CN108976279B - Theanyl amino acid benzyl ester modified curcumin, and synthesis, activity and application thereof - Google Patents
Theanyl amino acid benzyl ester modified curcumin, and synthesis, activity and application thereof Download PDFInfo
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Abstract
The invention discloses 1- (4-hydroxy-3-methoxyphenyl) -7- (4-oxyacetyltheanyl-AA-OBzl-3-methoxyphenyl) -1, 6-heptadiene-3, 5-diketone (AA in the formula is selected from L-Asp residue and L-Glu residue), discloses a preparation method thereof, discloses the anti-tumor growth activity thereof, discloses the activity of reversing the drug resistance of tumor cells and discloses the anti-inflammatory activity thereof, thus the invention discloses the application thereof in preparing anti-tumor drugs, drugs for reversing the drug resistance of tumor cells and anti-inflammatory drugs.
Description
Technical Field
The invention relates to 1- (4-hydroxy-3-methoxyphenyl) -7- (4-oxyacetyltheanyl-AA-OBzl-3-methoxyphenyl) -1, 6-heptadiene-3, 5-diketone, a preparation method thereof, antitumor growth activity thereof, tumor cell drug resistance reversing activity thereof and anti-inflammatory activity thereof, and thus, the invention relates to application thereof in preparing antitumor drugs, tumor cell drug resistance reversing drugs and anti-inflammatory drugs. The invention belongs to the field of biological medicine.
Background
Malignant tumors are a global problem that seriously jeopardizes human health. Most of patients with tumors are in the middle and late clinical stage, and the treatment method mainly comprises radiotherapy and chemotherapy, which are main treatment means when malignant tumors are in the middle and late stage. However, not only the resistance of tumor cells to anti-tumor drugs during chemotherapy seriously affects the effect of chemotherapy, but also inflammation further worsens the prognosis of tumor patients. The prior anti-tumor medicament has no effect of reversing the drug resistance of tumor cells and has no anti-inflammatory effect, so the clinical curative effect of tumor chemotherapy is not ideal. The invention relates to an anti-tumor medicament with anti-inflammatory activity and capable of reversing the drug resistance of tumor cells, which is an urgent clinical need. The inventors previously disclosed that 1- (4-hydroxy-3-methoxyphenyl) -7- (4-oxyacetamidobenzyl-3-methoxyphenyl) -1, 6-heptadiene-3, 5-dione has significant activity in inhibiting tumor cell proliferation. Later, the inventors further disclosed that anti-adhesion peptide modified curcumin could inhibit tumor growth in S180 mice and inhibit inflammation in ICR mice at a dose of 1 μmol/kg. However, the lowest effective dose is 1. mu. mol/kg. In order to reduce the lowest effective dose, the inventors have made various modifications to the phenolic hydroxyl group of curcumin. After 3 years of exploration, it was found that curcumin modified with theanyl-AA-OBzl (AA selected from L-Asp residues and L-Glu residues) not only reduced the minimum effective dose against tumor growth to 0.1. mu. mol/kg, but also reduced the minimum effective dose against inflammation to 0.1. mu. mol/kg. In addition, the compound has the activity of reversing drug resistance. The effective dosage for resisting tumor growth is reduced by 10 times, and the addition of the effects of reversing drug resistance and resisting inflammation shows that the structure modification has outstanding technical effect. Based on these findings, the inventors have proposed the present invention.
Disclosure of Invention
The first aspect of the present invention is to provide 1- (4-hydroxy-3-methoxyphenyl) -7- (4-acetoacetyl-theanyl-AA-OBzl-3-methoxyphenyl) -1, 6-heptadiene-3, 5-dione represented by the following formula (wherein AA is selected from the group consisting of L-Asp residue and L-Glu residue).
