CN107311986B - Tetrahydroisoquinoline-3-carboxylic acid heat shock protein 90 inhibitor and application thereof - Google Patents
Tetrahydroisoquinoline-3-carboxylic acid heat shock protein 90 inhibitor and application thereof Download PDFInfo
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Abstract
The invention discloses a tetrahydroisoquinoline-3-carboxylic acid heat shock protein 90 inhibitor, which is a compound with a general formula (I), or an optical isomer, a diastereoisomer or a racemate mixture thereof, or a pharmaceutically acceptable salt, solvate or prodrug thereof. The invention also discloses application of the inhibitor in preparing a medicament for preventing or treating diseases related to the heat shock protein 90. The experiment proves that: the tetrahydroisoquinoline-3-carboxylic acid heat shock protein 90 inhibitor has obvious inhibiting effect on tumor cells of various tissue types including mammary gland, reproductive system and respiratory system sources, but has low toxicity on normal human liver cells and better druggability. Has wide clinical application prospect.
Description
Technical Field
The invention relates to a heat shock protein 90 inhibitor and application thereof, in particular to a tetrahydroisoquinoline-3-carboxylic acid heat shock protein 90 inhibitor and a preparation method and application thereof, belonging to the technical field of organic compound synthesis and medical application.
Background
Heat shock protein 90(Hsp90) is a class of structurally highly conserved proteins during biological evolution. As a molecular chaperone, Hsp90 plays an important role in regulating cell growth, differentiation, apoptosis, and the like. Recent studies show that Hsp90 has close relationship with tumorigenesis, development, biological behavior and prognosis, and many oncogene proteins are target sites of Hsp 90. And the Hsp90 inhibitor can promote the degradation of Hsp90 effector protein which plays an important role in a tumor growth signal path, thereby blocking multiple targets of the tumor proliferation signal path and effectively preventing the growth of tumors. In vitro and in vivo experiments also demonstrated the antitumor activity of Hsp90 inhibitors. Meanwhile, the design of small molecule inhibitors based on related structures also draws increasing attention.
In previous studies, in order to find a structurally novel and potent Hsp90 inhibitor, the inventor found that the lead compound LCP1074 has a strong Hsp90 inhibitory activity through rational design and further structural modification of the compound (Chuan-Peng Liang, European Journal of Medicinal Chemistry 121(2016)272e 282). The LCP1074 has the following structural formula:
based on further structural modification of the compound, a compound with greatly improved Hsp90 inhibitory activity and anti-cell proliferation activity is expected to be obtained.
Disclosure of Invention
Aiming at the basis of the prior art, the invention aims to provide a tetrahydroisoquinoline-3-carboxylic acid heat shock protein 90 inhibitor and a preparation method and application thereof.
The invention relates to a tetrahydroisoquinoline-3-carboxylic acid heat shock protein 90 inhibitor, which is characterized in that: the heat shock protein 90 inhibitor is a compound with a general formula (I), or an optical isomer, a diastereoisomer or a racemic mixture thereof, or a pharmaceutically acceptable salt, solvate or prodrug thereof;
wherein:
R1selected from hydrogen radicals, halogens, or C1-8 alkyl radicals;
R2and R3Each independently selected from hydrogen, deuterium, or phosphonic acid;
ar is selected from substituted aromatic rings; the aromatic ring is selected from benzene ring, five-membered aromatic heterocycle, six-membered and five-membered heterocycle, six-membered and six-membered heterocycle, five-membered and five-membered heterocycle, benzo five-membered heterocycle or various substituted benzo six-membered heterocycle; the substitution represents mono-, di-or tri-substitution; the substituent is selected from hydrogen atom alkyl, halogen, hydroxyl, amino, dimethylamino, nitro, C1-8 alkoxy, C1-8 heteroalkoxy, trifluoromethoxy, cyano, carboxyl, sulfonic acid group or phosphoric acid group, or ester or salt thereof;
the phenyl ring A being optionally substituted by hydrogen, one or more R4X is substituted;
x is selected from N, O, S atom or acyl;
R4a cycloalkyl group selected from a hydrogen group, a C1-8 alkyl group or 5-6 ring members, the substituent being selected from a C1-2 alkoxy group, a mono-or di-C1-2 alkylamino group, a nitrogen-or oxygen-containing heterocyclic group of 5-6 ring members, a hydroxyl group, an amino group, a nitro group, a cyano group, an amide group, a methanesulfonylamino group, a methylsulfonyl group, a hydroxamic acid group, a methoxycarbonyl group, a hydrazide group, a carboxyl group, a sulfonic group, a phosphoric acid group, or an ester or salt thereof;
is S or R optical purity or racemic body thereof in spatial configuration.
