One class pyridine salt JAK inhibitor, preparation method and its usageTechnical field
The present invention relates to immunity, inflammatory, autoimmunity or allergic disease, or the medicine of the disease such as transplant rejectionField.In particular it relates to above-mentioned disease have a medicative class containing pyridiniujm structure JAK inhibitor,Its preparation method, and the purposes in pharmacy.
Background technology
The phosphorylation of kinase catalytic protein, lipid, sugar, nucleoside and other cell metabolites at eukaryotic cell physiologyAll aspects play a crucial role.Especially, protein kinase and lipid kinase participate in signal conductive process, and this process control is to carefullyThe outer instrumentality of born of the same parents or irritate the cell that thing (such as somatomedin, cytokine or chemotactic factor) responds activation, grow, break up andSurvival.Generally, protein kinase is divided into two classes, the protein kinase of preferential phosphorylation tyrosine residue and preferential phosphorylation serineAnd/or the protein kinase of threonine residues.Tyrosine kinase includes transmembrane growth factor receptor such as EGF-R ELISAAnd cytosolic non-receptor kinases such as Janus kinases (JAK) (EGFR).High protein kinase activity relates to numerous disease inadequately, bagInclude cancer, metabolic disease, autoimmunity or inflammatory disease.This effect can be directly or indirectly because the sudden change of enzyme, excessivelyExpress or inappropriate activate produce control mechanism fault and cause.In all these examples, it is desirable to kinase whose selectivity presses downThe useful effect of fixture.
Oneself is nonreceptor tyrosine kinase through becoming a class kinases of current drug development focus] anus kinases (JAK)Family.In mammal, this family has four members, JAK1, JAK2, JAK3 and tyrosine kinase 2 (TYK2).Each eggWhite matter all has kinases territory and catalytically inactive pseudokinase territory.JAK protein (carries 4.1 albumen by its amino terminal FERM(Band-4.1), ezrin (ezrin), radixin (radixin), moesin (moesin) territory are attached to cell factor receptorOn body.After cytokine is combined with its receptor, activates JAKs and make receptor phosphorylation, thus producing for signal transduction moleculeThe docking site (docking sites) of (the especially member of signal transducer and transcription activator (Stat) family)(Yamaoka etc., 2004, The Janus kinases (jaks) .Genome Biology, 5 (12), p253).MammalIn, JAK1, JAK2 and TYK2 generally express.On the contrary, the expression of JAK3 mainly in hematopoietic cell and by cell development with swashThe altitude mixture control (Musso etc., 1995,181 (4), p1425-1431) lived.JAK mono-deficient cells system and gene target miceBasic in cytokine signaling conducts of oneself disclosed JAKs of research, unduplicated function.JAK1 knock-out mice showsPerinatal stage lethal phenotype, may with stop its suckling effects on neural system relevant (Rodig etc., 1998, Ce1l, 93 (3): 373-383).Due to Dyserythropoiesis, the deletion of JAK2 gene causes producing embryonic lethal when embryo the 12.5th day(Neubauer etc., 1998, Ce1l, 93 (3), 397-409).Enjoyably, JAK3 defect is suffering from autosomal recessive weight firstIn the people of degree combined immunodeficiency (SCID) identified (Macchi etc., 1995, Nature, 377 (6544): 65-68).JAK3 strikesExcept mice displays that SCID but do not show non-immunity defect, show that JAK3 inhibitor will have in vivo as immunosuppressantLimited effect and therefore become for the promising medicine of immunosuppressant (Papageorgiou and Wikman, 2004, Trendsin Pharmacological Sciences,25(11),558-562).Acute megakaryoblastic leukemia (AMKL) patientIn oneself it be observed that the activated mutant (Walters etc., 2006, Cancer Cell, 10 (1), 65-75) of JAK3.These of JAK3Ba/F3 cells switch can be factor independent growth and induce megakaryoblastic leukemia in mouse model by mutant formFeature.
