium bicarbonate, 1, 8-diazabicylo [5.4.0] 11 carbon-7-alkene, triethylamine, diisopropylethylamine, pyridine or its combination etc.) existence under, compound 4 and compound 5 are carried out Suzuki linked reaction for some time (as 1-4 hour), obtain compound 6,
(3) under certain temperature (as 70-100 DEG C), in inert solvent (as methyl alcohol, ethanol, Virahol, propyl carbinol, the trimethyl carbinol, isopropylcarbinol, benzylalcohol etc.), under the existence of alkali (as salt of wormwood, sodium carbonate, sodium hydroxide, potassium hydroxide etc.), compound 6 is carried out deprotection reaction for some time (as 0.1-4 hour), obtains compound 1;
Above-mentioned various in, R, A, X, Y, Z definition is as front.
The method also can comprise following chiral separation step:
Pharmaceutical composition and application process
Because the compounds of this invention has the excellent inhibit activities to Tyrosylprotein kinase such as JAK2, therefore the compounds of this invention and steric isomer thereof, or pharmaceutically acceptable inorganic or organic salt etc., and can be used for treatment, prevention containing the pharmaceutical composition that the compounds of this invention is main active ingredient and alleviate by tyrosine kinase mediated disease.According to prior art, the compounds of this invention can be used for treating following disease: cancer etc.
Pharmaceutical composition of the present invention comprises the compounds of this invention in safe and effective weight range or its pharmacologically acceptable salt and pharmacologically acceptable vehicle or carrier.Wherein " safe and effective amount " refers to: the amount of compound is enough to obviously improve the state of an illness, and is unlikely to produce severe side effect.Usually, pharmaceutical composition contains 1-2000mg the compounds of this invention/agent, more preferably, containing 10-100mg the compounds of this invention/agent.Preferably, described " potion " is a capsule or tablet.
" pharmaceutically acceptable carrier " refers to: one or more biocompatible solid or liquid filler or gelatinous mass, and they are suitable for people and use, and must have enough purity and enough low toxicity." consistency " to referred to herein as in composition each component energy and compound of the present invention and they between mutually admix, and the drug effect of not obvious reduction compound.Pharmaceutically acceptable carrier part example has cellulose and its derivates (as Xylo-Mucine, ethyl cellulose sodium, cellulose ethanoate etc.), gelatin, talcum, solid lubricant (as stearic acid, Magnesium Stearate), calcium sulfate, vegetables oil (as soya-bean oil, sesame oil, peanut oil, olive wet goods), polyvalent alcohol (as propylene glycol, glycerine, N.F,USP MANNITOL, sorbyl alcohol etc.), emulsifying agent (as tween ), wetting agent (as sodium lauryl sulphate), tinting material, seasonings, stablizer, antioxidant, sanitas, apirogen water etc.
The method of application of the compounds of this invention or pharmaceutical composition is not particularly limited, and representational method of application comprises (but being not limited to): in oral, knurl, rectum, parenteral (intravenously, intramuscular or subcutaneous) and topical.
Solid dosage for oral administration comprises capsule, tablet, pill, powder and granule.In these solid dosages, active compound mixes with at least one conventional inert excipients (or carrier), as Trisodium Citrate or Si Liaodengji dicalcium phosphate feed grade, or mix with following compositions: (a) filler or expanding material, such as, starch, lactose, sucrose, glucose, N.F,USP MANNITOL and silicic acid; (b) tackiness agent, such as, Walocel MT 20.000PV, alginate, gelatin, Polyvinylpyrolidone (PVP), sucrose and gum arabic; (c) wetting Agent for Printing Inks, such as, glycerine; (d) disintegrating agent, such as, agar, calcium carbonate, yam starch or tapioca (flour), alginic acid, some composition silicate and sodium carbonate; (e) retarding solvent, such as paraffin; F () absorbs accelerator, such as, and quaternary ammonium compound; (g) wetting agent, such as hexadecanol and glyceryl monostearate; (h) sorbent material, such as, kaolin; (i) lubricant, such as, talcum, calcium stearate, Magnesium Stearate, solid polyethylene glycol, sodium lauryl sulphate, or its mixture.In capsule, tablet and pill, formulation also can comprise buffer reagent.