The second aspect of the present invention provides a method for synthesizing 1- (4-hydroxy-3-methoxyphenyl) -7- (4-acetoacetyl-theanyl-AA-OBzl-3-methoxyphenyl) -1, 6-heptadiene-3, 5-dione (wherein AA is selected from the group consisting of L-Asp residue, L-Glu residue and L-Phe residue), which comprises:
(1) preparing 6- (4-hydroxy-3-methoxyphenyl) -5, 6-hexene-2, 4-dione (1);
(2) preparation of benzyl 2- (4-formyl-2-methoxyphenoxy) -acetate (2);
(3) preparing 1- (4-hydroxy-3-methoxyphenyl) -7- (4-oxyacetobenzoyl-3-methoxyphenyl) -1, 6-heptadiene-3, 5-diketone (3) by taking the products obtained in the step (1) and the step (2) as reaction raw materials;
(4) saponifying 1- (4-hydroxy-3-methoxyphenyl) -7- (4-oxoacetoacetylbenzyl-3-methoxyphenyl) -1, 6-heptadiene-3, 5-dione to obtain 1- (4-hydroxy-3-methoxyphenyl) -7- (4-oxoacetoacetoxy-3-methoxyphenyl) -1, 6-heptadiene-3, 5-dione (4);
(5) coupling the compound 4 and L-benzyl theanine to obtain 1- (4-hydroxy-3-methoxyphenyl) -7- (4-oxyacetylbenzyl theanine-3-methoxyphenyl) -1, 6-heptadiene-3, 5-diketone (5);
(6) saponifying the compound 5 to obtain 1- (4-hydroxy-3-methoxyphenyl) -7- (4-oxyacetophytamic acid-3-methoxyphenyl) -1, 6-heptadiene-3, 5-dione (6);
(7) the compound 6 is coupled with L-amino acid benzyl ester to obtain 1- (4-hydroxy-3-methoxyphenyl) -7- (4-oxyacethyl theanyl-AA-OBzl-3-methoxyphenyl) -1, 6-heptadiene-3, 5-diketone.
The third aspect of the present invention is to evaluate the use of 1- (4-hydroxy-3-methoxyphenyl) -7- (4-acetoacetyl-theanyl-AA-OBzl-3-methoxyphenyl) -1, 6-heptadiene-3, 5-dione for inhibiting tumor growth in S180 mice.
The fourth aspect of the present invention is the use of 1- (4-hydroxy-3-methoxyphenyl) -7- (4-acetoacetyl-theanyl-AA-OBzl-3-methoxyphenyl) -1, 6-heptadiene-3, 5-dione for evaluating the reversal of drug resistance of drug-resistant tumor cells.
The fifth aspect of the present invention is to evaluate the inhibitory effect of 1- (4-hydroxy-3-methoxyphenyl) -7- (4-oxoacetyltheanyl-AA-OBzl-3-methoxyphenyl) -1, 6-heptadiene-3, 5-dione on the inflammation of ICR mice.
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FIG. 1 is 1- (4-hydroxy-3-methoxyphenyl) -7- (4-acetoacetyl-AA-OBzl-3-methoxyphenyl) -1, 6-heptadiene-3, 5-dione.7 a, wherein AA is an L-Asp residue; AA in 7b is L-Glu residue; i) boron trioxide (B)2O3) Acetylacetone, tri-n-butyl borate, n-butylamine, aqueous hydrogen chloride (1M); ii) potassium carbonate, benzyl bromoacetate; iii) boron trioxide (B)2O3) Tri-n-butyl borate, n-butylamine, 10% aqueous acetic acid; iv) aqueous sodium hydroxide (2M), acetone; v) Dicyclohexylcarbodiimide (DCC), 1-hydroxybenzotriazole (HOBt), N-methylmorpholine-quinoline (NMM), Tetrahydrofuran (THF).
Detailed Description
To further illustrate the invention, a series of examples are given below. These examples are purely illustrative and are intended to be a detailed description of the invention only and should not be taken as limiting the invention.
