C 14 Derivatives of tetrandrine substituted by sulfonic acid groups, preparation methods and applications thereof

Abstract

C 14 14-Sulfonic acid group-substituted tetrandrine derivatives, their preparation methods and applications belong to the fields of natural medicines and medicinal chemistry. The present invention aims to solve the problem of poor biological activity of tetrandrine. The general formula of the 14-carbon-substituted tetrandrine derivatives is #imgabs0# where X is NH or O, and R is H, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 mercapto, aryl or heteroaryl. In the present invention, tetrandrine is used as a lead compound, and 14-sulfonic acid group substitution is carried out at room temperature to generate the intermediate 14-sulfonic acid tetrandrine. Then, through the reaction of the intermediate 14-sulfonic acid group tetrandrine with compounds containing amino or hydroxyl groups, novel tetrandrine derivatives with sulfonamide and sulfonate structures are generated. It is proved by the MTT method that the anti-tumor activity of the new compounds is superior to that of the tetrandrine lead compound, and methods for preparing these two types of novel derivatives are provided.

Description

Technical Field

[0001] The present invention relates to the field of natural medicines and medicinal chemistry, and particularly relates to a preparation method of a C 14 -position sulfonic acid group-substituted tetrandrine derivative, and an application of the tetrandrine derivative in the preparation of an anti-tumor drug. Background Art

[0002] Tetrandrine, now also known as fangchinoline, is a bisbenzylisoquinoline alkaloid extracted from the root tuber of Stephania tetrandra S. Moore, a plant of the Menispermaceae family, and is the main active ingredient of traditional Chinese medicine Stephania tetrandra. Modern pharmacological studies have shown that tetrandrine has a wide range of pharmacological effects, such as antiplatelet aggregation, antibacterial, anti-tumor, antihypertensive, anti-inflammatory, anti-fibrotic, immunomodulatory, anti-arrhythmic, etc. In recent years, tetrandrine has shown great potential in the anti-tumor field. For example, it has varying degrees of inhibitory activity against human lung cancer cell lines and leukemia cell lines; it affects the proliferation and apoptosis of myeloma stem cells by activating apoptosis-related proteins and blocking the growth cycle of tumor cells; it inhibits the expression of β-catenin, thereby inducing apoptosis of glioma stem cells; there are also related studies on the cancer chemotherapy effect of tetrandrine at the molecular level, mainly including its effects on cell proliferation, apoptosis, angiogenesis, metastasis, autophagy, and multidrug resistance in different cancers, such as regulating various signaling molecules, such as cell cycle kinases, RAS pathways, tumor suppressor genes, autophagy-related proteins, and death receptors. In addition, tetrandrine shows reversal of drug resistance in different tumors by regulating the expression level of P-Glyco protein.

[0003] Sulfonamide and sulfonate drugs are two important types of drugs and have a wide range of biological activities. In recent years, a large number of sulfonamide and sulfonate compounds with anti-tumor activity have been reported. These two types of compounds have functions such as enhancing anti-tumor activity, improving water solubility, reducing compound toxicity, and enhancing compound stability. Among them, the mechanism of action of sulfonamide compounds shows diversity, such as interfering with tubulin polymerization, blocking the normal progress of the cell cycle, inhibiting carbonic anhydrase, folate-dependent enzymes, methionyl aminopeptidase, and histone deacetylase, and inhibiting vascular endothelial growth factor.

[0004] As a drug with a wide range of biological activities, tetrandrine has great application prospects in clinical practice. However, due to the disadvantages of poor water solubility, low bioavailability, and drug toxicity of the drug itself, its use is restricted. Therefore, the development of new tetrandrine derivatives with good biological activities and pharmacokinetic properties is still required in today's market. Summary of the Invention

[0005] The purpose of the present invention is to solve the problem of poor biological activity of tetrandrine, and to provide a C 1414 - Sulfonic Acid Group Substituted Tetrandrine Derivatives, Their Preparation Methods and Applications.

[0006] The C 14 -sulfonic acid group substituted tetrandrine derivatives of the present invention have the general formula of the 14 - carbon - substituted tetrandrine derivatives as

[0007] X is NH or O, and R is H, C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C 1 -C 6 mercapto, aryl or heteroaryl.

[0008] Furthermore, the C 1 -C 6 alkyl is -CH 3 、-CH 2 CH 3 or -CH 2 CH 2 CH 3 ; C 1 -C 6 alkoxy is -OCH 3 、-OCH 2 CH 3 or -OCH 2 CH 2 CH 3 ; C 1 -C 6 mercapto is -CH 2 SH、-CH 2 CH 2 SH or -CH 2 CH 2 CH 2 SH; C 1 -C 6 alkoxy is -OCH 3 、-OCH 2 CH 3 or -OCH 2 CH 2 CH 3 ; C 1 -C 6 mercapto is -CH 2 SH、-CH 2 CH 2 SH or -CH 2 CH 2 CH 2 SH.

