Rhein 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 a rhein 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 antioxidant drug effect substance rhein as a research object and selecting a No.3 carboxyl group as a modification site, introducing four active intermediate nitroxide free radicals into a rhein structure on the basis of an amide condensation and esterification reaction mechanism and a pharmacophore splicing principle, designing and synthesizing 30 rhein nitroxide free radical derivatives, a novel rhein nitroxide free radical derivative synthesis method which is mild in reaction condition, environmentally friendly and high in yield is established, and the provided novel rhein nitroxide free radical derivative has the capacity of scavenging DPPH free radicals and ABTS free radicals, has the protection effect on oxidative damage cells, has the capacity of delaying the life of caenorhabditis elegans and can be used for preparing the novel rhein nitroxide free radical derivative for treating caenorhabditis elegans. The action effect is better than that of rhein, and a new thought is provided for research and development of anti-oxidation and anti-aging drugs.

Description

Technical Field

[0001] The invention belongs to the technical field of organic chemical synthesis, and specifically relates to a rhein nitroxide free radical derivative and a synthesis method thereof, and also relates to the application of the derivative in antioxidant products. Background Art

[0002] Rhein is a trihydroxyanthraquinone compound extracted from rhubarb, a plant of the Polygonaceae family, and is the active ingredient of the Chinese herbal medicine rhubarb. Studies have found that rhein has antioxidant, anti-inflammatory, and anti-tumor effects; studies have shown that rhein has a protective effect on oxidatively damaged cells, and it is predicted that rhein is a precursor compound with antioxidant activity. However, rhein has poor water solubility, resulting in low bioavailability in the body.

[0003] Oxidative stress is related to the pathogenesis of many human diseases. The antioxidant system in the human body is defective under oxidative stress. Using antioxidants to inhibit the production of excessive reactive oxygen in the body, reduce the damage of reactive oxygen to normal cells in the body, and delay the development of related diseases is one way to cope with oxidative stress.

[0004] If rhein can be used as a research object and it can be appropriately modified to obtain substances with antioxidant activity and anti-aging effects, it will be of great significance to this field. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a rhein nitroxide free radical derivative and a synthesis method and application thereof. By modifying the No. 3 carboxyl group of rhein, based on the amide condensation reaction and esterification reaction mechanism, the nitroxide free radical is introduced into the rhein structure, and a new rhein nitroxide free radical derivative with high yield and significant antioxidant activity is synthesized.

[0006] The technical solution of the present invention is as follows:

[0007] In the first aspect of the present invention, a rhein nitroxide free radical derivative is provided, the structure of which is shown in Formula 1:

[0008]

[0009] R1 is a nitroxide free radical, and Linker represents different types of amino acids.

[0010] When the rhein nitroxide free radical derivative is of the general formula 1, the nitroxide free radical is selected from any one of Formulas 1 to 4:

[0011]

[0012] Preferably, the rhein nitroxide free radical derivative having the general formula 1 is prepared by the following method:

[0013] (1) Using Boc-amino acid and hydroxyl-containing nitroxide free radical as raw materials, an amide condensation reaction is carried out under the action of catalyst I to obtain intermediate I;

[0014] (2) Trifluoroacetic acid is added dropwise to the intermediate I, reacted, extracted, and the organic layer is collected and then dehydrated and dried to obtain the intermediate II;

[0015] (3) Using intermediate II and rhein as raw materials and tetrahydrofuran as solvent, reacting under the action of catalyst II, filtering out insoluble matter, spinning off the solvent, and separating by chromatography to obtain a rhein nitroxide free radical derivative having the structure described in general formula 1.

[0016] The reaction principle is as follows: using the amide condensation reaction principle, Boc-amino acid is condensed with hydroxyl-containing nitroxide free radicals, the Boc protecting group is removed by trifluoroacetic acid, the carboxyl group at position 3 of rhein is used as the modification site, and then using the esterification reaction principle, the hydroxyl-containing nitroxide free radical side chain is connected to position 3 to synthesize different rhein nitroxide free radical derivatives of general formula 1.

