CN108440513A - A kind of carboxamides derivatives and its application in antitumor drug - Google Patents

A kind of carboxamides derivatives and its application in antitumor drug Download PDF

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CN108440513A
CN108440513A CN201810469561.4A CN201810469561A CN108440513A CN 108440513 A CN108440513 A CN 108440513A CN 201810469561 A CN201810469561 A CN 201810469561A CN 108440513 A CN108440513 A CN 108440513A
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张玉叶
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Abstract

The invention discloses a kind of carboxamides derivatives formulas（Ⅰ）And its pharmaceutically acceptable salt, structure are, wherein R1Selected from H, OH or CH3, R2Selected from H, OH or CH3, R3Selected from H, OH or CH3.The invention discloses a kind of carboxamides derivatives formulas（Ⅰ）And synthetic method.Test example part of the present invention uses the inhibitory activity of the mTOR kinases of the activity detection kit vitro detection the compounds of this invention of mTOR protease, and measures the compounds of this invention with mtt assay and carry out in-vitro multiplication experiment confirmation the compounds of this invention with antitumor activity to 6 kinds of liver cancer, lung cancer, glioma, gastric cancer, oophoroma, breast cancer human cancer cells.

Description

A kind of carboxamides derivatives and its application in antitumor drug
Technical field
The invention belongs to pharmaceutical chemistry and area of pharmacology, it is related to a kind of carboxamides derivatives and its in antitumor drug In application.
Background technology
The morbidity of cancer and endanger the World Health Organization（WHO）Predict that 21 century malignant tumour will be as " the first of the mankind Killer ", therefore cancer control has become global health strategy emphasis.Though China is developing country, spectrum of disease has occurred Transformation, 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 gives Family, society, country cause white elephant, interfere economic construction of China and social development, are one very outstanding Social public health problem.In recent years, Incidence and death condition are more serious, year new cases be about 1,600,000, extremely Die up to 1,300,000, existing disease patient more than 200 ten thousand, it is average per dead 5 people in, just there is a people to die of cancer, in every 200 families, just It is hit there are one family because there is cancer patient.Moreover, with township industry, city of residence, aging of population into The acceleration of journey, environmental pollution, bad life habits and unreasonable life style generally existing, most cancers will also be in rise Gesture, 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）It is a kind of SARS Type serine/threonine protein kitase, belongs to phosphatidylinositol 3-kinase（Phosphoinositide3-kinase, PI3K）Phase Kinase families member is closed, is that the main signal of the cell functions such as intracellular synthesis and catabolism transmits molecule.MTOR signals are logical There is close relationship on road with nutrition, energy state and growth factor.It includes autophagy, albumen, lipid, lysosome that it, which is adjusted, Multiple cell processes such as synthesis and energetic supersession, cytoskeletal organization, cell survival.In mammalian cell periphery nutritional condition Constantly under variation, mTOR regulates and controls the conversion of synthesis and katabolism, so that cell can under different nutritional conditions Growth and survival.Due to important function of the mTOR in cell, abnormal or imbalance mTOR signals transmission can lead to human diseases Generation（Such as the diseases such as cancer）.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 evaluation The antitumor activity of this fixed compound.
