A kind of taxol and novel phthalazines ketone BTK inhibitor drug combination compositions andIt is appliedTechnical field
The invention belongs to medicinal chemistry arts, are related to phthalazines ketone BTK inhibitor and its application, and in particular to phthalazines ketoneBTK inhibitor, preparation method, the pharmaceutical composition containing such compound and its treatment bruton's tyrosine kinasePurposes in related disease.
Background technique
Bu Ludun histidine kinase (Bruton's tyrosine kinase, BTK) belongs to the member of Tec family.It is by onlyN- terminal domains, that is, PH (pleckstrin homology) structural domain, TH (Tec homology) homologous region, SH3 (Src of spyHomology3) structural domain, SH2 (Src homology2) structural domain and catalyst structure domain, also referred to as SH1/TK (SrcHomologyl/Tyrosine kinase) structural domain or kinase domain form (Akinleye et al:Ibrutiniband novel BTK inhibitors in clinical development.Journal of Hematology&Oncology2013,6:59).In bone-marrow-derived lymphocyte development process, the correct expression of BTK gene difference protein domain is in BThere is key effect in the function of cell and a variety of transduction pathway.
It is B cell class tumour such as leukaemia, hair property myeloma based on BTK signal transduction pathway exploitation small molecule targeted drugAnd the treatment of B cell para-immunity disease provides a completely new approach.The evidence of effect of the BTK in autoimmune disease is(Kil LP, et al:Bruton's tyrosine is provided by BTK- deletion form mouse and BTK- abundance type mouse model experimentkinase mediated signaling enhances leukemogenesis in a mouse model forChronic lymphocytic leukemia.Am J Blood Res2013,3 (1): 71-83.).It is white in chronic lymphocyticIn blood disease (CLL) mouse model, BTK- deletion form mouse abrogates chronic lymphocytic leukemia completely, and BTK overexpression can addFast leukaemia morbidity, increases the death rate.
The selectivity for being currently known BTK inhibitor is undesirable, in addition to inhibiting BTK, also inhibit other a variety of kinases (such as ETK,EGF, BLK, FGR, HCK, YES, BRK and JAK3 etc.), to generate more side effect；Meanwhile BTK binding site occurs to dash forwardIt frequently can lead to the generation of drug resistance after change.Therefore more BTK inhibitor are clinically needed, for treating the diseases such as tumour,Such adverse events can be overcome simultaneously.
Summary of the invention
The present invention the following technical schemes are provided:
In a first aspect, a kind of taxol provided by the invention and BTK inhibitor drug combination compositions, comprising activity atPoint and pharmaceutically acceptable auxiliary material, active constituent BTK inhibitor shown in taxol and formula (I) forms, describedThe molar ratio of BTK inhibitor shown in taxol and formula (I) is (0.14-0.20) in active constituent: 1；Wherein, the BTK suppressionPreparation, shown in chemical formula such as formula (I),
Wherein, the preparation method of the BTK inhibitor, includes the following steps:
Step A: the compound of formula (1) and the compound condensation of formula (2) obtain the compound of formula (3)；
Step B: the compound of formula (3) reacts to obtain the compound of formula (5) with the compound of formula (4)；
Step C: the compound of formula (5) sloughs protecting group and obtains the compound of formula (6)；
Step D: the compound of formula (6) and substituted alkene acyl chloride reaction obtain compounds of formula I, and reaction route is as follows:
Preferably, in step A,
It takes dimethoxy-methyl benzene in reaction flask, is added tetrahydrofuran dissolution, 60 DEG C, under nitrogen protection, s- is addedReaction solution is stirred to react by BuLi at -60 DEG C；It takes dry ice in reaction flask, tetrahydrofuran is added, n-BuLi, nitrogen is addedThe lower stirring of protection, adds the reaction solution after being stirred to react at -60 DEG C, continues to stir, stop reaction, water is added, uses concentrated hydrochloric acidPH to 2 is adjusted, organic phase is separated, water phase is extracted with ethyl acetate, and merges organic phase, saturated common salt water washing, anhydrous sodium sulfateIt is dry, it is recrystallized to give the compound of formula (8)；
The compound, acetic acid, hydrazine of formula (8) are weighed in reaction flask, isopropanol is added, under nitrogen protection, 100 DEG C of reflux are anti-It answers, stops reaction, addition ethyl acetate, water, extraction merges organic phase, and anhydrous sodium sulfate is dry, hangs and does, column chromatographic purifying obtainsThe compound of formula (4).