EXAMPLE 1 preparation of 6- (4-hydroxy-3-methoxyphenyl) -5, 6-hexene-2, 4-dione (1)
45.0mL (437.7mmol) of acetylacetone, 21.0g (301.6mmol) of boron oxide and 150.0mL of anhydrous ethyl acetate were refluxed at 60 ℃ for 1 h. Then, 22.5g (148.0mmol) of vanillin and 41mL (293.0mmol) of tributylborate were added thereto. The reaction mixture was stirred for 30min at 70 ℃. A solution of 15mL (205.1mmol) of n-butylamine in 135mL of ethyl acetate is added further during 30 min. The mixture was stirred at 100 ℃ for 3h, then cooled to room temperature, and 150mL of hydrochloric acid (1M) was added dropwise thereto. The mixture was stirred at 50 ℃ for 30min, allowed to stand, and the aqueous layer was extracted 3 times with ethyl acetate. The combined ethyl acetate layers were washed with saturated NaCl solution to neutrality, dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated to dryness under reduced pressure, and the residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate ═ 4/1) to give 10.05g (29%) of the title compound as a yellow solid. ESI-MS (m/e): 235[ M + H ]]+；1H NMR(300MHz,DMSO-d6):δ/ppm＝15.74(s,1H),9.64(s,1H),7.50(d,J＝15.9Hz,1H),7.30(s,1H),7.12(d,J＝8.1Hz,1H),6.82(d,J＝8.1Hz,1H),6.64(d,J＝15.9Hz,1H),5.84(s,1H),5.14(s,2H),3.83(s,3H),2.12(s,3H)。
EXAMPLE 2 preparation of 3-methoxy-4- (oxy-2-acetylcarbobenzoxy) benzaldehyde (2)
10g (65.8mmol) of vanillin are dissolved in 100mL of anhydrous tetrahydrofuran. To the solution was added 10.9g (79.0mmol) of potassium carbonate in portions and stirred for 3 h. Then, 9.3mL of benzyl bromoacetate was added dropwise to the solution, and the mixture was stirred at room temperature for 48 hours, followed by TLC (petroleum ether/ethyl acetate: 3/1) to show that the reaction was completed. The reaction mixture is filtered, the filtrate is concentrated under reduced pressure, the residue is triturated with 100mL of diethyl ether and left to stand for 12h before the diethyl ether is decanted off, 10mL of diethyl ether are triturated 3 times and the diethyl ether is removed to give 15.4g (78%) of the title compound as a colorless solid. ESI-MS (m/e): 301[ M + H]+；1H NMR(300MHz,DMSO-d6):δ/ppm＝9.86(s,1H),7.50(dd,J1＝8.4Hz,J2＝1.8Hz,1H),7.44(d,J＝1.8Hz,1H),7.39(s,5H),7.11(d,J＝8.4Hz,1H),5.21(s,2H),5.03(s,2H),3.84(s,3H)。
EXAMPLE 3 preparation of 1- (4-hydroxy-3-methoxyphenyl) -7- (4-oxoacetylbenzyloxy-3-methoxyphenyl) -1, 6-heptadiene-3, 5-dione (3)
A suspension of 5.55g (23.7mmol)6- (4-hydroxy-3-methoxyphenyl) -5, 6-hexene-2, 4-dione (1),0.83g (11.9mmol) boron oxide and 100mL ethyl acetate was refluxed at 70 ℃ for 1 h. Then, the mixture was concentrated under reduced pressure. The residue was dissolved in 100mL of anhydrous DMF. To the resulting solution were added 10.67g (35.6mmol) of 3-methoxy-4- (oxy-2-acetylcarbobenzoxy) benzaldehyde (2) and 11.15mL (41.0mmol) of tributyl borate. The resulting solution was stirred at 80 ℃ for 30 min. Thereafter, 0.98mL (6.4mmol) of n-butylamine was added dropwise thereto in 4 portions over 1 hour, and the resulting solution was stirred at 80 ℃ for 3 hours. Thereafter, 200mL of a 10% aqueous acetic acid solution preheated to 60 ℃ was added thereto. The resulting solution was stirred at 80 ℃ for a further 1 h. The reaction mixture was cooled to room temperature, filtered and the filter cake was purified by column chromatography (petroleum ether/ethyl acetate 3/1) to give 6.63g (53%) of the title compound as a yellow solid. ESI-MS (m/e): 517[ M + H]+；1H NMR(300MHz,DMSO-d6):δ/ppm＝9.69(s,1H),8.51(d,J＝7.5Hz,1H),7.80(t,J＝5.7Hz,1H),7.59(d,J＝3.0Hz,1H),7.54(d,J＝3.0Hz,1H),7.37(m,7H),7.16(m,1H),6.89(m,4H),6.09(s,1H),5.14(s,2H),4.62(s,2H),4.37(m,1H),3.87(s,3H),3.84(s,3H),3.04(qd,J1＝7.2Hz,J2＝5.7Hz,2H),2.15(m,2H),2.05(m,1H),1.90(m,1H),0.98(t,J＝7.2Hz,3H)。