Invention content
The purpose of the present invention is to provide a kind of new structural carboxamides derivatives formulas（Ⅰ）, structure is
, wherein R1Selected from H, OH or CH3, R2Selected from H, OH or CH3, R3 Selected from H, OH or CH3。
Further, the carboxamides derivatives formula（Ⅰ）The specific preferred structure of some represented is as follows：
Another object of the present invention is to provide a kind of carboxamides derivatives formulas（Ⅰ）And its pharmaceutically acceptable salt is as mTOR Application of the kinase inhibitor 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 is being controlled Treat 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 its The form of its derivative is for treating.Terms used herein " pharmaceutically acceptable salt " refer in this way some from medical angle Judge 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 invention The 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 suitable Alkali or acid react.Pharmaceutically acceptable nontoxic acid-addition salts amino and inorganic acid or organic acid be formed by amino its Its pharmaceutically acceptable salt includes adipate, alginates, ascorbate, aspartate, benzene sulfonate, benzoic acid Salt, disulfate, borate, butyrate, camphor hydrochlorate, camsilate, citrate, pentamethylene, digluconate, 12 Alkyl sulfate, esilate, formates, fumarate, gluceptate, glycerophosphate, gluconate, hemisulfic acid Salt, hydriodate, 2- isethionates, Lactobionate, lactate, laruate, lauryl sulfate, malate, horse Carry out hydrochlorate, malonate, methane sulfonates, 2- naphthalene sulfonates, nicotinate, nitrate, oleate, oxalates, palmitate is double Hydroxynaphthoate, pectate, persulfate, excessively 3- phenylpropionic acid salt, phosphate, picrate, Pivalate, propionate, firmly Resin acid salt, succinate, sulfate, tartrate, rhodanate, tosilate, undecanoate, pentyl sulfonate The 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 protease MTOR 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 formulas（Ⅰ）, synthetic route For：
,
Wherein, R1Selected from H, OH or CH3, R2Selected from H, OH or CH3, R3Selected from H, OH or CH3。
Another object of the present invention is to provide a kind of new structural carboxamides derivatives formulas（Ⅰ）Synthesis step For：
1) using toluene as solvent, by the Pd (PPh of suitable equivalent3)4It is added to containing the chloro- 5- trifluoromethylnicotinates of 2-, 2- In the toluene solution of furyl boronic acid and inorganic base, heated 2 hours under high temperature.It is post-treated to obtain 2- (2- furyls) -5- Trifluoromethylnicotinate；
2) by PtO2It is added in the EtOH solution of 2- (2- furyls) -5- trifluoromethylnicotinates that step 1) obtains, makes With Parr shaking machines hydrogenated mixture 1 hour under a certain pressure, then 2- (2- furyls) -5- trifluoros are obtained through subsequent processing Methyl 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 catalytic amount DMF, reaction is stirred at room temperature 2 hours, synthesis step 2 is then added) obtained 2- (2- furyls) -5- trifluoromethyls Piperidines -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 H2CrO in O3 Slurry in, obtain formic acid analog derivative, it is then again that it is molten with the dichloromethane of 4- methyl-3-trifluoromethyl phenylamines and triethylamine Liquid is reacted, and 1- n-propyl phosphoric anhydride T3P, and stirring 1.5 hours at room temperature are slowly added to, after purification through subsequent processing, Obtain 1- (5- bromopyridines acyl group) -2- (furans -2- bases)-N- (4- methyl -3- (trifluoromethyl) phenyl) -5- (trifluoromethyl) piperazine Pyridine -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 with corresponding boric acid and potassium carbonate in reaction bulb In react, mixture is vigorously stirred 10 minutes in air atmosphere in 60 DEG C, corresponding first is obtained through subsequent processing Amide.
Further, the amount range of the substance of Pd (PPh3) 4 can be 2-5mmol in the step 1), preferably 2.6mmol。
