A rhein nitroxide free radical derivative and its synthesis method and application

Abstract

The present invention belongs to the technical field of organic chemical synthesis, and specifically relates to a rhein nitroxide derivative, a synthesis method, and an application thereof. The present invention uses rhein, an antioxidant pharmacological substance from the traditional Chinese medicine rhubarb, as a research object, selects its carboxyl group at position 3 as a modification site, and introduces four active intermediate nitroxides into the rhein structure based on the amide condensation and esterification reaction mechanisms and the pharmacophore splicing principle. Thirty rhein nitroxide derivatives were designed and synthesized, thereby establishing a method for synthesizing novel rhein nitroxide derivatives with mild reaction conditions, environmental friendliness, and high yield. The provided novel rhein nitroxide derivatives have the ability to scavenge DPPH and ABTS free radicals, have a protective effect on oxidatively damaged cells, and have the ability to delay the lifespan of Caenorhabditis elegans. Their effects are superior to those of rhein, providing a new approach for the development of antioxidant and anti-aging drugs.

Description

Technical Field

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

[0002] Rhein, a trihydroxyanthraquinone compound extracted from the Polygonaceae plant Rheum officinale, is the active ingredient in the traditional Chinese medicine rhubarb. Studies have shown that rhein exhibits antioxidant, anti-inflammatory, and anti-tumor properties. Studies have also shown that rhein protects cells from oxidative damage, suggesting that it is a precursor compound with antioxidant activity. However, rhein is poorly water-soluble, resulting in low bioavailability in vivo.

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

[0004] If rhein can be used as a research object and 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 its synthesis method and application. 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 solutions of the present invention are 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 as raw materials, an amide condensation reaction is carried out in the presence of catalyst I to obtain intermediate I;

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

[0015] (3) Using intermediate II and rhein as raw materials and tetrahydrofuran as solvent, reacting in the presence of catalyst II, filtering to remove insoluble matter, removing the solvent by vortexing, 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 a hydroxyl-containing nitroxide free radical, 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 the 3 position to synthesize different rhein nitroxide free radical derivatives of general formula 1.

[0017] A more specific method is:

[0018] Using Boc-amino acid and hydroxyl-containing nitroxide free radical as raw materials, N,N-dicyclohexylcarbodiimide and 4-dimethylaminopyridine as catalyst I, and dichloromethane as solvent, the reaction is carried out at room temperature for 5-12 hours under Ar protection, and then the solvent is recovered by distillation under reduced pressure and separated by chromatography; using the intermediate as raw material and dichloromethane as solvent, trifluoroacetic acid is added dropwise in an ice bath, and the reaction is carried out at 0°C for 0.5-1 hour. Thin layer chromatography is performed until the reaction is complete, saturated sodium bicarbonate is added to adjust the pH to alkaline, and the product is extracted with dichloromethane. The organic layer is collected, dehydrated, and dried to obtain intermediate II; then using the intermediate 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 Ar protection, the insoluble matter is removed by filtration, the solvent is removed by swirl, and separated by chromatography to finally obtain the rhein nitroxide free radical derivative of general formula 1.

[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 nitrogen oxide free radical derivatives in anti-oxidation and / or anti-aging drugs.

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

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

[0025] (2) The novel rhein nitrogen oxide 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 effect of compounds 16, 16b and rhein on the heat stress resistance of wild-type Caenorhabditis elegans;

[0028] Figure 3 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, 16, 16b and Rhein in the treatment group; 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 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, nsp>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 will be further described in conjunction with specific embodiments.

[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) in 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 by thin layer chromatography. Stop the reaction until the reaction is complete, spin dry the solvent, and purify by 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 rod was placed, and dichloromethane (92.53 mmol, 4 mL) was added to dissolve the mixture. The mixture was placed in an ice bath, and trifluoroacetic acid (2 mL) was slowly added dropwise. The mixture was reacted in an ice bath for 1 h, and thin-layer chromatography was performed. When the reaction was complete, 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 dried to obtain intermediate II.

[0041] (3) Rhein (0.36 mmol) was weighed into a round-bottom flask, a magnetic rod was placed, and tetrahydrofuran (156.01 mmol, 10 mL) was added to dissolve it. 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.70 mmol) and 4-dimethylaminopyridine (0.60 mmol) were added to the reaction solution in sequence. Intermediate II (0.48 mmol) was added, and triethylamine (100 μL) was added. The reaction was allowed to react at room temperature for 24 h, and the reaction was detected by thin-layer chromatography until the reaction was complete. The reaction was stopped, the insoluble matter was removed by filtration, the solvent was removed by vortexing, and the product was purified by silica gel column chromatography (dichloromethane:methanol v:v=150:1) to 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 radicals to finally obtain the target products shown in Table 1. 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 the in vitro antioxidant activity of rhein nitroxide derivatives The in vitro antioxidant capacity of rhein and its nitroxide derivatives in Example 1 was determined with reference 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 nitroxides, rhein and its nitroxide derivatives for scavenging DPPH free radicals and ABTS free radicals 50

