A caffeic acid derivative, a preparation method and application thereof

Abstract

The application belongs to the technical field of antioxidant drug development, and discloses a caffeic acid derivative, a preparation method and application thereof. The caffeic acid 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 caffeic acid derivative is a novel compound, and the compounds are designed and successfully synthesized for the first time, and the structures thereof are characterized. The preparation method of the caffeic acid derivative is simple, convenient to operate, and capable of rapidly synthesizing the compounds. Research finds that the caffeic acid derivative has good antioxidant activity, and has a good application prospect in the treatment of oxidative stress.

Description

Technical Field

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

[0002] Caffeic acid, chemically named 3-(3,4-dihydroxyphenyl)acrylic acid, has the molecular formula C9H8O4, a molecular weight of 180.157, CAS number 331-39-5, and structural formula as shown in Formula 1. Caffeic acid is a natural phenolic acid compound found in various plants, such as coffee, vegetables, grapes, olive oil, plant seeds, and medicinal plants, such as cockscomb, artichoke, and honeysuckle.

[0003]

[0004] Caffeic acid, as a major representative of the most abundant polyphenols in natural plants, possesses various biological activities, such as anti-inflammatory, antibacterial, cardiovascular protective, immunomodulatory, and antioxidant pharmacological activities. The benzene ring structure of caffeic acid contains two phenolic hydroxyl groups, which form p-π conjugation with the double bond of the benzene ring, resulting in a uniform electron cloud distribution in the caffeic acid molecule and thus stabilizing the phenolic hydroxyl groups of the benzene ring. The hydroxyl groups of caffeic acid are excellent hydrogen atom donors, giving it good antioxidant activity; it has been shown to be effective against DPPH and O2. 2- and OH - Free radicals possess excellent scavenging capabilities. However, caffeic acid is structurally unstable, and there is relatively little research and analysis on its molecular modifications and conformational relationships with its derivatives, both domestically and internationally. Therefore, designing and developing novel caffeic acid derivatives with stronger biological activity and better drug-like properties is of great significance.

[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, glycine or β-alanine is introduced as a linker at the carboxyl side chain of caffeic acid to connect caffeic acid with different phenolic compounds to form new compounds, aiming to improve their biological activity and drug-likeness. 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 caffeic acid derivative that can enhance the antioxidant activity of caffeic acid.

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

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

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

[0010] A caffeic acid 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 caffeic acid derivative includes the following steps:

[0020] (1) Reacting caffeic acid with thionyl chloride yields intermediate I with the structure shown in Formula 3;

[0021]

[0022] (2) Boc-glycine or Boc-β-alanine is reacted with carbodiimide (EDCI), 4-dimethylaminopyridine (DMAP) and phenolic compounds with different substituents to obtain intermediate II with the structure shown in Formula 4.

[0023]

[0024] Where X = 1, 2

[0025] R1 is any one of the following groups;

[0026]

[0027] (3) Intermediate II was reacted with trifluoroacetic acid to obtain intermediate III with the structure shown in Formula 5;

[0028]

[0029]

[0030] Where X = 1, 2

[0031] R1 is any one of the following groups;

[0032]

[0033] (4) Using dichloromethane as solvent, intermediate I and intermediate III as raw materials, react with triethylamine to obtain a caffeic acid derivative with the structure shown in Formula 2.

[0034] The molar ratio of caffeic acid to thionyl chloride (SOCl2) in step (1) is 1:22.7;

[0035] The molar ratio of Boc-glycine or Boc-β-alanine mentioned in step (2) to carbodiimide (EDCI) and 4-dimethylaminopyridine (DMAP) is 1:0.9:0.14.

[0036] The molar ratio of trifluoroacetic acid and dichloromethane in step (3) is 1:3;

[0037] The molar ratio of intermediate I to intermediate III in step (4) is 1:1.

[0038] Step (1) is specifically performed as follows: thionyl chloride is added dropwise to caffeic acid, and then the reaction system is placed in an oil bath and heated and stirred, then refluxed for 2 hours. When the solution changes from yellow and turbid to clear and transparent, intermediate I is obtained.

[0039] The specific steps (2) are as follows: using dichloromethane as a solvent, add Boc-glycine or Boc-β-alanine, as well as carbodiimide (EDCI) and 4-dimethylaminopyridine (DMAP), stir the solution until clear, add phenolic compounds with different substituents, stir at room temperature for 2 hours; after extraction with distilled water and ethyl acetate, collect the organic phase, wash with saturated sodium bicarbonate solution, dry, evaporate the solvent with a rotary evaporator to obtain an oily crude product; redissolve the oily crude product with ethyl acetate, add silica gel and mix thoroughly, and separate by column chromatography to obtain intermediate II.

