Emodin nitroxide free radical derivative as well as synthesis method and application thereof

Abstract

The invention belongs to the technical field of organic chemical synthesis, and particularly relates to an emodin nitroxide free radical derivative as well as a synthesis method and application thereof. The preparation method comprises the following steps: by taking a traditional Chinese medicine rheum officinale lipid-lowering pharmacodynamic substance emodin as a research object and selecting a No.3 hydroxyl group as a modification site, introducing an active intermediate nitroxide free radical into an emodin structure on the basis of reaction mechanisms of substitution of carbon chains with different lengths, A3 coupling, esterification and the like and a pharmacophore splicing principle; the emodin nitroxide free radical derivative and other derivatives are designed and synthesized, a plurality of synthesis methods of the novel emodin nitroxide free radical derivative which are mild in reaction condition, environment-friendly and high in yield are established, and the provided novel emodin nitroxide free radical derivative is low in toxicity to normal hepatocytes and high in yield. The compound has obvious anti-proliferation activity on hepatoma carcinoma cells, has an effect superior to that of a novel active molecule of emodin, and provides a new thought for preparation of anti-tumor drugs.

Description

[0001] The present application is a divisional application of the patent application with application number 2024110445734; the original application has an application date of July 31, 2024, an application number of 2024110445734, and a title of A emodin nitroxide radical derivative and a synthesis method and application thereof. TECHNICAL FIELD

[0002] The present application belongs to the technical field of organic chemical synthesis, and specifically relates to a emodin nitroxide radical derivative and a synthesis method and application thereof. BACKGROUND

[0003] Emodin is a trihydroxy anthraquinone compound extracted from Polygonum cuspidatum, and is an effective component of Chinese herbal medicine rhubarb. Research has found that emodin not only has the effect of lowering blood lipids, but also has an inhibitory effect on lung cancer, bladder cancer and other cancer cells. Although emodin has an inhibitory effect on a variety of cancer cells, its intrinsic anticancer activity is low.

[0004] Hyperlipidemia is one of the main factors inducing liver cancer, and searching for an active substance against liver cancer from hypolipidemic drugs has become a new strategy for researching anti-liver cancer drugs. As a hypolipidemic drug, emodin has a good effect on lowering blood lipids, and based on its inhibitory effect on cancer cells, it can be used as a new breakthrough point for exploring active substances against liver cancer.

[0005] If emodin can be used as a research object and appropriately modified to obtain a substance with good anti-liver cancer activity, it will have important significance for the field. SUMMARY

[0006] In order to solve the above technical problems, the present application provides a emodin nitroxide radical derivative and a synthesis method and application thereof. By modifying the 3-hydroxyl group of emodin, based on different length carbon chain substitution, A 3 coupling, esterification reaction mechanism, a new type of emodin nitroxide radical derivative with high yield and significant anti-liver cancer activity is synthesized by introducing a nitroxide radical into the structure of emodin.

[0007] The structure of the emodin nitroxide radical derivative in the present application is selected from any one of the following:

[0008]

[0009] The synthesis method of the above emodin nitroxide radical derivative comprises the following steps:

[0010] The emodin nitroxide radical derivative with the structure as claimed in claim 1 is synthesized by using emodin and carboxyl-containing nitroxide radical as raw materials, DMF as solvent, EDCI and DMAP as catalysts, reacting for 6-8 hours under Ar protection, tracking and monitoring by TLC until the reaction is completed, and then extracting, washing, drying, filtering, and separating and purifying by silica gel column chromatography.

[0011] The carboxyl-containing nitroxide radical is selected from any one of formulae 1-4.

[0012]

[0013] The synthesis route of the emodin nitroxide radical derivative is as follows:

[0014]

[0015] The application of the emodin nitroxide radical derivative in the anti-tumor drug is also within the scope of protection of the present application.

[0016] Preferably, the anti-tumor drug is an anti-hepatoma drug.