[0065]

[0066] As shown in Table 2, the IC of rhein for scavenging DPPH free radicals is 50 The IC value of scavenging ABTS free radicals was 26.63±5.77mM. 50 The concentration of rhein nitroxide derivatives was 9.37±0.07mM. The scavenging ability of rhein nitroxide derivatives to DPPH free radical and ABTS free radical increased with the increase of 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] The data in Table 3 show that the protective effect of rhein nitroxide derivatives on H2O2-induced oxidative damage in L02 cells. The experimental results show that the cell survival rate of the oxidative damage model group was 50.95%, while the cell survival rate of the rhein group increased to 56.76%. The cell survival rates of compounds 3h and 7d were lower than those of the model group, indicating no protective effect. The cell survival rates of compounds 7f and 12d were higher than those of the model group, indicating a protective effect, but lower than that 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, with a significantly increased cell survival rate of 95.42%. Figure 1 As shown in Table 3 (model represents the model group, Rhein refers to rhein).

[0071] Test Example 2 Evaluation of the Antioxidant Activity of Rhein Nitrogen Oxygen Free Radical Derivatives in Vivo

[0072] After preliminary in vitro antioxidant screening, compound 16b with good in vitro antioxidant activity was selected. In this experiment, rhein, rhein nitroxide derivative 16b and nitroxide 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] The data in Tables 4-6 show that compounds 16, 16b, and rhein all increased the average lifespan of C. elegans within a concentration range of 50-300 μM in a concentration-dependent manner. While the average lifespan of the control group was 10.93±0.20 h, high concentrations of compounds 16, 16b, and rhein significantly increased the average lifespan. Compound 16b, at the highest concentration, achieved a maximum lifespan of 27.0±0.82 h, with an average lifespan of 15.41±0.29 h, representing a 41.09% increase compared to the control group, a highly significant difference (p<0.001). The effects of compounds 16, 16b, and rhein on the nematodes' heat shock resistance and antioxidant capacity were determined. Survival time at 35°C was measured. The results showed that the average survival time of the control group was 6.33±0.19 h, with the longest survival time being 11.33±0.47 h. Compared with the control group, the maximum and average survival times of the treated groups were prolonged to varying degrees. The maximum and average survival times of the maternal nematodes treated with rhein were significantly increased compared to the control group, reaching 13 hours and 8.62±0.28 hours, respectively (p<0.01). Compound 16b, on the other hand, had a maximum survival time of 14.33±0.47 hours, and an average survival time of 9.38±0.26 hours, a 48.09% increase compared to the control group (p<0.01). The effects of compound 16b and rhein on survival 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±0.49 hours, with a maximum survival time of 26±0 hours. The average survival time of the treated groups 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 the blank control group, Rhein refers to rhein).

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

[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 glutathione content 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 GSH activity in nematodes (p<0.05), while compound 16b significantly increases GSH activity (p<0.01). Rhein and compound 16b can reduce MDA content (p<0.05). Therefore, compound 16b can effectively reduce ROS accumulation, increase GSH activity and reduce MDA levels. Figures 4-6 As shown in Table 7 (Control represents the 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 selected from one of the following:

2. The method for synthesizing a nitrogen oxide free radical derivative of rhein according to claim 1, wherein: The method comprises the following steps: (1) Boc-amino acid and hydroxyl-containing nitroxide free radicals are used as raw materials, N,N-dicyclohexylcarbodiimide and 4-dimethylaminopyridine are used as catalyst I, and dichloromethane is used as solvent. The reaction is carried out at room temperature for 5-12 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 the raw material and dichloromethane as the solvent, trifluoroacetic acid was added dropwise in an ice bath, and the reaction was carried out at 0°C for 0.5-1h. Thin layer chromatography was performed to detect when 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 removed by filtration, the solvent is removed by spin-drying, and the reaction is separated by chromatography to obtain the rhein nitroxide free radical derivative having the structure described in claim 1; The hydroxyl-containing nitroxide radical is selected from any one of Formulas 1 to 4:

3. The method for synthesizing a nitrogen oxide free radical derivative of rhein according to claim 2, wherein: In (1), the molar ratios of Boc-amino acid, hydroxyl-containing nitroxide radical, N,N-dicyclohexylcarbodiimide and 4-dimethylaminopyridine are 1:1:2:1 to 5:1:6:1, respectively; and the molar ratio of solvent dichloromethane to Boc-amino acid is 100:1 to 200:

1.

4. The method for synthesizing a nitrogen oxide free radical derivative of rhein according to claim 2, wherein: In (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.

5. The method for synthesizing a nitrogen oxide free radical derivative of rhein according to claim 2, wherein: 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.

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

Images