Preparation method and application of piperine analogue with anti-inflammatory activity

Abstract

The invention relates to the field of chemical synthesis, and particularly provides a preparation method and application of a piperine analogue with anti-inflammatory activity. The invention relates to a compound as shown in a formula I, and a stereoisomer or pharmaceutically acceptable salt or ester thereof, and the piperine analogue has an anti-inflammatory effect, has the advantages of mild reaction, high process safety and the like, and has a wide application prospect in the aspect of biochemical preparations.

Description

Technical Field

[0001] The present invention belongs to the technical field of chemical synthesis, and relates to a preparation method and application of a piperine analogue with anti-inflammatory activity. Background Art

[0002] Natural products, due to the diversity and novelty of their chemical structures and biological activities, continuously provide key molecular skeletons for bioactive lead compounds. In recent years, research on their derivatives has been carried out in view of the structural characteristics of natural products. However, traditional structural derivatization strategies often face the dual problems of activity attenuation and toxicity enhancement, which is not conducive to the utilization of derivatives. Constructing natural product-like compounds with targeted selectivity, which can act more precisely on specific targets and have the advantages of reducing toxicity, increasing activity and yield, has become a research hotspot in this field.

[0003] Piperine is the main active ingredient of pepper and has functions such as antibacterial, anti-inflammatory, anti-cancer, antioxidant, and anti-convulsant effects, but its activity still cannot meet the needs of production applications. Based on the structural characteristics of piperine, designing and synthesizing piperine analogues is an important way to obtain compounds with higher activity. However, due to the complexity of the compound structure, there is a delicate balance relationship in the compound synthesis process, and specific target products can only be completed under specific conditions. There are few reports on piperine analogues in the prior art. Based on the structural characteristics of piperine and the previous research foundation, the present invention uses 3-(4-hydroxyphenyl)acrolein and 1-acetylpyrrolidine to design and synthesize 5-(4-hydroxy)phenyl-1-pyrrolidinyl-2,4-pentadien-1-one with strong anti-inflammatory activity through simple chemical synthesis. Summary of the Invention

[0004] The purpose of the present invention is to provide a piperine analogue, its preparation method and application. The chemical structural formula of the piperine analogue is shown in Formula I. This piperine analogue has an anti-inflammatory effect, and its preparation process has the advantages of mild reaction, high process safety, high yield, etc., and has a wide application prospect in biochemical preparations.

[0005] To achieve the above purpose, the present invention provides the following technical solutions: The first aspect of the present invention provides a compound of Formula I, or a pharmaceutically acceptable salt or ester thereof: .

[0006] In the second aspect of the present invention, a method for preparing a piperine analogue is provided. In a specific embodiment, the technical personnel of the present invention mixed 3-(4-hydroxyphenyl)acrolein with the structure shown in Formula II and 1-acetylpyrrolidine with the structure shown in Formula III in a molar ratio of 1:1, dissolved them in ethanol, stirred evenly, and then slowly added dropwise a 1.0 mmol / L sodium hydroxide solution. After reacting at 45 °C for 6 h, the pH value was adjusted to 6-7 with a 10% V / V hydrochloric acid solution to obtain a reaction mixture; the reaction mixture was extracted with ethyl acetate, rotary evaporated, and dried to obtain a crude product; the crude product was separated and purified by silica gel column chromatography, eluted with ethyl acetate and petroleum ether as eluents, and the volume ratio of ethyl acetate:petroleum ether = 10:1, and the eluate was collected; the eluate was concentrated and dried to prepare 5-(4-hydroxy)phenyl-1-pyrrolidinyl-2,4-pentadien-1-one, the chemical structural formula of which is shown in Formula I, and the specific synthesis route is as follows, and the yield of the main product reached 52.32%: 。

[0007] The technical personnel of the present invention also used the piperine analogue prepared by the above method to conduct an anti-inflammatory experiment on RAW264.7 and found that the piperine analogue has strong anti-inflammatory activity.

[0008] In the third aspect of the present invention, there is provided the use of the compound of Formula I above, or a pharmaceutically acceptable salt or ester thereof in the preparation of an anti-inflammatory preparation.

[0009] In the fourth aspect of the present invention, a composition is provided, which comprises the compound of Formula I above, or a pharmaceutically acceptable salt or ester thereof as an active ingredient, and one or more excipients.

[0010] Advantages of the present invention: (1) The present invention successfully synthesized a piperine analogue 5-(4-hydroxy)phenyl-1-pyrrolidinyl-2,4-pentadien-1-one with a completely new structure, enriching the types of piperine analogues. This unique structure endows it with anti-inflammatory properties, providing a new choice for the field of anti-inflammatory compounds.