EXAMPLE 4 preparation of 1- (4-hydroxy-3-methoxyphenyl) -7- (4-oxoacetoxy-3-methoxyphenyl) -1, 6-heptadiene-3, 5-dione (4)
5g (9.7mmol) of 1- (4-hydroxy-3-methoxyphenyl) -7- (4-oxoacetylbenzyloxycarbonyl-3-methoxyphenyl) -1, 6-heptadiene-3, 5-dione (3) were dissolved in acetone. Aqueous NaOH (2M) was added thereto at room temperature, and the reaction solution was adjusted to pH 13 and stirred for 6 hours. TLC (petroleum ether/ethyl acetate 3/1) showed the reaction was complete. The reaction mixture was saturated with KHSO4Adjusting the pH of the aqueous solution to 7, concentrating under reduced pressure, and subjecting the residue to saturated KHSO4The aqueous solution was adjusted to pH 2. After that, it was extracted 3 times with ethyl acetate. The ethyl acetate layers were combined, washed with a saturated NaCl solution to neutrality, and dried over anhydrous sodium sulfate. Filtration, concentration of the filtrate under reduced pressure and trituration of the residue with dry ether gave 2.64g (64%) of the title compound as a red solid. ESI-MS (m/e): 425[ M-H]-；1H NMR(300MHz,DMSO-d6):δ/ppm＝9.55(s,1H),7.57(m,2H),7.37(m,2H),7.20(m,2H),6.79(m,4H),6.06(s,1H),4.74(s,2H),3.85(s,6H)。
EXAMPLE 5 preparation of 1- (4-hydroxy-3-methoxyphenyl) -7- (4-oxyacetyltheanine-benzyl ester group-3-methoxyphenyl) -1, 6-heptadiene-3, 5-dione (5)
2g (4.7mmol) of 1- (4-hydroxy-3-methoxyphenyl) -7- (4-oxoacetoxy-3-methoxyphenyl) -1, 6-heptadiene-3, 5-dione (4),1.69g (5.6mmol) of benzyl theanine hydrochloride and 0.761g (5.64mmol) of N-hydroxybenzotriazole (HOBt) are dissolved in 50mL of anhydrous tetrahydrofuran under ice bath. To the solution was added dropwise a solution composed of 1.16g (5.63mmol) of Dicyclohexylcarbodiimide (DCC) and 10mL of anhydrous tetrahydrofuran. The reaction solution was adjusted to pH 8 with N-methylmorpholine and stirred at room temperature for 12 h. TLC (petroleum ether/ethyl acetate 3/1) showed the reaction was complete. The reaction mixture was concentrated to dryness under reduced pressure, and the residue was dissolved in 150mL of ethyl acetate. Filtering, washing the filtrate with saturated sodium bicarbonate water solution for 3 times, saturated sodium chloride water solution for 3 times, saturated potassium bisulfate water solution for 3 times, saturated sodium chloride water solution for 3 times, saturated sodium bicarbonate water solution for 3 times, and saturated sodium chloride water solution for 3 times. The combined ethyl acetate layers were dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (dichloromethane/methanol ═ 80/1) to give 2.24g (71%) of the title compound as a yellow solid. ESI-MS (m/e): 673[ M + H]+；1HNMR(300MHz,DMSO-d6):δ/ppm＝9.69(s,1H),8.51(d,J＝7.5Hz,1H),7.80(t,J＝5.7Hz,1H),7.59(d,J＝3.0Hz,1H),7.54(d,J＝3.0Hz,1H),7.37(m,7H),7.16(m,1H),6.89(m,4H),6.09(s,1H),5.14(s,2H),4.62(s,2H),4.37(m,1H),3.87(s,3H),3.84(s,3H),3.04(qd,J1＝7.2Hz,J2＝5.7Hz,2H),2.15(m,2H),2.05(m,1H),1.90(m,1H),0.98(t,J＝7.2Hz,3H)。
