Silybin C-5 chemical modifier with anti-tumor activity and preparation method of silybin C-5 chemical modifier

Abstract

The invention discloses a silybin C-5 site chemical modifier with anti-tumor activity and a preparation method thereof, and relates to an anti-tumor activity modifier prepared by modifying the structure of a natural product and a preparation method of the anti-tumor activity modifier. Silybin is used as a lead compound, firstly, silybin is dehydrogenated and oxidized into 2, 3-dehydrogenated silybin, and then the 2, 3-dehydrogenated silybin is added into the 2, 3-dehydrogenated silybin to prepare the silybin C-5 site chemical modifier with the anti-tumor activity. The method comprises the following steps: carrying out alkylation on silybin by using iodomethane on the basis of silybin to obtain 3, 5, 7, 20, 23-pentamethoxy-2, 3-dehydrosilybin, and further selectively demethylating a methoxy group at a C-5 site by using AlCl3 to obtain 5-hydroxy-3, 7, 20, 23-tetramethoxy-2, 3-dehydrosilybin, and further selectively demethylating a methoxy group at the C-5 site by using AlCl3 to obtain 5-hydroxy-3, 7, 20, 23-tetramethoxy-2, 3-dehydrosilybin. And finally, introducing different acyl groups on 5-OH to obtain the novel silybin derivatives I1-I19. Pharmacological experiment research shows that the compound has an obvious anti-proliferation effect on gastric cancer cells SGC-7901 and breast cancer cells MCF-7, and the anti-proliferation effect is better than that of silibinin. The structure of the silybin derivative is shown in the figure.

Description

Technical Field

[0001] The invention relates to the technical field of structural modification of a natural compound silibinin, in particular to a silibinin C-5 chemical modification product with anti-tumor activity and a preparation method thereof. Background Art

[0002] Silybin is a natural flavonoid compound extracted and separated from the fruit of milk thistle and is the main active ingredient in silymarin. [2] Silybin is a mixture of diastereoisomers Silybin A and Silybin B (1:1). Molecular formula: C 25 H 22 O 10 , molecular weight: 482.436. Melting point: 164-174℃, density: 1.527g / cm 3 . Silybin pure product is off-white crystalline powder, odorless, slightly bitter, relatively low solubility in water, but high solubility in organic solvents (such as ethanol, dimethyl sulfoxide, etc.). Silybin has anti-fibrosis, anti-inflammatory, antioxidant and other effects, and is often used as a liver-protecting drug. Recent studies have found that silybin also has a certain anti-proliferative effect on cancer cells and can be used as a lead compound for the development of anti-tumor drugs.

[0003] The chemical structure of silybin:

[0004] Summary of the invention

[0005] The purpose of the present invention is to provide a chemically modified compound at the C-5 position of silybin with anti-tumor activity and a preparation method thereof. The method uses silybin as a lead compound to design a series of silybin derivatives. The compounds have obvious anti-proliferation effects on gastric cancer cells SGC-7901 and breast cancer cells MCF-7, and are stronger than silybin.

[0006] The present invention is achieved through the following technical solutions:

[0007] A chemically modified silibinin C-5 with anti-tumor activity, wherein the modified silibinin is obtained by structural modification of the C-5 hydroxyl group to obtain a series of silibinin analogs I1 to I 19 , the silybin chemical modification is as follows:

[0008]

[0009] The C-23 hydroxyl group R is shown in the following table:

[0010]

[0011]

[0012] 2. Preparation method of modified substance:

[0013] (1) Silybin is dehydrogenated to form 2,3-dehydrosilybin;

[0014] (2) 2,3-dehydrosilybin is then alkylated with CH3I to obtain 3,5,7,20,23-pentamethoxy-2,3-dehydrosilybin;

[0015] (3) selectively demethylating the C-5 methoxy group using AlCl3 to obtain 5-hydroxy-3,7,20,23-tetramethoxy-2,3-dehydrosilybin;

[0016] (4) 5-Hydroxy-3,7,20,23-tetramethoxy-2,3-dehydrosilybin was acylated with different acyl chlorides to obtain 5-acyloxy-3,7,20,23-tetramethoxy-2,3-silybin derivatives (I1 to I 19 ). BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The figure is a structural diagram of the chemically modified silybin of the present invention. DETAILED DESCRIPTION

[0018] The present invention will be further described in detail below in conjunction with examples.