[0009] Further, the H atom in the aryl or heteroaryl group is substituted by a halogen, amino group, nitro group, cyano group, hydroxyl group, halogen-substituted alkyl group, halogen-substituted alkoxy group, carboxyl group, C 1 -C 6 alkoxy group, hydroxyl-substituted C 1 -C 6 alkyl group or mercapto-substituted C 1 -C 6 alkyl group.

[0010] Further, the halogen is F, Cl or Br; the halogen-substituted alkyl group is -CH 2 Cl, -CH 2 CH 2 Cl or -CH 2 CH 2 CH 2 Cl; the halogen-substituted alkoxy group is -OCH 2 Cl, -OCH 2 CH 2 Cl or -OCH 2 CH 2 CH 2 Cl; C 1 -C 6 alkoxy group is -OCH 3 、-OCH 2 CH 3 or -OCH 2 CH 2 CH 3 ; the hydroxyl-substituted C 1 -C 6 alkyl group is -CH 2 OH, -CH 2 CH 2 OH or -CH 2 CH 2 CH 2 OH; the mercapto-substituted C 1 -C 6 alkyl group is -CH 2 SH, -CH 2 CH 2 SH or -CH 2 CH 2 CH 2 SH.

[0011] The preparation method of the C 14 -position sulfonic acid group-substituted tetrandrine derivative of the present invention, the C 14 -position sulfonic acid group-substituted tetrandrine derivative is prepared as follows:

[0012] Using tetrandrine as a lead compound, at room temperature, carry out C14 Substitute a sulfonic acid group at the position to generate intermediate 14-sulfonic acid tetrandrine, and then react intermediate 14-sulfonic acid tetrandrine with a compound containing an amino group or a hydroxyl group to generate a novel tetrandrine derivative with sulfonamide and sulfonate structures, thus obtaining the described C 14 sulfonic acid group-substituted tetrandrine derivative at the position.

[0013] Furthermore, the preparation method of the intermediate 14-sulfonic acid tetrandrine is as follows: Take tetrandrine, dissolve it in dichloromethane, dropwise add concentrated sulfuric acid, and react at room temperature for 12 h; after the reaction is completed, concentrate under reduced pressure, wash successively with dichloromethane, an aqueous solution of NaOH with a concentration of 0.1 - 0.3 mol / L, and saturated brine, and then use anhydrous Na 2 SO 4 to dry, filter, concentrate under reduced pressure to obtain a crude product; purify by column chromatography with a dichloromethane / methanol / ethyl acetate = 5:1:1 system to prepare the intermediate 14-sulfonic acid tetrandrine; the molar ratio of tetrandrine to concentrated sulfuric acid is 1:5 - 15; the mass-volume ratio of tetrandrine to dichloromethane is 40 - 60 mg:1 mL.

[0014] Furthermore, dissolve tetrandrine in dichloromethane, add concentrated sulfuric acid under ice bath, warm the mixture to room temperature and stir for 12 hours, and detect by TLC; concentrate under reduced pressure, adjust the pH to neutral with an aqueous sodium hydroxide solution, wash with dichloromethane and saturated brine, and use anhydrous Na 2 SO 4 to dry; pour the mixture into an aqueous sodium hydroxide solution and extract with DCM for 3 times; wash the combined organic phases with brine and dry with anhydrous sodium sulfate, filter, and concentrate under reduced pressure to obtain a crude product.

[0015] Furthermore, the reaction of intermediate 14-sulfonic acid tetrandrine with a compound containing an amino group or a hydroxyl group to generate a novel tetrandrine derivative with sulfonamide and sulfonate structures, thus obtaining the described C 14 sulfonic acid group-substituted tetrandrine derivative at the position is carried out as follows:

[0016] Take intermediate 14-sulfonic acid tetrandrine, add thionyl chloride, and react at a temperature of 70 - 90 °C for 4 - 7 h; after the reaction is completed, concentrate under reduced pressure, add dichloromethane to remove residual thionyl chloride; successively add a compound containing an amino group, pyridine, DMAP, and dichloromethane to the reaction system, react at room temperature for 1 - 5 h, and detect by TLC after the reaction; wash successively with dichloromethane, an aqueous hydrochloric acid solution, and saturated brine, and use anhydrous Na 2 SO 4Dry, filter, and concentrate under reduced pressure to obtain the crude product; purify by column chromatography using a dichloromethane / methanol / ethyl acetate = 5:1:3 system to prepare the described C 14 Position-sulfonic acid group substituted tetrandrine derivative; the mass-volume ratio of intermediate 14-sulfonic acid tetrandrine to thionyl chloride is 40 - 60 mg:1 mL.

