A kind of carboxamides derivatives, preparation method and its application in antitumor drugTechnical field
The invention belongs to pharmaceutical chemistry and area of pharmacology, be related to a kind of carboxamides derivatives, preparation method and itsApplication in antitumor drug.
Background technology
The morbidity of cancer predicts that 21 century malignant tumour will be as " the first of the mankind with the World Health Organization (WHO) is endangeredKiller ", therefore cancer control has become global health strategy emphasis.Though China is developing country, spectrum of disease has occurredTransformation, China have become the first in the world cancer big country, and cancer not only seriously threatens the life and health of our people, but also givesFamily, society, country cause white elephant, interfere economic construction of China and social development, are one very outstandingSocial public health problem.Moreover, as township industry, city of residence, the acceleration of aging of population process, environment are dirtyDye, bad life habits and unreasonable life style generally existing, most cancers will also be in rising trend, especially lung cancer,Liver cancer, intestinal cancer steeply rise, and are worth paying much attention to.
Mammal rapamycin target protein (mammalian target of rapamycin, mTOR) is a kind of SARSType serine/threonine protein kitase belongs to phosphatidylinositol 3-kinase (phosphoinositide3-kinase, PI3K)Associated kinase family member is that the main signal of the cell functions such as intracellular synthesis and catabolism transmits molecule.MTOR signalsAccess has close relationship with nutrition, energy state and growth factor.It includes autophagy, albumen, lipid, lyase that it, which is adjusted,Body synthesizes and multiple cell processes such as energetic supersession, cytoskeletal organization, cell survival.In mammalian cell periphery nutrition barUnder part constantly variation, mTOR regulates and controls the conversion of synthesis and katabolism, so that cell energy under different nutritional conditionsEnough growths and survival.Due to important function of the mTOR in cell, abnormal or imbalance mTOR signals transmission can lead to mankind's diseaseThe generation (such as the diseases such as cancer) of disease.Therefore mTOR signal paths are increasingly becoming an important target spot of design anticancer drug.
The present invention obtains new structural carboxamides derivatives by chemical synthesis means, and true by pharmacological evaluationThe antitumor activity of this fixed compound.
Invention content
The purpose of the present invention is to provide a kind of new structural carboxamides derivatives formula (I), structure isWherein, R1Selected from H, F or CH3, R2Selected from H, F or CH3, R3It is selected fromH, F or CH3。
Further, some specific preferred structures represented by the carboxamides derivatives formula (I) are as follows：
Another object of the present invention is to provide a kind of carboxamides derivatives formula (I) and its pharmaceutically acceptable salt conductsApplication of the mTOR kinase inhibitors in preventing and/or treating disease.
Another object of the present invention is to provide a kind of carboxamides derivatives formulas (I) and its pharmaceutically acceptable salt to controlTreat the application in cancer.
Further, the cancer is liver cancer, lung cancer, glioma, gastric cancer, oophoroma, breast cancer.
The compound of the present invention can in a free form, or in the appropriate case with pharmaceutically acceptable salt or itsThe form of its derivative is for treating.Terms used herein " pharmaceutically acceptable salt " refer in this way some from medical angleJudge available salt, when it is in contact with the cell tissue of the mankind and lower animal, not will produce excessive toxicity, irritation,And allergic reaction etc., and when it is used to treat, there is rational interests/Hazard ratio.In the art, such as amine, carboxylic acid,The preparation method of the pharmaceutically acceptable salts such as phosphonic acids and certain form of compound is well-known.Chemical combination in the present inventionThe salt of object can be made in the separation and purifying of the compound, and the compound of the present invention can also individually be made to dissociate with suitableAlkali or acid react.Pharmaceutically acceptable nontoxic acid-addition salts amino and inorganic acid or organic acid be formed by amino itsIts pharmaceutically acceptable salt includes adipate, alginates, ascorbate, aspartate, benzene sulfonate, benzoic acidSalt, disulfate, borate, butyrate, camphor hydrochlorate, camsilate, citrate, pentamethylene, digluconate, 12Alkyl sulfate, esilate, formates, fumarate, gluceptate, glycerophosphate, gluconate, hemisulfic acidSalt, hydriodate, 2- isethionates, Lactobionate, lactate, laruate, lauryl sulfate, malate, horseCarry out hydrochlorate, malonate, methane sulfonates, 2- naphthalene sulfonates, nicotinate, nitrate, oleate, oxalates, palmitate is doubleHydroxynaphthoate, pectate, persulfate, excessively 3- phenylpropionic acid salt, phosphate, picrate, Pivalate, propionate, firmlyResin acid salt, succinate, sulfate, tartrate, rhodanate, tosilate, undecanoate, pentyl sulfonateThe amido cation formed with arylsulphonate.