Further, the heat shock protein 90 inhibitor is preferably one of the following compounds:
R1selected from halogens; r2And R3Are all hydrogen radicals; at the 7-position of the phenyl ring A by R4X is substituted; x is selected from O atoms; is the stereoconfiguration R optical purity.
The most preferred embodiments are: the heat shock protein 90 inhibitor is compound L10, L15, L22 or L41, in the order of chemical name:
r-2- (5-chloro-2, 4-dihydroxybenzoyl) -3- (N-p-cyanobenzyl-amido) -1,2,3, 4-tetrahydroisoquinoline (L10);
r-2- (5-chloro-2, 4-dihydroxybenzoyl) -3- (N- (pyridine-4-methyl) -amido) -1,2,3, 4-tetrahydroisoquinoline (L15);
r-2- (5-chloro-2, 4-dihydroxybenzoyl) -7- (2-methoxyethoxy) -3- (N- (pyridin-4-ylmethyl) -amido) -1,2,3, 4-tetrahydroisoquinoline (L22);
r-2- (5-chloro-2, 4-dihydroxybenzoyl) -7- (2-morpholinoethoxy) -3- (N-p-cyanobenzyl-amido) -1,2,3, 4-tetrahydroisoquinoline (L41).
The preparation method of the tetrahydroisoquinoline-3-carboxylic acid heat shock protein 90 inhibitor is as follows, namely a synthetic route 1 or a synthetic route 2, and the specific reaction steps and the reaction formula are as follows:
scheme 1:
2, 4-dihydroxy methyl benzoate and optical purity of tetrahydroisoquinoline-3-carboxylic acid as starting materials. Hydrolyzing 2, 4-dihydroxy methyl benzoate after being protected by 3-chloro and benzyl, forming amide with tetrahydroisoquinoline-3-methyl carboxylate, reacting with various amines after ester hydrolysis, and debenzylating to obtain a target compound L1-20; the reaction formula is as follows:
wherein Ar is selected from substituted aromatic rings; the aromatic ring is selected from benzene ring, five-membered aromatic heterocycle, six-membered and five-membered heterocycle, six-membered and six-membered heterocycle, five-membered and five-membered heterocycle, benzo five-membered heterocycle or various substituted benzo six-membered heterocycle; the substitution represents mono-, di-or tri-substitution; the substituent is selected from hydrogen atom alkyl, halogen, hydroxyl, amino, dimethylamino, nitro, C1-8 alkoxy, C1-8 heteroalkoxy, trifluoromethoxy, cyano, carboxyl, sulfonic acid group or phosphoric acid group, or ester or salt thereof;
reagents in the above synthetic scheme: (a) sulfonyl chloride, anhydrous dichloromethane; (b) benzyl bromide, potassium carbonate, N-dimethylformamide; (c) aqueous potassium hydroxide, methanol; (d) thionyl chloride, methanol; (e) 1-hydroxybenzotriazole, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, dichloromethane; (f) aqueous sodium hydroxide solution, methanol; (g) various arylcyclomethylamines, 1-hydroxybenzotriazole, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, N-dimethylformamide; (h) boron trichloride, anhydrous dichloromethane.