[0040] The specific steps (3) are as follows: Dissolve intermediate II in dichloromethane, add trifluoroacetic acid, and stir at room temperature for 0.5 h; evaporate the resulting reaction solution by rotary evaporation to remove dichloromethane and trifluoroacetic acid, and precipitate a white solid to obtain intermediate III.

[0041] The specific steps (4) are as follows: intermediate I is dissolved in CH3CN, then intermediate III is added, and 3-4 drops of triethylamine solution are added to the reaction system. The reaction is carried out at room temperature for 2 hours. The resulting reaction solution is extracted with saturated NaHCO3 solution and ethyl acetate, and the organic phase is collected. The solvent is removed from the organic phase by rotary evaporation to obtain the crude product. The crude product is redissolved with ethyl acetate, silica gel is added and mixed thoroughly, and the target compound caffeic acid derivative is separated by column chromatography.

[0042] The synthetic route for the above-mentioned caffeic acid derivatives is shown in the following formula, where the R groups are all... Figure 1 Groups in:

[0043]

[0044] The above-mentioned caffeic acid derivatives are used in the preparation of drugs for preventing and treating oxidative stress.

[0045] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0046] (1) The caffeic acid 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.

[0047] (2) The preparation method of the caffeic acid derivative of the present invention is simple and convenient to operate, and can quickly synthesize this type of compound.

[0048] (3) Through extensive and in-depth research, this invention has synthesized a large number of novel caffeic acid derivatives with antioxidant activity and screened for antioxidant activity. For the first time, it was discovered that this type of compound has good in vitro anti-inflammatory and antioxidant activity, and is suitable as a new drug for the prevention and treatment of oxidative stress. Attached Figure Description

[0049] Figure 1 This is the NMR spectrum of compound 1.

[0050] Figure 2 This is the NMR spectrum of compound 2.

[0051] Figure 3 This is the NMR spectrum of compound 3.

[0052] Figure 4 This is the NMR spectrum of compound 4.

[0053] Figure 5This is the NMR spectrum of compound 15.

[0054] Figure 6 This is the NMR spectrum of compound 16.

[0055] Figure 7 The MTT assay was used to determine the cytotoxicity of caffeic acid derivatives on RAW264.7 cells.

[0056] Figure 8 , 9 10 is a test to determine the scavenging ability of caffeic acid derivatives against DPPH free radicals using the DPPH method. Detailed Implementation

[0057] 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.

[0058] Example 1 Preparation of Intermediate I

[0059] Weigh 2.0 g (11.10 mmol) of caffeic acid and add it to a gaiwan-shaped reaction flask. Place the reaction system in an oil bath and slowly add 18 mL of SOCl2 dropwise along the wall of the gaiwan-shaped flask. Heat and stir, then reflux for 2 h. When the solution changes from yellow turbidity to clear transparency, use thin-layer chromatography (TLC) to spot the starting material and reaction solution on a silica gel plate. When the caffeic acid starting material spot disappears from the reaction mixture, it indicates that the caffeic acid reaction is complete, producing 1.85 g of intermediate I (intermediate I, structure shown in Formula 3), a yellow solid, with a yield of 92.5%.

[0060]

[0061] Example 2 Preparation of Intermediate II

[0062] 4 g (22.83 mmol) of Boc-glycine was dissolved in 20 mL of dichloromethane. The solution was stirred until the Boc-glycine was completely dissolved. Then, 4 g (20.87 mmol) of EDCI solid was added, followed by 0.4 g (3.27 mmol) of DMAP solid. The solution was stirred until clear. Phenolic compounds with different substituents were added, and the mixture was stirred at room temperature for 2 h. The reaction was monitored by TLC. After observing the reaction, the reaction system was transferred from a flask to a separatory funnel and extracted with distilled water and ethyl acetate. The separatory funnel was shaken up and down to fully extract the solution. After standing, the upper aqueous phase was discarded. The organic phase was collected in an Erlenmeyer flask, washed with saturated sodium bicarbonate solution, dried, and the solvent was evaporated using a rotary evaporator to obtain an oily crude product. The intermediate mixture was redissolved in 20 mL of ethyl acetate, and an appropriate amount of silica gel was added and mixed thoroughly. The mixture was separated by a column chromatography to obtain intermediate II (intermediate II, structure shown in Formula 4), a white solid, with a yield of 81%–93%.

