Paeonol imidazo ring derivative as well as chlorination preparation method and application thereof

Abstract

The invention belongs to the technical field of biological medicine, and relates to a paeonol imidazo ring derivative and a chlorination preparation method and application thereof. The chemical structure is shown as a formula I # imgabs0 #, X is selected from H, Cl and Br, and Y is C or N. The compound provided by the invention is novel in structure, not only has relatively high anti-inflammatory activity, but also can eliminate side effects such as pungent smell and pungency and improve compliance; meanwhile, the preparation method of the compound is simple to operate and suitable for industrial production.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine and relates to a paeonol imidazoline derivative and a chlorinated preparation method and application thereof. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance understanding of the overall background of the invention and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to those skilled in the art.

[0003] Paeonol is a natural product extracted from the dried root bark of the Ranunculaceae plant, Paeonia lactiflora. As the main active ingredient in Paeonia lactiflora, it is of great research value, possessing multiple activities such as antibacterial, anti-inflammatory, antiplatelet, and antitumor. Paeonol and its derivative, sodium paeonol sulfonate, have anti-inflammatory, antipruritic and anti-inflammatory effects, are effective, and are widely used in the fields of medicine and cosmetics. The inventors have found that medicines and cosmetics containing paeonol have a pungent odor during use and side effects such as a pungency when used, thereby affecting the compliance of special populations such as children and pregnant women, making the effects of related medicines and cosmetics less than expected, resulting in low market consumption. Therefore, paeonol, as a natural extract, has been structurally modified and transformed on its phenolic hydroxyl group to construct a novel, structurally stable, and excellent-performance lead compound, hoping to avoid the pungent odor and pungency side effects of paeonol in use. Summary of the Invention

[0004] In order to address the deficiencies of the prior art, the present invention aims to provide a paeonol imidazoline derivative and a chlorinated preparation method and application thereof. The compound provided by the present invention has a novel structure and not only has high anti-inflammatory activity, but also can eliminate side effects such as pungent odor and spicy sensation, thereby improving compliance; at the same time, the preparation method of the compound is simple to operate and is suitable for industrial production.

[0005] In order to achieve the above object, the technical solution of the present invention is:

[0006] In the first aspect, a paeonol imidazoline derivative has a chemical structure as shown in Formula I.

[0007]

[0008] Wherein, X is selected from H, F, Cl, Br, and Y is C or N.

[0009] The present invention unexpectedly discovered through experiments that when an imidazolin-based compound is introduced into the skeleton of paeonol, the obtained new compound not only has high anti-inflammatory activity, but also can eliminate the side effects of paeonol itself, such as the pungent smell and spicy feeling.

[0010] Preferably, Y is C and X is H or F.

[0011] Preferably, Y is N, and X is H or F.

[0012] In a second aspect, a method for preparing the chlorinated imidazoline derivatives of paeonol described in the first aspect comprises the steps of obtaining the compound of formula I by using compound 1 according to the following reaction scheme:

[0013]

[0014] Wherein, X and Y are respectively as described in the paeonol imidazoline derivatives in the first aspect.

[0015] In some embodiments, compound 1 is reacted with an acyl chloride reagent and an acid binding agent under heating reflux to obtain compound 2.

[0016] Specifically, the acyl chloride reagent is one of thionyl chloride, phosphorus oxychloride, triphosgene, and Vilsmeier reagent.

[0017] Specifically, the acid binding agent is pyridine or triethylamine.

[0018] Specifically, the molar ratio of compound 1 to the acyl chloride reagent is 1.0:2.0-8.0.

[0019] In some embodiments, a solution of compound 2 is added dropwise to an alkaline solution of paeonol at 0-20° C. to react and obtain a crude product.

[0020] Specifically, the solvent of the solution of compound 2 is one of acetone, acetonitrile, isopropyl ether, and diethyl ether.

[0021] Specifically, the alkaline solution of paeonol is adjusted with sodium hydroxide or potassium hydroxide.

[0022] Specifically, the pH of the alkaline solution of paeonol is 8.5 to 10.5.

[0023] Specifically, the molar ratio of compound 2 to paeonol is 1.0:1.5-3.0.

[0024] In some embodiments, the crude product of the compound of Formula I obtained according to the reaction route is recrystallized.

[0025] Specifically, during the recrystallization process, the crystallization solvent is one of methanol, ethanol, and acetonitrile.