[0011] (2) The piperine analogue provided by the present invention can be used in the biochemical field to develop novel anti-inflammatory preparations.

[0012] (3) For the preparation method provided by the present invention, the raw materials are 3-(4-hydroxyphenyl)acrolein with the structure shown in Formula II and 1-acetylpyrrolidine with the structure shown in Formula III, without a catalyst, and the solvent is a common solvent, so the raw materials are easily available.

[0013] (4) For the preparation method provided by the present invention, the conditions are mild, precisely controllable, with low energy consumption, high feasibility, and high process safety.

[0014] (5)The preparation method provided by the present invention has a main product yield of 52.32%, indicating a relatively high yield. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 It is a schematic diagram of the synthetic route of the piperine analog provided by the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0016] The following further elaborates on the concept and technical effects of the present invention in combination with specific embodiments to fully understand the purpose, features, and effects of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative efforts fall within the scope of protection of the present invention. The methods are conventional methods unless otherwise specified. The materials can be obtained from public commercial channels unless otherwise specified.

[0017] Example 1 Preparation of 5-(4-Hydroxyphenyl)-1-pyrrolidinyl-2,4-pentadien-1-one

[0018] 1.07 g (6.0 mmol) of 3-(4-hydroxyphenyl)acrolein, 0.68 g (6.0 mmol) of 1-acetylpyrrolidine, and 20 mL of ethanol were added to a 50 mL three-necked flask. After stirring evenly, 15 mL of sodium hydroxide solution (1.0 mmol / mL) was slowly added dropwise, and the reaction was carried out at 45 °C for 6 h. The pH value was adjusted to 6-7 with 10% V / V hydrochloric acid solution to obtain a reaction mixture; the reaction mixture was extracted with ethyl acetate, rotary evaporated, and dried to obtain a crude product; the crude product was separated and purified by silica gel column chromatography, eluted with ethyl acetate and petroleum ether as eluents, and the volume ratio of ethyl acetate:petroleum ether = 10:1. The eluate was collected; the eluate was concentrated and dried to obtain a product with a yield of 53.32% (the synthetic route is as Figure 1 shown).

[0019] Example 2 Preparation of 5-(4-Hydroxyphenyl)-1-pyrrolidinyl-2,4-pentadien-1-one

[0020] 1.07 g (6.0 mmol) of 4-hydroxy-3-methoxycinnamaldehyde, 0.52 g (4.6 mmol) of 1-acetylpyrrolidine, and 20 mL of ethanol were added to a 50 mL three-necked flask, stirred, and reacted at room temperature for 6 hours. The pH value was adjusted to 6-7 with 10% hydrochloric acid solution to obtain a reaction mixture; the reaction mixture was extracted with ethyl acetate, rotary evaporated, and dried to obtain a crude product; the crude product was separated and purified by silica gel column chromatography, eluted with ethyl acetate:petroleum ether (10:1, V / V), and the eluate was collected; the eluate was concentrated and dried to obtain a product with a yield of 1.35%, and the reaction failed.

[0021] Example 3 Preparation of 5-(4-Hydroxyphenyl)-1-pyrrolidinyl-2,4-pentadien-1-one

[0022] 1.07 g (6.0 mmol) of 3-(4-hydroxyphenyl)acrolein, 0.68 g (6.0 mmol) of 1-acetylpyrrolidine, and 20 mL of ethanol were added to a 50 mL three-necked flask. After stirring evenly, 15 mL of sodium hydroxide solution (1.0 mmol / mL) was slowly added dropwise. The reaction was carried out at room temperature for 6 h, and the pH value was adjusted to 6 - 7 with 10% V / V hydrochloric acid solution to obtain a reaction mixture. The reaction mixture was extracted with ethyl acetate, rotary evaporated, and dried to obtain a crude product. The crude product was separated and purified by silica gel column chromatography, eluted with ethyl acetate: petroleum ether (10:1, V / V), and the eluate was collected. The eluate was concentrated and dried to obtain the product, with a yield of 10.72%, and the experiment failed.

[0023] Example 4 Preparation of 5-(4-Hydroxyphenyl)-1-pyrrolidinyl-2,4-pentadien-1-one

[0024] 1.07 g (6.0 mmol) of 3-(4-hydroxyphenyl)acrolein, 0.68 g (6.0 mmol) of 1-acetylpyrrolidine, and 20 mL of ethanol were added to a 50 mL three-necked flask. After stirring evenly, 15 mL of sodium hydroxide solution (1.0 mmol / mL) was slowly added dropwise. The reaction was carried out at 45 °C for 6 hours to obtain a reaction mixture. The reaction mixture was extracted with ethyl acetate, dried with anhydrous magnesium sulfate, rotary evaporated, and separated and purified by column chromatography, with a yield of 8.52%, and the experiment failed.