Further, the inorganic base in the step 1) can be sodium hydroxide, potassium carbonate, sodium carbonate, potassium hydroxide, carbon Sour 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- (furans -2- bases) picolinoyl)-N- (4- methyl -3- (fluoroforms Base) 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 molten In liquid, and reaction mixture is heated 2 hours at 100 DEG C.After reaction mixture is cooled to room temperature, under reduced pressure by solvent It removes, 30 ml water is added and stir 5 minutes.Gained aqueous layer with ethyl acetate extracts, and combined organic layer is done with anhydrous sodium sulfate It 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 (400 MHz, 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 (125 MHz, 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- trifluoromethyl nicotinic acids that synthesis step 1-1 is obtained In EtOH (60mL) solution of ethyl ester (2.56g, 8.98mmol).It is small in 40-45psi hydrogenated mixtures 1 using Parr shaking machines When.Then reaction mixture is filtered by diatomite, plug of celite is washed with EtOH, and filtrate decompression is concentrated.It will be remaining Object CH2Cl2It dilutes and uses saturation NaHCO3Solution washs.Pass through flash chromatography column (SiO2, 0-20%MeOH/CH2Cl2) pure Change, obtains required product 2- (furans -2- bases) -5- (trifluoromethyl) piperidines -3- carboxylic acid, ethyl esters, 2.22g, yield 85%.1H-NMR (400 MHz, 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 (125 MHz, 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 conjunction of 1-3 1- (5- bromopyridines acyl group) -2- (furans -2- bases) -5- (trifluoromethyl) piperidines -3- Ethyl formates At：
At room temperature, oxalyl chloride (3.2mL, 30.25mmol) is added in reaction flask and is dissolved in CH2Cl2In (20mL) 5- bromopyridines formyl chloride (3.79g, 17.19mmol) solution in, then be added catalytic amount DMF.It will react at room temperature Stirring 2 hours.Solvent and extra oxalyl chloride are removed under vacuum, residue is dried 20 minutes under a high vacuum.It will obtain Acyl chlorides be dissolved in anhydrous CH2Cl2In (20mL) and it is cooled to 0 DEG C, the 2- (2- furyls)-that synthesis step 1-2 is obtained then is added 5- trifluoromethyl piperidine -3- carboxylic acid, ethyl esters (2.22g, 7.62mmol) and Et3N (8.6mL, 62.04mmol).Mixture is heated up To room temperature and it is stirred overnight.Reaction mixture CH2Cl2Dilution, after adding water, the solution that is layered.Water layer CH2Cl2 Extraction, by combined organic layer with anhydrous MgSO4It dries and is concentrated under reduced pressure, pass through flash chromatography column (SiO2, 10-35%'s EtOAc/ hexanes) purifying, obtain 3.59g 1- (5- bromopyridines acyl group) -2- (furans -2- bases) -5- (trifluoromethyl) piperidines -3- Ethyl formate, off-white powder, yield 99%.1H-NMR (400 MHz, 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 (125 MHz, 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- (three Methyl 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 1-3 at 0 DEG C Obtain 1- (5- bromopyridines acyl group) -2- (furans -2- bases) -5- (trifluoromethyl) piperidines -3- Ethyl formates (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 dropwise 15%NaOH aqueous solutions (625 μ L) to quench the reaction, then add the H of 625 μ L2O.It is added into muddy colloid admixture The water of 1.85mL, and mixture is stirred at room temperature 1 hour.Then mixture is filtered by plug of celite, and by filtrate It is concentrated under reduced pressure.It is purified by flash chromatography column (the EtOAc/ hexanes of SiO2,33-67%), obtains (the 5- of 3.04g yellow powders Bromopyridine -2- bases) (2- (furans -2- bases) -3- (methylol) -5- (trifluoromethyl) piperidin-1-yl) ketone, yield 93%.② At room temperature, (5- bromopyridine -2- the bases) (2- (furans -2- bases)-from synthesis step 1. being dissolved in acetic acid (65mL) 3- (methylol) -5- (trifluoromethyl) piperidin-1-yl) solution of ketone (3.04g, 7.01mmol) is added in H2O(16mL) In CrO3In (2.61g, 26.10mmol) slurry.Gained mixture is stirred at room temperature 90 minutes until the reaction was complete.So Reaction mixture is filtered by plug of celite afterwards, and filtrate decompression is concentrated.By flash chromatography column (SiO2,3-10%'s CH2Cl2：MeOH, then using the EtOAc/ hexanes of 50-67%) purifying, obtain dry 2.19g off-white powder shape products 1- (5- bromopyridines acyl group) -2- (furans -2- bases) -5- (trifluoromethyl) piperidines -3- formic acid, yield 70%.3. by 4- methyl- 3- 5-trifluoromethylanilines (1.62g, 9.25mmol) be added above-mentioned preparation acid (2.19g, 4.91mmol) and triethylamine (1.56g, In dichloromethane (5mL) solution 15.42mmol).Be