[0017] A more specific method is:

[0018] Boc-amino acid and hydroxyl-containing nitroxide free radical are used as raw materials, N,N-dicyclohexylcarbodiimide and 4-dimethylaminopyridine are used as catalyst I, dichloromethane is used as solvent, and the reaction is carried out at room temperature for 5 to 12 hours under the protection of Ar gas, and then the solvent is recovered by distillation under reduced pressure, and the reaction is separated by chromatography; the intermediate is used as raw material, dichloromethane is used as solvent, trifluoroacetic acid is added dropwise in an ice bath, and the reaction is carried out at 0°C for 0.5 to 1 hour, and thin layer chromatography is used to detect until the reaction is complete, saturated sodium bicarbonate is added to adjust the pH to alkaline, dichloromethane is used for extraction, the organic layer is collected, water is removed, and the intermediate II is obtained by rotary drying; then the intermediate and rhein are used as raw materials, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 4-dimethylaminopyridine, and triethylamine are used as catalyst II, and tetrahydrofuran is used as solvent, and the reaction is carried out at room temperature under the protection of Ar gas, the insoluble matter is removed by filtration, the solvent is removed by rotary drying, and the rhein nitroxide free radical derivative of general formula 1 is finally obtained.

[0019] Preferably, the Boc-amino acid is selected from any one of Boc-glycine, Boc-β-alanine, Boc-L-alanine, Boc-L-valine, Boc-L-leucine, Boc-L-methionine, Boc-L-phenylalanine and N-tert-butyloxycarbonyl-2-methylalanine;

[0020] The synthetic route of the above-mentioned rhein nitrogen oxide free radical derivative is as follows:

[0021]

[0022] The present invention also aims to protect the use of the above-mentioned rhein nitroxide free radical derivatives in anti-oxidation and / or anti-aging products, which may be medicines or skin care products.

[0023] The beneficial effects of the present invention are:

[0024] (1) The present invention uses rhein, an antioxidant substance of traditional Chinese medicine rhubarb, as the research object, selects its No. 3 carboxyl group as the modification site, introduces four active intermediate nitroxide free radicals into the structure of rhein based on the mechanisms of amide condensation reaction, esterification reaction and pharmacophore splicing principle, designs and synthesizes 30 rhein nitroxide free radical derivatives, and establishes a new method for synthesizing rhein nitroxide free radical derivatives with mild reaction conditions, environmental friendliness and high yield;

[0025] (2) The novel rhein nitroxide free radical derivative provided by the present invention has the ability to scavenge DPPH free radicals and ABTS free radicals, has a protective effect on oxidatively damaged cells, and has the ability to delay the lifespan of Caenorhabditis elegans. The effect is better than rhein, providing a new idea for the development of antioxidant and anti-aging drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The protective effect of rhein nitroxide free radical derivatives on L02 cells induced by H2O2;

[0027] Figure 2 The effects of compounds 16, 16b and rhein on the heat stress resistance of wild-type Caenorhabditis elegans;

[0028] Figure 3 The effects of compounds 16, 16b and rhein on the oxidative stress resistance of wild-type Caenorhabditis elegans;

[0029] Figure 4 Effects of compounds 16, 16b and rhein on ROS in wild-type Caenorhabditis elegans: A ROS fluorescence images of the control group, treatment groups 16, 16b and Rhein; B ROS fluorescence grayscale analysis;

[0030] Figure 5 The effects of compounds 16, 16b and rhein on GSH in wild-type Caenorhabditis elegans;

[0031] Figure 6 The effects of compounds 16, 16b and rhein on MDA in wild-type Caenorhabditis elegans;

[0032] Note: Figure 1 Compared with the model group, Figures 4 to 6 Compared with the blank control group, *p<0.05, **p<0.01, ***p<0.001,ns p>0.05; in the figure, model represents the model group, Control represents the blank control group, and Rhein refers to rhein. DETAILED DESCRIPTION

[0033] In order to enable those skilled in the art to better understand the present invention, the present invention is further described in conjunction with specific implementation methods.

[0034] In this application, unless otherwise specified, all solvents involved are analytical grade solvents.

