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; a plurality of emodin nitroxide free radical derivatives and other derivatives are designed and synthesized, a plurality of synthesis methods of the novel emodin nitroxide free radical derivatives which are mild in reaction condition, environment-friendly and high in yield are established, and the provided novel emodin nitroxide free radical derivatives are low in toxicity to normal hepatocytes, low in toxicity to liver cells 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 date of which is July 31, 2024, the application number is 2024110445734, and the invention name is "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 particularly 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 to 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 technical solution of the present application is as follows:

[0008] In the first aspect of the present application, a emodin nitroxide radical derivative is provided, and the structure of the emodin nitroxide radical derivative is selected from one of the following general formula 1 to general formula 3:

[0009]

[0010] R is a nitroxide radical, and R1 and R2 are different active functional groups.

[0011] Preferably, when the emodin nitroxide radical derivative is general formula 1 or general formula 3, the nitroxide radical is selected from any one of formula 1-4:

[0012]

[0013] When the emodin nitroxide radical derivative is general formula 2, the nitroxide radical is selected from any one of formula 5-6:

[0014]

[0015] Preferably, the emodin nitroxide radical derivative general formula 1 is prepared by the following method:

[0016] The hydroxyl group at the 3rd position of emodin is used as a modification site, a bromo alcohol is used to introduce different length hydroxyl alkyl side chains at the 3rd position through substitution reaction, and an esterification reaction is used to connect a carboxyl-containing nitroxide radical to the side chain, thereby synthesizing emodin nitroxide radical derivatives general formula 1 containing different carbon chain lengths.

[0017] Preferably, the method is specifically:

[0018] Emodin and a bromo alcohol are used as raw materials, Cs2CO3 is used as a catalyst, DMF (N,N-dimethylformamide) is used as a solvent, heating is performed under Ar gas protection to 50-70°C constant temperature reaction for 8-10h, then the solvent is recovered by reduced pressure distillation, dissolved with water, the pH is adjusted to less than 7 with acid, precipitates are allowed to separate out, filtration is performed, and different length carbon chain substituted emodin derivatives intermediates are obtained by washing with ethyl acetate; then the intermediates and carboxyl-containing nitroxide radicals are used as raw materials, EDCI (carbodiimide hydrochloride) and DMAP (4-dimethylaminopyridine) are used as catalysts, DMF is used as a solvent, reaction is performed at room temperature under Ar gas protection, the reaction liquid is extracted with ethyl acetate, water washing, drying, and chromatographic separation are performed, and finally emodin nitroxide radical derivatives general formula 1 are obtained.

[0019] Preferably, the bromo alcohol is selected from any one of 2-bromoethanol, 3-bromo-1-propanol, 4-bromo-1-butanol, 5-bromo-1-pentanol, and 6-bromo-1-hexanol;

[0020] Preferably, the carboxyl-containing nitroxide radical is selected from any one of formula 1-4:

[0021]

[0022] The synthesis route of the emodin nitroxide radical derivative is as follows:

[0023]

[0024] Preferably, the emodin nitroxide radical derivative general formula 2 is prepared by the following method:

[0025] With the hydroxyl group at the 3rd position of emodin as the modification site, propargyl is introduced into the structure by bromo-propargyl substitution, and then A 3 The coupling reaction dehydrates and condenses the synthesized aldehyde-containing nitroxide radical and the secondary amine three components under the action of a transition metal catalyst to generate the emodin propargyl amine nitroxide radical derivative general formula 2.

[0026] Preferably, the method is specifically:

[0027] First, emodin and bromo-propargyl are used as raw materials, DMF is used as a solvent, anhydrous K2CO3 is used as a catalyst, and the reaction is heated to 60-80°C under Ar protection for 4h. TLC detection until the reaction is complete, and then extraction, washing, drying, and column chromatography separation and purification are performed to obtain propargyl-substituted emodin derivatives. Then, propargyl-substituted emodin derivatives, aldehyde-containing nitroxide radicals, and secondary amines are used as raw materials, toluene is used as a solvent, CuBr is used as a catalyst, and the reaction is heated to 70-90°C under Ar protection for 7-9h. TLC detection until the reaction is complete, and then column chromatography is used for separation and purification to finally obtain emodin nitroxide radical derivatives general formula 2.