Preferably, in step B,
Triphosgene is weighed in reaction flask, toluene is added, the tetrahydrofuran for being dissolved with parachlorophenol and pyridine is added dropwise at 0 DEG CSolution, drop finish, and the reaction was continued at room temperature, concentration of reaction solution, and methylene chloride is added, and hang and do, and chloro-carbonic acid is made to benzyl chloride ester, directlyFor in next step；
- 2 (1H)-t-butyl formate of 5- amino -3,4- dihydro-isoquinoline and DIPEA are weighed in reaction flask, dichloro is addedMethane is stirred at room temperature down and chloro-carbonic acid is slowly added dropwise to benzyl chloride ester, and drop finishes, continues to stir at room temperature, stops reaction, and concentration reaction is mixedClose object, ethyl acetate be added, diluted hydrochloric acid aqueous solution and saturated common salt water washing, anhydrous sodium sulfate dry, filter, concentration to getThe compound of formula (3).
Preferably, in step C, the compound of modus ponens (4), formula (3) compound in reaction flask, DMF is added, at 55 DEG CReaction overnight, stops reaction, and water, methylene chloride is added, and extraction separates organic phase, and water phase continues to be extracted with dichloromethane, and mergesOrganic phase, anhydrous sodium sulfate is dry, and column chromatographic purifying obtains the compound of formula (5).
Preferably, in step D, the compound of modus ponens (5) is added trifluoroacetic acid, stirs at room temperature in reaction flask, depressurizesIt being concentrated to dryness, ethyl acetate is added, successively use disodium hydrogen phosphate aqueous solution and saturated common salt water washing, anhydrous sodium sulfate is dry,Filtering, be concentrated under reduced pressure formula (6) compound.
Preferably, in step E, the compound of modus ponens (6) is added methylene chloride 100ml and dissolves, at 0 DEG C in reaction flaskDIEA is added, after stirring, acryloyl chloride is added dropwise in continuation at 0 DEG C, and drop finishes, is stirred at room temperature, and is stopped reaction, is added water, methylene chlorideExtraction merges organic phase, and anhydrous sodium sulfate is dry, and column chromatographic purifying obtains the compound of formula (I).
The present invention also provides a kind of novel phthalazines ketone BTK inhibitor drug combination compositions, include active constituentWith pharmaceutically acceptable auxiliary material, active constituent BTK inhibitor group as shown in taxol, lenalidomide, formula (I)Molar ratio at BTK inhibitor shown in, taxol in the active constituent, lenalidomide, formula (I) is (0.14-0.20):(0.08-0.12): 1.
Pharmaceutical composition of the invention and pharmaceutically acceptable carrier, diluent or excipient can be prepared by mixing intoPharmaceutical preparation, to be suitable for oral or parenteral.Medication includes, but are not limited in intradermal, intramuscular, peritonaeum, veinInterior, subcutaneous, intranasal and peroral route.The preparation can be applied by any approach, such as by being transfused or inject, pass through throughThe approach application that epithelium or mucocutaneous (such as oral mucosa or rectum etc.) absorb.Administration can be whole body or local.The example of oral administration preparation includes solid or liquid dosage form, specifically, include tablet, pill, granula, pulvis, capsule,Syrup, emulsion, suspension etc..The preparation can be prepared by methods known in the art, and include field of pharmaceutical preparations routineCarrier, diluent or the excipient used.
The pharmaceutical composition can be in preparation prevention and/or treatment disease medicament relevant to bruton's tyrosine kinaseApplication, the compound of the logical formula (I) of the present invention is applied including the tumor disease patient that over-expresses to bruton's tyrosine kinaseOr its pharmaceutically acceptable salt, isomers, solvate, crystallization or prodrug or compound comprising the logical formula (I) of the present invention orThe pharmaceutical composition of its pharmaceutically acceptable salt, isomers, solvate, crystallization or prodrug, effectively to inhibit Bu Ludun junket ammoniaAcid kinase overexpression, prevents disease progression.
Specific embodiment
The preparation of 1 2H- phthalazines -1- ketone of embodiment
Step 1: dimethoxy-methyl benzene (500mmol) is weighed in reaction flask, and tetrahydrofuran (800ml) dissolution is added,60 DEG C, under nitrogen protection, be added s-BuLi (565mmol), reaction solution stirred into 1h at -60 DEG C.