[0017] The present application has the following advantages:

[0018] (1) The present application takes emodin, a blood lipid-lowering effective substance of traditional Chinese medicine rhubarb, as the research object, selects the hydroxyl group at the 3rd position as the modification site, and introduces six active intermediates of nitroxide radical into the emodin structure based on the reaction mechanisms of carbon chain substitution, coupling and esterification and the principle of pharmacophore splicing, so as to design and synthesize 50 emodin nitroxide radical derivatives of three series and eight other derivatives, and establish multiple new synthesis methods of the emodin nitroxide radical derivatives with mild reaction conditions, environmental friendliness and high yield. 3 Coupling, esterification and other reaction mechanisms and the principle of pharmacophore splicing, so as to design and synthesize 50 emodin nitroxide radical derivatives of three series and eight other derivatives, and establish multiple new synthesis methods of the emodin nitroxide radical derivatives with mild reaction conditions, environmental friendliness and high yield.

[0019] (2) The new emodin nitroxide radical derivative provided by the present application is low-toxic to normal liver cells and has significant anti-proliferation activity on hepatoma cells, and the effect is better than that of emodin, which provides a new idea for the preparation of anti-tumor drugs. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 Effect of emodin nitroxide radical derivative 8e on migration ability of HepG2 cells;

[0021] Figure 2 Effect of emodin nitroxide radical derivative 8e on cycle distribution of HepG2 cells;

[0022] Figure 3Effects of emodin and its nitroxide radical derivatives on the morphology of HepG2 cells, wherein A represents the blank control group, B-D represent emodin and its nitroxide radical derivatives 1e and 8e groups, the left graph is magnified 3000x, and the right graph is magnified 8000x;

[0023] Figure 4 Effects of emodin and its nitroxide radical derivatives on the Fe 2+ level in HepG2 cells;

[0024] Figure 5 Effects of emodin and its nitroxide radical derivatives on the expression of MDA in HepG2 cells;

[0025] Figure 6 Effects of emodin and its nitroxide radical derivatives on the expression of GSH in HepG2 cells;

[0026] Figure 7 Effects of emodin and its nitroxide radical derivatives on the ROS level in HepG2 cells;

[0027] Figure 8 Effects of emodin and its nitroxide radical derivatives 8e on the expression of SLC7A11 and GPX4 mRNA in HepG2 cells, wherein A represents the effect on GPX4, and B represents the effect on SLC7A11;

[0028] Figure 9 Effects of emodin and its nitroxide radical derivatives 8e on the expression of xCT and GPX4 in HepG2 cells;

[0029] Note: Figures 4-6 、 Figures 8-9 Compared with the blank control group, *p<0.05, **p<0.01; in the figure, Control represents the blank control group, Sorafenib represents sorafenib, and Emodin represents emodin. DETAILED DESCRIPTION

[0030] In order to enable those skilled in the art to better understand the present application, the present application will be further described in conjunction with specific embodiments.

[0031] Example 1

[0032] An emodin nitroxide radical derivative has the structure of general formula 1:

[0033]

[0034] The emodin nitroxide radical derivative of general formula 1 is prepared by the following method:

[0035] (1) emodin and 2-bromoethanol, 3-bromo-1-propanol, 4-bromo-1-butanol, 5-bromo-1-pentanol, 6-bromo-1-hexanol, respectively, as raw materials, Cs2CO3 as catalyst, DMF as solvent, heated to 60°C under Ar protection, constant temperature stirring for 9h, then reduced pressure distillation to recover the solvent, dissolved in water, concentrated hydrochloric acid to adjust pH=5, precipitate, filtration, ethyl acetate washing to obtain powder, which is emodin derivatives with different length of carbon chain intermediates z1-z5;

[0036] (2) four kinds of carboxyl-containing nitroxyl radicals a-d such as 4-carboxyl-TEMPO, as raw materials, EDCI, DMAP as catalyst, DMF as solvent, under Ar protection, room temperature reaction for 24h, ethyl acetate extraction of the reaction solution, water washing, anhydrous Na2SO4 drying, silica gel column chromatography separation, finally the target compound was obtained.

