Antitumoral compounds and preparation method thereof and purposesTechnical field
The present invention relates to antitumoral compounds and preparation method thereof and purposes, specifically, the antitumoral compounds are phoneticPyridine class compound, belongs to pharmaceutical technology field.
Background technique
Leukaemia is the Hematological malignancies that a kind of marrow and peripheral blood are infiltrated by hematopoietic stem/progenitor, is mainly shown asLeukaemia cell's hyper-proliferative, and there is dysdifferentiation more, ill juvenile cell largely infiltrates marrow and peripheral blood, causes peopleThere are the symptoms such as anaemia, bleeding, infection and fever in body hematological abnormalities.Leukaemia is often clinically divided into the white blood of myelocyteDisease and lymphocytic leukemia two major classes, compared with lymphocytic leukemia, myelocytic leukemia has higher disease incidence and deadDie rate.In China's mortality of malignant tumors seniority among brothers and sisters, leukaemia is the 8th, but in 35 years old or less person between twenty and fifty and pediatric populationHigh to rank first, and still risen with annual 10% or so ratio, seriously endanger the life and health of our people.
Currently, chemotherapy is the first-line treatment mode of domestic and international treatment leukaemia, main mechanism is by inhibiting answering for DNASystem, hinders synthesis of RNA etc. to inhibit the proliferation of tumour cell.The main therapy of acute myelocytic leukemia is cytarabine(Ara-C) combine other chemotherapeutics, in addition there are the treatment methods of interferon chemotherapeutics combination, but above method is because lackingWeary targeting, specificity also have certain toxic effect to normal cell, and during treatment, patient often will appear de-The side effects such as hair, canker sore, vomiting, constipation or diarrhea for the patient weaker for some bodies, tolerance is poor, are led toIt often needs to reduce dose, shortens the course for the treatment of, thus be difficult to reach ideal therapeutic effect, and be easy to appear after long-term use resistance toSituations such as medicine.Therefore, it finds the stronger treatment method of targeting and receives extensive attention in recent decades, also there are many make usSatisfied achievement, the all-trans retinoic acid (ATRA) as China scientist has found and promotes treat acute promyelocytic leukemia(M3 type), the therapy have become the first-line treatment method for the treatment of acute promyelocytic leukemia, greatly improve patientPrognosis, reduce mortality risk.But it is poor to other kinds of leukaemia effect because it is with stronger gene selectable,It is difficult to be generalized in other kinds of leukemia treating.Currently, FLT3 targeted drug Midostaurin is ratified by FDAListing, in addition to this, there are many more the drugs of kind magnetic target therapy acute myelocytic leukemia (AML) to come into clinical researchStage brings new vigor for tumor patient.But compared to other entity tumors, the progress of the relevant targeted drug of AMLIt is all relatively backward with clinical application.
Replacing Buddhist nun (Ibrutinib) according to Shandong is a kind of small molecule Bu Ludunshi tyrosine kinase (BTK) inhibitor, FDA approvalIts indication treated is lymphoma mantle cell (MCL), but after the drug emerges, due to its outstanding targeting and preferablyTherapeutic effect is widely used in the research of all kinds of oncotherapies including AML.There is research to confirm most of suffer from firstThere is the patient's body of AML all to there is the high expression of BTK, and a variety of leukemia cell lines including HL-60 have BTKHigh expression, then there is research to confirm that Ibrutinib may inhibit the functions such as proliferation and the migration of AML cell successively.TheseResearch provides new approaches for the targeted therapy of AML.
In view for the treatment of cancer, there is an urgent need to, this field, it is necessary to develop novel mechanism of action and better effect is goodAnti-tumor drug.
Summary of the invention
One of the objects of the present invention is to provide an antitumoral compounds or its pharmaceutically acceptable salts, this is antitumorCompound is specially pyrimidines, such compound has good anti-tumor activity.
Another object of the present invention is to provide the preparation methods of aforementioned antitumoral compounds.
Another object of the present invention is to provide the drugs containing the pyrimidines or its pharmaceutically acceptable saltComposition.
A further object of the present invention is to provide the pyrimidines or its pharmaceutically acceptable salt or described groupsClose the purposes of object.