The structural formula of the target compound of scheme 1 is as follows:
scheme 2:
optically pure 3, 5-diiodotyrosine is used as a raw material, a compound 10 is obtained by Pictet-Spengler cyclization, and the compound 10 is subjected to Boc protection secondary amine and hydrogenation reduction deiodination sequentially to obtain a key intermediate 12. The intermediate 12 and various aromatic methylamines are connected with different Ar groups through polypeptide condensation to obtain a compound 13.13, and Boc is removed to be condensed with the intermediate 4 respectively to obtain a corresponding compound 15. Carrying out Williams condensation reaction or mitsunobu reaction on the compound 15 and various halides or alcohols to obtain ether, and then carrying out debenzylation to obtain a target compound L21-45; the reaction formula is as follows:
wherein Ar is selected from substituted aromatic rings; the aromatic ring is selected from benzene ring, five-membered aromatic heterocycle, six-membered and five-membered heterocycle, six-membered and six-membered heterocycle, five-membered and five-membered heterocycle, benzo five-membered heterocycle or various substituted benzo six-membered heterocycle; the substitution represents mono-, di-or tri-substitution; the substituent is selected from hydrogen atom alkyl, halogen, hydroxyl, amino, dimethylamino, nitro, C1-8 alkoxy, C1-8 heteroalkoxy, trifluoromethoxy, cyano, carboxyl, sulfonic acid group or phosphoric acid group, or ester or salt thereof; r4Selected from hydrogen radical, C1-8 alkyl or cycloalkyl of 5-6 ring members, the substituents being selected from C1-2 alkoxy, mono-or di-C1-2Alkylamino, a nitrogen-or oxygen-containing heterocyclic group of 5 to 6 ring members, hydroxyl, amino, nitro, cyano, amido, methylsulfonamido, methylsulfonyl, hydroxamic acid, methoxycarbonyl, hydrazide, carboxyl, sulfonic acid, phosphoric acid, or an ester or salt thereof;
reagents in the above synthetic scheme: (a) paraformaldehyde, concentrated hydrochloric acid, ethylene glycol dimethyl ether; (b) l mol/L sodium hydroxide solution, di-tert-butyl carbonate and tetrahydrofuran; (c) palladium carbon, hydrogen, triethylamine and methanol; (d) various arylcyclomethylamines, 1-hydroxybenzotriazole, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, N-dimethylformamide; (e) trifluoroacetic acid, triethylamine, dichloromethane; (f) the intermediate 4, 1-hydroxybenzotriazole, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, N-dimethylformamide; (g) various halides, cesium carbonate, N-dimethylformamide; (h) boron trichloride, anhydrous dichloromethane.
The structural formula of the target compound of scheme 2 is as follows:
the specific procedures for the preparation of the above compounds are described in detail in the examples.
The skilled person can vary the above steps to increase the yield, and can determine the synthetic route according to the basic knowledge in the art, such as choice of reactants, solvents and temperature, and can increase the yield by using various conventional protecting groups to avoid side reactions.
The invention relates to application of a tetrahydroisoquinoline-3-carboxylic acid heat shock protein 90 inhibitor in preparing a medicament for preventing or treating diseases related to heat shock protein 90.
Wherein: the disease associated with heat shock protein 90 is: cancer, neurodegenerative disease, viral infection, inflammation, leukemia, malaria or diabetes.
The present invention also discloses a pharmaceutical composition suitable for oral administration to a mammal for preventing or treating a disease associated with heat shock protein 90, characterized in that: the pharmaceutical composition contains effective dose of the tetrahydroisoquinoline-3-carboxylic acid heat shock protein 90 inhibitor and one or more pharmaceutically acceptable carriers or excipients.
The invention also discloses a pharmaceutical composition suitable for parenteral administration to a mammal for preventing or treating a disease associated with heat shock protein 90, characterized in that: the pharmaceutical composition contains a therapeutically effective amount of the tetrahydroisoquinoline-3-carboxylic acid heat shock protein 90 inhibitor of claim 1,2 or 3 and one or more pharmaceutically acceptable carriers or excipients.
The carrier may be saline, buffered saline, dextrose, water, glycerol, ethanol or combinations thereof; or the solid carrier lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate or stearic acid, etc.
The invention screens and obtains a compound with remarkably improved Hsp90 inhibitory activity and anti-cell proliferation activity based on further structural modification of a compound LCP1074, and provides a tetrahydroisoquinoline-3-carboxylic acid heat shock protein 90 inhibitor and a pharmaceutical composition which is suitable for oral administration to mammals or parenteral administration to mammals and is used for preventing or treating diseases related to the heat shock protein 90. The experiment proves that: the tetrahydroisoquinoline-3-carboxylic acid heat shock protein 90 inhibitor has obvious inhibiting effect on tumor cells of various tissue types including mammary gland, reproductive system and respiratory system sources, but has low toxicity on normal human liver cells and better druggability. Has wide clinical application prospect.
Drawings
FIG. 1: compound L15 was evaluated for intracellular inhibition of Hsp90 activity.
