A resveratrol derivative, and a preparation method and application thereof

Abstract

The application belongs to the technical field of anti-inflammatory and antioxidant drug development, and discloses a resveratrol derivative, a preparation method and application thereof. The resveratrol derivative is a compound shown in formula 2 or a pharmaceutically acceptable salt thereof, and a solvate of the compound shown in formula 2 or the pharmaceutically acceptable salt thereof. The resveratrol derivative is a novel compound, and the compound is designed and successfully synthesized for the first time, and the structure of the compound is characterized. The preparation method of the resveratrol derivative is simple, convenient to operate, and capable of rapidly synthesizing the compound. Research shows that the resveratrol derivative has good anti-inflammatory and antioxidant activities, and has a good application prospect in the treatment of inflammation and oxidative stress.

Description

Technical Field

[0001] This invention belongs to the field of anti-inflammatory and antioxidant drug development technology, and specifically relates to a resveratrol derivative, its preparation method and application. Background Technology

[0002] Resveratrol, chemically named 3,4',5-trihydroxy-1,2-diphenylethylene, has the molecular formula C2. 14 H 12 O3, with a molecular weight of 228.23 and CAS number 501-36-0, has the structure shown in Formula 1. Resveratrol was first isolated from the roots of Veratrum nigrum by Japanese scientists. It is an antitoxin secreted by the plant in response to stress or pathogen attack. Common sources include peanuts, grapes, mulberries, and cranberries.

[0003]

[0004] Resveratrol, a non-flavonoid polyphenol compound, possesses various biological activities, including anti-inflammatory, antioxidant, anticancer, and cardiovascular protective effects. Studies have shown that resveratrol can scavenge free radicals to protect biomolecules from damage and can also increase the expression of antioxidant enzymes through multiple pathways, activate SIRT1, and improve mitochondrial function. In terms of anti-inflammation, resveratrol can inhibit cyclooxygenases COX1 and COX2, as well as tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) to combat inflammation. However, the oral bioavailability of resveratrol is low, approximately 12%, requiring relatively large doses to achieve effective concentrations in plasma or tissues. Therefore, rapid absorption, low bioavailability, and poor water solubility are key factors limiting the clinical use of resveratrol.

[0005] Molecular hybridization refers to the chemical synthesis of two active molecular fragments to form new active compounds. Molecular hybridization can enhance the pharmacological activity of active molecules and reduce their toxic side effects. Therefore, by extending an aldehyde group onto the benzene ring of resveratrol, and using this as a basis to link with benzoyl hydrazides to form new compounds, the aim is to improve its pharmacological activity and bioavailability. Summary of the Invention

[0006] In order to overcome the shortcomings and deficiencies of the prior art, the primary objective of this invention is to provide a resveratrol derivative that can enhance the anti-inflammatory and antioxidant activity of resveratrol.

[0007] Another object of the present invention is to provide a method for preparing the above-mentioned resveratrol derivative.

[0008] Another object of the present invention is to provide the application of the above-mentioned resveratrol derivative, which, due to its good in vitro anti-inflammatory and antioxidant activity, is suitable as a novel drug for inflammatory or oxidative stress diseases.

[0009] The objective of this invention is achieved through the following technical solution:

[0010] A resveratrol derivative, said derivative being a compound with the structure shown in Formula 2 or a pharmaceutically acceptable salt thereof, and a solvent compound of said compound with the structure shown in Formula 2 or a pharmaceutically acceptable salt thereof:

[0011]

[0012] Wherein, R is one of the following groups:

[0013]

[0014] The specific functional groups of the above compounds are summarized in Table 1:

[0015] Table 1. Compound Numbers and Structures

[0016]

[0017]

[0018] The pharmaceutically acceptable salt is preferably a salt formed by the compound of Formula 2 with hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, acetic acid, fumaric acid, maleic acid, oxalic acid, malonic acid, succinic acid, citric acid, malic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, toluenesulfonic acid, glutamic acid, or aspartic acid.

[0019] The preparation method of the above-mentioned resveratrol derivative includes the following steps:

[0020] (1) Resveratrol was reacted with N,N-dimethylformamide and phosphorus oxychloride to obtain intermediate I with the structure shown in Formula 3;

[0021]

[0022] (2) Using intermediate I and different benzoyl hydrazine derivatives as raw materials, resveratrol derivatives with the structure shown in Formula 2 were obtained by heating reaction and purification.

[0023] The molar ratio of resveratrol, N,N-dimethylformamide and phosphorus oxychloride in step (1) is 1:1.1:1.1.