Thus, on the one hand, the present invention provides a kind of antitumoral compounds or its pharmaceutically acceptable salt, antitumorizationConjunction object is structure shown in formula (I):
Structural compounds shown in formula (I) are pyrimidines, are named as BY4009, and antitumor activity screening of the present invention is aobviousShow, compared with Ibrutinib (replacing Buddhist nun according to Shandong), Ara-C (cytarabine), Dasatinib (Dasatinib), BY4009 is to HL-60, tri- kinds of marrow series leukemia cells of K562, U937, especially acute myelocytic leukemia (HL-60, U937), there is preferable suppressionThe ability of cell Proliferation processed；BY4009 almost without toxic effect, has relatively strong Healthy People PBMC to tumour cell and normal cellResolution capability；Concentration dependent and time dependence is presented to the inhibited proliferation of three kinds of cells in BY4009.As oneThe novel molecule of class formation, research compound has exploitation at the potentiality of new and effective BTK target spot inhibitor in the present invention, to controllingTreating relevant tumor disease especially acute myelocytic leukemia has biggish application value.
Structure shown in aforementioned formula (I) has following title:
(I) N- [3- [[the chloro- 2- of 5- [4- ((1- (4- piperidine alcohols)) methyl) aniline] -4- pyrimidine] amino] phenyl] -2- thirdAcrylamide.
On the other hand, the present invention provides the preparation method of aforementioned antitumoral compounds, which presses following roadLine preparation:
Compound of the present invention is due to their possibility purposes in drug, the preferred medicine of salt of compound shown in formula (I)The acceptable salt of object.The compound of the present invention is alkali, wherein required salt form can pass through appropriate parties legal system known in the artIt is standby, including with mineral acid treatment free alkali, the inorganic acid such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid etc.；Or with organicAcid processing free alkali, the organic acids for example acetic acid, trifluoroacetic acid, maleic acid, succinic acid, mandelic acid, fumaric acid, malonic acid,Pyruvic acid, oxalic acid, hydroxyacetic acid, salicylic acid, pyranose thuja acid (pyranosidy1acid), such as glucuronic acid or galactolipinAldehydic acid, 'alpha '-hydroxy acids, such as citric acid or tartaric acid, amino acid, such as aspartic acid or glutamic acid, aromatic acid, such as benzene firstAcid or cinnamic acid, sulfonic acid, such as p- toluenesulfonic acid, methanesulfonic acid, ethanesulfonic acid etc..The embodiment of pharmaceutically acceptable salt includes sulphurHydrochlorate, pyrosulfate, disulfate, sulphite, bisulfites, phosphate, chloride, bromide, iodide, acetic acidSalt, propionate, caprate, caprylate, acrylates, formates, isobutyrate, caproate, enanthate, propionate(propiolates), oxalates, malonate, benzoate, chloro benzoate, methyl benzoic acid salt, dinitrobenzoic acidSalt, hydroxy benzoate, methoxy benzoic acid salt, phthalate, phenyl acetate salt, phenylpropionic acid salt, phenylbutyrate(phenylbutrates), citrate, lactate, gamma hydroxybutyrate, hydroxyl acetate, tartrate, amygdalateWith sulfonate, such as xylenesulfonate, mesylate, propane sulfonic acid salt, naphthalene -1- sulfonate and naphthalene-2-sulfonic acid salt.
On the other hand, the present invention provides a kind of pharmaceutical composition, shown in the formula of the present invention (I) containing effective doseCompound or its pharmaceutically acceptable salt and pharmaceutical carrier.
Pharmaceutical composition of the invention usually contains a kind of the compounds of this invention.However, in some embodiments, this hairBright pharmaceutical composition, which contains, has more than a kind of the compound of the present invention.In addition, pharmaceutical composition of the invention can also optionally includeOne or more other pharmaceutically active compounds.Pyrimidines of the present invention or its pharmaceutically acceptable carrier havePhosphorylation occurs for good inhibition BTK.Therefore, the present invention also provides the pyrimidines or its pharmaceutically acceptable loadsBody or described pharmaceutical composition are preparing the application in BTK tyrosine kinase inhibitor.
Present invention discover that the pyrimidines or its pharmaceutically acceptable carrier have good inhibition BTK junket ammoniaThe activity of acid kinase, therefore, the present invention also provides the pyrimidines or its pharmaceutically acceptable carrier or the medicinesCompositions are preparing the application in BTK tyrosine kinase inhibitor.