then slowly added into 1- n-propyl phosphoric anhydrides T3P (95.5mg, 30.38mmol), and by the solution it is stirred at room temperature 1.5 hours.Use CH2Cl2(5mL) diluted reaction mixture, is used successively HCl/water solution, the saturation NaHCO of 1mol/L3Aqueous solution washs.By organic layer separation, with anhydrous MgSO4It is dry, it is concentrated under reduced pressure. Pass through flash chromatography column (SiO2, the EtOAc/ hexanes of 5-40%) and it is purified, obtain 2.17g white solids 1- (5- bromopyridine acyls Base) -2- (furans -2- bases)-N- (4- methyl -3- (trifluoromethyl) phenyl) -5- (trifluoromethyl) piperidines -3- formamides, yield It is 73%.1H-NMR (400 MHz, 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 (125 MHz, 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- (furans -2- bases) picolinoyl)-N- (4- methyl -3- (trifluoromethyl) Phenyl) -5- (trifluoromethyl) piperidines -3- formamides synthesis：
1- (5- bromopyridines acyl group) -2- (furans -2- bases)-N- (4- methyl -3- (trifluoromethyl) that synthesis step 1-4 is obtained Phenyl) -5- (trifluoromethyl) piperidines -3- formamides (2.17g, 3.58mmol) are dissolved in the H of 96mL2O:EtOH(1:1) mixing In solvent.2- furyl boronic acids (0.26g, 2.32mmol) and potassium carbonate (0.29g, 2.10mmol) are added to the mixture In.Then PdNPs catalyst (0.4mmol%Pd) is added, and mixture is vigorously stirred 10 points in 60 DEG C in air atmosphere Clock.Reaction mixture is added in 0.2mol/L sodium hydroxide solutions (15mL) and is extracted twice with ethyl acetate (20mL). Organic layer is merged, and is dried in air, bright yellow solid product 2- (furans -2- bases) -1- (5- (furans -2- bases) are obtained Picolinoyl)-N- (4- methyl -3- (trifluoromethyl) phenyl) -5- (trifluoromethyl) piperidines -3- formamides, 1.91g, yield It is 90%.1H-NMR (400 MHz, CDCl3) δ: 2.29(s, 3H), 2.38(m, 1H), 2.70(m, 1H), 2.77 (m, 1H), 3.50(m, 1H), 3.83(m, 1H), 4.09(m, 1H), 5.28(d, 1H), 6.36-6.45(m, 2H), 6.61(t, 1H), 7.03(d, 1H), 7.07(d, 1H), 7.48(d, 1H), 7.56(d, 1H), 7.77(d, 1H), 7.98(s, 1H), 8.35(d, 1H), 8.61(d, 1H), 8.79(s, 1H), 9.21(s, 1H). 13C-NMR (125 MHz, CDCl3) δ: 19.65, 24.98, 35.94, 40.69, 46.09, 52.70, 103.74, 110.99, 111.54, 112.59, 118.31, 124.02, 124.63, 125.42, 129.94, 130.86, 131.19, 132.26, 132.52, 133.54, 135.14, 142.40, 143.63, 144.85, 147.97, 150.24, 155.08, 167.74, 173.69. LC-MS(ESI, pos, ion) m/z: 593[M+H]。
2 2- of embodiment (furans -2- bases) -1- (5- (3- methyl-ribofuranosyl -2- bases) picolinoyl)-N- (4- methyl - 3- (trifluoromethyl) phenyl) -5- (trifluoromethyl) piperidines -3- formamides synthesis
1- (5- bromopyridines acyl group) -2- (furans -2- bases)-N- (4- methyl -3- (three that 1 step 1-4 of synthetic example is obtained Methyl fluoride) 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- methylfuran -2- boric acid (3.94 mmol) and potassium carbonate (0.29g, 2.10mmol) are added to this It closes in object.Then PdNPs catalyst (0.4mmol%Pd) is added, and mixture is vigorously stirred 10 in 60 DEG C in air atmosphere Minute.Reaction mixture is added in 0.2mol/L sodium hydroxide solutions (15mL) and extracts two with ethyl acetate (20mL) Time.Organic layer is merged, and is dried in air, bright yellow solid product 2- (furans -2- bases) -1- (5- (3- methyl-is obtained Furans -2- bases) picolinoyl)-N- (4- methyl -3- (trifluoromethyl) phenyl) -5- (trifluoromethyl) piperidines -3- formamides, 1.97g, yield 91%.LC-MS(ESI, pos, ion) m/z: 606[M+H].
Test example 1：External mTOR Kinase activity assays
The activity detection kit of mTOR protease（Invitrogen）To detect.Its test principle is：MTOR kinases, glimmering The first antibody of EDTA and terbium label is added after reacting in the substrate of light element label and ATP mixing.In mTOR kinases Learn in reaction process, antibody identification phosphorylation has occurred and by fluorescein-labeled substrate after, enhance that " time-resolved fluorescence is total It shakes energy transfer "（TR-FRET）Effect.TR-FRET effects be by the ratio of acceptor fluorescence element signal and donor terbium signal come It calculates.The amount for the antibody being incorporated on tracer with react after phosphorylation substrate amount direct proportionality, by this The activity of mode, kinases can be detected.In this test, the substrate of mTOR kinases is the 4E being connected with green fluorescent protein Binding Protein 1（GFP-4EBP1）.