[0024] The specific steps (1) are as follows: using acetonitrile as a solvent, N,N-dimethylformamide and phosphorus oxychloride are added under stirring in an ice-salt bath, followed by resveratrol, and the reaction is carried out overnight; a red solid is obtained by filtration and washed with cold acetonitrile; the obtained solid is transferred to a new reaction flask, water is added, and the reaction is carried out at 55°C for 3-5 hours, and intermediate I is obtained by filtration.

[0025] The molar ratio of intermediate I and benzoyl hydrazine derivatives in step (2) is 1:1; the reaction conditions are 70℃ for 1 to 5 hours.

[0026] The synthetic route for the resveratrol derivative is shown below, where all R groups are groups listed in Table 1:

[0027]

[0028] The above-mentioned resveratrol derivatives are used in the preparation of drugs for the prevention and treatment of inflammatory diseases and oxidative stress.

[0029] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0030] (1) The resveratrol derivatives provided by this invention are novel types of compounds that have never been reported before. This invention designs and successfully synthesizes these compounds for the first time, and characterizes their structures.

[0031] (2) The preparation method of the resveratrol derivatives of the present invention is simple and convenient to operate, and can quickly synthesize these compounds.

[0032] (3) Through extensive and in-depth research, this invention has synthesized a large number of resveratrol derivatives with novel structures and anti-inflammatory and antioxidant activities, and screened for anti-inflammatory and antioxidant activities. For the first time, it was discovered that this type of compound has good in vitro anti-inflammatory and antioxidant activities and is suitable as a new drug for the prevention and treatment of inflammatory diseases and oxidative stress. Attached Figure Description

[0033] Figure 1 This is the NMR spectrum of compound 1.

[0034] Figure 2 This is the NMR spectrum of compound 2.

[0035] Figure 3 This is the NMR spectrum of compound 19.

[0036] Figure 4 The MTT assay was used to determine the cytotoxicity of resveratrol and its derivatives on RAW264.7 cells.

[0037] Figure 5The inhibitory effect of resveratrol and its derivatives on NO content in RAW264.7 cells was determined using the Griess method.

[0038] Figure 6 The inhibitory effect of resveratrol and its derivatives on reactive oxygen species (ROS) production in RAW264.7 cells was analyzed by flow cytometry. Detailed Implementation

[0039] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0040] Example 1 Preparation of Intermediate I

[0041] Add 30 mL of acetonitrile to a single-diameter round-bottom flask, incubate in an ice-salt bath for 5 min, then add N,N-dimethylformamide (1.76 g, 24.1 mmol) and phosphorus oxychloride (3.69 g, 24.1 mmol). Stir for 5 min, then add resveratrol (5 g, 21.9 mmol). Stir overnight, then filter to collect the solid and wash with cold acetonitrile. Transfer the obtained solid to a new single-diameter round-bottom flask, add 30 mL of water, and react at 55 °C for 3–5 h. Filter to collect the solid, wash with cold water, and dry to obtain intermediate I with the structure shown in Formula 3, in 80% yield.

[0042] Example 2 Synthesis of N'-(Z)-2,4-dihydroxy-6-((E)-4-hydroxystyryl)benzyl)benzoylhydrazine (Compound 1)

[0043] Intermediate I (0.5 g, 1.95 mmol) and benzoyl hydrazide (0.26 g, 1.95 mmol) were added to a single-diameter round-bottom flask and dissolved in 5 mL of anhydrous ethanol. The mixture was stirred at 70 °C for 1–5 h, during which a solid precipitated. The reaction was monitored by TLC. After the reaction was complete, the solid was obtained by filtration and purified by recrystallization to give the product N'-(Z)-2,4-dihydroxy-6-((E)-4-hydroxystyryl)benzyl)benzoyl hydrazide. The NMR spectrum of the product is shown below. Figure 1 As shown, 1H NMR (600MHz, DMSO-d6) δ12.41(s,1H),11.94(s,1H),10.02(s,1H),9.67(s,1H),9.00(s,1H),8.02–7.89(m,2H),7.65–7.59(m,1H),7.55(dd,J=8. 3,6.9Hz,2H),7.51–7.46(m,2H),7.32(d,J=16.0Hz,1H),6.94(d,J=16.0 Hz,1H),6.84–6.77(m,2H),6.58(d,J=2.3Hz,1H),6.28(d,J=2.3Hz,1H). The number and shift of hydrogen atoms in compound 1 are similar to the predictions, thus the structure of the product is N'-(Z)-2,4-dihydroxy-6-((E)-4-hydroxystyryl)benzylidene)benzoylhydrazine.