Detailed Description
In order to make the present invention comprehensible to those skilled in the art, several embodiments of the present invention are described below with reference to examples, and although the description is specific and detailed, the present invention is not limited thereto.
Example 1: synthesis of Compound L1-20, exemplified by L1.
1) 5-chloro-2, 4-dihydroxybenzoic acid methyl ester 2
Compound 1, methyl 2, 4-dihydroxybenzoate (8.0g,47.6mmol) was dissolved in anhydrous dichloromethane, cooled to 0 deg.C and sulfuryl chloride (4.0mL,49.4mmol) was added, after stirring at room temperature for 13 hours, sulfuryl chloride (2mL,24.7mmol) was added again, and after stirring for 6 hours, the reaction was quenched by addition of saturated NaHCO3(7.5 mL). Extracting and collecting an organic phase, extracting a water phase with dichloromethane for three times, combining the organic phases, drying the organic phases with anhydrous sodium sulfate, evaporating the solvent to dryness, and purifying the solvent by silica gel column chromatography to obtain 5.78g of a white solid compound 2, wherein the yield is as follows: 60 percent.1H NMR(600MHz,CDCl3,r.t.)δ10.82(s,1H),7.82(s,1H),6.61(s,1H),5.94(s,1H),3.93(s,3H).
2)2, 4-dibenzyloxy-5-chloro-benzoic acid methyl ester 3
Compound 2(5.78g,28.56mmol) and potassium carbonate (8.68g,62.83mmol) were dissolved in 50mL of DMF, stirred for 5min and then 8.20mL of benzyl bromide was added. Reacting at 60 ℃ for 6 hours, cooling to room temperature, adding water and ethyl acetate, extracting and layering, washing an organic phase with saturated salt water, drying with anhydrous sodium sulfate, evaporating the solvent to dryness, and purifying by silica gel column chromatography to obtain 9.43g of a white solid 3. The yield thereof was found to be 86.2%.1H NMR(600MHz,CDCl3,r.t.)δ7.94(s,1H),7.44-7.31(m,10H),6.56(s,1H),5.12(s,2H),5.10(s,2H),3.87(s,3H).HR ESIMS:m/z 405.0869[M+Na]+(calcd.405.0870).
3)2, 4-dibenzyloxy-5-chloro-benzoic acid 4
After compound 3(9.43g, 24.62mmol) was added to 50mL of methanol, 20mL of an aqueous solution of potassium hydroxide (7.06g) was added, and the mixture was stirred under reflux for 24 hours. Cooling the reaction solution to room temperature, adding 5% hydrochloric acid solution to acidity, extracting with ethyl acetate, treating the organic phase with saturated saline solution, drying with anhydrous sodium sulfate, evaporating the solvent to obtain 8.8g of white solid 4 in total, and obtaining the yield: 97 percent.1HNMR(600MHz,CDCl3,r.t.)δ8.19(s,1H),7.41-7.36(m,10H),6.61(s,1H),5.19(s,2H),5.18(s,2H).HR ESIMS:m/z 367.0739[M-H]-(calcd.367.0737).
4)1, 2,3, 4-tetrahydroisoquinoline-3-carboxylic acid methyl ester hydrochloride 6
Compound 5(4.27g,20mmol) was added to 20mL of methanol and cooledAfter reaching 0 ℃, 5.2mL of thionyl chloride is added dropwise, the mixture is stirred at room temperature for 100 hours, then the mixture is dried by spinning under reduced pressure, and the residue is washed with diethyl ether and dried to obtain 4.3g of a light yellow solid compound 6, with the yield: 94.4 percent.1H NMR(300MHz,D2O,r.t.)δ7.45-7.24(m,4H),4.60(d,J＝5.5Hz,1H),4.56(d,J＝5.4Hz,1H),4.53(br s,1H),3.93(s,3H),3.53(dd,J＝17.4,5.5Hz,1H),3.53(dd,J＝17.4,5.5Hz,1H),3.32(dd,J＝17.3,10.9Hz,1H),3.32(dd,J＝17.3,10.9Hz,1H).HRESIMS:m/z192.1028[M+H]+(calcd.192.1025).