[0017] Furthermore, the molar ratio of the intermediate 14-sulfonic acid tetrandrine to thionyl chloride is 1:3 - 7; the molar ratio of the intermediate 14-sulfonic acid tetrandrine to the amino compound, pyridine, and DMAP is 1:1 - 2:2 - 4:0.1 - 1.

[0018] The C 14 Position-sulfonic acid group substituted tetrandrine derivative of the present invention is used in the preparation of anti-tumor drugs.

[0019] The present invention uses tetrandrine as a lead compound, substitutes the 14-position with a sulfonic acid group at room temperature to generate the intermediate 14-sulfonic acid tetrandrine, and then reacts the intermediate 14-sulfonic acid tetrandrine with a compound containing an amino or hydroxyl group to generate a new tetrandrine derivative with sulfonamide and sulfonate structures. Determined by the MTT method, it is proved that the anti-tumor activity of the new compound is superior to that of the tetrandrine lead compound, and a method for preparing these two types of new derivatives is provided. Description of the Drawings

[0020] Figure 1 For the 1 H-NMR of compound TET-XA-01 in Example 1 and 13 C diagram;

[0021] Figure 2 For the 1 H-NMR of compound TET-XA-07 in Example 2 and 13 C-NMR diagram;

[0022] Figure 3 For the 1 H-NMR of compound TET-XA-08 in Example 3 and 13 C-NMR diagram;

[0023] Figure 4 For the 1 H-NMR of compound TET-XA-11 in Example 4 and 13 C-NMR diagram;

[0024] Figure 5 For the 1 H-NMR of compound TET-XA-13 in Example 5 and 13 C-NMR diagram;

[0025] Figure 6 For the compound TET-SZ-03 of Example 6 1 H-NMR and 13 C-NMR spectra;

[0026] Figure 7 Bar graph of the tumor weights of xenograft tumor tissues excised from tumor-bearing mice at the treatment endpoint;

[0027] Figure 8 Curve of the body weight changes of tumor-bearing mice during treatment. Detailed implementation manners

[0028] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer and more understandable, the spirit of the content disclosed by the present invention will be described in detail below. After any person skilled in the art understands the embodiments of the content of the present invention, the techniques taught by the content of the present invention can be changed and modified, which does not deviate from the spirit and scope of the content of the present invention.

[0029] The schematic embodiments of the present invention and their descriptions are used to explain the present invention, but do not limit the present invention.

[0030] Example 1

[0031] Preparation of TET-XA-01: Take tetrandrine-14-sulfonate (50.00 mg, 0.07 mmol) in a 25.00 ml round-bottom flask, and add 3.00 ml of thionyl chloride. React at 80.00 °C for 6 h. After the reaction is completed, concentrate under reduced pressure, and add dichloromethane (10.00 ml × 3) to remove the residual thionyl chloride. Add ethanolamine (4 μL, 0.07 mmol), pyridine (12.00 μL, 0.14 mmol), DMAP (4.00 mg, 0.04 mmol) and 5.00 mL of dichloromethane to the reaction system in sequence, and react at room temperature for 3 h. Detect by TLC until the ethanolamine reaction is complete. Wash with dichloromethane (30.00 mL × 3), hydrochloric acid aqueous solution (2 M, 4 mL × 3), saturated brine (10.00 mL), and anhydrous Na 2 SO 4 Dry, filter, concentrate under reduced pressure to obtain a crude product. Purify by column chromatography with dichloromethane: methanol: ethyl acetate = 5:1:3 to prepare the compound TET-XA-01. The 1 H-NMR and 13 C are as Figure 1 shown. White solid; 84% yield, 1 H NMR (600 MHz, CDCl 3)δ 7.56 (s, 1H), 7.36 (dd, J = 8.2, 2.2 Hz, 1H), 7.16 (dd, J = 8.2, 2.6 Hz, 1H), 6.91 (s, 1H), 6.75 (dd, J = 8.3, 2.6 Hz, 1H), 6.50 (s, 1H), 6.31 (s, 1H), 6.21 (dd, J = 8.4, 2.2 Hz, 1H), 5.96 (s, 1H), 4.06 (dd, J = 14.5, 10.1 Hz, 1H), 3.98 (s, 5H), 3.85–3.79 (m, 1H), 3.74 (s, 4H), 3.69 (ddd, J = 11.0, 6.5, 4.0 Hz, 1H), 3.51–3.44 (m, 1H), 3.35 (s, 3H), 3.31 (dd, J = 12.4, 6.2 Hz, 1H), 3.16 (ddd, J = 13.5, 6.5, 4.2 Hz, 1H), 3.08 (s, 3H), 2.96–2.84 (m, 5H), 2.81 (dd, J = 12.5, 10.6 Hz, 1H), 2.73 (dd, J = 15.6, 4.7 Hz, 1H), 2.66 (s, 3H), 2.55–2.49 (m, 2H), 2.39 (s, 3H). 13 C NMR (151 MHz, CDCl 3 )δ 152.7, 152.2, 151.4, 149.3, 148.7, 146.9, 144.4, 138.2, 135.6, 133.5, 132.9, 131.7, 130.5, 128.4, 127.4, 127.2, 121.9, 121.8, 121.3, 120.7, 120.1, 112.6, 112.5, 106.2, 63.5, 62.2, 61.5, 60.0, 56.5, 55.7, 55.7, 45.9, 45.1, 43.6, 42.3, 41.1, 38.5, 38.2, 25.0, 20.9。