[0019] 1. After pre-defatting the silybum seed meal with n-hexane, use anhydrous ethanol as the extraction solvent, heat and dissolve, decolorize with activated carbon, let the filtrate stand to precipitate white crystals, and recrystallize from ethyl acetate-methanol to obtain silybin.

[0020] 2. Silybin is used as raw material and pyridine is used as solvent. The mixture is heated to reflux at 100°C for 24 hours. After the reaction is completed, pyridine is recovered under reduced pressure and the residue is extracted with 40 mL of ethyl acetate. The extract is allowed to stand overnight, filtered with 10 g of silica gel, and ethyl acetate is recovered under reduced pressure. Finally, 2,3-dehydrosilybin (A) is obtained by recrystallization with methanol.

[0021]

[0022] 3. Compound (A) was dissolved in DMF, NaH was added, and the mixture was reacted at 0°C for 30 min, then CH3I was added, TBAI was used as a catalyst, and the mixture was reacted at 80°C for 3 h, and the reaction endpoint was monitored by TLC. After the reaction, dilute hydrochloric acid was added to precipitate crystals, which were filtered and dried to obtain 3,5,7,20,23-pentamethoxy-2,3-dehydrosilybin (B).

[0023]

[0024] 4. The obtained compound (B) was dissolved in a solvent of 1,2-dichloroethane: acetonitrile = 1:1, AlCl3 was added, TBAI was used as a catalyst, and the mixture was heated to reflux at 70°C for 3h, and the reaction end point was monitored by TLC. After the reaction was completed, dilute hydrochloric acid was added and stirred overnight, and the mixture was extracted with water and dichloromethane, and the solvent was removed by vacuum rotary evaporation to obtain 5-hydroxy-3,7,20,23-tetramethoxy-2,3-dehydrosilybin (C).

[0025]

[0026] 5. The obtained 5-hydroxy-3,7,20,23-tetramethoxy-2,3-dehydrosilybin (C) was dissolved in DCM, NaH was added, and the reaction was continued for 30 minutes. Then, acyl chloride was added and the reaction was continued at 0°C for 1.5 hours. The reaction was monitored by TLC. After the reaction was completed, DCM and dilute hydrochloric acid were used for extraction, and the solvent was removed by rotary evaporation under reduced pressure to obtain 5-acyloxy-3,7,20,23-tetramethoxy-2,3-dehydrosilybin (D, I1-I 16 ,I 18 -I 19 ).

[0027] 6. Compound (C) was dissolved in DMF, NaH was added, and the reaction was continued for 30 min. Then benzyl bromide was added, and TBAI was used as a catalyst, and the reaction was continued at 80°C for 3 h. After the reaction was completed, dilute hydrochloric acid was added to precipitate crystals, and dried to obtain 5-O-benzyl-3,7,20,23-tetramethoxy-2,3-dehydrosilybin (D, I 17 ).

[0028]

[0029] Wherein R is p-toluenesulfonyl, benzoyl, o-methylbenzoyl, p-chlorobenzoyl, p-trifluoromethylbenzoyl, phenylpropionyl, p-methoxybenzoyl, phenylacetyl, m-chlorobenzoyl, 2,4-dichlorobenzoyl, 2, difluorobenzoyl, cyclohexylcarbonyl, hexanoyl, furanoyl, isobutyryl, n-butyryl, benzyl, propionyl, acetyl.