The disease relevant with JAK3 suppression and disease are further described in such as WO01/42246 and WO2008/060301In.In document oneself report some JAK3 inhibitor can be used for medical domain (O ' Shea etc., 2004, Nat.Rev.DrugDiscov.3(7):555-564).It is reported, effective JAK3 inhibitor (CP-690,550) is at the animal model of organ transplantation(Changelian etc., 2003, Science, 302 (5646), 875-888) and clinical trial (reference: Pesu etc., 2008,Immunol.Rev.223,132-142) display effect in.The substituted pyrimidine compound of fluorine is described in WO-as JAK3 inhibitorIn A2010/118986.Heterocyclic radical Pyrazolopyrimidine analogs is described in WO-A2011/048082 as JAK inhibitor.WO-A2008/129380 relates to treat the sulfamide derivative of abnormal cell growth.WO-A2006/117560 and JournalOf Molecular Graphics and Modelling (29) 2010,309-320 describe the substituted pyrimidine of pyrazolyl amino andIts purposes in treatment cancer.EP1054004A1 describes pyrimidine derivatives and the purposes in inflammation thereof.
The invention discloses the class JAK inhibitor containing pyridiniujm structure, these compounds can be used for preparation treatment immunityProperty, inflammatory, autoimmunity or allergic disease, or the medicine of the disease such as transplant rejection.
Summary of the invention
It is an object of the present invention to provide the JAK inhibitor of a kind of excellent activity with formula I.
It is a further object to provide the method that preparation has compounds of formula I.
It is also another object of the present invention to provide containing compounds of formula I in treatment immunity, inflammatory, autoimmunityOr the application in terms of allergic disease, or the disease such as transplant rejection.
In conjunction with the purpose of the present invention, present invention is specifically described.
The present invention has compounds of formula I and has a following structural formula:
Wherein, R is selected from C1-C3Alkyl.
The compound of preferred formula I has following structure,
Compound of Formula I of the present invention can be synthesized by following route:
Compound II first processes with highly basic, then reacts with compound III, obtains compound IV；Compound IV first uses highly basicProcess, then with 1,2-dichloroethanes reacts, and obtains compound V；There is Intramolecular substitution reaction in compound V, obtains under heatingCompound I；Wherein, described highly basic is selected from n-BuLi, isobutyl group lithium, tert-butyl lithium, phenyl lithium, lithium diisopropylamine, instituteStating X=Cl, Br, I, R's is defined as described above.
Compound of Formula I of the present invention has JAK inhibitory action, can be used for preparing immunity, inflammation as effective ingredientProperty, autoimmunity or allergic disease, or the disease therapeuticing medicine such as transplant rejection.Compound of Formula I of the present inventionActivity is to be verified by the inhibition test of external JAK.
The compound of Formula I of the present invention is effective in comparatively wide dosage range.The dosage that such as every day takes is aboutIn the range of 1mg-700mg/ people, it is divided into once or is administered for several times.The actual dosage taking compound of Formula I of the present invention can be by curingTake root and determine according to relevant situation.
Detailed description of the invention
Below in conjunction with embodiment, the present invention is further illustrated.It should be noted that following embodiment is only forIllustrate, and be not intended to limit the present invention.Those skilled in the art all should according to the various changes that the teachings of the present invention is madeWithin the protection domain required by the application claim.