[0032] Example 2

[0033] Preparation of TET-XA-07: Take 14-sulfonic acid tetrandrine (50.00 mg, 0.07 mmol) in a 25.00 ml round-bottom flask, and add 3.00 ml of thionyl chloride. React at 80.00 °C for 6 h. After the reaction is completed, concentrate under reduced pressure, and add dichloromethane (10.00 ml × 3) to remove the residual thionyl chloride. Add p-bromobenzylamine (8.8 μL, 0.07 mmol), pyridine (12.00 μL, 0.14 mmol), DMAP (4.00 mg, 0.04 mmol) and 5.00 mL of dichloromethane to the reaction system in sequence, react at room temperature for 3 h, and detect by TLC until the ethanolamine reaction is complete. Wash with dichloromethane (30.00 mL × 3), hydrochloric acid aqueous solution (2M, 4 mL × 3), saturated brine (10.00 mL), and anhydrous Na 2 SO 4 Dry, filter, concentrate under reduced pressure to obtain the crude product. Purify by column chromatography with dichloromethane: methanol: ethyl acetate = 5:1:3 to prepare the compound TET-XA-07. The 1 1H-NMR and 13 13C-NMR are as Figure 2 shown. Yellow solid; 82% yield, 1 1H NMR (600 MHz, CDCl 3 3) δ 9.82 (s, 1H), 7.59 (s, 1H), 7.43 (d, J = 8.4 Hz, 2H), 7.40 (dd, J = 8.2, 2.2 Hz, 1H), 7.20 (dd, J = 8.4, 6.4 Hz, 3H), 6.89 (s, 1H), 6.79 (dd, J = 8.3, 2.6 Hz, 1H), 6.52 (s, 1H), 6.28 (s, 1H), 6.23 (dd, J = 8.4, 2.2 Hz, 1H), 5.96 (s, 1H), 4.29 (d, J = 14.0 Hz, 1H), 4.19–4.02 (m, 3H), 4.00 (s, 3H), 3.89 (d, J = 10.0 Hz, 1H), 3.73 (s, 5H), 3.69 (d, J = 13.9 Hz, 2H), 3.45 (d, J = 14.6 Hz, 1H), 3.32 (s, 3H), 3.11 (s, 4H), 3.00 (dt, J = 17.8, 9.3 Hz, 1H), 2.90–2.71 (m, 8H), 2.65–2.59 (m, 1H), 2.51–2.45 (m, 2H), 1.86 (s, 3H). 13 13C NMR (151 MHz, CDCl 3)δ166.8,152.1,151.3,149.1,146.8,144.8,138.1,137.1,133.4,132.8,132.0,131.5,130.7,129.7,129.5,127.6,121.8,121.6,121.6,121.3,120.8,120.0,112.6,112.5,106.2,63.6,61.5,60.1,56.5,55.7,55.7,47.0,44.8,43.3,40.3,38.5,31.9,31.6,29.7,29.6,22.7,20.7。