[0026] Specifically, during the recrystallization process, the crystallization temperature is 0-10°C.

[0027] In a third aspect, a pharmaceutical composition comprises the paeonol imidazoline derivative or a pharmaceutically acceptable salt thereof according to the first aspect.

[0028] The pharmaceutically acceptable salts of the present invention include salts formed by the paeonol imidazoline derivatives with inorganic acids (such as hydrochloric acid, sulfuric acid, nitric acid, etc.), or salts formed with organic acids (such as formic acid, acetic acid, oxalic acid, lactic acid, citric acid, etc.).

[0029] In some embodiments, pharmaceutical excipients are also included. The pharmaceutical excipients include pharmaceutical carriers and / or excipients. The excipients include, but are not limited to, binders, fillers, disintegrants, lubricants, wetting agents, etc.

[0030] In some embodiments, the dosage form is one or more of tablets, capsules, granules, suspensions, injections, and the like.

[0031] In a fourth aspect, a use of the paeonol imidazoline derivative described in the first aspect or the pharmaceutical composition described in the third aspect in the preparation of anti-inflammatory drugs and / or cosmetics with anti-inflammatory efficacy.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] 1. The paeonol imidazoline derivatives in the present invention are new compounds, and there is no report on this compound at present.

[0034] 2. The synthesis method provided by the present invention has a short reaction route, simple operation, and the product can be directly obtained by recrystallization, which is suitable for industrial production.

[0035] 3. The imidazolin derivatives of paeonol provided by the present invention introduce an imidazolin heterocyclic structure into the phenolic hydroxyl skeleton of paeonol, thereby avoiding the side effects such as the pungent odor and spicy feeling caused by the original phenolic hydroxyl group of paeonol during use. The new compounds of the present invention show relatively good activity in anti-inflammatory biological activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] The accompanying drawings, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0037] Figure 1 The figure shows the effect of paeonol imidazopyridine-3-carboxylate on RAW264.7 cell viability at different concentrations in the examples of the present invention; compared with the control group, ****p<0.0001.

[0038] Figure 2 The figure shows the effect of 6-fluoroimidazopyridine-3-carboxylic acid paeonol ester at different concentrations on the viability of RAW264.7 cells in the examples of the present invention; compared with the control group, ****p<0.0001.

[0039] Figure 3 The figure shows the effect of 6-chloroimidazopyridazine-3-carboxylic acid paeonol ester at different concentrations on the viability of RAW264.7 cells in the example of the present invention; compared with the control group, ****p<0.0001.

[0040] Figure 4 The figure shows the effect of 6-bromoimidazopyridazine-3-carboxylic acid paeonol ester at different concentrations on the viability of RAW264.7 cells in the example of the present invention; compared with the control group, ****p<0.0001.

[0041] Figure 5 This is the effect of imidazopyridine-3-carboxylic acid paeonol ester in the examples of the present invention on the release of NO from RAW264.7 cells stimulated by LPS; compared with the control group, ****p<0.0001; compared with the model group, ##p<0.01, ####p<0.0001.

[0042] Figure 6 This is the effect of 6-fluoroimidazopyridine-3-carboxylic acid paeonol ester in the examples of the present invention on the release of NO from RAW264.7 cells stimulated by LPS; compared with the control group, ****p<0.0001; compared with the model group, ##p<0.01, ####p<0.0001.

[0043] Figure 7 This is the effect of 6-chloroimidazopyridazine-3-carboxylic acid paeonol ester in the examples of the present invention on the release of NO from RAW264.7 cells stimulated by LPS; compared with the control group, ****p<0.0001; compared with the model group, ##p<0.01, ####p<0.0001.

[0044] Figure 8 This is the effect of 6-bromoimidazopyridazine-3-carboxylic acid paeonol ester in the examples of the present invention on the release of NO from RAW264.7 cells stimulated by LPS; compared with the control group, ****p<0.0001; compared with the model group, ##p<0.01, ####p<0.0001. DETAILED DESCRIPTION

[0045] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0046]

[0047] Example 1: X=H, Y=C, imidazopyridine-3-carboxylic acid paeonol ester

[0048] 1) 10 g of imidazopyridine, 22 g of thionyl chloride, and 0.5 mL of pyridine were stirred and heated to reflux. The reaction was monitored by TLC until the reaction was complete. The mixture was cooled to room temperature and concentrated by rotary evaporation to remove unreacted thionyl chloride. The concentrate was imidazopyridine-3-carbonyl chloride, which was dissolved in 30 mL of acetone for later use.

