CN108727354A - 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 PDFInfo
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- CN108727354A CN108727354A CN201810470302.3A CN201810470302A CN108727354A CN 108727354 A CN108727354 A CN 108727354A CN 201810470302 A CN201810470302 A CN 201810470302A CN 108727354 A CN108727354 A CN 108727354A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
- C07D405/14—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing three or more hetero rings
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- A—HUMAN NECESSITIES
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- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
Abstract
The invention discloses a kind of carboxamides derivatives formulas（Ⅰ）And its pharmaceutically acceptable salt, structure are, wherein R1、R2、R3It is independently selected 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
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 predicts that 21 century malignant tumour will be as " the first of the mankind with the World Health Organization (WHO) is endangered
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.Moreover, as township industry, city of residence, the acceleration of aging of population process, environment are dirty
Dye, 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 SARS
Type 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 signals
Access 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 bar
Under part constantly variation, mTOR regulates and controls the conversion of synthesis and katabolism, so that cell energy under different nutritional conditions
Enough growths and survival.Due to important function of the mTOR in cell, abnormal or imbalance mTOR signals transmission can lead to mankind's disease
The 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 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 formula (I), structure isWherein, R1、R2、R3It is independently selected from H, OH or CH3.Further, described
Some specific preferred structures represented by 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 conducts
Application 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 control
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 formula (I), synthetic routes
For：
Wherein, R1、R2、R3It is independently selected from H, OH or CH3。
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) obtains
In, using Parr shaking machines hydrogenated mixture 1 hour under a certain pressure, then 2- (2- furyls)-is obtained through subsequent processing
5- 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 catalysis
Reaction is stirred at room temperature 2 hours, synthesis step 2 is then added by the DMF of amount) obtained 2- (2- furyls) -5- fluoroforms
Phenylpiperidines -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 O
CrO3Slurry in, obtain formic acid analog derivative, it is then again that it is molten with the dichloromethane of substituted furans -2- amine 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 corresponding carboxamides derivatives；
5) using PdNPs as catalyst, by synthesis step 4) obtained carboxamides derivatives and 4- isoquinolyls boric acid and carbon
Sour potassium reacts in reaction bulb, and mixture is vigorously stirred 10 minutes in air atmosphere in 60 DEG C, is obtained through subsequent processing
To corresponding substitution formamide.
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- (isoquinolin -4- bases) picolinoyl)-N- (furans -2- bases) -5-
The synthesis of (trifluoromethyl) piperidines -3- formamides
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, 30ml water is added and stirs 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 (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 obtained
In EtOH (60mL) solution of ethyl nicotinate (2.56g, 8.98mmol).Using Parr shaking machines in 40-45psi hydrogenated mixtures
1 hour.Then reaction mixture is filtered by diatomite, plug of celite is washed with EtOH, and filtrate decompression is concentrated.It will be residual
Excess 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 temperature
Lower stirring 2 hours.Solvent and extra oxalyl chloride are removed under vacuum, residue is dried 20 minutes under a high vacuum.Will
To 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 added
Base) -5- trifluoromethyl piperidine -3- carboxylic acid, ethyl esters (2.22g, 7.62mmol) and Et3N (8.6mL, 62.04mmol).It will mixing
Object is warming up to room temperature and is stirred overnight.Reaction mixture CH2Cl2Dilution, after adding water, the solution that is layered.Water layer is used
CH2Cl2Extraction, 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) piperazine
Pyridine -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- (furans -2- bases) -5- (trifluoromethyl) piperidines -3-
The synthesis of formamide
1. solutions of lithium aluminium hydride (2.0mol/L, 8.2mL, 16.4mmol in THF) is added to synthesis step at 0 DEG C
1- (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 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 3.04g yellow powders
(5- bromopyridine -2- bases) (2- (furans -2- bases) -3- (methylol) -5- (trifluoromethyl) piperidin-1-yl) ketone, yield are
93%.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 to
H2CrO 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 colors
Powdery product 1- (5- bromopyridines acyl group) -2- (furans -2- bases) -5- (trifluoromethyl) piperidines -3- formic acid, yield 70%.