EXAMPLE 6 preparation of 1- (4-hydroxy-3-methoxyphenyl) -7- (4-oxoacetyltheanine-3-methoxyphenyl) -1, 6-heptadiene-3, 5-dione (6)
2.24g (3.3mmol) of 1- (4-hydroxy-3-methoxyphenyl) -7- (4-oxyacetophtalotin carbobenzoxy-3-methoxyphenyl) -1, 6-heptadiene-3, 5-dione (5) was dissolved in acetone, an aqueous NaOH solution (2M) was added dropwise to the solution at room temperature until the reaction solution pH was 13, and the mixture was stirred for 6 hours, and TLC (petroleum ether/ethyl acetate) ═ 3/1 indicated that the reaction was complete. With saturated KHSO4Adjusting pH of the reaction solution to 7 with water solution, concentrating under reduced pressure, and adding saturated KHSO to the residue4The aqueous solution was adjusted to pH 2. Thereafter, extraction was carried out 3 times with ethyl acetate, and the combined ethyl acetate layers were washed with a saturated aqueous NaCl solution to neutrality, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give 1.07g (55%) of the title compound as a red syrup. ESI-MS (m/e): 581[ M-H]-；1H NMR(300MHz,DMSO-d6):δ/ppm＝9.42(s,1H),8.1(m,1H),7.77(m,1H),7.57(m,1H),7.37(m,1H),7.24(m,2H),7.10(m,2H),6.96(d,J＝5.1Hz,1H),6.79(m,3H),6.06(s,1H),4.57(s,2H),4.20(m,1H),3.82(s,6H),3.01(m,2H),2.09(m,3H),1.77(m,1H),0.95(t,J＝4.5Hz,3H)。
EXAMPLE 7 preparation of 1- (4-hydroxy-3-methoxyphenyl) -7- (4-oxyacetyltheanyl-Asp (OBzl) -OBzl-3-methoxyphenyl) -1, 6-heptadiene-3, 5-dione (7a)
2.32g (3.98mmol) of 1- (4-hydroxy-3-methoxyphenyl) -7- (4-oxoacetyltheanylamino-3-methoxyphenyl) -1, 6-heptadiene-3, 5-dione (6),1.67g (4.78mmol) of HCl. Asp. (OBzl) -OBzl and 0.65g (4.78mmol) of N-hydroxybenzotriazole (HOBt) were dissolved in 20mL of anhydrous tetrahydrofuran under ice bath. Thereafter, a solution of 0.98g (4.78mmol) of Dicyclohexylcarbodiimide (DCC) and 5mL of anhydrous tetrahydrofuran was added dropwise thereto. The reaction solution was adjusted to pH 8 with N-methylmorpholine (NMM), stirred at room temperature for 12h, and TLC (dichloromethane/methanol-50/1) indicated completion of the reaction. The reaction solution was concentrated to dryness under reduced pressure, the residue was dissolved in 150mL of dichloromethane, filtered, and the filtrate was washed with a saturated aqueous sodium bicarbonate solution 3 times, a saturated aqueous sodium chloride solution 3 times, a saturated aqueous potassium bisulfate solution 3 times, a saturated aqueous sodium chloride solution 3 times, a saturated aqueous sodium bicarbonate solution 3 times, and a saturated aqueous sodium chloride solution 3 times in this order. The combined dichloromethane layers were dried over anhydrousDried over sodium sulfate, filtered, and the filtrate concentrated to dryness under reduced pressure. The residue was purified by column chromatography (dichloromethane/methanol-50/1) to yield 1.98g (56.7%) of the title compound as a yellow solid. Mp 136-138 ℃;
(c ═ 0.1, methanol); ESI-MS (M/e):878[ M + H]+；IR(cm-1):3285,2936,1731,1640,1585,1507,1454,1423,1381,1359,1267,1212,1164,1134,1030,964,696；1HNMR(300MHz,DMSO-d6):δ/ppm＝9.68(s,1H),8.70(d,J＝8.1Hz,1H),8.10(d,J＝8.1Hz,1H),7.75(t,J＝5.7Hz,1H),7.57(dd,J1＝15.6Hz,J2＝1.5Hz,2H),7.31(m,12H),7.22(dd,J1＝8.4Hz,J2＝1.8Hz,1H),6.98(d,J＝8.4Hz,1H),6.72(m,3H),6.09(s,1H),5.10(s,2H),5.08(s,2H),4.77(m,1H),4.60(s,2H),4.40(m,1H),3.86(s,3H),3.85(s,3H),3.04(qd,J1＝7.2Hz,J2＝5.7Hz,2H),2.87(dd,J1＝19.8Hz,J2＝6.3Hz,2H),2.11(m,2H),1.92(m,1H),1.75(m,1H),0.99(t,J＝7.2Hz,3H)。