5)2- (2, 4-dibenzyloxy-5-chloro-benzoyl) -1,2,3, 4-tetrahydroisoquinoline-3-carboxylic acid methyl ester 7
Compound 4(2.213g,6mmol) and compound 6(1.639g,7.2mmol) were dissolved in 30mL of anhydrous dichloromethane, and 1-hydroxybenzotriazole (HOBt,0.973g,7.2mmol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDCI,1.38g,7.2mmol), and N-methylmorpholine (NMM,2.012mL,18mmol) were added in this order and stirred at room temperature for 12 hours. The reaction solution was treated with water, 5% hydrochloric acid solution, saturated NaHCO3, and saturated brine in this order, the organic phase was separated, dried over anhydrous sodium sulfate, and purified by silica gel column chromatography after evaporation of the solvent to dryness to obtain 2.89g of compound 7, with yield: 81 percent.1H NMR(300MHz,CDCl3,r.t)δ7.52–7.05(m,16H),5.30–5.12(m,4H),4.41(s,1H),3.59–3.45(m,2H),3.30(s,3H),3.14–3.00(m,2H).HR ESIMS:m/z 542.1731[M+H]+(calcd.542.1734).
6) R-2- (2, 4-dibenzyloxy-5-chloro-benzoyl) -1,2,3, 4-tetrahydroisoquinoline-3-carboxylic acid 8
Compound 7(2.89g,5.338mmol) was dissolved in 16mL of methanol, and 0.62mL of a 2mol/L aqueous solution of sodium hydroxide was added. Stirred at room temperature for 12 h. After the reaction is finished, adding acid liquor to adjust the pH value to weak acid, extracting with ethyl acetate, drying with anhydrous sodium sulfate, and evaporating to dryness to obtain 2.68g of a compound 8, wherein the yield is as follows: 95 percent.1H NMR(300MHz,CDCl3,r.t)δ12.85(s,1H),7.52–7.05(m,16H),5.30–5.12(m,4H),4.41(s,1H),3.59–3.45(m,2H),3.14–3.00(m,2H).HR ESIMS:m/z526.1418[M-H]-(calcd.526.1421).
7) R-2- (5-chloro-2, 4-dihydroxybenzoyl) -3- (N-p-chlorobenzyl-amido) -1,2,3, 4-tetrahydroisoquinoline L1
Compound 8(100mg,0.19mmol), 1-hydroxybenzotriazole (HOBt,30.8mg,0.228mmol), and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDCI,43.7mg,0.228mmol) were dissolved in 3mL of DMF, stirred for 3 minutes, added with 115. mu.L of p-chlorobenzylamine, and stirred at room temperature for 12 hours. The reaction solution was treated with water, saturated NaHCO3, 5% hydrochloric acid solution, and saturated brine in this order, the organic phase was separated, dried over anhydrous sodium sulfate, and the solvent was evaporated to dryness, and the obtained compound was used in the next step without purification. Dissolving the obtained compound in anhydrous dichloromethane, cooling to 0 ℃ under the protection of N2, adding 0.57mL of boron trichloride solution (3eq), stirring at room temperature for 2 hours after 20min, adding saturated sodium bicarbonate solution to weak base under ice bath, quenching reaction, removing a dichloromethane layer, extracting an aqueous phase with ethyl acetate, washing with brine, drying with anhydrous Na2SO4, evaporating the solvent, and purifying with silica gel column chromatography to obtain 81.3mg of a white solid A1. yield: 76 percent.1H NMR(400MHz,Acetone)δ7.70(br s,1H),7.36(s,1H),7.27–7.20(m,3H),7.19(br s,1H),7.10(s,1H),7.09(s,1H),7.04-7.00(m,1H),6.88(brs,1H),6.61(s,1H),5.14(br s,1H),4.80-4.70(m,2H),4.37(dd,J＝15.2,5.7Hz,1H),4.28(dd,J＝15.3,5.3Hz,1H),3.37(dd,J＝15.4,4.9Hz,1H),3.27(dd,J＝15.5,6.0Hz,1H).HRESIMS:m/z for 471.0846[M+H]+,(calcd.471.0878)。
Example 2: synthesis of Compound L21-45, exemplified by L21.