[0035] Example 1

[0036] A rhein nitroxide free radical derivative, wherein the rhein nitroxide free radical derivative has a structure of general formula 1:

[0037]

[0038] The rhein nitroxide free radical derivative of general formula 1 is prepared by the following method:

[0039] (1) Weigh Boc-amino acid (1.00 mmol) and hydroxyl-containing nitrogen oxide free radical (0.50 mmol) into a 50 mL round-bottom flask, place a magnetic bar, add dichloromethane (156.01 mmol, 10 mL) to dissolve, add N,N-dicyclohexylcarbodiimide (2.39 mmol) and 4-dimethylaminopyridine (0.55 mmol) to the reaction solution in sequence, react at room temperature, and detect with thin layer chromatography until the reaction is complete. Stop the reaction, spin dry the solvent, and purify with silica gel column chromatography (dichloromethane: methanol v:v = 100:1) to obtain intermediate I;

[0040] (2) The intermediate I obtained in (1) was used as a raw material (0.67 mmol) in a 50 mL round-bottom flask, a magnetic bar was placed, dichloromethane (92.53 mmol, 4 mL) was added to dissolve, and the mixture was placed in an ice bath. Trifluoroacetic acid (2 mL) was slowly added dropwise, and the mixture was reacted in an ice bath for 1 h. Thin layer chromatography was performed until the reaction was complete, and the reaction was stopped. Saturated sodium bicarbonate was added to adjust the pH to alkaline, and the mixture was extracted with dichloromethane. The organic layer was collected, anhydrous sodium sulfate was added to remove water, and the mixture was spin-dried to obtain intermediate II.

[0041] (3) Weigh rhein (0.36 mmol) into a round-bottom flask, place a magnetic bar, add tetrahydrofuran (156.01 mmol, 10 mL) to dissolve, add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.70 mmol) and 4-dimethylaminopyridine (0.60 mmol) to the reaction solution, add intermediate II (0.48 mmol), add triethylamine (100 μL) and react at room temperature for 24 h, and detect by thin layer chromatography until the reaction is complete. Stop the reaction, filter out insoluble matter, spin off the solvent, and purify by silica gel column chromatography (dichloromethane: methanol v:v = 150:1) to finally obtain the target product.

[0042] Example 1-1

[0043] The only difference from Example 1 is:

[0044] In (1), the molar mass of Boc-amino acid and nitroxide radical is 1:1;

[0045] (2), the volume ratio of dichloromethane to trifluoroacetic acid is 1:1;

[0046] The other steps are the same as those in Example 1.

[0047] Example 1-2

[0048] The only difference from Example 1 is:

[0049] In (1), the molar mass ratio of Boc-amino acid to nitroxide radical is 5:1;

[0050] (2), the volume ratio of dichloromethane to trifluoroacetic acid is 3:1;

[0051] The other steps are the same as those in Example 1.

[0052] Examples 1-3

[0053] The only difference from Example 1 is:

[0054] (3), the solvent is N,N-dimethylformamide;

[0055] The other steps are the same as those in Example 1.

[0056] The conditions in Examples 1-1, 1-2, and 1-3 can also achieve the purpose of synthesizing the target product, but the highest yield among them is only about 30%. The yield of Example 1 is higher than that of Examples 1-1, 1-2, and 1-3, which is about 50%. Therefore, Example 1 is preferred as the reaction condition.

[0057] The reaction conditions in Example 1 were adopted, and only the reaction raw materials were replaced with different types of Boc-amino acids and hydroxyl-containing nitroxide free radicals, and finally the target products shown in Table 1 were obtained. The specific structures of the raw materials are shown in Table 1:

[0058] Table 1 Rhein nitrogen oxide free radical derivatives general formula 1 and raw material structure

[0059]

[0060]

[0061]

[0062]

[0063]

[0064] Test Example 1 Evaluation of in vitro antioxidant activity of rhein nitroxide free radical derivatives The in vitro antioxidant capacity of rhein and its nitroxide free radical derivatives in Example 1 was determined by referring to the DPPH method and ABTS method described in GB / T 39100-2020. The results are shown in Table 2: Table 2 IC values ​​of nitroxide free radicals, rhein and its nitroxide free radical derivatives for scavenging DPPH free radicals and ABTS free radicals 50

[0065]

[0066] As shown in Table 2, the IC of rhein to scavenge DPPH free radicals is 50 The IC value of scavenging ABTS free radicals was 26.63±5.77mM. 50 The concentration of rhein nitroxide free radical derivatives was 9.37±0.07mM. The scavenging ability of rhein nitroxide free radical derivatives on DPPH free radical and ABTS free radical increased with the concentration. The antioxidant activity of the modified derivatives was significantly improved. Compound 16b showed the strongest antioxidant activity in both methods. Its IC 50 They are 0.51±0.09mM and 0.12±0.03mM respectively.