[0030]

[0031] The MTT method was used, Lapatinib was used as the positive control drug, and two cancer cells, breast cancer MCF-7 and gastric cancer SGC-7901, were selected to evaluate the in vitro anti-tumor activity of the new silybin derivatives. The experimental results showed that the synthesized compounds had a certain degree of in vitro anti-proliferative activity against SGC-7901 gastric cancer cells and MCF-7 breast cancer cells. The compound structure and in vitro experimental results are shown in the following table:

[0032]

[0033]

[0034] Note: a. Inhibition rate measured at a compound concentration of 10 μM. b. IC 50 It indicates the half effective inhibitory concentration.

[0035] The anti-tumor activity of the target compound was tested by MTT method. The results of two groups of cell experiments showed that the synthesized compounds had a certain degree of in vitro anti-proliferation activity against SGC-7901 gastric cancer cells and MCF-7 breast cancer cells. Compared with other compounds, the compounds with aromatic ring groups introduced on 5-OH had better in vitro anti-cancer cell proliferation activity, and the compounds without any substituents on the aromatic ring (I2, I 14 ,I 17 ) showed high anti-tumor activity. Among them, compound I2 had the best anti-proliferative activity against MCF-7 cells, IC 50 The value was 8.41 μmol / L, compound I 14 The antiproliferative activity against SGC-7901 cells was the best, IC 50 The value was 9.16 μmol / L, which was comparable to the positive control drug Lapatinib and superior to the parent silybin. When there were substituents on the aromatic ring, the antitumor activity of the compounds containing halogen substituents was higher than that of the compounds substituted with alkyl.

[0036] The present invention is further described below with reference to examples.

[0037] Example 1

[0038] Synthesis of 2,3-dehydrosilybin

[0039] 3g of silybin was dissolved in 30mL of pyridine, heated under reflux at 100℃ for 24h, and the reaction endpoint was monitored by silica gel thin layer chromatography (TLC). After the reaction was completed, pyridine was recovered under reduced pressure, and the residue was extracted with 40mL of ethyl acetate. The extract was allowed to stand overnight, filtered with 10g of silica gel, and ethyl acetate was recovered under reduced pressure. Finally, it was recrystallized with methanol to obtain 1.86g of pure 2,3-dehydrosilybin with a yield of 62%. mp230.2℃~233.5℃. 1HNMR(500MHz,DMSO-d6)δ12.42(s,1H),10.81(s,1H),9.56(s,1H),9.16(s,1H ),7.77(t,J=2.1Hz,1H),7.75(d,J=2.2Hz,1H),7.12(d,J=8.5Hz,1H),7.05(d,J =2.0Hz,1H),6.89(dd,J=8.2,2.0Hz,1H),6.82(d,J=8.0Hz,1H),6.46(d,J=2.0 Hz,1H),6.20(d,J=2.0Hz,1H),4.99(t,J=5.6Hz,1H),4.97(d,J=7.9Hz,1H),4.2 8-4.26(m,1H),3.80(s,3H),3.58(ddd,J=12.3,5.0,2.5Hz,1H),3.40-3.35(m, 1H).13CNMR(125MHz,Chloroform-d)δ176.94,165.06,161.65,157.55,148.16, 147.30,145.78,145.35,144.51,136.45,128.61,124.54,122.88,121.28,116.85,116.13,115.56,112.00,102.98,99.88,94.59,78.04,76.90,61.51,56.16.