[0037] Example 1-1

[0038] The difference from Example 1 is only that:

[0039] (1) in Ar protection, heated to 50°C constant temperature stirring for 9h;

[0040] (2) in Ar protection, room temperature reaction for 12h.

[0041] The other steps are the same as Example 1.

[0042] Example 1-2

[0043] The difference from Example 1 is only that:

[0044] (1) in Ar protection, heated to 70°C constant temperature stirring for 9h;

[0045] (2) in Ar protection, room temperature reaction for 18h. The other steps are the same as Example 1.

[0046] Example 1-3

[0047] The difference from Example 1 is only that:

[0048] (2) in Ar protection, room temperature reaction for 30h. The other steps are the same as Example 1.

[0049] The conditions in Examples 1-1, 1-2, 1-3 can also achieve the purpose of synthesizing the target product, but the yield of only about 45% in them, Example 1 relative to the yield of Examples 1-1, 1-2, 1-3 is higher, about 55%, therefore, the preferred embodiment 1 as the reaction conditions. Similarly, in the following examples, the highest yield is also selected as the reaction condition, see Examples 2, 3.

[0050] The specific structure of the emodin nitroxide radical derivative of general formula 1 and the raw material obtained in Example 1 is shown in Table 1:

[0051] Table 1 emodin nitroxide radical derivative of general formula 1 and its raw material structure

[0052]

[0053]

[0054]

[0055] Example 2

[0056] An emodin nitroxide radical derivative, which has the structure of general formula 2:

[0057]

[0058] The emodin nitroxide radical derivative of general formula 2 is prepared by the following method:

[0059] (1) First, emodin and bromo-propyne are used as raw materials, DMF is used as a solvent, anhydrous K2CO3 is used as a catalyst, and the reaction is heated to 70°C under Ar protection and stirred magnetically for 4h, TLC detection until the reaction is complete, and then extracted, washed, dried and column chromatography separation and purification to obtain a propynyl-substituted emodin derivative;

[0060] (2) Propynyl-substituted emodin derivative, 4-aldehyde-TEMPO (e) and 3-aldehyde-2, 2, 5, 5-tetramethylpyrrolidine-1-nitroxide (f) and 20 kinds of secondary amines such as di-n-propylamine, 4-(trifluoromethyl) piperidine and morpholine are used as raw materials, toluene is used as a solvent, CuBr is used as a catalyst, and the reaction is heated to 80°C under Ar protection and stirred magnetically for about 8h, TLC detection until the reaction is complete, and then separated and purified by column chromatography to obtain the target compound.

[0061] Example 2-1

[0062] The difference between Example 2 is only:

[0063] (1) In Ar, under the protection of gas, heated to 60°C and stirred magnetically for 4h;

[0064] (2) in Ar gas protection, heated to 60 °C magnetic stirring reaction about 8 h.

[0065] Example 2-2

[0066] The difference from example 2 is only:

[0067] (1) in Ar gas protection, heated to 80 °C magnetic stirring reaction 4 h; (2) in Ar gas protection, heated to 80 °C magnetic stirring reaction about 6 h.

[0068] Example 2-3

[0069] (1) in Ar gas protection, heated to 100 °C magnetic stirring reaction 4 h; (2) in Ar gas protection, heated to 60 °C magnetic stirring reaction about 7 h.

[0070] Example 2-4

[0071] (1) in Ar gas protection, heated to 120 °C magnetic stirring reaction 4 h; (2) in Ar gas protection, heated to 60 °C magnetic stirring reaction about 6 h.

[0072] Example 2-5

[0073] (1) in Ar gas protection, heated to 80 °C magnetic stirring reaction 4 h; (2) in Ar gas protection, heated to 80 °C magnetic stirring reaction about 6 h.

[0074] Example 2-6

[0075] The difference from example 2 is only:

[0076] (1) in Ar gas protection, heated to 80 °C magnetic stirring reaction 4 h; (2) in Ar gas protection, heated to 80 °C magnetic stirring reaction about 7 h.