EXAMPLE 8 preparation of 1- (4-hydroxy-3-methoxyphenyl) -7- (4-oxoacetyltheanyl-Glu-OBzl-3-methoxyphenyl) -1, 6-heptadiene-3, 5-dione (7b)
Using the method of example 7, 880mg (17.0%) of the title compound were obtained as a yellow solid from 2.5g (5.87mmol) of 1- (4-hydroxy-3-methoxyphenyl) -7- (4-oxoacetyltheanylamino-3-methoxyphenyl) -1, 6-heptadiene-3, 5-dione (6) and 3.59g (8.80mmol) of HCl. Glu (OBzl) -OBzl. Mp 140-142 deg.C;
(c ═ 0.1, methanol); ESI-MS (M/e):892[ M + H ]]+；IR(cm-1):3284,3066,2937,1731,1665,1639,1585,1543,1508,1449,1422,1390,1336,1265,1215,1162,1135,1032,964,845,799,754,741,696；1HNMR(300MHz,DMSO-d6):δ/ppm＝9.69(s,1H),8.56(d,J＝8.1Hz,1H),8.12(d,J＝8.1Hz,1H),7.76(t,J＝5.7Hz,1H),7.57(m,2H),7.36(m,12H),7.20(dd,J1＝8.4Hz,J2＝1.8Hz,1H),6.98(d,J＝8.4Hz,1H),6.85(m,3H),6.11(s,1H),5.13(s,2H),5.08(s,2H),4.61(s,2H),4.40(m,2H),3.87(s,3H),3.85(s,3H),3.06(qd,J1＝7.2Hz,J2＝5.7Hz,2H),2.45(m,2H),2.09(m,2H),1.94(m,1H),1.80(m,1H),1.01(t,J＝7.2Hz,3H)。
Example 9 determination of the anti-tumor growth Activity of Compounds 7a, b
Doxorubicin, compound 6 and compounds 7a, b were dissolved in physiological saline prior to assay for administration to S180 mice. Taking S180 ascites tumor liquid which is inoculated in a male ICR mouse and grows vigorously for 10 days in a sterile environment, diluting the S180 ascites tumor liquid into liquid (1:2) by using normal saline, fully mixing the liquid, dyeing the tumor cell suspension by using freshly prepared 0.2% trypan blue, uniformly mixing the liquid and the liquid, counting the liquid according to a white cell counting method, wherein the blue-dyed cell is a dead cell, and the non-dyed cell is a live cell. The cell concentration is 4-large-grid viable cell number/4 × 104The cell density was calculated as x dilution factor ═ cell number/mL, and the cell survival rate was calculated as live cell number/(live cell number + dead cell number) × 100%. Homogenizing tumor solution with survival rate of more than 90% to density of 2.0 × 107Cell suspension per mL. This cell suspension was inoculated subcutaneously (0.2 mL/mouse) in the right axilla of a mouse to prepare S180 tumor-bearing mice. 24h after inoculation, S180 tumor-bearing mice were intraperitoneally injected daily with a normal saline solution of doxorubicin (dose 2. mu. mol/kg/day g), or with a normal saline solution of compound 6 (dose 1. mu. mol/kg/day), or with a normal saline solution of compounds 7a, b (dose 0.1. mu. mol/kg/day), 10 per group. The administration is once daily for 12 days. The next day of the last administration, cervical spine was removed under ether anesthesia, and then the right axillary tumor growth site of the mouse was fixed with forceps, and the skin was excised and the tumor was blunt-stripped and weighed. Efficacy was expressed as tumor weight (mean ± SD g), and data were analyzed by t-test and variance. The results are shown in Table 1. Not only was compound 7a, b effective in inhibiting tumor growth at a dose of 0.1 μmol/kg, but there was no significant difference in activity from compound 6 at a dose 10-fold greater than them. These data indicate that the present invention has significant technical effects.
TABLE 1 Effect of Compounds 7a, b on tumor growth in S180 mice
a) P <0.01 to saline, p <0.05 to compound 6; b) p <0.01 compared to saline group, p <0.05 compared to compound 6, and p >0.05 compared to doxorubicin; n is 10.