[0049] 2) 20.5 g of paeonol (with side effects such as a pungent odor and a spicy sensation) and 100 mL of water were stirred and the temperature was controlled at 0-10° C., and 31 mL of 6 M sodium hydroxide solution was added dropwise and stirred to dissolve; a solution of imidazopyridine-3-carbonyl chloride was added dropwise and the temperature was controlled at 0-10° C. During the addition, a white solid was produced; after the addition was completed, 200 mL of water was added, the pH was adjusted to 9.5, the mixture was stirred for 0.5 h, filtered, and dried to obtain 11.7 g of crude imidazopyridine-3-carboxylate paeonol, with a yield of 61%.

[0050] 3) 10 g of crude imidazopyridine-3-carboxylic acid paeonol ester was added to 150 mL of methanol, heated to dissolve, and the temperature was controlled at 0-10° C. The crystallized product was filtered and dried to obtain 8.3 g of a white solid, which was imidazopyridine-3-carboxylic acid paeonol ester.

[0051] Structure confirmation:

[0052] Melting point: 137.4-141.6℃

[0053] 1 H-MHR(CDCl3,400MHz)δ:9.26-9.27(d,1H),8.56(s,1H),7.92-7.90(d,1H),7.80-7.82(d,1H),7.4 7-7.49(d,1H),7.07(d,1H),6.88-6.91(d,1H),6.78(d,1H),6.78(d,1H),3.88(s,3H),2.53(s,3H);

[0054] 13 C-NMR(CDCl3,101MHz)δ195.66,163.77,158.53,150.70,143.12,132.43,128 .38,127.87,123.78,118.05,115.11,114.80,112.07,109.48,55.78,29.56.

[0055] High-resolution mass spectrometry: molecular formula C 17 H 14 The mass number of N2O4 is 310.0974, the target mass is 310.0954, the error is 6.54ppm, which is consistent.

[0056] Example 2: X=H, Y=C, imidazopyridine-3-carboxylic acid paeonol ester

[0057] 1) 10 g of imidazopyridine, 18.9 g of phosphorus oxychloride, and 15.6 g of triethylamine were stirred and heated at 70-80°C. After TLC monitoring, the reaction mixture was cooled to room temperature and dissolved in 50 mL of acetonitrile.

[0058] 2) 20.5 g of paeonol (with side effects such as a pungent odor and a spicy sensation) and 100 mL of water were stirred and the temperature was controlled at 0-10° C., and 45 mL of 6 M sodium hydroxide solution was added dropwise and stirred to dissolve; a solution of imidazopyridine-3-carbonyl chloride was added dropwise and the temperature was controlled at 0-10° C. During the addition, a white solid was produced; after the addition was completed, 200 mL of water was added, the pH was adjusted to 10.5, and the mixture was stirred for 0.5 h, filtered, and dried to obtain 10.2 g of crude imidazopyridine-3-carboxylate paeonol, with a yield of 53%.

[0059] 3) 8 g of crude imidazopyridine-3-carboxylic acid paeonol ester was added to 100 mL of methanol, heated to dissolve, and the temperature was controlled at 0-10°C. The mixture was crystallized, filtered, and dried to obtain 6.1 g of a white solid, which was imidazopyridine-3-carboxylic acid paeonol ester.

[0060] Example 3: X=H, Y=C, imidazopyridine-3-carboxylic acid paeonol ester

[0061] 1) 10 g of imidazopyridine, 36.6 g of triphosgene, and 13.7 g of triethylamine were stirred and heated at 60-70°C under TLC monitoring. After the reaction was complete, the mixture was cooled to room temperature and dissolved in 50 mL of acetone;

[0062] 2) 25.8 g of paeonol (with side effects such as a pungent odor and a spicy sensation) and 100 mL of water were stirred and the temperature was controlled at 0-10° C., and 50 mL of 6 M potassium hydroxide solution was added dropwise and stirred to dissolve; a solution of imidazopyridine-3-carbonyl chloride was added dropwise and the temperature was controlled at 0-10° C. During the addition, a white solid was produced; after the addition was completed, 200 mL of water was added, the pH was adjusted to 8.5, and the mixture was stirred for 0.5 h, filtered, and dried to obtain 10.2 g of crude imidazopyridine-3-carboxylate paeonol, with a yield of 53%.