Solid dosage such as tablet, sugar-pill, capsule, pill and granule can adopt dressing and the preparation of shell material, as casing and other material well known in the art.They can comprise opacifying agent, and in this composition, the release of active compound or compound can discharge in certain part in a delayed fashion in digestive tube.The example of adoptable embedding component is polymeric material and Wax.If desired, active compound also can form microencapsulation form with one or more in above-mentioned vehicle.
Liquid dosage form for oral administration comprises pharmaceutically acceptable emulsion, solution, suspension, syrup or tincture.Except active ingredient beyond the region of objective existence, liquid dosage form can comprise the conventional inert diluent adopted in this area, as water or other solvent, solubilizing agent and emulsifying agent, example is known, the mixture etc. of ethanol, Virahol, ethyl-carbonate, ethyl acetate, propylene glycol, 1,3 butylene glycol, dimethyl formamide and oil, particularly Oleum Gossypii semen, peanut oil, maize germ, sweet oil, Viscotrol C and sesame oil or these materials.
Except these inert diluents, composition also can comprise auxiliary agent, as wetting agent, emulsifying agent and suspension agent, sweeting agent, correctives and spices.
Except active ingredient beyond the region of objective existence, suspension can comprise suspension agent, such as, and the mixture etc. of ethoxylation isooctadecane alcohol, polyoxyethylene sorbitol and Isosorbide Dinitrate, Microcrystalline Cellulose, aluminum methylate and agar or these materials.
Composition for parenteral injection can comprise physiologically acceptable sterile, aqueous or anhydrous solution, dispersion liquid, suspension or emulsion, and for being again dissolved into aseptic Injectable solution or the sterilized powder of dispersion liquid.Suitable moisture and nonaqueous carrier, thinner, solvent or vehicle comprise water, ethanol, polyvalent alcohol and suitable mixture thereof.
Formulation for the compounds of this invention of topical comprises ointment, powder, patch, propellant and inhalation.Activeconstituents aseptically with physiologically acceptable carrier and any sanitas, buffer reagent, or the propelling agent that may need if desired is mixed together.
The compounds of this invention can be individually dosed, or with other pharmaceutically acceptable compound Combined Preparation.
When making pharmaceutical composition, it is the Mammals (as people) being applicable to the compounds of this invention of safe and effective amount need treatment, when wherein using, dosage is the effective dosage pharmaceutically thought, for the people of 60kg body weight, day dosage is generally 1 ~ 2000mg, preferably 10 ~ 100mg.Certainly, concrete dosage also should consider the factor such as route of administration, patient health situation, and these are all within skilled practitioners skill.
The present invention mainly has the following advantages:
Compare with existing compound (Ruxolitinib), compound of the present invention improves the metabolic stability of medicine greatly, and its exposed amount (AUC) in animal body improves 3-5 doubly, and also there is obvious prolongation the transformation period.Reach the research expection that we improve its stability.
Below in conjunction with concrete enforcement, set forth the present invention further.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually conveniently condition, or according to the condition that manufacturer advises.Unless otherwise indicated, otherwise per-cent and number calculate by weight.
Embodiment 1
Compound 2 (250mg, 2.1mmol) and compound 10 (400mg, 2.1mmol) are placed in 50mL single port bottle, under nitrogen protection, drip DBU (1g, 4.2mmol) under room temperature, stir.Then 80 DEG C are heated to, reaction 8h.After completion of the reaction, be spin-dried for solvent, column chromatography (ethyl acetate: sherwood oil=5:1) obtains white solid 250mg, productive rate 36%.
1HNMR(CDCl 3，400MHz)δppm7.67(s,1H),4.04(td,1H,J＝10.2Hz,4.0Hz),3.03(dd,1H,J＝8.4Hz,17.2Hz),2.84(dd,J＝4Hz,17.2Hz),2.49(m,1H),2.38(s,3H),1.89(m,1H),1.45-1.70(m,4H),1.20-1.32(m,15H).