1) R-7-hydroxy-6, 8-diiodo-1, 2,3, 4-tetrahydroisoquinoline-3-carboxylic acid hydrochloride 10
To 8mL of concentrated HCl were added 3, 5-diiodo-D-tyrosine (1.0g,2.31mmol), ethylene glycol dimethyl ether (0.7mL) and paraformaldehyde (0.26g,8.67mmol) and the temperature was gradually raised to 72 ℃. After 0.5 hour, concentrated hydrochloric acid (1.7mL), ethylene glycol dimethyl ether (0.33mL) and poiyiformic acid (0.17g,5.78mmol) were added and the reaction was continued for 18 hours with the oil bath controlled at 72-75 ℃. The reaction suspension is cooled in ice bath and filtered, and the filter cake is washed with glycol dimethyl ether to obtain 0.53g of light yellow powder after drying.1H NMR(300MHz,DMSO-d6,r.t)δ3.07(dd,J＝16.8Hz,10.8Hz,1H),3.22(dd,J＝16.8Hz,4.8Hz,1H),4.02(d,J＝16.2Hz,1H),4.15(d,J＝16.2Hz,1H),4.32(dd,J＝4.8Hz,10.8Hz,1H),7.73(s,1H),9.68(s,1H),10.00(br s,2H),14.17(br s,1H),ESI-MS m/z:446.2[M+H]+
2) R-2-Boc 7-hydroxy-6, 8-diiodo-1, 2,3, 4-tetrahydroisoquinoline-3-carboxylic acid hydrochloride 11
Compound 10(0.53g) was dissolved in 2.5mL of sodium oxy oxide solution and 0.6mL of a solution of di-tert-butyl carbonate (0.27g) in tetrahydrofuran was added. In the reaction process, the pH of the reaction solution is controlled to be 9-11 by using 1mol/L sodium hydroxide solution. After 6 hours of reaction at room temperature, tetrahydrofuran in the reaction solution was evaporated, the reaction solution was extracted with petroleum acid 3 times, acidified to pH4-5 with 1mol/L citric acid solution, extracted with ethyl acetate three times, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was evaporated to dryness to obtain 0.8g of yellow powder 11. The yield of the product is 92 percent,1H NMR(300MHz,DMSO-d6,r.t)δ1.34+1.40(s,9H),2.87-3.00(m,2H),4.13-4.41(m,2H),4.61-4.75(m,1H),7.57(s,1H),9.41(brs,1H),12.71(br s,1H)。
3) R-2-Boc 7-hydroxy-1, 2,3, 4-tetraoxyisoquinoline-3-carboxylate 12
Compound 11(0.8g) was dissolved in 10mL of anhydrous methanol, and triethylamine (480. mu.L) and 10% palladium on carbon (0.08g) were added thereto. After reacting for 5 hours by introducing hydrogen, filtering the catalyst by using kieselguhr, distilling off methanol, adding 1mol/L citric acid solution to the pH value of 4-5, extracting for three times by using ethyl acetate, combining ethyl acetate layers, washing paint by using saturated common salt water, drying by using anhydrous sodium sulfate, and evaporating the solvent to dryness to obtain 0.36g of yellow powder 12. The yield is 75 percent,1H NMR(300MHz,DMSO-d6,r.t)δ1.39+1.45(s,9H),2.92-3.04(m,2H),4.26-4.51(m,2H),4.57-4.82(m,1H),6.52(s,1H),6.57(d,J＝8.4Hz,1H),6.97(d,J＝8.4Hz,1H),9.28(s,1H),12.60(s,1H)。
4) R-2-tert-Butoxycarbonyl-7-hydroxy-3- (N- (pyridine-4-methyl) -amido) -1,2,3, 4-tetrahydroisoquinoline 13
Compound 12(0.36g) and 4-methylaminopyridine (500. mu.L) were dissolved in 10mL of DMF, and 1-hydroxybenzotriazole (HOBt,0.2g) and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDCI,0.28g) were added in this order, followed by stirring at room temperature for 12 hours. The reaction solution is sequentially added with water and saturated NaHCO3Treating with 10% citric acid solution and saturated saline, separating organic phase, drying with anhydrous sodium sulfate, evaporating solvent, and performing silica gel column chromatographyWhite solid 0.26g, yield 75%,1H NMR(300MHz,Acetone-d6,r.t)δ1.39+1.45(s,9H),2.92-3.04(m,2H),3.57-3.82(m,2H),4.26-4.51(m,2H),4.57-4.82(m,1H),6.72-6.80(m,4H),7.02(d,J＝8.1Hz,1H),7.68(s,1H),8.31-8.37(m,2H).