[0077] Example 2-7

[0078] The difference from example 2 is only:

[0079] (1) in Ar gas protection, heated to 80 °C magnetic stirring reaction 4 h; (2) in Ar gas protection, heated to 100 °C magnetic stirring reaction about 6 h.

[0080] Example 2-8

[0081] The difference from example 2 is only:

[0082] (1) in Ar gas protection, heated to 60 °C magnetic stirring reaction 4 h;

[0083] (2) In Ar gas protection, heated to 120℃ magnetic stirring reaction for about 6h.

[0084] Example 2-9

[0085] The difference from Example 2 is only:

[0086] (1) In Ar gas protection, heated to 100℃ magnetic stirring reaction for 4h;

[0087] (2) In Ar gas protection, heated to 60℃ magnetic stirring reaction for about 6h.

[0088] As an example of Example 2, the specific structures of the obtained emodin nitroxide radical derivatives of general formula 2 and raw materials are shown in Table 2 and Table 3:

[0089] Table 2 Emodin propargylamine nitroxide radical derivative of general formula 2 and its raw material structure

[0090]

[0091]

[0092]

[0093] Table 3 Emodin propargylamine nitroxide radical derivative of general formula 2 and its raw material structure

[0094]

[0095]

[0096]

[0097] Example 3

[0098] An emodin nitroxide radical derivative, which has the following general formula 3 structure:

[0099]

[0100] The emodin nitroxide radical derivative of general formula 3 is prepared by the following method:

[0101] Take emodin and 4-carboxyl-TEMPO, etc. a-d four kinds of carboxyl-containing nitroxide radicals as raw materials, DMF as solvent, EDCI and DMAP as catalyst, magnetic stirring reaction under Ar gas protection at room temperature for 7h, TLC monitoring until the reaction is complete, after stopping the reaction, extraction, washing, drying and filtration, silica gel column chromatography separation and purification, finally the target compound is obtained.

[0102] Example 3-1

[0103] The difference from Example 3 is only that:

[0104] The reaction was stirred at room temperature under Ar protection for 5 h, and other steps were the same as in Example 3.

[0105] Example 3-2

[0106] The reaction was stirred at room temperature under Ar protection for 9 h, and other steps were the same as in Example 3.

[0107] Example 3-3

[0108] The reaction was stirred at room temperature under Ar protection for 12 h, and other steps were the same as in Example 3.

[0109] The specific structures of the emodin nitroxide radical derivatives of general formula 3 and the raw materials obtained in Example 3 are shown in Table 4:

[0110] Table 4: Structures of emodin nitroxide radical derivatives of general formula 3 and their raw materials

[0111]

[0112]

[0113] Test Example 1: Evaluation of the anti-hepatoma activity of emodin nitroxide radical derivatives

[0114] The anti-proliferative activity of the nitroxide radicals, emodin and its nitroxide radical derivatives in Example 1, Example 2 and Example 3 on hepatoma cells and the cytotoxicity on normal cells L02 were determined by MTT method, and the results are shown in Table 5:

[0115] Table 5: Anti-proliferative activity of nitroxide radicals, emodin and its nitroxide radical derivatives on hepatoma cells and cytotoxicity on normal cells L02

[0116]

[0117]

[0118]

[0119] As can be seen from Table 5, the six nitroxide radicals and emodin all showed good anti-proliferative activity on hepatoma cells HepG2, with IC 50 values of the nitroxide radicals being between 25.40 and 50.01 μM, the IC 50 value of emodin being 59.55 μM, and the anti-proliferative abilities of the new emodin nitroxide radical derivatives being different, with 31 new compounds having IC 50The values of the 5 new compounds 1e, 8e, etc. were less than 59.55 μM, and the anti-proliferation ability of emodin was significantly improved. The IC 50 values of 26 of the 31 new compounds were less than 10 μM, showing good anti-proliferation activity. The cytotoxicity results showed that the IC 50 values of 26 of the 31 new compounds were less than 10 μM, showing good anti-proliferation activity. The cytotoxicity results showed that the IC 50 values of 26 of the 31 new compounds were less than 10 μM, showing good anti-proliferation activity. The cytotoxicity results showed that the IC