By compound 11 (160mg; 0.5mmol); compound 12 (153mg, 0.5mmol), tetra-triphenylphosphine palladium (29mg; 0.025mmol) with salt of wormwood (207mg; 1.5mmol), 50mL single port reaction flask is placed in, under nitrogen protection; Isosorbide-5-Nitrae-dioxane (4mL) and H is added under room temperature 2o (1mL), is stirred in the system of being dispersed in, and is then warming up to 80 DEG C, reacts 2h under nitrogen protection.After completion of the reaction, ethyl acetate 30mL and H is added 2o (10mL), extraction separatory.Organic phase with sodium sulfate is dry, is spin-dried for, and column chromatography (sherwood oil: ethyl acetate=3:1-1:1) obtains white solid 132mg, productive rate 55%.
1HNMR(CDCl 3，400MHz)δppm9.00(s,1H),8.10(d,2H,J＝7.2Hz),8.08(s,1H)7.78(d,1H,J＝4.0Hz),7.32(d,2H,7.2Hz),4.14(td,1H,J＝0.2Hz,4.0Hz),3.10(dd,1H,J＝8.4Hz,17.2Hz),2.90(dd,J＝8.4Hz,17.2Hz),2.60(m,1H),2.59(s,3H),2.40(s,3H),1.90(m,1H),1.45-1.76(m,4H),1.20-1.32(m,3H).
Compound 13 (132mg, 0.28mmol) and salt of wormwood (77mg, 0.56mmol) are placed in the single port bottle of 50mL, add methyl alcohol 6mL and water 1.5mL, stir, be heated to 60 DEG C of reactions 1 hour.After completion of the reaction, after completion of the reaction, ethyl acetate 20mL and H is added 2o (10mL), extraction separatory.Organic phase with sodium sulfate is dry, is spin-dried for, and column chromatography (sherwood oil: ethyl acetate=1:1) obtains white solid 72mg, productive rate 81%.
1HNMR(CDCl 3,400mHz),δppm0.92(br,1H),8.90(s,1H),8.17(m,1H),7.39(m,1H),6.69(m,1H),4.20(td,1H,J＝10.2Hz,4.0Hz),3.13(dd,1H,J＝8.4Hz,17.2Hz),2.93(dd,J＝8.4Hz,17.2Hz),2.63(m,1H),2.62(s,3H),1.90(m,1H),1.45-1.76(m,4H),1.20-1.32(m,3H).
500mg compound 14 is dissolved in moving phase, separation condition: instrument: Gilson281, pillar: CHIRALPAKIC30*250mm, 5um (Daicel), moving phase: normal hexane (0.1% diethylamine): ethanol (0.1% diethylamine)=70:30, wavelength: 214nm & 254nm, flow velocity: 1.0ml/min temperature: 40 DEG C.A cycling time is: 19 minutes.Collect compound 15 (174mg) and compound 16 (210mg) respectively.
Embodiment 2:
Compound 17 (190mg, 0.7mmol) and compound 2 (169mg, 1.4mmol) are placed in 50mL single port bottle, under nitrogen protection, drip DBU (425mg, 2.8mmol) under room temperature, stir.Then 80 DEG C are heated to, reaction 8h.After completion of the reaction, be spin-dried for solvent, column chromatography (ethyl acetate: sherwood oil=5:1) obtains colorless oil 60mg, productive rate 17%.
1HNMR(CDCl 3，400MHz)δppm7.56(s,1H),4.21(td,1H,J＝10.2Hz,4.0Hz),3.05(m,1H),2.85(m,1H),2.53(m,1H),1.93(m,1H),1.45-1.80(m,4H),1.20-1.32(m,15H).
By compound 18 (60mg; 0.16mmol); compound 12 (48mg, 0.16mmol), tetra-triphenylphosphine palladium (9mg; 0.008mmol) with salt of wormwood (207mg; 1.5mmol), 50mL single port reaction flask is placed in, under nitrogen protection; Isosorbide-5-Nitrae-dioxane (4mL) and H is added under room temperature 2o (1mL), is stirred in the system of being dispersed in, and is then warming up to 80 DEG C, reacts 2h under nitrogen protection.After completion of the reaction, ethyl acetate 30mL and H is added 2o (10mL), extraction separatory.Organic phase with sodium sulfate is dry, is spin-dried for, and column chromatography (sherwood oil: ethyl acetate=3:1-1:1) obtains white solid 24mg, productive rate 30%.