[0034] Example 3

[0035] Preparation of TET-XA-08: Take 14-sulfonic acid tetrandrine (50.00 mg, 0.07 mmol) in a 25.00 ml round-bottom flask, and add 3.00 ml of thionyl chloride. React at 80.00 °C for 6 h. After the reaction is completed, concentrate under reduced pressure, and add dichloromethane (10.00 ml × 3) to remove the residual thionyl chloride. Add 3-methylbenzylamine (8.7 μL, 0.07 mmol), pyridine (12.00 μL, 0.14 mmol), DMAP (4.00 mg, 0.04 mmol) and 5.00 mL of dichloromethane to the reaction system in sequence, and react at room temperature for 3 h. Detect by TLC until the reaction of ethanolamine is complete. Wash with dichloromethane (30.00 mL × 3), hydrochloric acid aqueous solution (2M, 4 mL × 3), saturated brine (10.00 mL), and anhydrous Na 2 SO 4 dry, filter, concentrate under reduced pressure to obtain the crude product. Purify by column chromatography with dichloromethane: methanol: ethyl acetate = 5:1:3 to prepare the compound TET-XA-08. The 1 1H-NMR and 13 13C-NMR are as Figure 3 shown. White solid; 87% yield, 1 1H NMR (600 MHz, CDCl 3)δ9.64(s,1H),7.59(s,1H),7.38(dd,J=8.2,2.2Hz,1H),7.20–7.13(m,2H),7.09–7.06(m,1H),7.04–7.01(m,2H),6.90(s,1H),6.78(dd,J=8.3,2.6Hz,1H),6.50(s,1H),6.27(s,1H),6.23(dd,J=8.3,2.2Hz,1H),5.97(s,1H),4.30(d,J=13.7Hz,1H),4.11–4.01(m,3H),3.99(s,3H),3.89(d,J=10.0Hz,1H),3.79–3.75(m,2H),3.72(s,4H),3.55(d,J=15.0Hz,1H),3.37(dd,J=12.8,6.1Hz,1H),3.30(s,3H),3.09(s,3H),2.97(ddd,J=17.3,11.9,7.6Hz,2H),2.84–2.68(m,8H),2.64(dd,J=14.3,6.4Hz,1H),2.51–2.46(m,1H),2.44(d,J=5.2Hz,1H),2.28(s,3H),1.80(s,3H). 13 C NMR(151MHz,CDCl 3 )δ152.9,152.1,151.2,149.2,148.9,146.8,144.6,138.1,138.0,137.8,133.4,132.8,132.3,130.6,128.8,128.3,128.1,127.6,125.1,121.9,121.9,121.5,120.7,120.0,112.6,112.5,106.2,63.5,61.4,60.0,56.4,55.7,55.6,47.7,45.0,43.2,42.0,40.1,38.5,38.3,29.7,21.3,20.8。

[0036] Example 4

[0037] Preparation of TET-XA-11: Take 14-sulfonic acid tetrandrine (50.00 mg, 0.07 mmol) in a 25.00 ml round-bottom flask, and add 3.00 ml of thionyl chloride. React at 80.00 °C for 6 h. After the reaction is completed, concentrate under reduced pressure, and add dichloromethane (10.00 ml × 3) to remove the residual thionyl chloride. Add 3-chlorobenzylamine (8.7 μL, 0.07 mmol), pyridine (12.00 μL, 0.14 mmol), DMAP (4.00 mg, 0.04 mmol) and 5.00 mL of dichloromethane to the reaction system in sequence, and react at room temperature for 3 h. Detect by TLC until the reaction of ethanolamine is complete. Wash with dichloromethane (30.00 mL × 3), hydrochloric acid aqueous solution (2 M, 4 mL × 3), saturated brine (10.00 mL), and anhydrous Na 2 SO 4 Dry, filter, concentrate under reduced pressure to obtain the crude product. Purify by column chromatography with dichloromethane:methanol:ethyl acetate = 5:1:3 to prepare the compound TET-XA-11. The 1 1H-NMR and 13 13C-NMR are as Figure 4 shown. Yellow solid; 88% yield, 1 1H NMR (700 MHz, CDCl 3 ) δ 9.86 (s, 1H), 7.55 (s, 1H), 7.37 (dd, J = 8.2, 2.2 Hz, 1H), 7.23–7.19 (m, 4H), 7.17 (dd, J = 8.1, 2.6 Hz, 1H), 6.91 (s, 1H), 6.80 (dd, J = 8.3, 2.6 Hz, 1H), 6.50 (s, 1H), 6.28 (s, 1H), 6.22 (dd, J = 8.4, 2.2 Hz, 1H), 5.96 (s, 1H), 4.31 (d, J = 14.3 Hz, 1H), 4.08 (dd, J = 15.0, 10.1 Hz, 1H), 4.04–4.00 (m, 1H), 3.97 (s, 3H), 3.95 (d, J = 10.1 Hz, 1H), 3.83–3.76 (m, 2H), 3.73 (s, 3H), 3.51 (d, J = 16.2 Hz, 1H), 3.37–3.33 (m, 1H), 3.31 (s, 3H), 3.08 (s, 3H), 2.97 (dq, J = 16.3, 7.1 Hz, 2H), 2.84–2.76 (m, 3H), 2.69 (s, 3H), 2.68–2.65 (m, 1H), 2.52–2.46 (m, 2H), 1.90 (s, 3H). 13 13C NMR (176 MHz, CDCl 3)δ166.7,152.8,152.2,151.4,149.2,148.9,146.8,144.5,140.1,138.2,134.3,133.4,132.8,132.2,130.6,129.7,128.0,127.5,127.4,126.1,121.9,121.8,121.4,120.8,120.1,112.5,112.5,106.2,63.5,61.4,60.0,56.4,55.7,55.7,47.2,45.0,43.3,42.2,40.3,38.5,38.3,29.7,20.8。