[0063] 3) 8 g of crude imidazopyridine-3-carboxylic acid paeonol ester was added to 150 mL of methanol, heated to dissolve, and the temperature was controlled at 0-10°C. The mixture was crystallized, filtered, and dried to obtain 5.8 g of a white solid, which was imidazopyridine-3-carboxylic acid paeonol ester.

[0064] Example 4: X=F, Y=C, 6-fluoroimidazopyridine-3-carboxylic acid paeonol ester

[0065] 1) 10 g of 6-fluoroimidazopyridine, 33 g of thionyl chloride, and 0.5 mL of pyridine were stirred and heated to reflux. The reaction was monitored by TLC until the reaction was complete. The mixture was cooled to room temperature and concentrated by rotary evaporation to remove unreacted thionyl chloride. The concentrate was 6-fluoroimidazopyridine-3-carbonyl chloride, which was dissolved in 50 mL of acetone for later use.

[0066] 2) 23 g of paeonol (with side effects such as a pungent odor and a spicy sensation) and 100 mL of water were stirred and temperature controlled at 0-10° C., and 28 mL of 6M sodium hydroxide solution was added dropwise and stirred to dissolve; a solution of 6-fluoroimidazopyridine-3-carbonyl chloride was added dropwise, and the temperature was controlled at 0-10° C. During the addition, a white solid was produced; after the addition was complete, 200 mL of water was added, the pH was adjusted to 9.0, and the mixture was stirred for 0.5 h, filtered, and dried to obtain 11.6 g of crude 6-fluoroimidazopyridine-3-carboxylate, with a yield of 57%.

[0067] 3) 10 g of crude 6-fluoroimidazopyridine-3-carboxylic acid paeonol ester was added to 150 mL of methanol, heated to dissolve, and the temperature was controlled at 0-10° C. The crystallized product was filtered and dried to obtain 7.7 g of a white solid, which is 6-fluoroimidazopyridine-3-carboxylic acid paeonol ester.

[0068] Structural confirmation:

[0069] Melting point: 147.3-149.6℃

[0070] 1 H-MHR(CDCl3,400MHz)δ:9.14-9.15(d,1H),8.46(s,1H),7.81-7.83(d,1H),7.67-7.71(d ,1H),7.30-7.32(d,1H),6.80-6.83(d,1H),6.68-6.69(d,1H),3.79(s,3H),2.44(s,3H);

[0071] 13 C-NMR (101MHz, CDCl3) δ194.58,162.80,157.42,154.48,152.10,149.49,145.52,142.45,131.53 ,122.46,119.04,118.79,117.41,117.32,115.31,114.43,114.01,111.05,108.52,54.77,28.29.

[0072] High-resolution mass spectrometry: molecular formula C 17 H 13 The mass number of FN2O4 is 328.088, the target mass is 328.0859, the error is 6.42ppm, which is consistent.

[0073] Example 5: X=Cl, Y=N, 6-chloroimidazopyridazine-3-carboxylic acid paeonol ester

[0074] 1) 10 g of 6-chloroimidazopyridazine, 23 g of thionyl chloride, and 0.5 mL of pyridine were stirred and heated to reflux. The reaction was monitored by TLC. The reaction mixture was cooled to room temperature and concentrated by rotary evaporation to remove unreacted thionyl chloride. The concentrate was 6-chloroimidazopyridazine-3-carbonyl chloride, which was dissolved in 30 mL of acetone for later use.

[0075] 2) 21 g of paeonol (with side effects such as a pungent odor and a spicy sensation) and 110 mL of water were stirred and temperature controlled at 0-10°C, and 33 mL of 6M sodium hydroxide solution was added dropwise and stirred to dissolve; a solution of dissolved 6-chloroimidazopyridazine-3-carbonyl chloride was added dropwise and the temperature was controlled at 0-10°C. During the addition, a white solid was produced; after the addition was complete, 200 mL of water was added, the pH was adjusted to 9.5, and the mixture was stirred for 0.5 h, filtered, and dried to obtain 12.5 g of crude 6-chloroimidazopyridazine-3-carboxylate, with a yield of 71.5%.

[0076] 3) 10 g of crude 6-chloroimidazopyridazine-3-carboxylic acid paeonol ester was added to 120 mL of methanol, heated to dissolve, and the temperature was controlled at 0-10° C. The crystallized product was filtered and dried to obtain 8.5 g of a white solid, which is 6-chloroimidazopyridazine-3-carboxylic acid paeonol ester.