1HNMR(CDCl 3，400MHz)δppm9.04(s,1H),8.12(d,2H,J＝7.2Hz),8.06(s，1H)7.81(d,1H,J＝4.0Hz),7.36(d,2H,j＝7.2Hz),4.30(m,1H,),3.10(m,1H),2.94(m,J1H),2.64(m,1H),2.40(s,3H),1.99(m,1H),1.45-1.77(m,4H),1.20-1.32(m,3H).
Compound 19 (24mg, 0.045mmol) and salt of wormwood (12mg, 0.09mmol) are placed in the single port bottle of 50mL, add methyl alcohol 3mL and water 0.75mL, stir, be heated to 80 DEG C of reactions 1 hour.After completion of the reaction, after completion of the reaction, ethyl acetate 20mL and H is added 2o (10mL), extraction separatory.Organic phase with sodium sulfate is dry, is spin-dried for, and column chromatography (sherwood oil: ethyl acetate=1:1) obtains white solid 12mg, productive rate 70%.
1HNMR(CDCl 3,400MHz)δppm10.40(br,1H),8.99(s,1H),8.17(m,1H),7.45(m,1H),6.68(m,1H),4.34(m,1H),3.13(dd,1H,J＝8.0Hz,16.8Hz),2.93(dd,J＝8.0Hz,16.8Hz),2.66(m,1H),2.0(m,1H),1.45-1.76(m,4H),1.20-1.32(m,3H).
Following compound can synthesize according to the similar method of above-mentioned example
Embodiment 3: compound is in the impact of molecular level on JAK enzymic activity
This enzyme lives reaction experiment based on FRET (fluorescence resonance energy transfer) (FRET) principle; two ends are respectively with the enzyme reaction substrate of a donor fluorophore and an acceptor fluorophore; modification can be phosphorylated under ATP and kinase whose acting in conjunction; protection substrate is not cut by substrates enzymes A; thus realize FRET (fluorescence resonance energy transfer); after adding inhibitor, then this phenomenon reduces.The reagent that this experiment is used is as follows:
Enzyme reaction substrate: starting point concentration is 1mM, the tyrosine residues on this polypeptide can be phosphorylated under kinases, ATP acting in conjunction; Purchased from Invitrogen;
100% phosphorylated substrate: starting point concentration is 1mM, the tyrosine residues on this polypeptide is by complete phosphorylation modification; Purchased from Invitrogen;
ATP: starting point concentration is 10mM, purchased from Invitrogen;
Reaction buffer: containing the HEPES (PH is 7.5) of 250mM, the MgCl of 50mM 2, the EGTA of 5mM, the Brij-35 of 0.05%; Purchased from Invitrogen;
Substrates enzymes A: enzyme can be carried out to the enzyme reaction substrate be not phosphorylated and cut; Purchased from Invitrogen;
Stop buffer: purchased from Invitrogen.
Experiment concrete steps are summarized as follows: balance enzyme reaction substrate, 100% phosphorylated substrate, reaction buffer, ATP, substrates enzymes A, stop buffer to room temperature respectively.Select jak kinase territory recombinant protein JAK1, JAK2JH1JH2, JAK2JH1JH2V617F, JAK3 (purchased from Invitrogen), make final concentration be respectively 0.1ng/ μ L, 1ng/ μ L, 0.2ng/ μ L, 0.08ng/ μ L with reaction buffer dilution, be placed in and prepare application of sample on ice.