5) r-2- (2, 4-dibenzyloxy-5-chloro-benzoyl) -7-hydroxy-3- (N- (pyridine-4-methyl) -amido) -1,2,3, 4-tetrahydroisoquinoline 14
To 3mL of a dichloromethane solution of Compound 13(410mg) was added 1mL of trifluoroacetic acid, and after the reaction was completed, excess triethylamine was added to the reaction solution until basic. To 3mL of an anhydrous tetrahydrofuran solution containing intermediate 4(413mg,1eq) was added triethylamine, 1-hydroxybenzotriazole (HOBt,182mg) and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDCI258mg), and the mixture was stirred at room temperature for 15 minutes, and then poured into the above dichloromethane solution. After the reaction was carried out overnight, the solvent was distilled off, ethyl acetate was added, and water and saturated NaHCO were added in this order3Treating with saturated saline solution, separating organic phase, drying with anhydrous sodium sulfate, evaporating solvent, and performing silica gel column chromatography to obtain white solid 600mg.
7) R-2- (5-chloro-2, 4-dihydroxybenzoyl) -7-methoxy-3- (N-p-chlorobenzyl-amido) -1,2,3, 4-tetrahydroisoquinoline L21
Cesium carbonate powder (150 mg) and N- (2-chloroethyl) morpholine hydrochloride (37mg) were added to a 4ml dmf solution of compound 14(50 mg), stirred at room temperature overnight, extracted with ethyl acetate three times, the ethyl acetate layers were combined, washed with saturated brine, dried over anhydrous magnesium sulfate, and the solvent was evaporated to dryness to give a crude product. The resulting compound was used in the next step without purification. The resulting compound was dissolved in anhydrous dichloromethane, N2Cooling to 0 ℃ under protection, adding 0.57mL of boron trichloride solution, stirring at room temperature for 2 hours after 20min, adding saturated sodium bicarbonate solution to weak base under ice bath, quenching, removing dichloromethane layer, extracting water phase with ethyl acetate, washing with brine, and adding anhydrous Na2SO4Drying, evaporating the solvent, and purifying by silica gel column chromatography to obtain 81.3mg of white solid A1. yield: 76 percent.1HNMR(400MHz,Acetone-d6,r.t)δ7.70(br s,1H),7.36(s,1H),7.27–7.20(m,3H),7.19(brs,1H),7.10(s,1H),7.09(s,1H),7.04-7.00(m,1H),6.61(s,1H),5.14(br s,1H),4.80-4.70(m,2H),4.37(dd,J＝15.2,5.7Hz,1H),4.28(dd,J＝15.3,5.3Hz,1H),3.76(s,3H),3.37(dd,J＝15.4,4.9Hz,1H),3.27(dd,J＝15.5,6.0Hz,1H)。
Example 3: four hydrogen isoquinoline-3-carboxylic acid derivatives in vitro antitumor cell activity screening test
The test method comprises the following steps:
the antitumor activity of each compound was evaluated by the SRB method. Cells in logarithmic growth phase were taken, seeded at a density of 2000 and 8000cells/well in 96-well plates, dosed 12h after cell attachment (compounds L1-L45), and cultured for a further 72 h. After the cell culture is finished, 100 mu L of 10% trichloroacetic acid (TCA) is added into each hole for fixation, the mixture is kept stand for 5min, and the mixture is transferred to 4 ℃ for standing for 1 h. After the cells were fixed, the TCA was discarded, washed with distilled water 5 times, spun-dried, and allowed to stand in air for 5min to dry. Add 50. mu.L of 4mg/mLSRB solution to each well, stain at room temperature for 10 minutes, wash away the SRB not bound to the protein with 1% glacial acetic acid, spin dry 5 times, and dry in air. Protein-bound SRB were solubilized by adding 200. mu.L of 10mM Tris unbuffered solution to each well and tapping with hand for 1 min. Finally, the OD value was measured with a spectrophotometer at a measurement wavelength of 570 nm.
The inhibition rate of tumor cell proliferation was calculated as follows:
IC50the concentration at which the sample inhibited tumor cells by 50% was expressed.
Testing materials:
human breast cancer cells MDA-MB-231, MDA-MB-453; human neuroblastoma cell SHSY 5Y; human glioma cell U251; human colon cancer cells HCT116, SW 480; human lung cancer cells H1299, a 549; human cervical cancer cell HeLa, human prostate cancer PC 3; human liver cancer cell HepG 2; human normal liver cell HL 7702. Purchased from ATCC.