One, solution and reagent prepare
1,1 × detection buffer storage liquid：50mM 4- hydroxyethyl piperazineethanesulfonic acids（HEPES）PH7.5,1mM ethylene glycol-bis-- （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 L ATP（10mM）.Ultimate density：0.4μM GFP-4E-BP1；10M ATP。
3, mTOR working solutions：4mL 2.5×mTOR（Invitrogen, 1000 reactions）：4mL.
4,1 × detection liquid；7.5μL mTOR（0.4mg/mL storing liquids）, ultimate density is 0.3 μ g/mL.
5, working solution is detected：10mL 2 × detection buffer solution（1000 reactions）：9.6mL TR-FRET dilutions, 11.5 μ L Tb-anti-p4E-BP1 antibody（stock 3.49μM）, 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（Include 47.5 μ L detections liquid/every hole）, rock several seconds Clock.
4,4 μ L mTOR 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 has 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% DMSO Diluted 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 is 495nm's, transmitting light 2 is 520nm.Ratio=520nm/495nm is TR-FRET values
13, data analysis and the calculating of 50% inhibiting rate（IC50）：
50% inhibiting rate is calculated with Nonlinear regression equation：
The bottoms Y=+（Top-Bottom）/（1+10^（（LogIC50-X）*Hill Slope））, X：The concentration of compound（It is with 10 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 log units as X）.
Three, experimental result
The compounds of this invention see the table below the inhibitory activity of mTOR kinases
As seen from the above table, the IC of compound listed in table to the inhibitory activity of mTOR kinases50Equal ＜ 10nM, illustrate this hair Bright compound 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, human gastric adenocarcinoma 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）；
Control group（Compared with drug test group, the drug to be measured that concentration gradient is added is changed to that the RPMI 1640 of not drug containing is added Cell culture fluid）；
Blank group（Compared with the control group, it 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 is 6 × 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, cell patch Wall.
2. removing 1640 cell culture fluids of RPMI, 1640 cell culture fluids of RPMI of the drug to be measured of concentration gradient are added 100 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 inverted The 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% MTT 1640 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, formazan is made to tie Brilliant fully dissolving.
5. measuring the optical density in each hole at enzyme-linked immunosorbent assay instrument 490nm（OD values）.
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, compound listed in table has good cancer cell in vitro inhibitory activity, and has part chemical combination IC of the object to some human cancer cells501 μ g/ml of value ＜ but in table only indicated with 10 μ g/ml of ＜.The compounds of this invention is not in vitro to Inhibition experiment with cancer cell illustrates that the compounds of this invention has growth of cancer cells inhibiting effect, can be used as anticancer drug candidate More goed deep into extensive pharmacological effect or even Pharmaceutical study.

Claims (5)

1. a kind of carboxamides derivatives formula（Ⅰ）And its pharmaceutically acceptable salt,
, wherein R1Selected from H, OH or CH3, R2Selected from H, OH or CH3, R3Choosing From H, OH or CH3。
2. carboxamides derivatives formula as described in claim 1（Ⅰ）And its pharmaceutically acceptable salt, characterized in that formula（Ⅰ）Choosing From：
Carboxamides derivatives formula as described in claim 1（Ⅰ）And its pharmaceutically acceptable salt is as mTOR kinase inhibitors Application in preventing and/or treating disease.
3. carboxamides derivatives formula as described in claim 1（Ⅰ）And its pharmaceutically acceptable salt answering in treating cancer With.
4. cancer as claimed in claim 4 is selected from liver cancer, lung cancer, glioma, gastric cancer, oophoroma, breast cancer.
5. carboxamides derivatives formula as described in claim 1（Ⅰ）, synthetic route is：
,
Wherein, R1Selected from H, OH or CH3, R2Selected from H, OH or CH3, R3Selected from H, OH or CH3。
CN201810469561.4A 2018-05-16 2018-05-16 A kind of carboxamides derivatives and its application in antitumor drug Withdrawn CN108440513A (en)

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