[0038] Example 5

[0039] Preparation of TET-XA-13: Take 14-sulfonic acid tetrandrine (50.00 mg, 0.07 mmol) in a 25.00 ml round-bottom flask, and add 3.00 ml of thionyl chloride. React at 80.00 °C for 6 h. After the reaction is completed, concentrate under reduced pressure, and add dichloromethane (10.00 ml × 3) to remove the residual thionyl chloride. Add acrylic acid (8.7 μL, 0.07 mmol), pyridine (12.00 μL, 0.14 mmol), DMAP (4.00 mg, 0.04 mmol) and 5.00 mL of dichloromethane to the reaction system in sequence, and react at room temperature for 3 h. Detect by TLC until the reaction of ethanolamine is complete. Wash with dichloromethane (30.00 mL × 3), hydrochloric acid aqueous solution (2M, 4 mL × 3), saturated brine (10.00 mL), and anhydrous Na 2 SO 4 dry, filter, concentrate under reduced pressure to obtain the crude product. Purify by column chromatography with dichloromethane: methanol: ethyl acetate = 5:1:3 to prepare the compound TET-XA-13. The 1 1H-NMR and 13 13C-NMR are as Figure 5 shown. White solid; 86% yield, 1 1H NMR (600 MHz, CDCl 3)δ9.82(s,1H),7.57(s,1H),7.36(dd,J=8.1,2.3Hz,1H),7.17(dd,J=8.2,2.7Hz,1H),6.89(s,1H),6.74(dd,J=8.3,2.6Hz,1H),6.50(s,1H),6.40(dd,J=17.3,1.4Hz,1H),6.31(s,1H),6.20(dd,J=8.3,2.3Hz,1H),6.09(dd,J=17.3,10.5Hz,1H),5.95(s,1H),5.78(dd,J=10.4,1.4Hz,1H),4.29(ddd,J=11.9,6.7,5.6Hz,1H),4.20(dt,J=11.6,6.0Hz,1H),4.06(dd,J=14.8,10.0Hz,1H),3.98(s,3H),3.87–3.80(m,1H),3.74(s,3H),3.52–3.47(m,1H),3.34(s,3H),3.33–3.30(m,1H),3.09(s,3H),3.05–2.99(m,2H),2.98–2.93(m,2H),2.90–2.71(m,5H),2.68(s,3H),2.54(dd,J=16.7,5.4Hz,1H),2.49(d,J=14.7Hz,1H),2.39(s,3H). 13 C NMR(151MHz,CDCl 3 )δ166.0,152.9,152.2,151.3,149.3,148.8,147.0,144.5,138.3,133.3,132.8,132.6,132.3,131.4,130.5,130.2,128.0,127.4,121.9,121.8,121.3,120.6,120.3,116.2,112.5,112.4,106.3,63.5,61.3,60.0,56.5,55.7,55.6,45.0,43.5,42.3,42.2,40.8,38.5,38.3,29.7,24.9,20.8。

[0040] Example 6

[0041] Preparation of TET-SZ-03: Take 14-sulfonic acid tetrandrine (50.00 mg, 0.07 mmol) in a 25.00 ml round-bottom flask, and add 3.00 ml of thionyl chloride. React at 80.00 °C for 6 h. After the reaction is completed, concentrate under reduced pressure, and add dichloromethane (10.00 ml × 3) to remove the residual thionyl chloride. Add N-(2-hydroxyethyl)piperazine (8.7 μL, 0.07 mmol), triethylamine (5.5 μL, 0.04 mmol) and 5.00 mL of dichloromethane to the reaction system in sequence, and react at room temperature for 3 h. Detect by TLC until the ethylene glycol reaction is complete. Adjust the pH to neutral with dilute hydrochloric acid aqueous solution, wash with dichloromethane (30.00 mL × 3), wash with saturated brine (10.00 mL), and anhydrous Na 2 SO 4 Dry, filter, concentrate under reduced pressure to obtain the crude product. Purify by column chromatography with dichloromethane:methanol:ethyl acetate = 5:1:3 to prepare TET-SZ-03. The 1 1H-NMR and 13 13C-NMR are as Figure 6 shown. White solid; 84% yield, 1 1H NMR (600 MHz, CDCl 3 3) δ 7.35 (dd, J = 8.1, 2.3 Hz, 1H), 7.15 (dd, J = 8.1, 2.6 Hz, 1H), 6.86 (d, J = 1.2 Hz, 2H), 6.80 (dd, J = 8.3, 2.6 Hz, 1H), 6.54–6.52 (m, 2H), 6.31 (dd, J = 8.3, 2.2 Hz, 1H), 6.02 (s, 1H), 4.12 (q, J = 7.1 Hz, 1H), 3.93 (s, 5H), 3.80 (d, J = 10.2 Hz, 1H), 3.75 (d, J = 3.3 Hz, 4H), 3.56–3.49 (m, 2H), 3.48–3.42 (m, 2H), 3.38 (s, 3H), 3.28 (dt, J = 12.9, 6.6 Hz, 2H), 3.22 (s, 3H), 3.00 (dt, J = 17.4, 8.7 Hz, 3H), 2.95–2.89 (m, 2H), 2.84–2.68 (m, 7H), 2.66 (s, 3H), 2.54 (dd, J = 17.9, 5.4 Hz, 2H), 2.48 (dt, J = 14.2, 2.8 Hz, 2H), 2.30 (s, 3H). 13 13C NMR (151 MHz, CDCl 3)δ153.8, 149.4, 148.4, 147.8, 147.2, 147.1, 143.4, 142.8, 134.8, 134.6, 134.4, 132.6, 130.2, 128.0, 127.1, 126.6, 122.8, 122.0, 122.0, 120.9, 120.3, 116.2, 112.2, 111.5, 63.8, 61.4, 60.7, 60.5, 56.1, 55.5, 45.1, 43.3, 42.5, 42.1, 41.4, 37.9, 31.9, 31.6, 29.7, 29.7, 29.6, 25.2, 22.7, 19.8。