[0077] Structure confirmation:

[0078] Melting point: 177.6-178.8℃;

[0079] 1 H-MHR(CDCl3,400MHz)δ:8.61(s,1H),8.06-8.08(d,1H),7.89-7.91(d,1H),7. 31-7.34(d,1H)6.88-6.90(d,1H),6.79-6.80(d,1H),3.88(s,3H),2.53(s,3H);

[0080] 13 C-NMR(CDCl3,101MHz)δ195.54,163.77,156.12,150.63,148.70,142.42,141 .39,132.52,127.54,123.25,122.20,119.82,112.22,109.46,55.81,29.30.

[0081] High-resolution mass spectrometry: molecular formula C 16 H 12The mass number of ClN3O4 is 345.0535, the target mass is 345.0516, the error is 5.5ppm, which is consistent.

[0082] Example 6: X=Cl, Y=N, 6-chloroimidazopyridazine-3-carboxylic acid paeonol ester

[0083] 1) 10 g of 6-chloroimidazopyridazine, 18.9 g of phosphorus oxychloride, and 15.6 g of triethylamine were stirred and heated at 70-80°C. After the reaction was completed, the reaction mixture was monitored by TLC. The mixture was cooled to room temperature and dissolved in 50 mL of acetonitrile.

[0084] 2) 20.5 g of paeonol (with side effects such as a pungent odor and a spicy sensation) and 100 mL of water were stirred and the temperature was controlled at 0-10° C., and 45 mL of 6M sodium hydroxide solution was added dropwise and stirred to dissolve; a solution of dissolved 6-chloroimidazopyridazine-3-carbonyl chloride was added dropwise and the temperature was controlled at 0-10° C. During the addition, a white solid was produced; after the addition was complete, 200 mL of water was added, the pH was adjusted to 10.5, and the mixture was stirred for 0.5 h, filtered, and dried to obtain 10.8 g of crude 6-chloroimidazopyridazine-3-carboxylate paeonol, with a yield of 61.7%.

[0085] 3) 8 g of crude 6-chloroimidazopyridazine-3-carboxylic acid paeonol ester was added to 100 mL of ethanol, heated to dissolve, and the temperature was controlled at 0-10° C. The crystallized product was filtered and dried to obtain 6.7 g of a white solid, which is 6-chloroimidazopyridazine-3-carboxylic acid paeonol ester.

[0086] Example 7: X=Cl, Y=N, 6-chloroimidazopyridazine-3-carboxylic acid paeonol ester

[0087] 1) Stir and heat 10 g of 6-chloroimidazopyridazine, 36.6 g of triphosgene, 13.7 g of triethylamine, and 80 mL of 1,2-dichloroethane. Control the temperature at 60-70°C and monitor the reaction by TLC. Cool to room temperature, concentrate the solvent, and dissolve in 50 mL of acetone for later use.

[0088] 2) 25.8 g of paeonol (with side effects such as a pungent odor and a spicy sensation) and 100 mL of water were stirred and the temperature was controlled at 0-10° C., and 50 mL of 6M potassium hydroxide solution was added dropwise and stirred to dissolve; a solution of 6-chloroimidazopyridazine-3-carbonyl chloride was added dropwise and the temperature was controlled at 0-10° C. During the addition, a white solid was produced; after the addition was complete, 200 mL of water was added, the pH was adjusted to 8.5, and the mixture was stirred for 0.5 h, filtered, and dried to obtain 10.3 g of crude 6-chloroimidazopyridazine-3-carboxylate paeonol, with a yield of 58.9%.

[0089] 3) 8 g of crude 6-chloroimidazopyridazine-3-carboxylic acid paeonol ester was added to 150 mL of acetonitrile, heated to dissolve, and the temperature was controlled at 0-10° C. The crystallized product was filtered and dried to obtain 5.6 g of a white solid, which is 6-chloroimidazopyridazine-3-carboxylic acid paeonol ester.

[0090] Example 8: X = Br, Y = N, 6-bromoimidazopyridazine-3-carboxylic acid paeonol ester

[0091] 1) 10 g of 6-bromoimidazopyridazine, 33 g of thionyl chloride, and 0.5 mL of pyridazine were stirred and heated to reflux. The reaction was monitored by TLC. The reaction mixture was cooled to room temperature and concentrated by rotary evaporation to remove unreacted thionyl chloride. The concentrate was 6-bromoimidazopyridazine-3-carbonyl chloride, which was dissolved in 50 mL of acetone for later use.