The present invention also provides the formula (I) compound represented or its pharmaceutically acceptable salts or of the present inventionPurposes of the pharmaceutical composition in the drug of preparation treatment tumour.Preferably, the tumour is selected from acute myelocytic leukemia.MorePreferably, the purposes, which mainly passes through, inhibits the realization of BTK tyrosine kinase.
Detailed description of the invention
Fig. 1 is to indicate that BY4009 different disposal group makees the inhibition of tri- kinds of marrow series leukemia cells of HL-60, U937, K562With changing over time situation.
Fig. 2 is to detect BY4009 to the toxic effect of healthy human peripheral blood PBMC.
Fig. 3 be Western Blotting method detect various concentration BY4009 handle HL-60 cell 48h after BTK, p-BTK,The expression of Bcl-2 and Bax protein level changes.
Specific embodiment
The explanation present invention is further described below in conjunction with specific embodiment, but these embodiments are not meant as limiting this hairBright range.
Test method without specific conditions in the embodiment of the present invention, usually according to normal condition, or according to raw material orCondition proposed by commodity manufacturer.The reagent in specific source is not specified, for the conventional reagent of market purchase.
The preparation of 1 target molecule of embodiment
Tlc silica gel plate uses Yantai Huanghai Sea GF254 or Qingdao GF254 silica gel plate, and thin-layered chromatography (TLC) usesThe specification that uses of silicon amine plate be 0.15mm-0.2mm, the specification that thin-layer chromatography isolates and purifies product use is 0.4mm-0.5mm.
The raw material that the present invention uses mainly is purchased from commercially available from Sinopharm Chemical Reagent Co., Ltd., Beijing coupling science and technologyCo., Ltd, reaches the companies such as auspicious chemicals at Aladdin chemical reagent Co., Ltd.
Without specified otherwise in embodiment, solution refers to aqueous solution.
Without specified otherwise in embodiment, it is 20 DEG C -30 DEG C that the temperature of reaction, which is room temperature,.
The technical solution adopted by the invention is as follows:
The synthetic route reagent and condition of compound (I): (a) 4- hydroxy piperidine, K2CO3,KI,CH3CN,80℃,12h,91%；(b)Fe-NH4Cl,MeOH-H2O, 2h, 80 DEG C, 72%；(c) acryloyl chloride, NaHCO3,CH3CN,NaHCO3,CH3CN,0DEG C, 0.5h, 95%；(d)Fe-NH4Cl,MeOH-H2O, 70 DEG C, 2h, 81%；(e) 2,4,5 ,-trichloropyrimidine, DIPEA, 1,4- bis-Six ring of oxygen, 60 DEG C, 2h, 82-91%；(f) TFA, 2-BuOH, 100 DEG C, 12h, 18-31%.
Target molecule is synthesized according to above method, the physicochemical data of synthesized target molecule is as follows:
(I) N- [3- [[the chloro- 2- of 5- [4- ((1- (4- piperidine alcohols)) methyl) aniline] -4- pyrimidine] amino] phenyl] -2- thirdAcrylamide；Yield 30.4%；White-yellowish solid.1H NMR(DMSO-d6):δ10.20(s,1H),9.38(s,1H),8.91(s,1H), 8.07 (s, 1H), 7.77 (s, 1H), 7.48 (d, J=8.4Hz, 3H), 7.28 (d, J=6.4Hz, 2H), 7.02 (d, J=8.4Hz, 2H), 6.42 (dd, J=17.2,10.0Hz, 1H), 6.20 (dd, J=17.2,2.0Hz, 1H), 5.70 (dd, J=10.0,2.0Hz,1H),3.60(s,2H),3.50(m,1H),3.28(m,4H),2.10(m,4H).HRMS(ESI)forC25H27ClN6O2,[M+H]+Theoretical calculation: 479.1957, actual measurement: 469.0233.