Select black 384 hole micro sample-adding plate (PerkinElmer), every hole adds the enzyme reaction substrate 2.5 μ L with reaction buffer dilution, and final concentration is 2 μMs.Testing compound (compound prepared by the embodiment of the present invention) 4%DMSO is diluted to suitable concentration (initial concentration is generally set to 3 μ Μ, and the dilution proportion by 1/3 becomes 9 concentration gradients), and every hole adds 2.5 μ L.Every hole adds the ATP solution of reaction buffer dilution, final concentration corresponding JAK1, JAK2JH1JH2, JAK2JH1JH2V617F, JAK3 are respectively 75 μMs, 50 μMs, 50 μMs, 10 μMs, the jak kinase territory recombinant protein 2.5 μ L adding reaction buffer dilution again starts reaction, need arrange 0% phosphorylation control group at every turn, 100% phosphorylation control group, 0% suppresses control group.Each reaction group is defined as follows:
0% phosphorylation control group: containing enzyme reaction substrate, ATP, but do not add kinases, or containing enzyme reaction substrate, kinases, but do not add ATP, namely this control wells ensures that enzyme reaction substrate is not phosphorylated, can by substrates enzymes A complete degestion;
100% phosphorylation control group: add 100% phosphorylated substrate, namely when not adding kinases and ATP, this control group ensures that substrate is phosphorylated completely, can not be cut by substrates enzymes A enzyme;
0% suppresses control group: containing enzyme reaction substrate, kinases, ATP, inhibitor solvent, but not containing inhibitor, this control group is used to indicate in this kinase reaction system, and the phosphorylation degree of substrate is in linearity range, advises between 20 ~ 50%;
After application of sample completes, vibration plate mixes 1 minute, puts 27 DEG C of lucifuges and reacts 1 hour.After having reacted, add the substrates enzymes A that 5 μ l dilute through reaction buffer in all reacting holes, thinning ratio is 1:1067, and after application of sample completes, vibration plate mixes 1 minute, puts 27 DEG C of lucifuges and reacts 1 hour.5 μ l stop buffer termination reactions are added in the most backward all reacting holes, the vibration plate rear Synergy2MicroplateReader of mixing (BioTec) detects fluorescent signal (excitation wavelength is 400nm, and wavelength of transmitted light is 445nm, 520nm).
Calculated the inhibiting rate in each hole by complete active hole and background signal hole, data analysing method is as follows:
The strength of signal of the strength of signal of emission ratios=donor fluorophore (be 445nm place at wavelength)/acceptor fluorophore (be 520nm place at wavelength)
Percent phosphorylation=100 × { 1-, (strength of signal of the donor fluorophore of strength of signal-100% phosphorylation control group of the acceptor fluorophore of emission ratios * 100% phosphorylation control group)/[strength of signal+emission ratios of the donor fluorophore of strength of signal-100% phosphorylation control group of the donor fluorophore of 0% phosphorylation control group ×, (strength of signal of the acceptor fluorophore of strength of signal-0% phosphorylation control group of the acceptor fluorophore of 100% phosphorylation control group)] }
Inhibiting rate=100 × (1-(percent phosphorylation/0% of test compounds group suppresses the percent phosphorylation of control group))
With software GraphPrism6, half inhibit activities (IC is carried out to testing compound simultaneously 50) matching, the results are shown in Table 1.Experiment in triplicate.
Embodiment 4: the impact of compound on intracellular propagation
Test method: compound detects with CCK-8 Cell counting Kit (purchased from Dojindo) the inhibited proliferation of BaF3 (mouse pre B cell), HEL (human erythroleukemia cell).
Concrete steps are as follows: be in the BaF3 of logarithmic phase, hel cell is seeded in 96 well culture plates by proper density (10,000/hole), every hole 90 μ L, cultivate after 12 hours, (initial concentration is generally set to 50 μ Μ to add different concns, dilution proportion by 1/3 becomes 9 concentration gradients) testing compound (compound prepared by the embodiment of the present invention) 10 μ L act on 72 hours, and setting solvent control group (negative control).Until compound effects cell after 72 hours, the impact of compound on intracellular propagation adopts CCK-8 Cell counting Kit to detect, every hole adds 10 μ LCCK-8 reagent, be placed in after 37 DEG C of incubators place 2-4 hour, with long orifice plate microplate reader SpectraMax190 (the purchased from American MolecularDevices company) reading that declines of all-wave, mensuration wavelength is 450nm.
Press with the inhibiting rate of following formulae discovery drug on tumor Growth of Cells:
Inhibiting rate (%)=(OD control wells-OD dosing holes)/OD control wells × 100%.
With software GraphPrism6, half inhibit activities (IC is carried out to testing compound simultaneously 50) matching.Experiment in triplicate, the results are shown in Table 1.Visible, part of compounds of the present invention reaches nano level.
Table 1: the activity data of testing compound
Compound Molecular level activity (IC 50) Cell levels activity (BaF3, IC 50)
14 <100nM <500nM
15 <100nM <500nM
16 <100nM 500-10000nM
20 100-500nM N/A
24 <100nM 500-10000nM
25 100-500nM 500-10000nM
26 500-10000nM N/A
Note: N/A represents undetermined.