Test example part of the present invention uses the activity detection kit vitro detection the compounds of this invention of mTOR proteaseMTOR kinases inhibitory activity, and with mtt assay measure the compounds of this invention to liver cancer, lung cancer, glioma, gastric cancer,6 kinds of oophoroma, breast cancer human cancer cells, which carry out in-vitro multiplication experiment confirmation the compounds of this invention, has antitumor activity.
Another object of the present invention is to provide a kind of new structural carboxamides derivatives formula (I), synthetic routesFor：
Another object of the present invention is to provide a kind of synthesis steps of new structural carboxamides derivatives formula (I)For：
1) using toluene as solvent, by the Pd (PPh of suitable equivalent3)4It is added to containing the chloro- 5- trifluoromethylnicotinates of 2-,In the toluene solution of 2- furyl boronic acids and inorganic base, heated 2 hours under high temperature.It is post-treated to obtain 2- (2- furyls)-5- trifluoromethylnicotinates；
2) by PtO2It is added to the EtOH solution of 2- (2- furyls) -5- trifluoromethylnicotinates that step 1) obtainsIn, using Parr shaking machines hydrogenated mixture 1 hour under a certain pressure, then 2- (2- furyls)-is obtained through subsequent processing5- trifluoromethyl piperidine -3- carboxylic acid, ethyl esters；
3) under low temperature, oxalyl chloride is added to and is dissolved in CH2Cl2In 5- bromopyridine formyl chlorides solution in, be added catalysisReaction is stirred at room temperature 2 hours, synthesis step 2 is then added by the DMF of amount) obtained 2- (2- furyls) -5- fluoroformsPhenylpiperidines -3- carboxylic acid, ethyl esters and Et3N after purification through subsequent processing obtains 1- (5- bromopyridines acyl group) -2- (furans -2- bases) -5- (trifluoromethyl) piperidines -3- Ethyl formates；
4) intermediate product obtained with solutions of lithium aluminium hydride reduction synthesis step 3) at 0 DEG C, is then added in H2In OCrO3Slurry in, formic acid analog derivative is obtained, then again by the dichloro of itself and 4- methyl-3-trifluoromethyl phenylamines and triethylamineDichloromethane is reacted, and is slowly added to 1- n-propyl phosphoric anhydride T3P, and stir 1.5 hours at room temperature, pure through subsequent processingAfter change, 1- (5- bromopyridines acyl group) -2- (furans -2- bases)-N- (4- methyl -3- (trifluoromethyl) phenyl) -5- (trifluoros are obtainedMethyl) piperidines -3- formamides；
5) using PdNPs as catalyst, by synthesis step 4) obtained 1- (5- bromopyridines acyl group) -2- (furans -2- bases) -N- (4- methyl -3- (trifluoromethyl) phenyl) -5- (trifluoromethyl) piperidines -3- formamides exist with substituted phenyl boric acid and potassium carbonateIt reacts in reaction bulb, mixture is vigorously stirred 10 minutes in air atmosphere in 60 DEG C, is obtained finally through subsequent processingThe corresponding carboxamides derivatives of product.
Further, the amount range of the substance of Pd (PPh3) 4 can be 2-5mmol in the step 1), preferably2.6mmol。
Further, the inorganic base in the step 1) can be sodium hydroxide, potassium carbonate, sodium carbonate, potassium hydroxide, carbonSour hydrogen sodium, cesium carbonate, lithium hydroxide, preferably potassium carbonate.
Further, the pressure limit of Parr shaking machines is 30-60psi, preferably 40-45psi in the step 2).
Further, the low temperature in the step 3) refers to 0-10 DEG C, preferably 0 DEG C.