[0092] 2) 23 g of paeonol (with side effects such as a pungent odor and a spicy sensation) and 100 mL of water were stirred and temperature controlled at 0-10° C., and 28 mL of 6M sodium hydroxide solution was added dropwise and stirred to dissolve; a solution of dissolved 6-bromoimidazopyridazine-3-carbonyl chloride was added dropwise, and the temperature was controlled at 0-10° C. During the addition, a white solid was produced; after the addition was complete, 200 mL of water was added, the pH was adjusted to 9.0, and the mixture was stirred for 0.5 h, filtered, and dried to obtain 11.1 g of crude 6-bromoimidazopyridazine-3-carboxylate paeonol, with a yield of 68.9%.

[0093] 3) 10 g of crude 6-bromoimidazopyridazine-3-carboxylic acid paeonol ester was added to 150 mL of ethanol, heated to dissolve, and the temperature was controlled at 0-10° C. The mixture was crystallized, filtered, and dried to obtain 7.6 g of a white solid, which is 6-bromoimidazopyridazine-3-carboxylic acid paeonol ester.

[0094] Structural confirmation:

[0095] Melting point: 173.4-175.1℃;

[0096] 1 H-MHR(CDCl3,400MHz)δ:8.51(s,1H),7.90-7.93(d,1H),7.68-7.69(d,1H), 7.55-7.57(d,1H),6.90-6.92(d,1H),6.77(d,1H),3.89(s,3H),2.53(s,3H);

[0097] 13C-NMR(CDCl3,101MHz)δ195.63,163.85,158.44,150.53,147.46,143.04,132.59 ,131.77,128.13,123.48,118.59,115.48,112.15,109.78,109.52,55.82,29.30.

[0098] High-resolution mass spectrometry: molecular formula C 16 H 12 The mass number of BrN3O4 is 390.0187, which is consistent with the target.

[0099] The compounds prepared in the above examples have no pungent odor or spicy feeling.

[0100] To illustrate the anti-inflammatory activity of the compounds prepared in the above examples of the present invention, pharmacological activity experiments were conducted on the compounds prepared in the above examples. The specific experimental data are as follows:

[0101] Cytotoxicity assay:

[0102] RAW264.7 cells (1×10 5 Cells were inoculated into 96-well plates (100 μL / well, 100 μg / mL) and cultured in complete medium for 24 hours. The original medium was then replaced with fresh medium (100 μL / well), and the test samples were added at different concentrations of 6.25, 12.5, 25, 50, 100, 200, 400, and 800 μg / mL, with 3 replicates for each concentration. After incubation for 24 hours, the cells were washed three times with PBS, and fresh medium containing 10% CCK8 reagent (100 μL / well) was added to each well and cultured for another 2 hours at 37°C. Cell viability was determined by measuring the absorbance at 450 nm. The concentration that had no significant effect on cell growth was selected as the concentration for subsequent NO content detection.

[0103] NO content detection:

[0104] RAW264.7 cells (4×10 5 Cells were inoculated with 100 μL / well (100 μL / well) in a 96-well culture plate and cultured for 24 hours. The original culture medium was replaced with fresh medium (100 μL / well). Various concentrations of compound were added and incubated for 2 hours. LPS was then added to each well to a final concentration of 1.5 μg / mL. After incubation at 37°C, 5% CO₂ for 24 hours, an equal volume of Griess reagent (Griess A:Griess B = 1:1) was added. The reaction was allowed to proceed at room temperature for 30 minutes. The NO content was determined by measuring the absorbance at 548 nm. A standard curve was generated according to the kit instructions, and NO content was calculated based on the OD value.

[0105] In the cytotoxicity test and NO content detection experiment, n=3, three independent experiments were repeated. The experimental data were analyzed using GraphPadPrism, and the data were compared and tested using one-way ANOVA. The experimental data are expressed as mean±standard deviation (mean±SD).