Step 2: dry ice (50mmol) is weighed in reaction flask, is added tetrahydrofuran (200ml), is added n-BuLi (5ml),After stirred under nitrogen atmosphere 2h, the mixture of step 1 is added, continues to stir 30min, stops reaction, water 1000ml is added, use is denseSalt acid for adjusting pH separates organic phase, water phase is extracted with ethyl acetate, and merges organic phase, saturated common salt water washing, anhydrous sulphur to 2Sour sodium is dry, is recrystallized to give 2- dimethoxy-methyl benzoic acid.
Step 3: weighing step 2 gains (400mmol), acetic acid (93mmol), hydrazine (600mmol) in reaction flask, addEntering isopropanol 300ml, under nitrogen protection, 100 DEG C of back flow reaction 2h stop reaction, ethyl acetate 300ml, water 500ml is added,Extraction merges organic phase, and anhydrous sodium sulfate is dry, hangs and does, column chromatographic purifying obtains title compound.
ESI–MS:[M+H]+m/z 147。
Embodiment 2 (3,4- dihydro-isoquinoline -2 (1H)-t-butyl formate -5- base) preparation of-carbamic acid to benzyl chloride ester
Triphosgene (5mmol) is weighed in reaction flask, toluene 100ml is added, is added dropwise at 0 DEG C and is dissolved with parachlorophenolThe tetrahydrofuran solution 20ml of (5mmol) and pyridine (10ml), drop finish, the reaction was continued at room temperature 8h, concentration of reaction solution, addition twoChloromethanes 40ml is hanged and is done, and chloro-carbonic acid is made to benzyl chloride ester, is directly used in next step.
Weigh -2 (1H)-t-butyl formate (50mmol) of 5- amino -3,4- dihydro-isoquinoline and DIPEA (100mmol) inIn reaction flask, methylene chloride 300ml is added, is stirred at room temperature down and chloro-carbonic acid is slowly added dropwise to benzyl chloride ester (51mmol), drop finishes, room temperatureUnder continue stir 1h, stop reaction, concentrated reaction mixture, be added ethyl acetate 70ml, diluted hydrochloric acid aqueous solution (0.2-0.3N)With saturated common salt water washing, anhydrous sodium sulfate is dried, filtered, and is concentrated to give title compound, is directly used in next step.
ESI–MS:[M+H]+m/z 417。
Embodiment 3 (3,4- dihydro-isoquinoline -2 (1H)-t-butyl formate -5- base)-carbamic acid -4- (2H)-phthalazines -1-The preparation of ketone group benzyl ester
2H- phthalazines -1- ketone and (3,4- dihydro-isoquinoline -2 (1H)-t-butyl formate -5- base)-carbamic acid are weighed to chlorineBenzyl ester (195mmol) is added DMF100ml, is reacted overnight at 55 DEG C, stop reaction, water 100ml, dichloro is added in reaction flaskMethane 200ml, extraction separate organic phase, and water phase continues to be extracted with dichloromethane (3*50ml), merge organic phase, anhydrous slufuric acidSodium is dry, and column chromatographic purifying obtains title compound.
ESI–MS:[M+H]+m/z 527。
Embodiment 4 [2 (1H)-acryloyl group -3,4- dihydro-isoquinoline -5- base]-carbamic acid -4- [(2H)-phthalazines -1-Ketone group] benzyl ester preparation
Weigh (3,4- dihydro-isoquinoline -2 (1H)-t-butyl formate -5- base)-carbamic acid -4- [(2H)-phthalazines -1- ketoneBase] in reaction flask, addition trifluoroacetic acid 20ml stirs 1h at room temperature, is concentrated to dryness, acetic acid is added benzyl ester (50mmol)Ethyl ester 80ml is successively dried, filtered with 1.5M disodium hydrogen phosphate aqueous solution and saturated common salt water washing, anhydrous sodium sulfate, is depressurizedIt is concentrated to give intermediate (1,2,3,4- tetrahydroisoquinoline -5- base) carbamic acid -2- (2H)-phthalazines -1- ketone group benzyl ester, is directly castOne step.