Specific implementation mode
Embodiment 1：2- (furans -2- bases) -1- (5- (2,4,5- trifluorophenyls) picolinoyl)-N- (4- methyl -3-(trifluoromethyl) phenyl) -5- (trifluoromethyl) piperidines -3- formamides synthesis
The synthesis of 1-1 2- (furans -2- bases) -5- (trifluoromethyl) ethyl nicotinate
By Pd (PPh3)4(3.0g, 2.60mmol) be added to containing the chloro- 5- trifluoromethylnicotinates of 2- (2.5g,9.86mmol), 2- furyl boronic acids (2.10g, 18.77mmol) and K2CO3The toluene (200mL) of (5.5g, 39.80mmol) is moltenIn liquid, and reaction mixture is heated 2 hours at 100 DEG C.After reaction mixture is cooled to room temperature, under reduced pressure by solventIt removes, 30ml water is added and stirs 5 minutes.Gained aqueous layer with ethyl acetate extracts, and combined organic layer is done with anhydrous sodium sulfateIt after dry, is filtered by diatomite, plug of celite is washed with EtOAc, is then concentrated under reduced pressure.Pass through automatic flash chromatography column method(SiO2, the EtOAc- hexanes of 10% to 100% gradient) purifies residue, and 50 DEG C of dryings of vacuum obtain 2- (furans -2- bases) -5- (trifluoromethyl) ethyl nicotinate, 2.56g, yield 91%.1H-NMR (400MHz,CDCl3)δ:1.30(t,3H),4.29(t,2H),6.98(t,1H),7.59(d,1H),8.07(d,1H),8.62(s, 1H),8.88(s,1H).13C-NMR(125MHz,CDCl3)δ:14.68,61.22,106.67,110.99,124.92, 127.33,129.39,135.39,143.63,146.88,148.69,150.27,167.28.LC-MS(ESI,pos,ion) m/z:286[M+H]。
The synthesis of 1-2 2- (furans -2- bases) -5- (trifluoromethyl) piperidines -3- carboxylic acid, ethyl esters
By PtO2(800mg, 3.52mmol) is added to 2- (2- furyls) -5- trifluoromethyls that synthesis step 1-1 is obtainedIn EtOH (60mL) solution of ethyl nicotinate (2.56g, 8.98mmol).Using Parr shaking machines in 40-45psi hydrogenated mixtures1 hour.Then reaction mixture is filtered by diatomite, plug of celite is washed with EtOH, and filtrate decompression is concentrated.It will be residualExcess CH2Cl2It dilutes and uses saturation NaHCO3Solution washs.Pass through flash chromatography column (SiO2, 0-20%MeOH/CH2Cl2)Purifying, obtains required product 2- (furans -2- bases) -5- (trifluoromethyl) piperidines -3- carboxylic acid, ethyl esters, 2.22g, yield 85%.1H-NMR(400MHz,CDCl3)δ:1.22(t,3H),1.87(m,1H), 1.91(s,1H),2.36-2.72(m,4H),3.52(t,1H),4.21(q,2H),4.50(d,1H),6.35-6.45(m,2H), 7.56(d,1H).13C-NMR(125MHz,CDCl3)δ:14.68,25.88,34.50,42.23,43.92,55.24, 61.74,109.88,112.59,129.43,142.40,159.25,173.81.LC-MS(ESI,pos,ion)m/z: 292[M+H]。
The synthesis of 1-3 1- (5- bromopyridines acyl group) -2- (furans -2- bases) -5- (trifluoromethyl) piperidines -3- Ethyl formates
At room temperature, oxalyl chloride (3.2mL, 30.25mmol) is added in reaction flask and is dissolved in CH2Cl2(20mL)In 5- bromopyridines formyl chloride (3.79g, 17.19mmol) solution in, then be added catalytic amount DMF.It will react in room temperatureLower stirring 2 hours.Solvent and extra oxalyl chloride are removed under vacuum, residue is dried 20 minutes under a high vacuum.WillTo acyl chlorides be dissolved in anhydrous CH2Cl2In (20mL) and it is cooled to 0 DEG C, 2- (the 2- furans that synthesis step 1-2 is obtained then is addedBase) -5- trifluoromethyl piperidine -3- carboxylic acid, ethyl esters (2.22g, 7.62mmol) and Et3N (8.6mL, 62.04mmol).It will mixingObject is warming up to