[0106] Experimental results:

[0107] Effects of compounds on the activity of macrophage RAW264.7 cells:

[0108] In order to determine whether each compound has toxic effects on RAW264.7 at different concentrations, the CCK-8 kit was used to detect the effects of different concentrations on RAW264.7 activity after 24 hours. Figures 1 to 4 As shown, 6-fluoroimidazopyridine-3-carboxylic acid paeonol ester had no significant inhibitory effect on cell growth at a concentration of 6.25-25 μg / mL, but had a significant inhibitory effect on cell growth at a concentration of 50-100 μg / mL; imidazopyridine-3-carboxylic acid paeonol ester, 6-fluoroimidazopyridine-3-carboxylic acid paeonol ester, and 6-bromoimidazopyridazine-3-carboxylic acid paeonol ester had no significant inhibitory effect on cell growth at a concentration of 12.5-50 μg / mL, but had a significant inhibitory effect on cell growth at a concentration of 100 μg / mL.

[0109] Reduce NO secretion by macrophage RAW264.7:

[0110] The Griess kit was used to detect the level of NO secretion under the intervention of different concentrations of the compound. Figures 5 to 8 As shown, 1.5 μg / mL LPS can significantly increase the content of NO produced by cells (p<0.0001), and each compound can reduce the NO content at the selected concentration, and most concentrations can significantly reduce the NO content (p<0.0001).

[0111] Experimental Summary

[0112] The LPS-induced RAW264.7 cell inflammation model is a classic in vitro inflammation model. The effects of four compounds on NO release from RAW264.7 cells were evaluated through cell experiments. First, a toxicity experiment was performed using a CCK-8 kit to determine the concentration that had no significant toxicity to the cells. In subsequent inflammation experiments, the RAW264.7 cell inflammation model was used, and NO release was used as an indicator to evaluate inflammation. Concentrations that showed no significant toxicity in the toxicity experiment were selected for anti-inflammatory activity evaluation. The results showed that the compounds could effectively reduce NO release at the selected concentrations and had potential anti-inflammatory activity.

[0113] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A paeonol imidazoline derivative, characterized in that the chemical The structure is shown in Formula I, Wherein, X is selected from H, F, Cl, Br, and Y is C or N.

2. The paeonol imidazoline derivative according to claim 1, characterized in that: Y is C, X is H or F; Or, Y is N, and X is H or F.

3. A method for preparing the chlorinated paeonol imidazoline derivatives according to claim 1, characterized in that: The method comprises the steps of obtaining the compound represented by formula I by using compound 1 according to the following reaction scheme: Wherein, X and Y are respectively as described in the paeonol imidazoline derivatives according to claim 1.

4. The method for preparing the chlorinated paeonol imidazoline derivatives according to claim 3, wherein: Compound 1 is reacted with an acyl chloride reagent and an acid-binding agent under reflux to obtain compound 2; Preferably, the acyl chloride reagent is one of thionyl chloride, phosphorus oxychloride, triphosgene, and Vilsmeier reagent; Preferably, the acid binding agent is pyridine or triethylamine; Preferably, the molar ratio of compound 1 to the acyl chloride reagent is 1.0:2.0-8.

0.

5. The method for preparing the chlorinated paeonol imidazoline derivatives according to claim 3, wherein: At 0-20° C., the solution of compound 2 was added dropwise to the alkaline solution of paeonol to react to obtain a crude product; Preferably, the solvent of the solution of compound 2 is one of acetone, acetonitrile, isopropyl ether, and diethyl ether; Preferably, the alkaline solution of paeonol is adjusted with sodium hydroxide or potassium hydroxide; Preferably, the pH of the alkaline solution of paeonol is 8.5 to 10.5; Preferably, the molar ratio of compound 2 to paeonol is 1.0:1.5-3.

0.

6. The method for preparing the chlorinated paeonol imidazoline derivatives according to claim 3, wherein: Recrystallizing the crude product of the compound of formula I obtained according to the reaction route; Preferably, during the recrystallization process, the crystallization solvent is one of methanol, ethanol, and acetonitrile; Preferably, during the recrystallization process, the crystallization temperature is 0-10°C.

7. A pharmaceutical composition, characterized in that: It includes the paeonol imidazocyclic derivative or a pharmaceutically acceptable salt thereof as described in claim 1.

8. The pharmaceutical composition according to claim 7, wherein Also includes pharmaceutical excipients.

9. The pharmaceutical composition according to claim 7, wherein The dosage form is one or more of tablets, capsules, granules, suspensions, and injections.

10. Use of the paeonol imidazoline derivative according to claim 1 or 2 or the pharmaceutical composition according to any one of claims 7 to 9 in the preparation of anti-inflammatory drugs and / or cosmetics with anti-inflammatory efficacy.

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