[0067] Table 3 Protective effect of rhein nitroxide derivatives on H2O2-induced L02 cells

[0068]

[0069]

[0070] From the data in Table 3, it can be seen that the experimental results of the protective effect of rhein nitroxide free radical derivatives on H2O2-induced oxidative damage L02 cells showed that the cell survival rate of the oxidative damage model group was 50.95%, and the cell survival rate of the rhein group was increased to 56.76%. The cell survival rates of compounds 3h and 7d were lower than those of the model group and had no protective effect. The survival rates of compounds 7f and 12d were higher than those of the model group and had a protective effect, but lower than the cell survival rate of the parent rhein. The cell survival rates of the remaining compounds were higher than those of the rhein group and the model group. Among them, compound 16b had the strongest protective effect, and the cell survival rate was significantly improved to 95.42%. Specifically, Figure 1 As shown in Table 3 (model represents the model group, Rhein refers to rhein).

[0071] Test Example 2 Evaluation of the in vivo antioxidant activity of rhein nitroxide free radical derivatives

[0072] After preliminary in vitro antioxidant experiment screening, compound 16b with good in vitro antioxidant activity was selected. In this experiment, rhein, rhein nitroxide free radical derivative 16b and nitroxide free radical 16 (Formula 4) were applied to the Caenorhabditis elegans model.

[0073] Table 4 Effects of different concentrations of compounds 16, 16b and rhein on the lifespan of wild-type Caenorhabditis elegans

[0074]

[0075] Table 5 Effects of compounds 16, 16b and rhein on heat stress resistance of wild-type Caenorhabditis elegans

[0076]

[0077] Table 6 Effects of compounds 16, 16b and rhein on oxidative stress resistance of wild-type Caenorhabditis elegans

[0078]

[0079] From the data in Tables 4 to 6, it can be seen that within the concentration range of 50 to 300 μM, compounds 16, 16b and rhein all increased the average lifespan of Caenorhabditis elegans in a concentration-dependent manner. The average lifespan of the control group was 10.93±0.20 h, and the average lifespan of compounds 16, 16b and rhein at high concentrations was significantly increased. Among them, compound 16b had a maximum lifespan of 27.0±0.82 h at the highest concentration, and an average lifespan of 15.41±0.29 h, which was 41.09% higher than that of the control group, and the difference was extremely significant (p<0.001). The effects of compounds 16, 16b and rhein on the heat shock resistance and antioxidant capacity of nematodes were determined, and the survival time of nematodes at 35°C was determined. The results showed that the average survival time of nematodes in the control group was 6.33±0.19 h, and the longest survival time was 11.33±0.47 h. Compared with the control group, the longest survival time and average survival time of the treatment group were prolonged to varying degrees. The longest survival time and average survival time of the mother rhein were significantly higher than those of the control group, reaching 13h and 8.62±0.28h (p<0.01), respectively; while the longest survival time of compound 16b reached 14.33±0.47h, and the average survival time increased by 48.09% compared with the control group, reaching 9.38±0.26h (p<0.01). The effects of compound 16b and rhein on the survival rate under oxidative stress conditions were determined, and the results showed that the average survival time of the nematodes in the control group was 13.78±0.49h, and the maximum survival time was 26±0h. The average survival time of the treatment group was improved compared with the control group. The average lifespan of rhein was 15.6±0.28h, which was significantly different from the control group and extended by 13.44% (p<0.05). The average lifespan of compound 16b was extended by 31.91% compared with the control group, reaching 18.13±0.52h (p<0.05). Figures 2-3 As shown in Tables 4 to 6 (Control represents blank control group, Rhein refers to rhein).

[0080] The ROS level in nematodes was detected by the DCFH-DA fluorescent probe method; the GSH activity and MDA content in Caenorhabditis elegans were detected by ELISA kits.

[0081] Table 7 Effects of compounds 16, 16b and rhein on ROS levels, GSH levels and MDA content in wild-type Caenorhabditis elegans

[0082] project Relative level of reactive oxygen species Relative content of glutathione Relative content of malondialdehyde Control 1 1 1 Rhein 0.67 1.20 0.81 16 0.81 1.12 0.97 16b 0.46 1.28 0.65

[0083] The results in Table 7 show that the drug-treated group can significantly reduce the fluorescence intensity of ROS in nematodes, and compound 16, rhein and compound 16b can significantly reduce the accumulation of ROS in C. elegans (p<0.001). Compound 16 and rhein can significantly increase the activity of GSH in nematodes (p<0.05), while compound 16b significantly increases the activity of GSH (p<0.01). Rhein and compound 16b can reduce the content of MDA (p<0.05). Therefore, compound 16b can effectively reduce the accumulation of ROS, increase the activity of GSH, and reduce the level of MDA. Specifically, Figures 4 to 6 As shown in Table 7 (Control represents blank control group, Rhein refers to rhein).