Target molecule at salt method
The preparation method of inorganic acid salt: it takes target molecule (1mmol) to be dissolved in 10mL anhydrous methanol, under ice bath, slowly dripsThe 5mL absolute methanol solution for adding inorganic acid (1mmol), is added dropwise, and stirs 30 minutes at a temperature of this, then first is evaporated off in room temperatureAlcohol to get target molecule inorganic acid salt.By this method be prepared for the hydrochloride (I-1) of compound I, hydrobromate (I-2),Sulfate (I-3) and phosphate (I-4)；
The preparation method of acylate: it takes target molecule (1mmol) to be dissolved in 10mL anhydrous methanol, under ice bath, slowly dripsThe 5mL dry ether for adding organic acid (1mmol), is added dropwise, and stirs 30 minutes at a temperature of this, and then solvent is evaporated off in room temperature,Up to the acylate of target molecule.By this method be prepared for the maleate (I-5) of compound I, succinate (I-6) andFumarate (I-7).
2 target molecule biological evaluation of embodiment
2.1 cell culture
2.1.1HL-60 the culture of cell selects IMDM culture medium, in culture medium containing 20% FBS, 1% it is dual anti-,37 DEG C, 5%CO2Environment in cultivated, usually 1 pass 3,5ml culture medium be added, passage in 2-3 days, logarithmic growth phase is thinBorn of the same parents test.
2.1.2K562 cell uses 1640 culture medium cultures, contains 15% FBS, 1% penicillin/chain in culture mediumMycin is dual anti-, at 37 DEG C, 5%CO2It is cultivated in environment, usual 1 passes 5, and culture medium 5ml is passed on, logarithmic growth phase is thin for every 2 daysBorn of the same parents test.
2.1.3U937 cell uses 1640 culture medium cultures, in culture medium containing 15% FBS, 1% it is dual anti-, 37DEG C contain 5%CO2Incubator in cultivate, usually 1 pass 5, culture medium 5ml be added, every 2 days secondary cultures take logarithmic growthPhase cell is tested.
2.1.4PBMC extraction and culture
Fresh and healthy people anticoagulation 10ml-20ml is taken, after mixing with PBS1:1, is added to isometric separation of lymphocytesOn liquid liquid level, adition process will be operated slowly, and blood is avoided to break through the liquid level of lymphocyte separation medium, rapid 2000rpm centrifugation20min.Liquid after centrifugation is divided into four layers, is respectively as follows: plasma layer, mononuclearcell layer, lymphocyte separation medium from top to bottomLayer and red blood cell layer.Separating liquid in PBS cleaning cell for taking mononuclearcell layer to be added 2-3 times etc. 1-2 times, every time2000rpm is centrifuged 20min.The above operation carries out under iuntercellular gnotobasis, then with 1640 cultures containing 20%FBSBase is cultivated, and condition of culture is 37 DEG C, 5%CO2, tested in one week.
2.2CCK8 method detects drug to the Proliferation Ability ability and IC of HL-60, U937, K562 and PBMC cell50Calculating
2.2.1CCK8 method measure various concentration BY4009 for 24 hours, 48h, 72h it is thin to HL-60 cell, U937 cell, K562The growth inhibition ratio of born of the same parents
5×104The HL-60 cell inoculation of density is in 96 well culture plates, and 5 × 103K562, U937 cell inoculation of density in96 well culture plates make its final volume be 100 holes μ L/, if control group (0.5%DMSO is added), blank group (only adds full cultureLiquid), dosing group, as shown in table 1 (after many experiments, determining suitable adding consistency range), every group sets 3 to adding consistencyMultiple holes.Set 37 DEG C, 5%CO2Cultivated in incubator cultivate respectively for 24 hours, after 48h, 72h, 10 μ L CCK8 reagents are added in every hole, afterContinuous to be incubated for 0.5-4h, microplate reader detects absorbance value under 450nm wavelength, calculates each group proliferation inhibition rate.Experiment is repeated 3 times,Take its mean value.Proliferation inhibition rate=1- (A dosing group-A blank group)/(A control group-A blank group) %.Acquired results such as Fig. 1 instituteShow.