room temperature and is stirred overnight.Reaction mixture CH2Cl2Dilution, after adding water, the solution that is layered.Water layer is usedCH2Cl2Extraction, by combined organic layer with anhydrous MgSO4It dries and is concentrated under reduced pressure, pass through flash chromatography column (SiO2, 10-35% EtOAc/ hexanes) purifying, obtain 3.59g1- (5- bromopyridines acyl group) -2- (furans -2- bases) -5- (trifluoromethyl) piperazinePyridine -3- Ethyl formates, yield 99%.1H-NMR(400MHz,CDCl3)δ:1.22(t,3H),2.59-2.83(m,2H),3.49(t,1H),3.71(m,1H), 3.96(t,1H),4.21(q,2H),5.24(d,1H),6.35-6.45(m,2H),7.56(d,1H),8.30(d,1H), 8.73-8.78(m,2H).13C-NMR(125MHz,CDCl3)δ:14.68,26.96,35.94,40.42,46.09,54.31, 61.74,111.54,112.59,119.57,120.26,129.94,139.19,142.40,146.01,153.47,155.08, 167.74,173.51.LC-MS(ESI,pos,ion)m/z:476[M+H]。
1-4 1- (5- bromopyridines acyl group) -2- (furans -2- bases)-N- (4- methyl -3- (trifluoromethyl) phenyl) -5- (threeMethyl fluoride) piperidines -3- formamides synthesis
1. solutions of lithium aluminium hydride (2.0mol/L, 8.2mL, 16.4mmol in THF) is added to synthesis step at 0 DEG C1- (5- bromopyridines acyl group) -2- (furans -2- bases) -5- (trifluoromethyl) piperidines -3- Ethyl formates that 1-3 is obtained (3.59g,In THF (100mL) solution 7.54mmol), acquired solution is stirred 2 hours until the reaction was complete at 0 DEG C.It is added dropwise15%NaOH aqueous solutions (625 μ L) to quench the reaction, then add the H of 625 μ L2O.It is added into muddy colloid admixtureThe water of 1.85mL, and mixture is stirred at room temperature 1 hour.Then mixture is filtered by plug of celite, and by filtrateIt is concentrated under reduced pressure.It is purified by flash chromatography column (the EtOAc/ hexanes of SiO2,33-67%), obtains 3.04g yellow powders(5- bromopyridine -2- bases) (2- (furans -2- bases) -3- (methylol) -5- (trifluoromethyl) piperidin-1-yl) ketone, yield are93%.2. at room temperature, being dissolved in (5- bromopyridine -2- bases) (2- (furans from synthesis step 1. in acetic acid (65mL)Mutter -2- bases) -3- (methylol) -5- (trifluoromethyl) piperidin-1-yl) solution of ketone (3.04g, 7.01mmol) is added toH2CrO in O (16mL)3In (2.61g, 26.10mmol) slurry.Gained mixture is stirred at room temperature 90 minutes until anti-It should be complete.Then reaction mixture is filtered by plug of celite, and filtrate decompression is concentrated.By flash chromatography column (SiO2,The CH of 3-10%2Cl2：MeOH, then using the EtOAc/ hexanes of 50-67%) purifying, obtain dry 2.19g off-white colorsPowdery product 1- (5- bromopyridines acyl group) -2- (furans -2- bases) -5- (trifluoromethyl) piperidines -3- formic acid, yield 70%.3. 4- methyl-3-trifluoromethyl phenylamines (1.62g, 9.25mmol) to be added to the acid (2.19g, 4.91mmol) and three of above-mentioned preparationIn dichloromethane (5mL) solution of ethamine (1.56g, 15.42mmol).It is then slowly added into 1- n-propyl phosphoric anhydrides T3P(95.5mg, 30.38mmol), and the solution is stirred at room temperature 1.5 hours.Use CH2Cl2(5mL) diluted reaction mixture,HCl/water solution, the saturation NaHCO of 1mol/L are used successively3Aqueous solution washs.By organic layer separation, with anhydrous MgSO4It is dry, subtractPressure concentration.Pass through flash chromatography column (SiO2, the EtOAc/ hexanes of 5-40%) and it is purified, obtain 2.17g white solid 1- (5-Bromopyridine acyl group) -2- (furans -2- bases)-N- (4- methyl -3- (trifluoromethyl) phenyl) -5- (trifluoromethyl) piperidines -3- firstAmide, yield 73%.1H-NMR(400MHz,CDCl3)δ:2.29(s,3H),2.41(m,1H),2.49(m, 1H),2.75(m,1H),3.53(m,1H),3.97-4.14(m,2H),4.74(d,1H),6.36-6.45(m,2H),7.07(d, 1H),7.48(d,1H),7.56(d,1H),7.98(s,1H),8.30(d,1H),8.62(s,1H),8.73-8.78(m,2H).13C-NMR(125MHz,CDCl3)δ:19.65,24.98,35.94,40.69,46.09,52.70,111.54,112.59, 118.31,119.57,120.26,124.63,125.42,129.94,130.86,131.19,133.54,135.14,139.19,142.40,146.01,153.47,155.08,167.74,173.69.LC-MS(ESI,pos,ion)m/z:605[M+H]。
1-5 2- (furans -2- bases) -1- (5- (2,4,5- trifluorophenyls) picolinoyl)-N- (4- methyl -3- (trifluorosMethyl) phenyl) -5- (trifluoromethyl) piperidines -3- formamides synthesis
1- (5- bromopyridines acyl group) -2- (furans -2- bases)-N- (4- methyl -3- (fluoroforms that synthesis step 1-4 is obtainedBase) phenyl) -5- (trifluoromethyl) piperidines -3- formamides (2.17g, 3.58mmol) are dissolved in the H of 96mL2O:EtOH(1:1)In the mixed solvent.2,4,5 trifluoro phenyl boric acids (0.26g, 2.32mmol) and potassium carbonate (0.29g, 2.10mmol) are added to thisIn mixture.Then PdNPs catalyst (0.4mmol%Pd) is added, and mixture is acutely stirred in 60 DEG C in air atmosphereIt mixes 10 minutes.Reaction mixture is added in 0.2mol/L sodium hydroxide solutions (15mL) and is extracted with ethyl acetate (20mL)Twice.Organic layer is merged, and is dried in air, obtain bright yellow solid product 2- (furans -2- bases) -1- (5- (2,4,5- trifluorophenyls) picolinoyl)-N- (4- methyl -3- (trifluoromethyl) phenyl) -5- (trifluoromethyl) piperidines -3- formylsAmine, 2.09g, yield 89%.1H-NMR(400MHz,CDCl3)δ:1.89(t,1H),2.39(t, 1H),2.50(s,3H),2.93(m,1H),3.53(m,1H),3.86(m,2H),5.20(d,1H),6.36(d,2H),7.13(s, 1H),7.35-7.41(m,3H),7.62(s,1H),7.78(m,2H),7.88(d,1H),8.87(d,1H),9.60(s, 1H).13C-NMR(125MHz,CDCl3)δ:20.43,26.07,37.54,44.29,46.24,57.38,104.83, 109.53,109.81,115.96,116.87,121.32,123.14,123.75,124.66,126.46,127.5,131.47, 132.65,133.3,133.36,138.42,142.21,146.88,148.17,149.08,149.7,149.99,158.96, 166.11,173.43.LC-MS(ESI,pos,ion)m/z:656[M+H]。
Embodiment 2：2- (furans -2- bases) -1- (5- (3- aminomethyl phenyls) picolinoyl)-N- (4- methyl -3- (trifluorosMethyl) phenyl) -5- (trifluoromethyl) piperidines -3- formamides synthesis
1- (5- bromopyridines acyl group) -2- (furans -2- bases)-N- (4- methyl -3- that 1 synthesis step 1-4 of embodiment is obtained(trifluoromethyl) phenyl) -5- (trifluoromethyl) piperidines -3- formamides (2.17g, 3.58mmol) are dissolved in the H of 96mL2O:EtOH(1:1) in the mixed solvent.It is mixed that 3- methylphenylboronic acids (3.94mmol) and potassium carbonate (0.29g, 2.10mmol) are added to thisIt closes in object.Then PdNPs catalyst (0.4mmol%Pd) is added, and mixture is vigorously stirred in 60 DEG C in air atmosphere10 minutes.Reaction mixture is added in 0.2mol/L sodium hydroxide solutions (15mL) and is extracted with ethyl acetate (20mL)Twice.Organic layer is merged, and is dried in air, bright yellow solid product 2- (furans -2- bases) -1- (5- (3- first is obtainedBase phenyl) picolinoyl)-N- (4- methyl -3- (trifluoromethyl) phenyl) -5- (trifluoromethyl) piperidines -3- formamides,1.96g, yield 89%.LC-MS(ESI,pos,ion)m/z:616[M+H].