EXAMPLE 10 determination of the Activity of Compounds 7a, b to reverse tumor cell resistance
Doxorubicin (ADR), paclitaxel (TAX), verapamil and compounds 7a, b were dissolved in DMSO and the culture diluted to the desired concentration (DMSO content less than 0.5%) for administration prior to assay. The MTT method is adopted for determination. Verapamil is a well-recognized tool with activity to reverse tumor cell resistance, used here as a positive control.
Paclitaxel-resistant adherent cells A549/TAX with 6 × 10 of good growth state in 3 days after drug removal and logarithmic growth phase4The cells were plated at a density of 100. mu.L/mL in 96-well plates. At 5% CO2Incubate at 37 ℃ for 6 hours in an incubator. Compound wells were first filled with 25. mu.L of 10. mu.M compound 7a, b, and then 25. mu.L of paclitaxel solution at the desired concentration. The blank control wells were first filled with 50. mu.L of vehicle and then with 25. mu.L of paclitaxel solution at the pre-set concentration. The positive control wells were first loaded with 25. mu.L verapamil at a concentration of 10. mu.M, and then 25. mu.L paclitaxel at the pre-set concentration. The incubation was continued for 48 hours, 25. mu.L of MTT solution at a concentration of 5mg/mL was added to each well and incubated at 37 ℃ for 4 hours. The supernatant was carefully removed and 100. mu.L of LDMSO was added to each well and the pellet was dissolved by shaking for 15 min. The absorbance (O.D.) value was immediately measured on a microplate reader at a detection wavelength of 570 nm.
The adriamycin-resistant suspension cells K562/ADR which have good growth state after 3 days of drug removal and are in logarithmic growth phase are 8 multiplied by 104The cells were plated at a density of 100. mu.L/mL in 96-well plates. At 5% CO2Incubate at 37 ℃ for 6 hours in an incubator. Compound wells were first filled with 25. mu.L of 10. mu.M compound 7a, b, and then 25. mu.L of paclitaxel solution at the desired concentration. The blank control wells were first filled with 50. mu.L of vehicle and then with 25. mu.L of paclitaxel solution at the pre-set concentration. The positive control wells were first loaded with 25. mu.L verapamil at a concentration of 10. mu.M, and then 25. mu.L paclitaxel at the pre-set concentration. The culture was continued for 48 hours with 25. mu.L/wellMTT solution at 5mg/mL was incubated at 37 ℃ for 4 hours. The supernatant was carefully removed, 100. mu.L DMSO was added to each well, and the pellet was dissolved by shaking for 15 min. Immediately, the absorbance (OD) was measured on a microplate reader at a detection wavelength of 570 nm.
The inhibition rate of compound 7a, b at a concentration of 10. mu.M on the growth of drug-resistant cells at different concentrations of doxorubicin and paclitaxel was determined as follows. Growth inhibition rate ═ OD of control well-OD of compound well/OD of blank well]X 100%. The experiment was repeated 3 times and IC was calculated50The value is obtained. The data in Table 2 show that compounds 7a-b are effective in reversing K562/ADR cell resistance and have superior activity to verapamil. The data in Table 3 show that compounds 7a-b can effectively reverse K562/TAX cell resistance and have better activity than verapamil. These data indicate that the present invention has significant technical effects.
TABLE 2 Effect of Compounds 7a, b (10. mu.M) on Adriamycin inhibition of drug resistant cells K562/ADR
a) A ratio p to doxorubicin + verapamil of < 0.01; n is 3.
TABLE 3 Effect of Compounds 7a, b (10. mu.M) on Adriamycin inhibition of drug resistant cells A562/TAX
a) A ratio p to doxorubicin + verapamil of < 0.01; n is 3.