[0025] Example 5 Preparation of 5-(4-Hydroxyphenyl)-1-pyrrolidinyl-2,4-pentadien-1-one

[0026] 1.07 g (6.0 mmol) of 3-(4-hydroxyphenyl)acrolein, 0.68 g (6.0 mmol) of 1-acetylpyrrolidine, and 20 mL of ethanol were added to a 50 mL three-necked flask. After stirring evenly, 20 mL of sodium hydroxide solution (1.0 mmol / mL) was slowly added dropwise. The reaction was carried out at room temperature for 6 h, and the pH value was adjusted to 6 - 7 with 10% V / V hydrochloric acid solution to obtain a reaction mixture. The reaction mixture was extracted with ethyl acetate, rotary evaporated, and dried to obtain a crude product. The crude product was separated and purified by silica gel column chromatography, eluted with ethyl acetate: petroleum ether (10:1, V / V), concentrated, and dried to obtain the product, with a yield of 38.57%.

[0027] Example 6 Preparation of 5-(4-Hydroxyphenyl)-1-pyrrolidinyl-2,4-pentadien-1-one

[0028] 1.07 g (6.0 mmol) of 3-(4-hydroxyphenyl)acrolein, 0.68 g (6.0 mmol) of 1-acetylpyrrolidine, and 20 mL of ethanol were added to a 50 mL three-necked flask. After stirring evenly, 15 mL of sodium hydroxide solution (1.0 mmol / mL) was slowly added dropwise. The reaction was carried out at 45 °C for 8 hours, and the pH value was adjusted to 6 - 7 with 10% hydrochloric acid solution to obtain a reaction mixture; the reaction mixture was extracted with ethyl acetate, dried over anhydrous magnesium sulfate, and rotary evaporated, with a yield of 29.47%.

[0029] Example 7 Preparation of Piperine

[0030] White pepper was crushed and passed through a 40-mesh sieve to obtain pepper powder, which was added to 80% V / V edible alcohol to obtain a mixed solution. The mixed solution was heated to 60 °C and continuously heated at a constant temperature for 60 min for extraction treatment to obtain a piperine extract. The material-liquid ratio of the pepper powder to 80% V / V edible alcohol was 0.04 g / mL; The piperine extract was concentrated, dried, and formulated into a crude piperine extract of 2 mg / mL. The crude piperine extract was loaded onto a column with HPD22 resin at a flow rate of 1.5 mL / min, and eluted with 90% V / V ethanol at a flow rate of 2.0 mg / mL to obtain a crude piperine pure product; The crude piperine pure product was concentrated, dried, and formulated into a refined piperine extract of 200 mg / mL with 100% V / V edible alcohol as the solvent. After crystallizing the refined piperine extract at 4 °C for 24 h, it was recrystallized twice, the crystals were collected, and vacuum freeze-dried to obtain high-purity piperine.

[0031] Experiment 1 Determination of Anti-inflammatory Effect

[0032] ① Cell culture. RAW 264.7 cells were cultured in a medium containing 10% fetal bovine serum and high-glucose DMEM medium (containing 10000 U / mL penicillin and 10000 μg / mL streptomycin) in a constant temperature incubator at 37 °C and 5% CO 2 2. The medium was changed every 1 - 2 days, and cells in the logarithmic growth phase were taken for subsequent experiments. The specific experimental results are shown in Table 1. In addition, the result of the blank control was 0, indicating no pollution.

[0033] ② Cell viability determination. Cells in the logarithmic growth phase were taken, and the cell concentration was adjusted to 1×10 4 / mL, inoculated on a 96-well plate, 100 μL per well, at 37 °C and 5% CO 2Cultivate for 24 h under the conditions, and discard the old culture medium. Divide RAW264.7 cells into 4 groups, namely the normal group (Control), the model group (lipopolysaccharide, LPS), and the experimental groups (with a concentration of 10 mg / L for all). The normal group and the model group are not intervened with piperine and LPS, and an equal volume of complete culture medium is added and cultured for 2 h; the experimental groups are successively added with sample solutions prepared in different examples at a final concentration and cultured for 2 h. Then, the model group and the experimental groups are added with an LPS solution at a final concentration of 0.5 mg / l and cultured. After culturing for 24 h, discard the culture medium, add 90 μL of complete culture medium and 10 μL of 5 mg / mL MTT to each well, continue to culture for 4 h, discard the upper liquid, add 110 μL of DMSO, shake until all the crystals are dissolved, detect the OD value of each well at 490 nm, and calculate the cell survival rate. Cell survival rate = (OD value of the experimental group - OD value of the blank group) / (OD value of the normal group - OD value of the blank group) × 100%.