Weigh intermediate (1,2,3,4- tetrahydroisoquinoline -5- base) carbamic acid -2- (2H)-phthalazines -1- ketone group benzyl ester(20mmol) is added methylene chloride 100ml dissolution, DIEA (40mmol) is added at 0 DEG C in reaction flask, after stirring 30min, afterContinue dropwise addition acryloyl chloride (20mmol) at 0 DEG C, drop finishes, and 3h is stirred at room temperature, and stops reaction, adds water 100ml, methylene chloride extraction(3*50ml) merges organic phase, and anhydrous sodium sulfate is dry, and column chromatographic purifying obtains title compound.
1H NMR(600MHz,CDCl3) (δ, ppm): 9.50 (s, 1H), 8.18~8.16 (m, 1H), 8.10 (s, 1H),8.01~7.99 (m, 1H), 7.77~7.70 (m, 2H), 7.54~7.53 (m, 2H), 7.34~7.33 (m, 2H), 7.21~7.18 (m, 2H), 7.15~7.13 (m, 1H), 6.62~6.60 (m, 1H), 5.96~5.94 (m, 1H), 5.36~5.34 (m,1H), 4.65 (s, 2H), 4.22 (s, 2H), 3.61~3.59 (m, 2H), 3.13~3.11 (m, 2H)
ESI–MS:[M+H]+m/z 481。
The evaluation of 1 the compound of the present invention vitro kinase activity of experimental example
The compound of the present invention of above embodiments preparation successively dilutes after each compound is diluted to 10mM with DMSOTo 1uM, 100nM, 10nM, 1nM, 0.1nM, 0.01nM.
Take the 10 μ l of compound solution of each concentration into 96 orifice plates, addition 90 μ l1 × kinase buffer liquid (50mMHEPES,PH7.5,0.0015%Brij-35,10mMMgCl2,2mM DTT, prepared before use)；Set up DMSO control group and without enzyme simultaneouslyControl group living, contains only 10 μ lDMSO and 90 μ l1 × kinase buffer liquid.Each group mixes 10min at room temperature, then shifts respectively5 μ l are into 384 orifice plates；Kinase b TK is dissolved in 1 × kinase buffer liquid, is configured to 2.5 × kinase solution, then shifts 10 μ l2.5× kinase solution is into above-mentioned 384 orifice plates containing each concentration compound；10 μ l2.5 × kinase solution is added in DMSO control group；Nothing1 × kinase buffer liquid that 10 μ l are free of kinases is added in enzyme activity control group.It is incubated for 10min at room temperature；By FAM label polypeptide andATP is dissolved in 1 × kinase buffer liquid, is configured to 2.5 × substrate solution, then shifts 10 μ l2.5 × substrate solution to above-mentioned 384 holeIn plate, 28 DEG C of incubation 1hr；25 μ l (100mMHEPES, pH7.5,0.015%Brij-35,0.2% are added in each holeCoatingReagent#3,50mMEDTA prepared before use), terminate liquid terminates reaction；It is placed on LabChipEZReader and readsConversion data, and inhibiting rate I% is calculated, calculation formula is I%=(Max-Conversion)/(Max-Min) × 100,Middle Max is the conversion ratio of DMSO control group, and Min is the conversion ratio of no enzyme activity control group, and Conversion is compound processing groupConversion ratio, data handle through XLfit, are fitted to obtain IC50。IC50Value indicates that compound presses down with not plus compared with compound processing groupMake corresponding compound concentration when 50% enzyme activity.IC50 the results are shown in Table 1.
Table 1
The external Romas cell activity evaluation of 2 the compound of the present invention of experimental example
It takes in exponential phase of growth in good condition one bottle of Raji cell, collects cell, low speed desk centrifuge, 1500Turn/min, is centrifuged 3min.Supernatant is abandoned, 5mL complete medium is added with pipettor and carries out cell resuspension.Use cell count instrument meterNumber, complete medium are diluted, adjustment cell density to 5 × 104A/mL.It is inoculated on 96 orifice plates using the volley of rifle fire, 100 μ L/Constant temperature CO is set in hole2It is cultivated 24 hours in incubator.Compound sample-adding, which is carried out, using nanoliter sample adding instrument adds CCK-8 after 72 hours,Its light absorption value is detected in 10 holes μ L/ at Envision microplate reader 450nm after 2 hours, calculate inhibiting rate, and calculate IC50, as a result seeTable 2.
Table 1