[0028] Preferably, the aldehyde-containing nitroxide radical is selected from any one of formula 5 to formula 6:

[0029]

[0030] Preferably, the secondary amine is selected from any one of formula 7 to formula 19:

[0031]

[0032] The synthesis route of the emodin nitroxide radical derivative is as follows:

[0033]

[0034] Preferably, the emodin nitroxide radical derivative general formula 3 is prepared by the following method:

[0035] With the hydroxyl group at the 3rd position of emodin as the modification site, the synthesized carboxyl-containing nitroxide radical is directly connected in the emodin structure by dehydration condensation to obtain the emodin nitroxide radical derivative.

[0036] Preferably, the method is specifically:

[0037] With emodin and carboxyl-containing nitroxyl radical as raw materials, DMF as solvent, EDCI and DMAP as catalysts, under Ar protection, the reaction is carried out 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 finally, the emodin nitroxyl radical derivative general formula 3 is obtained through silica gel column chromatography separation and purification;

[0038] Preferably, the carboxyl-containing nitroxyl radical is selected from any one of the above formula 1 to formula 4.

[0039] The synthesis route of the emodin nitroxyl radical derivative is as follows:

[0040]

[0041] In the second aspect of the present application, the emodin nitroxyl radical derivative is applied to an antitumor drug.

[0042] Preferably, the antitumor drug is an anti-liver cancer drug.

[0043] The present application has the following beneficial effects:

[0044] (1) The present application takes emodin, a traditional Chinese medicine hypolipidemic drug, as the research object, selects the 3-position hydroxyl group as the modification site, and based on different length carbon chain substitution, A 3 coupling, esterification and other reaction mechanisms and pharmacophore splicing principles, six active intermediates nitroxyl radicals are introduced into the emodin structure, and three series of 50 emodin nitroxyl radical derivatives and other derivatives 8 are designed and synthesized, and a plurality of new emodin nitroxyl radical derivative synthesis methods with mild reaction conditions, environmental friendliness and high yield are established;

[0045] (2) The new emodin nitroxyl radical derivative provided by the present application has low toxicity to normal liver cells, and has significant anti-proliferation activity to liver cancer cells, and the effect is better than that of emodin, which is a new active molecule, and provides a new idea for the preparation of antitumor drugs. BRIEF DESCRIPTION OF DRAWINGS

[0046] Figure 1 Effect of emodin nitroxyl radical derivative 8e on migration ability of HepG2 cells;

[0047] Figure 2 Effect of emodin nitroxyl radical derivative 8e on cell cycle distribution of HepG2 cells;

[0048] 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;

[0049] Figure 4 Effects of emodin and its nitroxide radical derivatives on the Fe 2+ level in HepG2 cells;

[0050] Figure 5 Effects of emodin and its nitroxide radical derivatives on the expression of MDA in HepG2 cells;

[0051] Figure 6 Effects of emodin and its nitroxide radical derivatives on the expression of GSH in HepG2 cells;

[0052] Figure 7 Effects of emodin and its nitroxide radical derivatives on the ROS level in HepG2 cells;

[0053] 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;

[0054] Figure 9 Effects of emodin and its nitroxide radical derivatives 8e on the expression of xCT and GPX4 in HepG2 cells;

[0055] Notes: Figures 4-6 、 Figures 8-9 In the above table, 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

[0056] 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.

[0057] Example 1

[0058] An emodin nitroxide radical derivative has the structure of general formula 1:

[0059]

[0060] The emodin nitroxide radical derivative of general formula 1 is prepared by the following method:

[0061] (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 pH = 5, precipitate, filtration, ethyl acetate washing to obtain powder, which is emodin derivatives with different length of carbon chain intermediates z1-z5;

[0062] (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, room temperature reaction for 24h under Ar protection, ethyl acetate extraction of the reaction solution, water washing, anhydrous Na2SO4 drying, silica gel column chromatography separation, finally the target compound was obtained.