EXAMPLE 11 determination of the anti-inflammatory Activity of Compounds 7a, b
Since xylene-induced ear swelling in mice is recognized as an acute inflammation model, the present invention measures the therapeutic effect of compounds 7a, b on a xylene-induced ear swelling model in mice. Because aspirin is a positive drug for treating acute inflammation, aspirin is selected as a positive control in the present invention. ICR male mice (22 + -3 g in weight) were allowed to rest for 2 days at 22 ℃ with free access to water and food. Thereafter, the mice were randomly divided into a saline group (dose of 0.2 mL/mouse), an aspirin group (dose of 1.11mmol/kg), a compound 6 group (dose of 1. mu. mol/kg) and a compound 7a, b group (dose of 0.1. mu. mol/kg), each of which was 10 mice. The mice are injected with normal saline or aspirin or compound 6 or 7a and b. After 30min of administration, the left auricle of the mouse was evenly smeared with 30 μ L of xylene, and after 2h, the mouse was subjected to ether anesthesia, the neck was cut off, the left and right ears were cut off, round ears were taken at the same positions of the two ears by a 7mm punch, and the difference in swelling between the two ears was weighed and found to be the swelling degree. Namely the swelling degree is equal to the weight of the left ear disk to the weight of the right ear disk. The data are shown in Table 4. Compound 7a, b was not only effective in inhibiting xylene-induced ear swelling in mice at a dose of 0.1 μmol/kg, but also had no significant difference in activity from compound 6 at a dose 10-fold greater than them. These data indicate that the present invention has significant technical effects.
TABLE 4 Effect of Compounds 7a, b on xylene-induced ear swelling in mice
a) P <0.01 to saline, p >0.05 to compound 6; n is 10.
Claims (5)
1. 1- (4-hydroxy-3-methoxyphenyl) -7- (4-oxyacetyltheanyl-AA-OBzl-3-methoxyphenyl) -1, 6-heptadiene-3, 5-dione represented by the following formula, wherein AA is selected from the group consisting of an L-Asp residue and an L-Glu residue,
2. a process for the preparation of 1- (4-hydroxy-3-methoxyphenyl) -7- (4-oxyacethyl-AA-OBzl-3-methoxyphenyl) -1, 6-heptadiene-3, 5-dione according to claim 1, which comprises:
(1) preparing 6- (4-hydroxy-3-methoxyphenyl) -5, 6-hexene-2, 4-dione (1);
(2) preparation of benzyl 2- (4-formyl-2-methoxyphenoxy) -acetate (2);
(3) preparing 1- (4-hydroxy-3-methoxyphenyl) -7- (4-oxyacetobenzoyl-3-methoxyphenyl) -1, 6-heptadiene-3, 5-diketone (3) by taking the products obtained in the step (1) and the step (2) as reaction raw materials;
(4) saponifying 1- (4-hydroxy-3-methoxyphenyl) -7- (4-oxoacetoacetylbenzyl-3-methoxyphenyl) -1, 6-heptadiene-3, 5-dione to obtain 1- (4-hydroxy-3-methoxyphenyl) -7- (4-oxoacetoacetoxy-3-methoxyphenyl) -1, 6-heptadiene-3, 5-dione (4);
(5) coupling the compound 4 and L-benzyl theanine to obtain 1- (4-hydroxy-3-methoxyphenyl) -7- (4-oxyacetylbenzyl theanine-3-methoxyphenyl) -1, 6-heptadiene-3, 5-diketone (5);
(6) saponifying the compound 5 to obtain 1- (4-hydroxy-3-methoxyphenyl) -7- (4-oxyacetophytamic acid-3-methoxyphenyl) -1, 6-heptadiene-3, 5-dione (6);
(7) coupling of compound 6 with benzyl L-amino acid ester gives 1- (4-hydroxy-3-methoxyphenyl) -7- (4-oxoacetyltheanyl-AA-OBzl-3-methoxyphenyl) -1, 6-heptadiene-3, 5-dione according to claim 1.
3. Use of 1- (4-hydroxy-3-methoxyphenyl) -7- (4-oxyacethyl-AA-OBzl-3-methoxyphenyl) -1, 6-heptadiene-3, 5-dione according to claim 1 for the preparation of an antitumor medicament.
4. Use of 1- (4-hydroxy-3-methoxyphenyl) -7- (4-oxyacetyltheanyl-AA-OBzl-3-methoxyphenyl) -1, 6-heptadiene-3, 5-dione according to claim 1 for the preparation of a medicament for reversing tumor cell resistance.
5. Use of 1- (4-hydroxy-3-methoxyphenyl) -7- (4-oxyacethyl-AA-OBzl-3-methoxyphenyl) -1, 6-heptadiene-3, 5-dione according to claim 1 for the preparation of an anti-inflammatory medicament.
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