Claims

1. A rhein nitroxide free radical derivative, characterized in that: The structure of the rhein nitroxide free radical derivative is shown in Formula 1: R1 is a nitroxide free radical, and Linker represents different types of amino acids.

2. The method for synthesizing the nitrogen oxide free radical derivative of rhein as claimed in claim 1, comprising the following steps: (1) Using Boc-amino acid and hydroxyl-containing nitroxide free radical as raw materials, an amide condensation reaction is carried out under the action of catalyst I to obtain intermediate I; (2) Trifluoroacetic acid is added dropwise to the intermediate I, reacted, extracted, and the organic layer is collected and then dehydrated and dried to obtain the intermediate II; (3) Using intermediate II and rhein as raw materials and tetrahydrofuran as solvent, reacting under the action of catalyst II, filtering out insoluble matter, spinning off the solvent, and separating by chromatography to obtain a rhein nitroxide free radical derivative having the structure described in general formula 1.

3. The synthesis method according to claim 2, characterized in that The method is specifically as follows: (1) Boc-amino acid and hydroxyl-containing nitroxide free radicals are used as raw materials, N,N-dicyclohexylcarbodiimide and 4-dimethylaminopyridine are used as catalysts I, and dichloromethane is used as solvent. The reaction is carried out at room temperature for 5 to 12 hours under the protection of Ar gas, and then the solvent is recovered by vacuum distillation and chromatographic separation is performed to obtain intermediate I; (2) Using intermediate I in (1) as a raw material and dichloromethane as a solvent, trifluoroacetic acid was added dropwise in an ice bath, and the reaction was carried out at 0°C for 0.5 to 1 h. The reaction was detected by thin layer chromatography until the reaction was complete. Saturated sodium bicarbonate was added to adjust the pH to alkaline, and dichloromethane was used for extraction. The organic layer was collected, dehydrated, and dried to obtain intermediate II; (3) Using intermediate II and rhein as raw materials, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 4-dimethylaminopyridine, and triethylamine as catalyst II, and tetrahydrofuran as solvent, the reaction is carried out at room temperature under the protection of Ar gas, the insoluble matter is filtered out, the solvent is removed by rotation, and the reaction is separated by chromatography to obtain a rhein nitroxide free radical derivative having the structure described in general formula 1.

4. The synthesis method according to claim 2, characterized in that The Boc-amino acid is selected from any one of Boc-glycine, Boc-β-alanine, Boc-L-alanine, Boc-L-valine, Boc-L-leucine, Boc-L-methionine, Boc-L-phenylalanine and N-tert-butyloxycarbonyl-2-methylalanine; The hydroxyl-containing nitroxide free radical is selected from any one of Formula 1 to Formula 4:

5. The synthesis method according to claim 3, characterized in that In (1), the molar ratio of Boc-amino acid, hydroxyl-containing nitroxide free radical, N,N-dicyclohexylcarbodiimide and 4-dimethylaminopyridine is 1:1:2:1 to 5:1:6:1, respectively; the molar ratio of solvent dichloromethane to Boc-amino acid is 100:1 to 200:

1.

6. The synthesis method according to claim 3, characterized in that (2), the molar ratio of the solvent dichloromethane to the intermediate I is 40:1 to 160:1; the volume ratio of the solution of trifluoroacetic acid to the solvent dichloromethane is 1:1 to 1:

3.

7. The synthesis method according to claim 3, characterized in that In (3), the molar ratios of intermediate II, rhein, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine are 1:1:1:1 to 1:3:1:2, respectively; the molar ratio of triethylamine to rhein is 0.1:1 to 0.5:1; and the molar ratio of solvent tetrahydrofuran to rhein is 400:1 to 500:

1.

8. Use of the rhein nitroxide free radical derivative according to claim 1 in the preparation of antioxidant and / or anti-aging products.

Images