Test example 1：External mTOR Kinase activity assays
The activity of mTOR protease is detected with detection kit (Invitrogen).Its test principle is：MTOR is swashedThe first antibody of EDTA and terbium label is added after reacting in enzyme, fluorescein-labeled substrate and ATP mixing.Swash in mTORIn enzymology reaction process, antibody identification phosphorylation has occurred and by fluorescein-labeled substrate after, enhance that " time resolution is glimmeringPhotoresonance energy transfer " (TR-FRET) effect.TR-FRET effects are the ratios by acceptor fluorescence element signal and donor terbium signalRate calculates.The amount for the antibody being incorporated on tracer with react after phosphorylation substrate amount direct proportionality, pass throughThe activity of this mode, kinases can be detected.In this test, the substrate of mTOR kinases is connected with green fluorescent protein4E Binding Protein 1s (GFP-4EBP1).
One, solution and reagent prepare
1,1 × detection buffer storage liquid：50mM 4- hydroxyethyl piperazineethanesulfonic acids (HEPES) pH7.5,1mM ethylene glycol-Double-(2- amino ethyl ethers) tetraacethyl (EGTA), 0.01%Tween-20,10mM MnCl2, 1mM Isosorbide-5-Nitrae dithiothreitol dithios(DTT)。
2, substrate working solution：2.5 × substrates of 4mL (1000 reactions)：3.8mL 1 × detection liquid, 191 μ L GFP-4E-BP1 (Invitrogen, 20.96 μM of storing liquids), 10 μ LATP (10mM).Ultimate density：0.4μM GFP-4E-BP1；10MATP。
3, mTOR working solutions：2.5 × mTOR of 4mL (Invitrogen, 1000 reactions)：4mL.
4,1 × detection liquid；7.5 μ LmTOR (0.4mg/mL storing liquids), ultimate density are 0.3 μ g/mL.
5, working solution is detected：10mL 2 × detection buffer solution (1000 reactions)：9.6mLTR-FRET dilutions, 11.5 μ LTb-anti-p4E-BP1 antibody (3.49 μM of stock), 400 μ L EDTA (storing liquid 500mM), ultimate density:2nM Tb-Anti-p4E-BP1 antibody, 10mM EDTA.
Two, test procedure：
1, a concentration of 100 μM of 50 μ L are added to be diluted in plate with the diluted the compounds of this invention of DMSO to 38 holes.
2, with DMSO with 1:3 ratio carrys out diluted compounds (the additional zero-dose of 10 dilutions).
3, the 2.5 diluted compounds of μ L are transferred to corresponding hole (including 47.5 μ L detections liquid/every hole), rocked several secondsClock.
4,4 μ LmTOR working solutions are added in 384 hole black Proxi plates.
5, the 2 diluted compounds of μ L are added in detection plate (each concentration there are 3 multiple holes).
6, it is incubated at room temperature 15 minutes.
7,4 μ L substrate working solutions are added.
8, final mTOR reaction densities:0.3 μ g/mL mTOR, 0.4 μM of GFP-4E-BP1,10 μM of ATP.With 1% DMSODiluted compounds are to a concentration of：1 μM, 0.33 μM, 0.11 μM, 0.037 μM, 0.0123 μM, 0.00411 μM, 0.00137 μM,0.000457 μM, 0.000152 μM, 0.000051 μM, 0 μM.
9, it is incubated at room temperature 30 minutes.
10,10 μ L are added and detect liquid, final working concentration：Tb-anti-p4E-BP1 antibody 2nM, EDTA 10mM. 11,It is incubated at room temperature 30 minutes.