[0120] The cell wound healing experiment results showed that emodin and its derivatives 1e and 8e had a certain inhibitory effect on the migration and repair of HepG2 liver cancer cells, and showed a dose-dependent effect. The cell cycle experiment results showed that emodin and its derivatives 1e and 8e could block HepG2 cells in the G1 / S phase in a dose-dependent manner, thereby exerting anti-tumor activity, as shown in Table 6 and Table 7. Figures 1-2

[0121] Table 6 Effect of emodin nitroxide derivative 8e on the migration ability of HepG2 cells

[0122]

[0123] Table 7 Effect of emodin nitroxide derivative 8e on the cell cycle distribution of HepG2 cells

[0124]

[0125] Test Example 2 Anti-liver cancer mechanism of emodin nitroxide derivatives

[0126] Transmission electron microscopy showed that the mitochondria of liver cancer cells in each group showed significant changes after intervention with emodin and its nitroxide derivatives 1e and 8e, with reduced volume, increased double membrane density, and reduced mitochondrial cristae, which was consistent with the characteristics of ferroptosis mitochondria, and the changes were more obvious at higher concentrations, as shown in Table 8. Figure 3 2+ , MDA, GSH and ROS levels in HepG2 cells before and after drug intervention, it was found that compared with the blank control group, emodin and its nitroxide derivatives 1e and 8e could significantly increase the Fe 2+ , MDA and ROS levels (p<0.01) and reduce the GSH level (p<0.01) in a dose-dependent manner, as shown in Table 8. Figures 4-7 ​​and Table 8 (Control represents blank control group, Sorafenib refers to sorafenib, and Emodin refers to emodin). The RT-qPCR and WB results show that SLC7A11 and GPX4 are expressed in HepG2 cells, and the mRNA and protein expression levels of SLC7A11 and GPX4 are significantly reduced after drug intervention (p<0.01), as shown in Table 8 (Control represents blank control group, Sorafenib refers to sorafenib, and Emodin refers to emodin). Figures 8-9 and Table 9, the effect of emodin nitroxide radical derivative 1e, 8e is more significant than that of emodin.

[0127] Table 8 Effect of emodin and emodin nitroxide radical derivative 8e on Fe 2+ level in HepG2 cells, MDA expression in HepG2 cells, GSH expression in HepG2 cells, and ROS level in HepG2 cells

[0128]

[0129] Table 9 Effect of emodin nitroxide radical derivative 8e on mRNA expression of SLC7A11 and GPX4 and protein xCT and GPX4 expression in HepG2 cells

[0130]

[0131] As can be seen from Tables 8 and 9, emodin nitroxide radical derivatives 1e and 8e down-regulate SLC7A11 by inhibiting System Xc-, reduce the uptake of cystine in cells, reduce the synthesis of GSH, indirectly inhibit the activity of GPX4, induce abnormal accumulation of lipid ROS, and up-regulate Fe 2+ level, ultimately leading to ferroptosis of liver cancer cells.

Claims

1. An emodin nitroxide radical derivative, characterized by, The structure of the emodin nitroxide radical derivative is selected from any one of the following:

2. The method for synthesizing a rhein nitroxide radical derivative as described in claim 1, characterized in that, The method comprises the following steps: The emodin and the carboxyl-containing nitroxide radical are used as raw materials, DMF is used as a solvent, EDCI and DMAP are used as catalysts, and the reaction is carried out under Ar protection for 6-8 hours; TLC is used for tracking and monitoring until the reaction is completed; after the reaction is terminated, extraction, washing, drying and filtration are carried out; and the final emodin nitroxide radical derivative with the structure of claim 1 is obtained through silica gel column chromatography separation and purification. The carboxyl-containing nitroxide radical is selected from any one of formulas 1-4:

3. The use of the emodin nitroxide radical derivative according to claim 1 in antitumor drugs, characterized in that, The antitumor drug is an anti-liver cancer drug.

Images