[0063] Example 1-1

[0064] The difference from Example 1 is only that:

[0065] (1) in Ar protection, heated to 50°C constant temperature stirring for 9h;

[0066] (2) in Ar protection, room temperature reaction for 12h.

[0067] The other steps are the same as Example 1.

[0068] Example 1-2

[0069] The difference from Example 1 is only that:

[0070] (1) in Ar protection, heated to 70°C constant temperature stirring for 9h;

[0071] (2) in Ar protection, room temperature reaction for 18h. The other steps are the same as Example 1.

[0072] Example 1-3

[0073] The difference from Example 1 is only that:

[0074] (2) in Ar protection, room temperature reaction for 30h. The other steps are the same as Example 1.

[0075] 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.

[0076] 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:

[0077] Table 1 emodin nitroxide radical derivative of general formula 1 and its raw material structure

[0078]

[0079]

[0080]

[0081] Example 2

[0082] An emodin nitroxide radical derivative having the structure of general formula 2:

[0083]

[0084] The emodin nitroxide radical derivative of general formula 2 is prepared by the following method:

[0085] (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;

[0086] (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.

[0087] Example 2-1

[0088] The difference between Example 2 is only:

[0089] (1) In Ar, under the protection of gas, heated to 60°C and stirred magnetically for 4h;

[0090] (2) in Ar gas protection, heated to 60 °C magnetic stirring reaction about 8 h.

[0091] Example 2-2

[0092] The difference from example 2 is only:

[0093] (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.

[0094] Example 2-3

[0095] (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.

[0096] Example 2-4

[0097] (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.

[0098] Example 2-5

[0099] (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.

[0100] Example 2-6

[0101] The difference from example 2 is only:

[0102] (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.

[0103] Example 2-7

[0104] The difference from example 2 is only:

[0105] (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.

[0106] Example 2-8

[0107] The difference from example 2 is only:

[0108] In (1), under the protection of Ar gas, heated to 60℃ magnetic stirring reaction 4h; in (2), under the protection of Ar gas, heated to 120℃ magnetic stirring reaction about 6h.

[0109] Examples 2-9

[0110] The difference from Example 2 is only:

[0111] In (1), under the protection of Ar gas, heated to 100℃ magnetic stirring reaction 4h;

[0112] In (2), under the protection of Ar gas, heated to 60℃ magnetic stirring reaction about 6h.

[0113] Example 2, the resulting emodin nitroxide radical derivative general formula 2 and the specific structure of raw materials are shown in Table 2 and Table 3:

[0114] Table 2 emodin propargylamine nitroxide radical derivative general formula 2 and the structure of raw materials

[0115]

[0116]

[0117]

[0118] Table 3 emodin propargylamine nitroxide radical derivative general formula 2 and the structure of raw materials

[0119]

[0120]

[0121]

[0122] Example 3

[0123] An emodin nitroxide radical derivative, which has the following general formula 3 structure:

[0124]

[0125] Emodin nitroxide radical derivative general formula 3 is prepared by the following method:

[0126] Respectively, emodin and 4-carboxyl-TEMPO, etc. a-d four kinds of carboxyl-containing nitroxide radical as raw material, DMF as solvent, EDCI, DMAP as catalyst, under the protection of Ar gas, magnetic stirring reaction at room temperature 7h, TLC monitoring to complete the reaction, after the reaction was stopped by extraction, washing, drying and filtration, silica gel column chromatography separation and purification, finally obtained the target compound.

[0127] Example 3-1

[0128] The difference from Example 3 is only that:

[0129] The reaction was stirred at room temperature under Ar protection for 5 h, and other steps were the same as in Example 3.

[0130] Example 3-2

[0131] The reaction was stirred at room temperature under Ar protection for 9 h, and other steps were the same as in Example 3.