[0040] Example 2

[0041] Synthesis of 3,5,7,20,23-pentamethoxy-2,3-dehydrosilybin

[0042] Weigh 0.5mmol (0.24g) 2,3-dehydrosilybin and dissolve it in DMF, add 5mmol NaH (0.12g), react at 0℃ for 30min, then add 6.5mmol CH3I (405μL), use TBAI as catalyst, react at 80℃ for 3h, and monitor the reaction end point by TLC. After the reaction, add dilute hydrochloric acid to precipitate crystals, and filter to obtain red powder. Purify by silica gel thin layer chromatography (petroleum ether: ethyl acetate = 2:1) to obtain 0.223g of pure 3,5,7,20,23-pentamethoxy-2,3-dehydrosilybin, with a yield of 93%. mp 98℃~102℃. 1HNMR(500MHz,Chloroform-d)δ7.77–7.70(m,2H),7.10(d,J=8.6Hz,1H),7.02(d,J=8.2Hz,1H),6.97–6.91(m,2H),6.47(d,J=2.3Hz,1H),6.3 2(d,J=2.3Hz,1H),5.05(d,J=8.0Hz,1H),4.20-4.18(m,1H),3.95(s,3H),3.92(s,6H),3.89(s,3H),3.87(s,3H),3.62(dd,J=5.5,5.0Hz,1H), 3.36(s,3H),3.32(dd,J=6.4,4.5Hz,1H).13CNMR(125MHz,DMSO-d6)δ178.84,165.49,160.95,156.75,155.35,149.76,149.41,146.01,143.7 5,139.20,128.53,123.69,122.44,120.13,117.47,111.32,110.20,1 06.09,97.91,92.05,77.28,76.32,71.22,59.77,59.08,56.08,55.97.

[0043] Example 3

[0044] Synthesis of 5-Hydroxy-3,7,20,23-Tetramethoxy-2,3-Dehydrosilybin

[0045] Weigh 0.5mmol (0.275g) 3,5,7,20,23-pentamethoxy-2,3-dehydrosilybin and dissolve it in a solvent of 1,2-dichloroethane: acetonitrile = 1:1, add 1.5mmol (0.2g) AlCl3, use TBAI as a catalyst, heat and reflux at 70℃ for 3h, and monitor the reaction end point by TLC. After the reaction is completed, add dilute hydrochloric acid and stir overnight, extract with water and dichloromethane, remove the solvent by vacuum rotary evaporation, separate and purify by silica gel thin layer chromatography (chloroform: acetone = 10:1) to obtain 0.231g of pure 5-hydroxy-3,7,20,23-tetramethoxy-2,3-dehydrosilybin, with a yield of 86%. mp114.3℃~119.5℃. 1HNMR(500MHz,Chloroform-d)δ12.61(s,1H),7.77(d,J=2.1Hz,1H),7.73(dd,J=8.6,2.2Hz,1H),7.12(d,J=8.6Hz,1H),7.02(d,J=8.3Hz,1H),6.94(s ,2H),6.41(d,J=2.1Hz,1H),6.34(d,J=2.2Hz,1H),5.07–5.04(m,1H),4.21 –4.18(m,1H),3.92(s,6H),3.88(s,3H),3.85(s,3H),3.62(dd,J=8.4,2.5Hz ,1H),3.37(s,3H),3.33(dd,J=8.5,2.0Hz,1H).13CNMR(125MHz,Chloroform-d)δ178.84,165.49,162.00,156.75,155.35,149.76,149.40,146.00,143 .76,139.19,128.53,123.69,122.44,120.13,117.47,111.32,110.20,106 .09,97.91,92.05,78.05,76.30,71.10,60.18,59.55,56.03,56.01,55.79.

[0046] Example 4

[0047] Compound I1: Synthesis of 5-p-toluenesulfonyloxy-3,7,20,23-tetramethoxy-2,3-dehydrosilybin