[0044] Example 3 Synthesis of compounds 2-20

[0045] Intermediate I (0.5 g, 1.95 mmol) and benzoylhydrazine derivatives 2–20 (1.95 mmol) were added to a single-diameter round-bottom flask and dissolved in 5 mL of anhydrous ethanol. The mixture was stirred at 70 °C for 1–5 h, during which solids precipitated. The reaction was monitored by TLC. After the reaction was complete, the solids were filtered and purified by recrystallization to obtain compounds 2–20. The NMR spectra of compounds 2 and 19 are shown below. Figure 2 and Figure 3 As shown.

[0046] The yields of the above compounds are summarized in Table 2.

[0047] Table 2 Yields of compounds 1–20

[0048]

[0049] Effect Example

[0050] 1. Experimental Methods

[0051] 1.1 DPPH free radical scavenging experiment

[0052] 190 μL of a pre-prepared 100 μmol / L DPPH solution was added to each well of a 96-well plate, followed by 10 μL of compound 1-20 (resveratrol derivatives prepared in Examples 2-3) at a final concentration of 5–50 μmol / L. A control group was also included, maintaining a total volume of 200 μL. The reaction was allowed to proceed in the dark at room temperature for 30 min, and the absorbance was measured at 517 nm. The DPPH radical scavenging rate was calculated as follows: DPPH scavenging rate = [1 - (Am - An) / As] × 100%. Am represents the OD value of the sample group, An represents the OD value of the methanol solution, and As represents the OD value of the negative control group. A standard curve was plotted with the concentration of the resveratrol derivative as the x-axis and the scavenging rate corresponding to each resveratrol derivative concentration as the y-axis, and the linear regression equation and correlation coefficient (R²) were obtained. 2 The sample concentration measured when 50% of DPPH free radicals were removed was expressed using IC50. 50 The values ​​are expressed as shown in Table 3. Compounds 5, 6, 7, 8, and 9 all exhibited stronger DPPH radical scavenging ability than resveratrol, with compounds 6 and 9 showing the strongest scavenging ability (IC50). 50 The values ​​were 37.8 and 39.8 μM, respectively.

[0053] 1.2 ABTS Free Radical Scavenging Experiment

[0054] A 2.45 mmol / L K₂S₂O₈ solution was prepared using 7 mmol / L ABTS solution and incubated at room temperature in the dark for 12–16 h to obtain an ABTS radical stock solution. The stock solution was diluted with methanol solution (50:1) to achieve an absorbance of 0.7 ± 0.02 at 734 nm, yielding the ABTS+ working solution. 195 μL of the prepared ABTS+ working solution was added to each well of a 96-well plate, followed by 5 μL of compound 1-20 (the resveratrol derivative prepared in Examples 2–3) at a final concentration of 2.5–30 μmol / L. A control group was also included. The reaction was allowed to proceed at room temperature in the dark for 20 min, and the absorbance was then measured at 734 nm. The ABTS cationic radical scavenging rate was calculated using the following formula: ABTS scavenging rate = [1 - (Am - An) / As] × 100%. Am represents the OD value of the sample group, An represents the OD value of the methanol solution, and As represents the OD value of the negative control group. The sample concentration measured when 50% of ABTS free radicals were removed was determined using IC50. 50 The values ​​are expressed as shown in Table 3. Compounds 5, 6, 7, 8, and 9 all exhibited stronger ABTS radical scavenging ability than resveratrol. Most of these compounds showed enhanced ABTS radical scavenging ability compared to resveratrol, with compounds 8 and 9 exhibiting the strongest scavenging abilities (IC50). 50 The values ​​are 9.7 and 8.5 μM, respectively.

[0055] Table 3

[0056]

[0057]

[0058] 1.3 Cytotoxicity assay

[0059] Mouse monocyte-macrophage RAW 264.7 cells were passaged in DMEM medium containing 10% fetal bovine serum, 100 mg / L penicillin, and 100 mg / L streptomycin at 37°C in a 5% CO2 incubator. RAW 264.7 cells in the logarithmic growth phase were harvested and the cell density was adjusted to 1 × 10⁻⁶ cells / year. 5 Cells were seeded at a density of 100 μL / ml in 96-well plates. After 12 h of cell adhesion, the culture medium was replaced with DMEM containing or without 50 μmol / L compound 1-20 (the resveratrol derivative prepared in Examples 2-3), with three replicates for each concentration. After 24 h of incubation, 100 μL of MTT was added to each well, and the cells were cultured for another 4 h. The supernatant was discarded, and 150 μL of dimethyl sulfoxide (DMSO) was added to each well. The plates were shaken for 10 min to allow the crystals to dissolve completely, and the absorbance (A) was measured at 490 nm. The experiment was repeated three times. Cell viability (%) was calculated as (Adetector well / Ablank well) × 100%. The experimental results are as follows: Figure 4 As shown, compounds 5, 7, 8, 18, 19 and 20 had no significant inhibitory effect on RAW 264.7 cells.