3. by furans -2- amine (5.40mmol) 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 1.91g white solids 1- (5- bromopyridines
Acyl group) -2- (furans -2- bases)-N- (furans -2- bases) -5- (trifluoromethyl) piperidines -3- formamides, yield 76%.1H-NMR
(400MHz,CDCl3)δ:2.11(t,2H),2.93(m,1H),3.42(m,1H),3.83(dd,2H),5.17(d,1H), 6.36
(m,2H),6.44(d,1H),6.60(dd,1H),7.20(m,1H),7.42(t,1H),7.80(d,1H),7.86(dd, 1H),
8.63(d,1H),9.54(s,1H).13C-NMR(125MHz,CDCl3)δ:24.96,37.81,44.46,47.34, 57.05,
100.85,109.27,109.81,110.43,122.87,124.48,126.48,137.33,139.72,142.2, 149.64,
150.75,150.93,151.17,165.67,171.81.LC-MS(ESI,pos,ion)m/z:512[M+H]。
1-5 2- (furans -2- bases) -1- (5- (isoquinolin -4- bases) picolinoyl)-N- (furans -2- bases) -5- (trifluoros
Methyl) piperidines -3- formamides synthesis
1- (5- bromopyridines acyl group) -2- (furans -2- bases)-N- (furans -2- bases) -5- (three that synthesis step 1-4 is obtained
Methyl fluoride) piperidines -3- formamides (10mmol) are dissolved in the H of 200mL2O:EtOH(1:1) in the mixed solvent.By 4- isoquinolyls
Boric acid (11mmol) and potassium carbonate (11mmol) are added in the mixture.Then PdNPs catalyst (0.4mmol% is added
Pd it), and by mixture is vigorously stirred in air atmosphere in 60 DEG C 10 minutes.Reaction mixture is added to 0.2mol/L hydrogen-oxygens
Change in sodium solution (50mL) and is extracted twice with ethyl acetate (60mL).Organic layer is merged, and is dried in air, is obtained bright
Yellow solid product 2- (furans -2- bases) -1- (5- (isoquinolin -4- bases) picolinoyl)-N- (furans -2- bases) -5- (trifluoros
Methyl) piperidines -3- formamides, 5.04g, yield 93%.1H-NMR(400MHz, CDCl3)δ:2.13(td,2H),2.94
(m,1H),3.54(m,1H),3.84(dd,2H),5.16(d,1H),6.35(m,2H), 6.52(dd,1H),6.58(dd,1H),
7.21(m,1H),7.40(m,1H),7.48(m,2H),7.69(m,1H),7.87(dd, 1H),7.99(t,2H),8.91(d,
1H),8.95(t,1H),9.14(d,1H),9.77(s,1H).13C-NMR(125MHz, CDCl3)δ:26.34,37.76,
44.33,47.22,57.38,100.78,109.41,109.91,110.46,123.66, 126.38,126.95,127.02,
127.65,128.92,131.05,131.28,133.39,133.96,135.44,139.33, 142.21,145.51,
147.63,149.62,150.23,151.17,165.92,171.95.LC-MS(ESI,pos,ion)m/z: 561[M+H]。
Embodiment 2：2- (furans -2- bases) -1- (5- (isoquinolin -4- bases) picolinoyl)-N- (5- hydroxyls-furans -2-
Base) -5- (trifluoromethyl) piperidines -3- formamides synthesis
2-1,2-2,2-3 synthetic method are respectively the same as the 1-1 in embodiment 1,1-2,1-3；Synthetic product is identical.