[0048] Weigh 0.268g (0.5mmol) of 5-hydroxy-3,7,20,23-tetramethoxy-2,3-dehydrosilybin and dissolve it in DCM. Add 2.2mmol (0.053g) of NaH. After reacting for 30min, add 1.5mmol (0.285g) of p-toluenesulfonyl chloride. React at 0℃ for 1.5h. Monitor the end point of the reaction by TLC. After the reaction, extract with DCM and dilute hydrochloric acid, remove the solvent by vacuum rotary evaporation, and purify by silica gel thin layer chromatography (petroleum ether: acetone = 3: 1) to obtain 0.307g of pure compound I1 with a yield of 89%. mp130.5℃~133.6℃. 1HNMR (500MHz, Chloroform-d) δ8.00 (s, 1H), 7.98 (s, 1H), 7.73 (d, J = 10.2Hz, 1H), 7. 69(dd,J=8.7,3.5Hz,1H),7.32(d,J=8.1Hz,2H),7.10(d,J=8.6Hz,1H),7.04–7.01(m ,1H),6.96–6.92(m,2H),6.83–6.81(m,2H),5.05(d,J=8.2Hz,1H),4.20–4.17(m,1H) ,3.92(s,6H),3.87(s,3H),3.79(s,3H),3.62(dd,J=6.5,2.5Hz,1H),3.36(s,3H),3. 32(dd,J=8.2,4.5Hz,1H),2.42(s,3H).13CNMR(125MHz,DMSO-d6)δ171.34,162.79,1 57.67,152.72,149.77,149.31,147.30,146.22,145.98,143.99,140.80,132.30,13 0.36,129.05,128.79,123.25,122.39,120.85,117.54,117.09,112.03,111.82,111.63,109.25,101.02,77.28,76.32,71.22,59.77,59.09,56.98,56.04,56.03,21.59.

[0049] Example 5

[0050] Compound I 17 :5-O-benzyl-3,7,20,23-tetramethoxy-2,3-dehydrosilybin (I 17 )

[0051] Weigh 0.5mmol (0.268g) 5-hydroxy-3,7,20,23-tetramethoxy-2,3-dehydrosilybin and dissolve it in DMF, add 1.5mmol (0.036g) NaH, react for 30min, then add 1.5mmol (171μL) benzyl bromide, use TBAI as catalyst, and react at 80℃ for 3h. After the reaction, add dilute hydrochloric acid to precipitate crystals, and filter. Purify by silica gel thin layer chromatography (petroleum ether: acetone = 3: 1) to obtain 0.288g of pure compound I17 with a yield of 94.0%. mp198.6℃~202.1℃1HNMR(500MHz,DMSO-d6)δ7.69(d,J=6.7Hz,2H),7.63(d,J=7.0Hz,2H),7.42(t,J =7.5Hz,2H),7.33(t,J=7.3Hz,1H),7.14(d,J=9.2Hz,1H),7.10(s,1H),7.02(s,2H),6.86(d,J=2.3 Hz,1H),6.57(d,J=2.3Hz,1H),5.23(s,2H),5.00(d,J=8.0Hz,1H),4.52–4.48(m,1H),3.86(s,3H), 3.79(s,6H),3.78(s,3H),3.46(dd,J=6.2,2.4Hz,1H),3.28(dd,J=6.5,2.2Hz,1H),3.23(s,3H).13C NMR(125MHz,DMSO-d6)δ172.73,164.09,159.46,158.64,151.62,149.77 ,149.32,145.61,143.93,141.04,137.29,128.86,128.80,127.97,127.3 1,123.72,122.12,120.82,117.46,116.89,112.04,111.63,109.25,97.7 9,93.80,77.26,76.31,71.24,70.36,59.80,59.09,56.47,56.08,55.97.

Claims

1. A chemically modified product of silybin at position C-5 having anti-tumor activity, characterized in that: The modified product is a structural modification of the C-5 hydroxyl group to obtain a series of silybin structural analogs I1~I 19 , the silybin chemically modified substances are as follows: The C-23 hydroxyl group R is shown in the following table:

2. A method for preparing a C-5 chemically modified substance of silybin with anti-tumor activity, characterized in that: The method comprises the following preparation steps: (1) Silybin is dehydrogenated to form 2,3-dehydrosilybin; (2) 2,3-dehydrosilybin is then alkylated with CH3I to obtain 3,5,7,20,23-pentamethoxy-2,3-dehydrosilybin; (3) selectively demethylating the C-5 methoxy group using AlCl3 to obtain 5-hydroxy-3,7,20,23-tetramethoxy-2,3-dehydrosilybin; (4) 5-Hydroxy-3,7,20,23-tetramethoxy-2,3-dehydrosilybin was acylated with different acyl chlorides to obtain 5-acyloxy-3,7,20,23-tetramethoxy-2,3-silybin derivatives (I1 to I 19 ).

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