[0034] ③ Determination of inflammatory factors TNF-α, IL-1β, IL-6. Take cells in the logarithmic growth phase, adjust the cell concentration to 7×10 5 / mL, inoculate on a 24-well plate, 500 μL per well, and culture at 37 °C and 5% CO 2 under the conditions for 10 h. Discard the old culture medium. Divide RAW264.7 cells into 4 groups, namely the normal group (Control), the model group (LPS), and the experimental groups (with a concentration of 10 mg / L for all). The normal group and the model group are not intervened with piperine and LPS, and an equal volume of complete culture medium is added and cultured for 2 h; the experimental groups are successively added with sample solutions prepared in different examples at a final concentration and cultured for 2 h. Then, the model group and the experimental groups are added with an LPS solution at a final concentration of 0.5 mg / l and cultured for 24 h. Then, centrifuge at 2000 r / min for 15 min at 4 °C. According to the Elisa kit instructions, determine the release levels of TNF-α, IL-1β, and IL-6.

[0035] Table 1 Effects of different groups on the survival rate of LPS-induced RAW264.7 cells and inflammation-related factors

[0036] Note: Different letters indicate significant differences between different groups ( p >0.05) As can be seen from Table 1: (1)Examples 1, 5, and 6 compared with the model group, 5-(4-hydroxy)phenyl-1-pyrrolidinyl-2,4-pentadien-1-one prepared by the method of the present invention showed significantly enhanced anti-inflammatory activity; among them, 5-(4-hydroxy)phenyl-1-pyrrolidinyl-2,4-pentadien-1-one prepared according to Example 1 had the strongest anti-inflammatory activity.

[0037] (2)Example 1 compared with Examples 2-4, adding 1.0 mmol / L sodium hydroxide solution and 10% hydrochloric acid solution to adjust the pH value to 6-7 and reacting at 45 °C for 6 h were the key steps in the preparation of 5-(4-hydroxy)phenyl-1-pyrrolidinyl-2,4-pentadien-1-one. Without any one of these three steps, 5-(4-hydroxy)phenyl-1-pyrrolidinyl-2,4-pentadien-1-one could not be synthesized.

[0038] (3)Experimental Example 1 compared with Examples 5 and 6, 5-(4-hydroxy)phenyl-1-pyrrolidinyl-2,4-pentadien-1-one prepared by the method of the present invention showed significantly enhanced anti-inflammatory activity. Changing the amount of sodium hydroxide added and lacking the column chromatography separation step were not conducive to the exertion of the anti-inflammatory activity of 5-(4-hydroxy)phenyl-1-pyrrolidinyl-2,4-pentadien-1-one, and lacking the column chromatography separation had a greater impact.

[0039] (4)Experimental Examples 1, 5, and 6 compared with Example 7, 5-(4-hydroxy)phenyl-1-pyrrolidinyl-2,4-pentadien-1-one prepared by the method of the present invention showed significantly enhanced anti-inflammatory activity, indicating that 5-(4-hydroxy)phenyl-1-pyrrolidinyl-2,4-pentadien-1-one had better anti-inflammatory effects.

[0040] The sample prepared in Experimental Example 1 was subjected to mass spectrometry analysis, HRMS [M+H] + : The theoretical value was 243.3060, and the measured value was 243.3058.

[0041] Although the present invention has been described in detail above with general descriptions, specific embodiments, and experiments, the present invention is not limited to the above embodiments. Based on the present invention, some modifications or improvements can be made, which are obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection required by the present invention.

Claims

1. A piperine analogue having anti-inflammatory activity, characterized in that: The piperine analog is a compound shown in formula I. The preparation method of the piperine analog is as follows: 3-(4-hydroxyphenyl) acrolein of the structure shown in formula II and 1-acetyl pyrrolidine of the structure shown in formula III are mixed in a molar ratio of 1:1, dissolved in ethanol, stirred evenly, and then slowly dripped with 1.0mmol / L sodium hydroxide solution, reacted at 45°C for 6 hours, and then adjusted the pH value to 6-7 with 10% V / V hydrochloric acid solution to obtain a reaction mixture; the reaction mixture is extracted with ethyl acetate, rotary evaporated, and dried to obtain a crude product; the crude product is separated and purified by silica gel column chromatography, eluted with ethyl acetate and petroleum ether as eluents, and the volume ratio of ethyl acetate: petroleum ether is 10:1, and the eluate is collected; the eluate is concentrated and dried to obtain the piperine analog, 5-(4-hydroxy)phenyl-1-pyrrolidinyl-2,4-pentadien-1-one, the specific chemical structure of which is shown in formula I: 。 2. Use of the compound of formula I as claimed in claim 1, or a pharmaceutically acceptable salt or ester thereof in the preparation of an anti-inflammatory preparation.

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