[0132] Example 3-3

[0133] The reaction was stirred at room temperature under Ar protection for 12 h, and other steps were the same as in Example 3.

[0134] 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:

[0135] Table 4: Structures of emodin nitroxide radical derivatives of general formula 3 and their raw materials

[0136]

[0137]

[0138] Test Example 1: Evaluation of the anti-hepatoma activity of emodin nitroxide radical derivatives

[0139] 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:

[0140] Table 5: Anti-proliferative activity of nitroxide radicals, emodin and its nitroxide radical derivatives on hepatoma cells and cytotoxicity on normal cells L02

[0141]

[0142]

[0143]

[0144] 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 other 26 compounds were less than 59.55 μM, and the anti-proliferation ability of emodin was significantly improved. The IC 50 values of 31 new compounds were less than 10 μM, showing good anti-proliferation activity. The cytotoxicity results showed that 26 of the 31 new compounds had IC 50 values greater than emodin (IC 50 = 44.63 μM), showing low toxicity, and the selectivity index SI was greater than that of emodin (0.75). The selectivity index of derivatives 1e and 8e was 8.13 and 14.87, respectively, showing good selective anti-hepatoma cell proliferation activity. The low-toxic and highly active anti-hepatoma activity molecules 1e and 8e were screened out.

[0145] 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 hepatoma cells HepG2, 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

[0146] Table 6 Effect of emodin nitroxide derivative 8e on the migration ability of HepG2 cells

[0147]

[0148]

[0149] Table 7 Effect of emodin nitroxide derivative 8e on the cell cycle distribution of HepG2 cells

[0150]

[0151] Test Example 2 Anti-hepatoma mechanism of emodin nitroxide derivatives

[0152] Transmission electron microscopy showed that the mitochondria of hepatoma 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. The greater the concentration, the more obvious the change, as shown in Table 8. Figure 3 The levels of Fe 2+ , MDA, GSH and ROS in HepG2 cells before and after drug intervention were determined, and it was found that compared with the blank control group, emodin and its nitroxide derivatives 1e and 8e could significantly increase the levels of Fe 2+ , MDA and ROS in HepG2 cells (p<0.01), and decrease the level of GSH (p<0.01), and showed a dose-dependent effect, as shown in Table 8.​Figures 4-7 and Table 8 (Control represents blank control group, Sorafenib represents sorafenib, and Emodin represents 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 and Figure 2. Figures 8-9 and Table 9 show that the effects of emodin nitroxide radical derivatives 1e and 8e are more significant than those of emodin.

[0153] Table 8 Effects 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

[0154]

[0155] Table 9 Effects of emodin nitroxide radical derivative 8e on mRNA expression of SLC7A11 and GPX4 and protein xCT and GPX4 expression in HepG2 cells

[0156]

[0157] 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 emodin nitroxide radical derivative of claim 1, wherein, The synthesis method of the emodin nitroxide radical derivative is as follows: emodin and bromo-propyne are used as raw materials, DMF is used as a solvent, anhydrous K2CO3 is used as a catalyst, heating is performed to 60-80 DEG C under the protection of Ar gas, and reaction is performed for 4h, TLC detection is performed until the reaction is complete, extraction, washing, drying and column chromatography separation and purification are performed, and a propynyl-substituted emodin derivative is obtained; then, the propynyl-substituted emodin derivative, an aldehyde group-containing nitroxide radical and a secondary amine are used as raw materials, toluene is used as a solvent, CuBr is used as a catalyst, heating is performed to 70-90 DEG C under the protection of Ar gas, and reaction is performed for 7-9h, TLC detection is performed until the reaction is complete, column chromatography is used for separation and purification, and finally the emodin nitroxide radical derivative with the structure of claim 1 is obtained; The aldehyde group-containing nitroxide radical is selected from any one of formula 5 to formula 6: The secondary amine is selected from any one of formula 7 to formula 19:

3. Use of the emodin nitroxide radical derivative according to any one of claims 1-2 in an antitumor drug, characterized in that, The antitumor drug is an anti-liver cancer drug.

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