[0063]

[0064] Where X = 1, 2

[0065] R1 is any one of the following groups;

[0066]

[0067] Example 3 Preparation of Intermediate III

[0068] 2 g of the obtained intermediate II was dissolved in dichloromethane, and trifluoroacetic acid was added at a ratio of dichloromethane:trifluoroacetic acid (TFA) of 3:1. The mixture was stirred at room temperature for 0.5 h. The reaction progress was monitored by TLC until most or all of the starting material of intermediate II disappeared. The reaction solution was poured into a rotary evaporator flask, and the dichloromethane and trifluoroacetic acid were removed using a rotary evaporator, precipitating a white solid to obtain intermediate III (intermediate III, structure shown in Formula 5), ​​with a yield of 90%–95%.

[0069]

[0070] Where X = 1, 2

[0071] R1 is any one of the following groups;

[0072]

[0073] Example 4: Synthesis of caffeic acid derivatives 1-16

[0074] Intermediate I (10.10 mmol) obtained in Example 1 and a different intermediate III (10.10 mmol) obtained in Example 3 were added to CH3CN, followed by 3-4 drops of triethylamine solution. The reaction was carried out at room temperature for 2 hours. The reaction was monitored by TLC. After the reaction was completed, the reaction solution was extracted with saturated NaHCO3 solution and ethyl acetate, and the organic phase was collected. The solvent was removed by rotary evaporation to obtain the crude product. The crude product was redissolved in ethyl acetate, and an appropriate amount of silica gel was added and mixed thoroughly. The target compounds 1-16 were separated by column chromatography. The NMR spectra of compounds 1, 2, 3, 4, 15, and 16 are shown below. Figures 1-6 As shown.

[0075] The yields of the above compounds are summarized in Table 2.

[0076] Table 2 Yields of compounds 1–16

[0077]

[0078]

[0079] Effect Example

[0080] 1. Experimental Methods

[0081] 1.1 Cytotoxicity assay

[0082] Mouse monocyte-macrophage RAW 264.7 cells were passaged in DMEM medium containing 10% fetal bovine serum at 37°C and 5% CO2. Log-phase RAW 264.7 cells were adjusted to a density of 1×10⁵ cells / ml and seeded into 96-well plates at 100 μL per well. After 24 h of cell adhesion, caffeic acid derivative 1-20 (the caffeic acid derivative prepared in Example 4) was prepared to a concentration of 4 μg / mL using DMEM basal solution. The original culture medium was replaced with working solution containing the caffeic acid derivative. A positive control group (no test compound) and a negative control group (cell-free group) were set up. Three parallel groups were set up for each caffeic acid derivative. After incubation for 12 h, 100 μL of MTT was added to each well and 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. The absorbance (A) was measured at 490 nm. The experiment was repeated three times. Cell viability (%) was calculated as follows: (A detection well / A blank well) × 100%. Experimental results are shown below. Figure 7 As shown, at a concentration of 20 μM, some caffeic acid derivatives are toxic to RAW 264.7 cells and have a slight inhibitory effect on their growth. Among them, some caffeic acid derivatives, such as derivatives 3, 5, 8, 10, 11, 13, and 14, have a greater impact on cell viability and are more toxic.

[0083] 1.2 Determination of DPPH scavenging rate

[0084] Caffeic acid derivatives, caffeic acid, and the positive control drug Trolox were prepared at concentrations of 5 μM, 10 μM, 20 μM, 30 μM, 40 μM, and 50 μM. First, 190 μL of DPPH solution was added to each well of a 96-well plate. Then, different concentrations of the prepared caffeic acid derivatives were added sequentially, with three replicates for each drug. Positive and negative control groups were also included. The plates were then incubated in a light-protected incubator for 0.5 h, and the absorbance at 517 nm was measured using a microplate reader. The scavenging rate of DPPH free radicals by caffeic acid derivatives was calculated using the following formula. The concentrations of the 16 caffeic acid derivatives were plotted on the X-axis, and the absorbance at 517 nm was plotted on the Y-axis. The linear regression equation and correlation coefficient (R²) were then obtained. 2 The DPPH scavenging rates of different caffeic acid derivatives, or at different concentrations of caffeic acid derivatives, were compared. In the DPPH method, a higher scavenging rate of caffeic acid derivatives for free radicals indicates higher antioxidant performance. The formula for calculating the DPPH scavenging rate is shown below:

[0085] DPPH clearance rate = [1-(A m -A n ) / A s ]×100%

[0086] Where A m A represents the absorbance value of the derivative group. n A represents the absorbance of the positive control group. s The absorbance values ​​are for the negative control group. A graph is plotted with drug concentration on the X-axis and clearance percentage on the Y-axis. The IC50 of DPPH clearance can be calculated by fitting the curve using Excel. 50 Value. Experimental results are as follows: Figure 8 , 9 As shown in Figures 1 and 10, derivatives 1, 2, and 13 exhibit strong DPPH radical scavenging ability at lower concentrations, while the other 13 derivatives show weaker radical scavenging ability.