The same 1-4 of 2-4 synthetic methods, synthetic method are as follows：
1. solutions of lithium aluminium hydride (2.0mol/L, 8.2mL, 16.4mmol in THF) is added to embodiment 1 at 0 DEG C
1- (5- bromopyridines acyl group) -2- (furans -2- bases) -5- (trifluoromethyl) piperidines -3- Ethyl formates that synthesis step 1-3 is obtained
In THF (100mL) solution of (3.59g, 7.54mmol), acquired solution is stirred 2 hours until the reaction was complete at 0 DEG C.By
It is added dropwise to 15%NaOH aqueous solutions (625 μ L) to quench the reaction, then adds the H of 625 μ L2O.Add again into muddy colloid admixture
Enter the water of 1.85mL, and mixture is stirred at room temperature 1 hour.Then mixture is filtered by plug of celite, and will filter
Liquid 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 is
93%.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 to
H2CrO 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 color powder
Last shape product 1- (5- bromopyridines acyl group) -2- (furans -2- bases) -5- (trifluoromethyl) piperidines -3- formic acid, yield 70%.③
By 5- hydroxyls-furans -2- amine (5.40mmol) 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
The HCl aqueous solutions of 1mol/L, saturation NaHCO3Aqueous 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 2.02g white solids 1- (5- bromopyridines
Acyl group) -2- (furans -2- bases)-N- (5- hydroxyls-furans -2- bases) -5- (trifluoromethyl) piperidines -3- formamides, yield is
78%.LC-MS(ESI,pos,ion)m/z:528[M+H].
The same 1-5 of 2-5 synthetic methods, synthetic method are as follows：
1- (5- bromopyridines acyl group) -2- (furans -2- bases)-N- (5- hydroxyls-furans -2- that synthesis step 2-4 is obtained
Base) -5- (trifluoromethyl) piperidines -3- formamides (10mmol) are dissolved in the H of 200mL2O:EtOH(1:1) in the mixed solvent.It will
4- isoquinolyls boric acid (11mmol) and potassium carbonate (11mmol) are added in the mixture.Then PdNPs catalyst is added
(0.4mmol%Pd), and mixture is vigorously stirred 10 minutes in air atmosphere in 60 DEG C.Reaction mixture is added to
It is extracted twice in 0.2mol/L sodium hydroxide solutions (50mL) and with ethyl acetate (60mL).Organic layer is merged, and in air
Middle drying obtains bright yellow solid product 2- (furans -2- bases) -1- (5- (isoquinolin -4- bases) picolinoyl)-N- (5- hydroxyls
Base-furans -2- bases) -5- (trifluoromethyl) piperidines -3- formamides, 5.24g, yield 91%.LC-MS(ESI,pos,ion)
m/z:577[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 swashed
The first antibody of EDTA and terbium label is added after reacting in enzyme, fluorescein-labeled substrate and ATP mixing.Swash in mTOR
In enzymology reaction process, antibody identification phosphorylation has occurred and by fluorescein-labeled substrate after, enhance that " time resolution is glimmering
Photoresonance energy transfer " (TR-FRET) effect.TR-FRET effects are the ratios by acceptor fluorescence element signal and donor terbium signal
Rate calculates.The amount for the antibody being incorporated on tracer with react after phosphorylation substrate amount direct proportionality, pass through
The activity of this mode, kinases can be detected.In this test, the substrate of mTOR kinases is connected with green fluorescent protein
4E 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；
10M ATP。
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 L
Tb-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 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 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% 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, 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 (with
10 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 log
units 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 | < 10 |
| c | < 10 |
| d | < 10 |
| h | < 10 |
| i | < 10 |
| j | < 10 |
As seen from the above table, the IC of compound listed in table to the inhibitory activity of mTOR kinases50Equal < 10nM, illustrate this
Invention 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, people's sdenocarcinoma of stomach are thin
Born 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 group
1640 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 are
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, carefully
Born 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 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, makes formazan
Crystallization 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, compound listed in table has good cancer cell in vitro inhibitory activity, and has part
IC of the compound to some human cancer cells501 μ g/ml of value < but in table only is indicated with 10 μ g/ml of <, in table < 100 expression
Range be greater than equal to 10 be less than 100 numerical value, some results with 10 very close to but still indicated with < 100.Chemical combination of the present invention
The outer inhibition experiment to different cancer cells of object illustrates that the compounds of this invention has growth of cancer cells inhibiting effect, can be used as anti-
Cancer drug candidate is 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 R1、R2、R3It is independently selected 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 3 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：
。
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