[0060] 1.4 Effects of Compounds on NO Content

[0061] Take a suspension of RAW264.7 cells in the logarithmic growth phase and adjust the cell density to 2 × 10⁻⁶. 5 NO was inoculated at a concentration of 100 μL / mL into a 96-well plate. After 12 h of adhesion, 100 μL of compound 1-20 (a resveratrol derivative prepared in Examples 2-3) at a concentration of 50 μmol / L was added and incubated for 3 h. Then, 100 ng / L LPS was added and incubated for 12 h. A blank group, an LPS group, and an LPS + drug treatment group were set up, with 3 replicates for each group. The NO content in the supernatant was detected by the Griess method.

[0062] 1.5 Effects of Compounds on ROS Expression

[0063] Take a suspension of RAW264.7 cells in the logarithmic growth phase and adjust the cell density to 2 × 10⁻⁶. 5The cells were seeded at a concentration of 1 mL / well in a 12-well plate. After 12 h of adhesion, compound 1-20 (a resveratrol derivative prepared in Examples 2-3) at a concentration of 50 μmol / L was added and incubated for 3 h. Then, 800 μmol / L H2O2 was added and incubated for 3 h. Finally, DCEH-DA was added and treated in the dark for 1 h. A blank group, an LPS group, and an LPS+ drug-treated group were set up, with 3 replicates for each group. The ROS production was detected by flow cytometry.

[0064] 1.6 Data Analysis

[0065] Data analysis was performed using Prism software. The t-test was used for comparisons between groups, and P < 0.05 was considered statistically significant.

[0066] 2. Results

[0067] Based on the MTT assay results, six compounds with no obvious toxicity were selected to study the effects of compounds 5, 7, 8, 18, 19, and 20 on LPS-induced NO release and ROS expression in RAW264.7 cells. The results are as follows: Figure 5 and 6 As shown.

[0068] The results showed that compound 5 of the present invention did not have a significant inhibitory effect on RAW264.7 cells, but it could significantly reduce the release level of the inflammatory factor NO induced by LPS in RAW264.7 cells and the expression level of ROS in RAW264.7 cells stimulated by H2O2. It has good anti-inflammatory and antioxidant activity and can be used as an active ingredient of anti-inflammatory and antioxidant drugs.

[0069] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A resveratrol derivative, characterized by: The derivative is a compound with the structure shown in Formula 2 or a pharmaceutically acceptable salt thereof: Formula 2 Wherein, R is one of the following groups: , , , or .

2. The resveratrol derivative according to claim 1, characterized in that: The pharmaceutically acceptable salt is a salt formed by the compound of Formula 2 with hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, acetic acid, fumaric acid, maleic acid, oxalic acid, malonic acid, succinic acid, citric acid, malic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, toluenesulfonic acid, glutamic acid, or aspartic acid.

3. The method of preparing a resveratrol derivative according to claim 1 or 2, characterized in that It includes the following steps: (1) Resveratrol was reacted with N,N-dimethylformamide and phosphorus oxychloride to obtain intermediate I with the structure shown in Formula 3; (2) Using intermediate I and different benzoyl hydrazine derivatives as raw materials, resveratrol derivatives with the structure shown in Formula 2 were obtained by heating reaction and purification.

4. The method of claim 3, wherein: The molar ratio of resveratrol, N,N-dimethylformamide and phosphorus oxychloride in step (1) is 1:1.1:1.

1.

5. The method of claim 3, wherein: The specific steps (1) are as follows: using acetonitrile as solvent, N,N-dimethylformamide and phosphorus oxychloride are added under stirring in an ice-salt bath, followed by resveratrol, and the reaction is carried out overnight; a red solid is obtained by filtration and washed with cold acetonitrile; the obtained solid is transferred to a new reaction flask, water is added, and the reaction is carried out at 55°C for 3-5 hours, and intermediate I is obtained by filtration.

6. The method of claim 3, wherein: The molar ratio of intermediate I and benzoyl hydrazine derivatives in step (2) is 1:1; the reaction conditions are 70℃ for 1 to 5 hours.

7. The use of the resveratrol derivative according to claim 1 or 2 in the preparation of drugs for the prevention and treatment of inflammatory diseases and oxidative stress.

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