[0042] In vitro activity experiment

[0043] 1. In vitro anti-tumor research on derivatives of Stephania tetrandra

[0044] 1) Materials

[0045] Cell lines: Human lung cancer cell line A549, human breast cancer cell line MDA-MB-231, mouse liver cancer cell line H22, and human melanoma cell line A375 were all purchased from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences and stored in a liquid nitrogen tank in our laboratory.

[0046] 2) Reagents: Tetrandrine was purchased from Macklin. A series of derivatives of tetrandrine were prepared from the above experiments. DMEM medium and 1640 medium were purchased from Corning. Trypsin, thiazolyl blue (MTT), and dimethyl sulfoxide (DMSO) were purchased from Biofroxx. Fetal bovine serum was purchased from Excell. Penicillin-streptomycin (double antibody) solution was purchased from Beyotime.

[0047] 3) Instruments: Cell culture incubator, water bath, centrifuge, microplate reader (Thermo Fisher Scientific, Ulm, Germany), and laminar flow hood.

[0048] 4) Cell resuscitation

[0049] Take out the A549, A375, H22, and MDA-MB-231 cell lines from the liquid nitrogen tank, quickly thaw them in a 37°C water bath, centrifuge (1000 rpm, 3 min), discard the supernatant, add 1 mL of complete medium (containing 10% fetal bovine serum and 1% double antibody) to resuspend the cells to make a single-cell suspension, and finally transfer all of them to a 10 mm culture dish and place it in a cell culture incubator for culture. (A549, H22, and MDA-MB-231 cells were cultured in DMEM medium, and A375 cells were cultured in 1640 medium).

[0050] 5) Cell passage

[0051] Take A549, A375, H22, and MDA-MB-231 cells in the logarithmic growth phase, discard the culture medium, add 1 mL of sterile culture medium to wash the culture dish, discard the PBS, add 1.5 mL of trypsin to digest the cells. After the cells become round and detached, add an equal volume of culture medium to terminate the digestion, transfer the cells into a centrifuge tube, centrifuge (1000 rpm, 3 min), discard the supernatant, add 1 mL of fresh culture medium to resuspend the cells into a single-cell suspension, transfer the cells to a new culture dish at a subculture ratio of 1:3, and place them in a cell culture incubator for culture.

[0052] 6) Determination of the in vitro anti-tumor activity of tetrandrine derivatives by the MTT method

[0053] Take A549, A375, H22, and MDA-MB-231 cells in the logarithmic growth phase, operate according to the cell subculture steps. After resuspending the cells into a single-cell suspension, count the cells with a hemocytometer, seed the cells in a 96-well plate at a cell density of 3000 cells / well, with 100 μL of culture medium in each well. After the cells are cultured overnight and adhere to grow, discard the original culture medium, add the drug diluted with a gradient concentration of culture medium, and continue to culture for 48 h. After 48 h, add 10 μL of MTT solution to each well, incubate in the dark at 37 °C for 2 h, then add 100 μL of DMSO, incubate on a shaker for 10 min, measure the OD value with an enzyme-linked immunosorbent assay (ELISA) reader at 490 nm, and finally calculate the cell proliferation inhibition rate and the corresponding IC50 value.

[0054] 7) Experimental results

[0055]

[0056]

[0057] 2. In vitro toxicity study of tetrandrine derivatives

[0058] 1) Materials

[0059] 2) Cell lines: Human normal liver cells WRL68 and human normal mammary epithelial cells MCF-10A were both purchased from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences and stored in this laboratory.

[0060] 3) Reagents: Tetrandrine was purchased from Macklin. A series of tetrandrine derivatives were prepared from the above experiments. DMEM medium and 1640 medium were purchased from Corning. Trypsin, thiazolyl blue (MTT), and dimethyl sulfoxide (DMSO) were purchased from Biofroxx. Fetal bovine serum was purchased from Excell. Penicillin-streptomycin (double antibody) solution was purchased from Beyotime.