[0087] 1.3 Data Analysis

[0088] Data analysis was performed using Prism software. The t-test was used for comparisons between groups, and P < 0.05 was considered statistically significant.

[0089] 2. Results

[0090] Based on the MTT assay results, some compounds showed some toxicity to RAW 264.7 cells. The DPPH scavenging rate of the compounds was investigated, and the results showed that compounds 1, 2, and 13 of this invention exhibited strong DPPH free radical scavenging ability at low concentrations. The results are as follows: Figure 4 and 5 As shown.

[0091] 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 caffeic acid derivative, characterized in that: The derivative is a compound with the structure shown in Formula 2 or a pharmaceutically acceptable salt thereof: Where R is or .

2. The caffeic acid 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 for preparing caffeic acid derivatives according to claim 1 or 2, characterized in that... It includes the following steps: (1) Reacting caffeic acid with thionyl chloride to obtain intermediate I with the structure shown in Formula 3; (2) Boc-glycine, carbodiimide, 4-dimethylaminopyridine and phenolic compounds with different substituents were reacted to obtain intermediate II with the structure shown in Formula 4; Where X=1; R1 is or ; (3) Intermediate II was reacted with trifluoroacetic acid to obtain intermediate III with the structure shown in Formula 5; Where X=1; R1 is or ; (4) Using dichloromethane as solvent, intermediate I and intermediate III as raw materials, react with triethylamine to obtain a caffeic acid derivative with the structure shown in Formula 2.

4. The preparation method according to claim 3, characterized in that: The molar ratio of caffeic acid to thionyl chloride in step (1) is 1:22.7; The molar ratio of Boc-glycine, carbodiimide and 4-dimethylaminopyridine in step (2) is 1:0.9:0.14; The molar ratio of intermediate I to intermediate III in step (4) is 1:

1.

5. The preparation method according to claim 3, characterized in that: The specific steps (1) are as follows: thionyl chloride is added dropwise to caffeic acid, and then the reaction system is placed in an oil bath and heated and stirred. The mixture is refluxed for 2 hours and the solution changes from yellow turbidity to clear and transparent to obtain intermediate I.

6. The preparation method according to claim 3, characterized in that: The specific steps (2) are as follows: using dichloromethane as solvent, add Boc-glycine, carbodiimide and 4-dimethylaminopyridine, stir the solution until clear, add phenolic compounds with different substituents, stir at room temperature for 2 h; after extraction with distilled water and ethyl acetate, collect the organic phase, wash with saturated sodium bicarbonate solution, dry, evaporate the solvent with a rotary evaporator to obtain an oily crude product; redissolve the oily crude product with ethyl acetate, add silica gel and mix thoroughly, and separate by column chromatography to obtain intermediate II.

7. The preparation method according to claim 3, characterized in that: The specific steps (3) are as follows: Dissolve intermediate II in dichloromethane, add trifluoroacetic acid, the molar ratio of trifluoroacetic acid to dichloromethane is 1:3; stir at room temperature for 0.5 h; evaporate the resulting reaction solution by rotary evaporation to remove dichloromethane and trifluoroacetic acid, and precipitate a white solid to obtain intermediate III.

8. The preparation method according to claim 3, characterized in that: The specific steps (4) are as follows: intermediate I is dissolved in CH3CN, then intermediate III is added, and 3-4 drops of triethylamine solution are added to the reaction system. The reaction is carried out at room temperature for 2 h. The resulting reaction solution is extracted with saturated NaHCO3 solution and ethyl acetate, and the organic phase is collected. The solvent in the organic phase is evaporated by rotary evaporator to obtain the crude product. The crude product is redissolved with ethyl acetate, silica gel is added and mixed thoroughly, and the target compound caffeic acid derivative is obtained by column chromatography.

9. The use of the caffeic acid derivative according to claim 1 or 2 in the preparation of drugs for preventing and treating oxidative stress.

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