And (3) testing results: see table 1.
Table 1: activity of partial tetrahydroisoquinoline-3-carboxylic acid derivatives on 12 cell lines
In conclusion, the compounds L10, L15, L22 and L41 have obvious inhibition effect on tumor cells of various tissue types including breast, reproductive system and respiratory system sources, but have low toxicity on normal human liver cells and better druggability.
Example 4: evaluation of Hsp90 inhibitory Activity
Representative compound L15 was selected and tested for its effect on Hsp 90-related proteins at the cellular level. Down-regulation of Hsp90 client protein levels and anaplerotic up-regulation of Hsp70 are two of the most common biological measures for the inhibition of Hsp90 by compounds. The results of Westernblot indicate that compound L15 acts intracellularly on Hsp 90.
The test method comprises the following steps:
and (3) inoculating MDA-MB231 cells in logarithmic growth into a cell culture dish, adding drugs for corresponding time when the cells grow to 70% of density, removing the culture medium, washing once with PBS precooled on ice, and adding cell lysate for treatment. The lysate was added to an equal volume of 2 x SDS sample buffer and heated to boil for 5min to completely denature the protein. The denatured samples were first subjected to 8% -12% SDS-PAGE and then electroporated onto methanol activated polyvinylidene fluoride (PVDF) membranes. The PVDF membrane after electrotransfer is placed in a blocking solution (5% BSA), and a decolorizing shaker is used for blocking for 1 hour at room temperature. Subsequently, a primary antibody solution diluted with 5% BSA in the corresponding ratio was added, blocked overnight at 4 ℃ and washed 3 times for 10min each with TBST. Adding corresponding second antibody solution coupled with horseradish peroxidase, placing in a decolorizing shaker, shaking for 1h, and washing for 3 times in TBST for 10min each time. And soaking the washed PVDF membrane by using a newly-prepared enhanced chemiluminescent solution, and placing the soaked PVDF membrane in a ChemDoc XRS + gel imager for photographing and observation.
And (3) testing results: see fig. 1.
Claims (1)
1. A tetrahydroisoquinoline-3-carboxylic acid heat shock protein 90 inhibitor is characterized in that: the heat shock protein 90 inhibitor is a compound with the following chemical names respectively:
r-2- (5-chloro-2, 4-dihydroxybenzoyl) -3- (N-p-cyanobenzyl-amido) -1,2,3, 4-tetrahydroisoquinoline, designated as L10;
r-2- (5-chloro-2, 4-dihydroxybenzoyl) -3- (N- (pyridine-4-methyl) -amido) -1,2,3, 4-tetrahydroisoquinoline, named L15;
r-2- (5-chloro-2, 4-dihydroxybenzoyl) -7- (2-methoxyethoxy) -3- (N- (pyridin-4-ylmethyl) -amido) -1,2,3, 4-tetrahydroisoquinoline, designated L22;
r-2- (5-chloro-2, 4-dihydroxybenzoyl) -7- (2-morpholinoethoxy) -3- (N-p-cyanobenzyl-amido) -1,2,3, 4-tetrahydroisoquinoline, designated as L29;
r-2- (5-chloro-2, 4-dihydroxybenzoyl) -7- (2-morpholinoethoxy) -3- (N-p-cyanobenzyl-amido) -1,2,3, 4-tetrahydroisoquinoline, named L41.
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| CN103724269A (en) * | 2012-10-11 | 2014-04-16 | 中国科学院上海药物研究所 | Phenyl 1,2-isoxazole or phenyl 1,2-pyrazole compounds and application thereof |
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| Title |
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| Design and synthesis of N-(5-chloro-2,4-dihydroxybenzoyl)-(R)-1,2,3,4-tetrahydroisoquinoline-3-carboxamides as novel Hsp90 inhibitors;Chuanpeng Liang et al.;《European Journal of Medicinal Chemistry》;20160520;第121卷;第272-282页 * |
| Synthesis and SAR of tetrahydroisoquinolines as Rev-erba agonists;Romain Noel et al.;《Bioorganic & Medicinal Chemistry Letters》;20120413;第22卷;第3739–3742页 * |
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