The synthesis of embodiment 1 compound I-1
The synthesis of step A. compound IV-1
Compound II-1 (1.08g, 10mmol) is dissolved in the THF that 10mL is dried, stirring, be cooled in nitrogen atmosphere-20 DEG C, then slowly drip the hexane solution (6.25mL, 10mmol) of the n-BuLi of 1.6M with syringe, after dropping,Reactant mixture continues to stir 1 hour at such a temperature.The most slowly drip III-1 (2.01g, 10mmol) to be dissolved in syringeThe solution that the THF that 3mL is dried makes, after dropping, reactant mixture stirs half an hour at such a temperature, is then warming up to roomTemperature is stirred for overnight.Reactant mixture carefully pours in 200mL frozen water, and stirring, with 50mL × 3CH2Cl2Extraction, merges extractionPhase, washs with saline, and anhydrous sodium sulfate is dried.Sucking filtration removes desiccant, and filtrate is evaporated on a rotary evaporator, and residue usesSilica gel column chromatography purification, obtains compound IV-1, white solid, ESI-MS, m/z=229 ([M+H]+).Step B. compound V-The synthesis of 1
Compound IV-1 (1.37g, 6mmol) is dissolved in the THF that 10mL is dried, stirring, is cooled to-20 in nitrogen atmosphereDEG C, the hexane solution (3.75mL, 6mmol) of the n-BuLi of 1.6M is then slowly dripped with syringe, after dropping, reactionMixture continues to stir 1 hour at such a temperature.1,2-dichloroethanes (0.99g, 10mmol) is the most slowly dripped molten with syringeThe solution that the THF being dried in 3mL makes, after dropping, reactant mixture stirs half an hour at such a temperature, is then warming up toRoom temperature is stirred for overnight.Reactant mixture carefully pours in 200mL frozen water, and stirring, with 50mL × 3CH2Cl2Extraction, merges extractionTaking phase, wash with saline, anhydrous sodium sulfate is dried.Sucking filtration removes desiccant, and filtrate is evaporated on a rotary evaporator, and residue makesPurify with silica gel column chromatography, obtain compound V-1, white solid, ESI-MS, m/z=291 ([M+H]+)。
The synthesis of step C. compound I-1
Compound V-1 (0.87g, 3mmol) is dissolved in the toluene that 10mL is dried, heated overnight at reflux in nitrogen atmosphere,TLC display reaction completes.After reactant mixture is cooled to room temperature, adds 10mL normal hexane, stir 1 hour, collected by suction solid,Ambient temperature in vacuum is dried, and obtains compound I-1, white solid, ESI-MS, m/z=255 ([M-Cl-]+)。
Embodiment 2-8
With reference to the method for embodiment 1, synthesize compound listed in Table.
Embodiment 9 Compound ira vitro inhibitory action to jak kinase
Using the test as hereafter specified, the ability for compound suppression JAK1, JAK2 and JAK3 carrys out SCREENED COMPOUND.
Use baculovirus expression system, make people JAKl (aa850-1154), JAK2 (aa826-1132), JAK3And the catalyst structure domain of TYK2 (aa871-1187) is expressed as N end gst fusion protein and it is purchased from Carna (aa795-1124)Biosciences.Use biotin labeled peptide--poly-(GT)-biotin (CisBio)--as substrate to measure the clean of enzymeProperty.Peptide concentration in reaction is 60nM for JAKl, is 20nM for JAK2, is 140nM for JAK3 and for TYK2 is50nM.By TR-FRET (time-resolved fluorescence energy transfer (time-resolved fluorescence energyTransfer)) method detects the degree of phosphorylation.For at 8mM MOPS (pH7.0), 10mM MgC12, 0.05% β-dredge baseEach kinases in the reactant mixture containing enzyme, ATP and peptide in ethanol, 0.45mg/mL BSA measures the IC of compound50.InsteadATP concentration in Ying is 3 μMs for JAK1, is 0.2 μM for JAK2, for JAK3 is 0.6 μM and is 1.8 μMs for TYK2.Enzyme process reaction is at room temperature carried out 30 minutes.Then 0.115 μ g/mL anti-valine phosphorylation (phosphoTyr) is contained with 20 μ L(PT66) the floating detection buffer (50mM that goes out of the SA-XL665 (CisBio) of-cryptate (CisBio) and variable concentrationsHEPES, 0.5M KF, EDTA 0.25M, 0.l% (w/v) BSA, pH7.5) non-stopped reaction to be to keep SA-B ratios constant.IncubateEducate 3 hours, be then set as reading the upper reading of Victor2V spectrofluorimeter (PerkinElmer) of fluorescence resonance energy transmissionTake.
From upper table result it can be seen that the compound of the present invention has the strongest inhibitory action to JAK, can be as preparationThe medicine of the diseases such as treatment immunity, inflammatory, autoimmunity or allergic disease, or transplant rejection.