2.2.2CCK8 method measurement BY4009, Ibrutinib, Dasatinib and Ara-C are thin to HL-60 cell, U937Born of the same parents, K562 cell IC50Value
5×104The HL-60 cell inoculation of density is in 96 well culture plates, and 5 × 103K562, U937 cell inoculation of density in96 well culture plates make its final volume be 100 holes μ L/, if control group (0.5%DMSO is added), blank group (only adds full cultureLiquid), dosing group, adding consistency is (after many experiments grope concentration, determining suitable adding consistency range) as shown in table 1,Every group sets 3 multiple holes.Set 37 DEG C, 5%CO2After cultivating 72h in incubator, 10 μ L CCK8 reagents are added in every hole, continue to be incubated for0.5-4h, microplate reader detect absorbance value under 450nm wavelength.Finally IC is acquired using SPSS23.0 software50.Acquired results are such asShown in table 2:
2.2.3 toxic effect of the measurement BY4009 to Healthy People PBMC
1×105In 96 well culture plates culture medium is added, making its final volume is 100 μ L/ in the PBMC cell inoculation of densityHole, if control group (0.5%DMSO is added), blank group (only adds full nutrient solution), dosing group, and the setting of dosing group concentration is equalAre as follows: 200 μM, 100 μM, 50 μM, 12.5 μM, 6.25 μM are all provided with three multiple holes.Set 37 DEG C, 5%CO248h is cultivated in incubatorAfterwards, 10 μ L CCK8 reagents are added in every hole, continue to be incubated for 0.5-4h, are detected under the wavelength of 450nm using microplate reader Gene5 eachHole absorbance value calculates the survival rate of group of cells.Cell survival rate=(A dosing group-A blank group)/(A control group-A blankGroup) %.Acquired results are as shown in Figure 2.
Table 1:CCK8 drug effect concentration
Table 2
2.3 protein immunoblottings (Western blot) method detects BY4009 to HL-60 cell in protein expressionEffect
2.3.1 total protein is extracted
Bed board HL-60 cell, every hole cell quantity is 5 × 10 in six orifice plates6It is a, setting following groups: blank control group,0.5 μM, 1 μM and 2 μM BY4009 processing group, the agent-feeding treatment time is 48h.Then collected with 1.5ml centrifuge tube with thinThe culture medium of born of the same parents, 800rpm are centrifuged 5min, abandon supernatant.PBS is washed 3 times.Configured cell pyrolysis liquid is added in each sample(RIPA:PMSF=100:1) 100-150 μ l, sample loading gun blow and beat mixing repeatedly, generally at 50 times or more, set 4 DEG C of environment waitings and splitSolution, 15min concussion is primary, and each 30s is repeated 4 times.Last 4 DEG C, 12,000rpm centrifugation 20min, supernatant, that is, institute's leach protein.InstituteThere is operation to carry out on ice, and the albumen extracted should use as early as possible, avoid multigelation.
2.3.2 determination of protein concentration
(1) working solution is prepared: working solution total amount needed for calculating, 200 μ l working solutions of each sample needs (BCA:Cu=50:1) it, using being prepared in one day, and mixes well.
(2) it dilution standard product: takes 20 μ l standard items to be diluted to 200 μ l with PBS, makes final concentration of 1mg/ml.Standard itemsIt is added in the standard sample wells of 96 orifice plates by the total amount of 0,1,2,3,4,6,8,10 μ g, PBS is added to complement to 20 μ l, be respectively provided with three againHole.
(3) sample dilutes: sample suitably being diluted, complements to 20 μ l with PBS.
(4) 200 μ l BCA working solutions, 37 DEG C of placement 30min are added in every hole.
(5) absorbance value in each hole is detected at 570nm wavelength.Protein concentration is calculated according to standard curve, determines WesternAlbumen volume needed for Blot, albumen applied sample amount is 20-40 μ g under normal circumstances, is adjusted according to specific destination protein.
2.3.3 protein immunoblotting (Western Blotting) is carried out
(1) glue: according to the separation gel of destination protein molecular weight BTK (76KD), p-BTK (76KD) selection 10%, (it is matchedSystem processed is as shown in table 3) and 5% concentration glue (its prepare system as shown in table 4).It is formulated as follows:
Table 3:10% separation gel prepares system (10ml)
Table 4:5% is concentrated glue and prepares system (5ml)
(2) electrophoresis: 4 × sample-loading buffer and testing protein 3:1 are mixed, and are supplemented to volume with 1 × sample-loading buffer30 μ l, 100 DEG C, 8min make albuminous degeneration.Loading in order, every hole add 30 μ l samples, Marker5 μ l.The glue stage is concentrated, usesVoltage 90V makes sample aggregation on one wire, and when sample enters separation gel, adjusting voltage is 130V, continues electrophoresis extremelyMarker band is separated to suitable degree, terminates electrophoresis, and the transferring film after being placed at room temperature for a period of time.