12, with the readings of Envision-2104 read plate machine testings TR-FRET.Exciting light is 340nm, and transmitting light 1 is495nm's, transmitting light 2 is 520nm.Ratio=520nm/495nm is TR-FRET values
13, the calculating (IC of data analysis and 50% inhibiting rate50)：
50% inhibiting rate is calculated with Nonlinear regression equation：
The bottoms Y=+(Top-Bottom)/(1+10^ ((LogIC50-X) * Hill Slope)), X：Compound concentration (with10 be the logarithm at bottom), Y：TR-FRET values (ratios of the 520nm to 495nm), top and bottom：Identical peak value is as Y(Plateaus in same units as Y), 50% inhibiting rate (logIC50)：Identical logarithm is as X (same logunits as X)。
Three, experimental result
The compounds of this invention see the table below the inhibitory activity of mTOR kinases.
| Compound | IC50(nM) |
| a | < 10 |
| b | < 100 |
| c | < 100 |
| d | < 10 |
| e | < 100 |
| f | < 10 |
| g | < 10 |
| h | < 10 |
| i | < 100 |
| j | < 100 |
As seen from the above table, the IC of compound listed in table to the inhibitory activity of mTOR kinases50Equal < 100nM, explanationThe compounds of this invention can carry out more deep research and development as mTOR kinase inhibitors.
Test example 2：Mtt assay measures inhibiting effect of the compounds of this invention to different cancer cells.
One, cell strain
Human lung cancer cell A549, Human hepatoma cell line Bel-7402, neuroglia cell of human oncocyte U251, people's sdenocarcinoma of stomach are thinBorn of the same parents SGC-7901, people ovary adenocarcinoma cells SK-OV-3, human breast cancer cell line Bcap-37.
Two, experiment packet：
Medicine group to be measured (referring to experimental procedure part)；
(compared with drug test group, the drug to be measured that concentration gradient is added is changed to that the RPMI of not drug containing is added control group1640 cell culture fluids)；
Blank group (compared with the control group, is not added with cell).
Three, experimental procedure：
1. the cell of logarithmic growth phase, trypsin digestion, 1640 cell culture fluid tune concentration of cell suspension of RPMI are6×104A/mL.Add 100 μ L of cell suspension per hole in 96 well culture plates, sets 37 DEG C, 5%CO2It is cultivated in incubator for 24 hours, carefullyBorn of the same parents are adherent.
2. removing 1640 cell culture fluids of RPMI, 1640 cell culture fluids of RPMI of the drug to be measured of concentration gradient are added100 μ L, each concentration set 6 parallel holes.96 orifice plates after dosing are placed in 37 DEG C, 5%CO248h is cultivated in incubator, is invertedThe function and effect of microscopically observation drug.
Culture solution is discarded after the centrifugation of 3.96 orifice plates, after carefully being rushed 2~3 times with PBS, adds the RPMI containing 0.5%MTT1640 cell culture fluid, 100 μ L continue to cultivate 4h.
4. removing supernatant, 150 μ L dimethyl sulfoxide (DMSO)s are added per hole, sets low-speed oscillation 10min on shaking table, makes formazanCrystallization fully dissolving.
5. measuring the optical density (OD values) in each hole at enzyme-linked immunosorbent assay instrument 490nm.
6. parallel hole OD values are indicated with mean ± SD, inhibiting rate formula is calculated：[(ODControl group-ODBlank group)-(ODDrug study group-ODBlank group)]/(ODControl group-ODBlank group) * 100%.
7. using 5 data processing softwares of GraphPad Prism, by drawing amount effect curve calculation of half inhibitory concentration(IC50)。
Four, experimental result
1 the compounds of this invention pair of table, 6 kinds of human cancer cell IC50Value
As seen from the above table, 6 kinds of human cancer cell IC of compound pair listed in table50Value is respectively less than 100 μ g/ml, partIC50Value is less than 10 μ g/ml, has good cancer cell in vitro inhibitory activity.The compounds of this invention is in vitro to different cancer cellsInhibit experiment to illustrate that the compounds of this invention has growth of cancer cells inhibiting effect, can more be deepened as anticancer drug candidateEnter extensive pharmacological effect or even Pharmaceutical study.