Embodiment 5: the metabolic characteristic research of compound in rat body
Test method: select SD rat 14, male, body weight 200-220g, is divided into 4 groups at random, often organize 4/3, gavage and vein give positive compound Ruxolitinib and compound 15 respectively.Rat is fasting 12h before the test, freely drinks water, with the 0.5%CMC-Na compounding pharmaceutical of 5%DMSO/5% tween 80/90%.Gastric infusion dosage is 50mg/kg, and intravenously administrable dosage is 10mg/kg, the unified feed of 2h after rat administration.Gastric infusion group upon administration 0.25,0.5,1.0,2.0,4.0,6.0,8.0 and 24h blood sampling, intravenously administrable group 5min upon administration, 0.25,0.5,1.0,2.0,4.0,6.0,8.0 and 24h blood sampling, through rat eye rear vein beard extracting vein blood 0.3ml, put in heparinised tubes, the centrifugal 5min of 11000rpm, separated plasma, freezes in-20 DEG C of refrigerator and cooled.Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) method is adopted to measure plasma sample drug concentration, plasma sample is after albumen precipitation process, be separated through C18 post, TSQQuantumUltra type triple quadrupole bar tandom mass spectrometer (purchased from Thermo) detects, ion source is heating electron spray ionisation (HESI) source, and positive ion mode detects.
Pharmacokinetic parameter after adopting the non-compartment model of Phoenix1.3 software (purchased from American Pharsight company) to calculate administration.The experimental result of gastric infusion group sees the following form 2, and the experimental result of intravenous administration group is in table 3.
Table 2: rat oral gavage gives the pharmacokinetic parameters after the different compound of 50mg/kg
Table 3: rat vein gives the pharmacokinetic parameters after the different compound of 10mg/kg
The all documents mentioned in the present invention are quoted as a reference all in this application, are just quoted separately as a reference as each section of document.In addition should be understood that those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.

Claims (8)

1. structure compound as shown in Equation 1 or its steric isomer, or its pharmacy acceptable salt,
Wherein, R is the straight-chain paraffin of C1-C6 or branched paraffin, the straight-chain paraffin of C1-C6 of halo or branched paraffin or halogen;
A is H or CH 3;
X and Y is selected from independently of one another: N, CH or C-CH 3;
Z is N, CH or CX, and wherein, X is halogen.
2. compound as claimed in claim 1, is characterized in that, the steric isomer of described compound such as formula shown in 1a,
Wherein, R is the straight-chain paraffin of C1-C4 or branched paraffin, the halogen of the branched paraffin of C1-C6, the straight-chain paraffin of the C1-C4 of halo or the C1-C6 of halo;
A is H or CH 3;
X and Y is selected from independently of one another: N, CH or C-CH 3;
Z is N, CH or CX, and wherein, X is halogen.
3. the preparation method of compound as claimed in claim 1 or its steric isomer, is characterized in that, comprise step:
(1) in inert solvent, in the presence of base, compound 2 and compound 3 are carried out addition reaction, obtains compound 4;
(2) in inert solvent, under the existence of catalyzer and alkali, compound 4 and compound 5 are carried out linked reaction, obtains compound 6;
(3) in inert solvent, in the presence of base, compound 6 is carried out deprotection reaction, obtains compound 1;
Above-mentioned various in, R, A, X, Y, Z definition as claim 1.
4. preparation method as claimed in claim 3, is characterized in that, also comprise chiral separation step:
In formula, R, A, X, Y, Z definition is as claim 1.
5. a pharmaceutical composition, is characterized in that, comprises (a) activeconstituents: compound as claimed in claim 1 or its steric isomer, or its pharmacy acceptable salt; And (b) pharmaceutically acceptable carrier.
6. compound or its steric isomer as claimed in claim 1, or the purposes of its pharmacy acceptable salt or pharmaceutical composition as claimed in claim 5, is characterized in that, for the preparation of kinase inhibitor or for the preparation of antitumor drug.
7. purposes as claimed in claim 6, it is characterized in that, described kinases is Tyrosylprotein kinase.
8. purposes as claimed in claim 7, it is characterized in that, described kinases is JAK2.
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