(3) transferring film: pvdf membrane is cut according to the size of glue, pvdf membrane first balances 15s in formaldehyde, then in transferring film liquidImpregnate 20s-30s.It by sequence from top to bottom in transferring film liquid, is sequentially placed foam-rubber cushion 1 and opens, filter paper 3 is opened, glue, pvdf membrane, filterPaper 3 is opened, and the sequence that sponge 1 opens stacks, and is placed in membrane-transferring device.Power supply is connected, adjusting electric current is 300mA, and the time is according to moleculeDepending on the size of amount, general 1KD=1min.Surrounding low temperature environment is kept when transferring film.
(4) close: 5% skimmed milk power closes pvdf membrane, and at least 1h is usually closed under room temperature.
(5) antibody incubation: diluting antibody with 5% skimmed milk power in proportion, and pvdf membrane is put into wherein 4 DEG C of overnight incubations,Continue to be incubated for 1h under next day room temperature, so that antibody be made preferably with the protein binding on film, then to use, PBST/TBST washes film 3It is secondary, each 15min.Secondary antibody after adding dilution is incubated at room temperature 1h；Secondary antibody is abandoned, PBST/TBST washes film 3 times, each 15min(selecting TBST when destination protein is p-BTK, other albumen select PBST).
2.3.4 image scanning and analysis
It is uniformly added on pvdf membrane after the A liquid of ECL detection reagent and B liquid are mixed by 1:1, developing machine is in dark field and light fieldPicture is obtained respectively, and purpose band is determined by Marker after synthesis.Gray scale point is carried out to purpose band using Image J softwareAnalysis.
3 data statistics
This research experiment data use average ± standard deviation (Mean ± SD) to indicate, use SPSS23.0 statistical software pairData are analyzed；T inspection carries out the comparison of mean between group；Think there is significant difference as p < 0.05.Acquired results are as schemedShown in 3.
The above bioactivity the result shows that:
(1) BY4009 has good growth and inhibited proliferation to three kinds of myeloid leukemia cells, and this makees apparatusThere are concentration and time dependence.
(2) BY4009 has preferable pharmacological activity to HL-60 and U937, and commonly uses treatment acute myeloid with clinicThe Ara-C of leukaemia is compared, IC50It is horizontal approximate even lower；And K562 is as chronic myelocytic leukemia, IC50Be worth it is slightly larger,Gap is larger compared with its clinical commonly used drug Dasatinib, but the IC compared with Ibrutinib50It is relatively low.
(3) BY4009 there's almost no growth inhibition to Healthy People PBMC, only just have when activity reaches 100 μMLesser toxic effect, i.e., the low concentration BY4009 employed in subsequent experimental is to normal cell almost without toxic effect.
(4) various concentration BY4009 assesses its protein expression level after handling HL-60 cell, and discovery BTK protein expression is simultaneouslyDo not occur significantly changing, and p-BTK is with the increase of drug concentration that its expression is gradually lowered, Bcl-2/Bax decline, showsBY4009 can be effectively suppressed BTK phosphorylation and then inhibit growth of tumour cell.In conjunction with Ibrutinib (replacing Buddhist nun according to Shandong), Ara-CThe IC of (cytarabine) and Dasatinib (Dasatinib) three kinds of drugs50, and the assessment to the related apoptosis factor, discoveryMolecule of the BY4009 as a kind of structure novel, research compound shows good BTK and inhibits potentiality in the present invention, can developAt efficient BTK target spot inhibitor, to treatment acute myelocytic leukemia (AML), acute lymphoblastic leukemia (ALL), chronicGranulocytic leukemia chronic phase, chronic lymphocytic leukemia, chronic grain or lymphocytic leukemia progressive stage, jacket cellThe related neoplasms such as lymthoma and small lymphocyte lymthoma have preferable application value.
The above is only preferred embodiments of the invention, it is noted that for the ordinary skill people of the artFor member, without departing from the technical principles of the invention, several improvements and modifications can also be made, these improvements and modificationsAlso it should be regarded as protection scope of the present invention.