[0061] 4) Instruments: cell culture incubator, water bath, centrifuge, microplate reader (Thermo Fisher Scientific, Ulm, Germany), laminar flow hood.

[0062] 5) Cell resuscitation

[0063] Take out the WRL68 and MCF-10A cell lines from the liquid nitrogen tank, quickly thaw them in a 37°C water bath, centrifuge (1000 rpm, 3 min), discard the supernatant, add 1 mL of complete medium (containing 10% fetal bovine serum and 1% double antibody) to resuspend the cells to make a single-cell suspension, and finally transfer all of them to a 10-mm culture dish and place it in the cell culture incubator for culture.

[0064] Take WRL68 and MCF-10A cells in the logarithmic growth phase, discard the medium, add 1 mL of sterile medium to wash the culture dish, discard the PBS, add 1.5 mL of trypsin to digest the cells, and when the cells become round and detached, add an equal volume of medium to terminate the digestion. Transfer the cells to a centrifuge tube, centrifuge (1000 rpm, 3 min), discard the supernatant, add 1 mL of fresh medium to resuspend the cells into a single-cell suspension, and transfer the cells to a new culture dish at a passage ratio of 1:3 and place it in the cell culture incubator for culture.

[0065] 6) Determination of the in vitro toxicity of tetrandrine derivatives by MTT method

[0066] Take WRL68 and MCF-10A cells in the logarithmic growth phase, operate according to the cell passage steps. After resuspending the cells into a single-cell suspension, count the cells with a hemocytometer, and seed them in a 96-well plate at a cell density of 3000 cells / well, 100 μL of medium per well. After the cells are cultured overnight and adhere to grow, discard the original medium, add the drug diluted with a gradient concentration of medium, and continue to culture for 48 h. After 48 h, add 10 μL of MTT solution to each well, incubate in the dark at 37°C for 2 h, then add 100 μL of DMSO, incubate on a shaker for 10 min, and measure the OD value with a microplate reader at 490 nm. Finally, calculate the cell proliferation inhibition rate and the corresponding IC50 value.

[0067] 7) Experimental results

[0068]

[0069] 3. In vivo activity study

[0070] 1) Materials

[0071] Animals: Balb / c-nu female mice, 6 - 7 weeks old, provided by Beijing Spearf Bio-Technology Co., Ltd., and housed in the experimental animal house of Chengdu University of Traditional Chinese Medicine (SPF level).

[0072] 2) Cell line: The human colon cancer cell line HCT116 was purchased from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences and stored in a liquid nitrogen tank in our laboratory.

[0073] 3) Reagents: Tetrandrine, a series of tetrandrine derivatives were prepared by the above experiments, DMSO, normal saline, and 5-fluorouracil were purchased from Macklin.

[0074] 4) Experimental method: Resuspend HCT116 cells with sterile PBS and subcutaneously inoculate the cell suspension into the right axilla of nude mice at a cell density of 5*10 6 cells / 100 μL. When the tumor volume reached 100 mm 3 , the mice were randomly divided into 10 groups: normal saline group, tetrandrine group (40 mg / kg), 5-fluorouracil group (25 mg / kg), TET-SO 3 H (40 mg / kg), TET-XA-01 (40 mg / kg), TET-XA-07 (40 mg / kg), TET-XA-08 (40 mg / kg), TET-XA-11 (40 mg / kg), TET-XA-13 (40 mg / kg), and TET-SZ-03 (40 mg / kg). Intraperitoneal injection was given once every other day. After continuous administration for eight times, the mice were euthanized, the tumors were dissected, and the tumor weights were measured.

[0075] 5) Experimental results

[0076] As Figure 7 shown, the tumor weights of the normal saline group without drug treatment and the mice treated with TET-SO 3 H were comparable. The TET-XA-01, TET-XA-07, TET-XA-08, TET-XA-11, TET-XA-13, and TET-SZ-03 groups all showed good anti-tumor activity, and the tumor weights were all less than that of the tetrandrine group. In addition, the tumor weights of the TET-XA-01, TET-XA-08, and TET-XA-13 groups were all lower than that of the 5-fluorouracil group. Among them, TET-XA-13 demonstrated the most superior anti-tumor ability. As Figure 8 shown, in all treatment groups of mice, except for the 5-fluorouracil group, their body weights did not change during the treatment period, which indirectly indicated that the tetrandrine derivatives showed low-toxic treatment effects. However, the body weight of the mice in the 5-fluorouracil group decreased significantly after 12 days of treatment.

Claims

1.C 14 Application of tetrandrine derivatives substituted with sulfonic acid groups, characterized in that The C 14 Application of tetrandrine derivatives substituted with sulfonic acid groups in the preparation of drugs for treating human colon cancer cell line HCT116; The C 14 The structural formula of the tetrandrine derivative substituted with sulfonic acid group is

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