JP2024505106A - Azulene compounds and their preparation methods and uses - Google Patents

Abstract

The present invention provides an azulene-based compound and a method for producing and using the same. [Solution] The azulene compound includes two compounds that are isomers of each other, and the chemical name thereof is 8-(1,2-dihydroxypropan-2-yl)-2-hydroxy-1,10-dimethyl-9. -dihydroazulene-3,4-dione. The preparation method of azulene-based compounds is easy to operate, pure compounds can be isolated and obtained, and experiments have shown that both compounds have no obvious effect on the proliferation of RAW264.7 macrophages within 24 h, and LPS The induced NO release of RAW264.7 cells and the excessive secretion of IL-1β, IL-6 and TNF-α in the cells were significantly suppressed, and there was a clear anti-inflammatory effect. [Selection diagram] Figure 1

Description

TECHNICAL FIELD The present invention belongs to the technical field of medicine, and specifically relates to azulene compounds and their preparation and use, particularly in the preparation of anti-inflammatory drugs.

Inflammation is a complex biological response of tissues to harmful stimuli, such as pathogens, damaged cells, and irritants. This is usually a protective attempt by the organism to remove harmful stimuli and allow tissue healing processes to occur. However, inflammation, if not sufficiently restricted, can lead to the development of various diseases. When the body's immune response status becomes abnormal, it can cause an inappropriate or excessive immune response, which can damage tissues and cells and cause inflammation. Tissue damage caused by immune reactions is caused by, for example, type I allergies such as allergic rhinitis and urticaria, type II allergies such as anti-basement membrane glomerulonephritis, type III allergies such as glomerulonephritis due to immune complex deposition, tuberculosis, It is most common in various types of hypersensitivity reactions, such as type IV allergies such as typhoid fever, and also in many autoimmune diseases such as lymphocytic thyroiditis and ulcerative colitis.

Lilac is a lilac (Syringa oblata Lindl.) as a plant of the genus Lilac of the family Oleaceae. Its roots, stems, leaves and heartwood can be used as medicine and have functions such as removing heat and humidity, antibacterial, and anti-hepatitis.Lilac roots and heartwood are used in Mongolian medicine to relieve heat and tingling in the heart It is used to treat dizziness, insomnia, palpitations, asthma, and "Khiigenous" disease. Lilac is widely cultivated in Japan as an ornamental flower and has abundant resources, but the development of its medicinal uses has focused mainly on the development of its leaves, and there are few studies and reports on its stems. In-depth research and development regarding the ingredients is necessary.

The purpose of the present invention is to deeply study the chemical components of lilac stems, and to provide a method for preparing azulene-based compounds in lilac stems and their use.

It is an azulene-based compound, comprising two compounds 1 and 2 that are isomerides with each other, and its chemical name is 8-(1,2-dihydroxypropan-2-yl)-2-hydroxy-1,10. -dimethyl-9-dihydroazulene-3,4-dione, and its structural formula is as follows.

Azulene compound.
Furthermore, the method for producing the azulene compound is as follows:

Step (1) of taking the medicinal material of the lilac stem, adding an ethanol solution to extract it, collecting the extract after filtration, and concentrating the extract as an extract;

The extract was taken, dispersed in water, extracted in turn with dichloromethane and ethyl acetate, the two solvent extracts and water were collected, and the solvent was concentrated under reduced pressure and dried to separate the dichloromethane extracted part, the ethyl acetate extracted part, and Step (2) of obtaining a water portion;
Step (3) of obtaining the azulene compound by taking the ethyl acetate extracted portion obtained in step (2) and separating and purifying it by chromatography.
Furthermore, the separation and purification by chromatography in step (3) specifically includes:

Take the ethyl acetate extracted part obtained in step (2), dissolve it in methanol, add silica gel and stir the sample, add the sample to a silica gel column, perform separation by column chromatography, and prepare different volume ratios. step A, performing gradient elution with petroleum ether-acetone to obtain 11 fractions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11;

Step B of taking fraction 4, performing chromatographic separation using ODS medium pressure, and performing gradient elution with methanol-water with different volume concentrations to obtain 5 fractions A, B, C, D, and E;

Fraction B was taken and separated by liquid phase chromatography, eluted with acetonitrile-water with a volume concentration of 13%, fractions were collected according to the peak time of each chromatography peak, and each fraction was concentrated. Step C of obtaining pure crystals of Compounds 1 and 2.

Further, the ethanol solution in step (1) is an ethanol aqueous solution with a volume concentration of 5 to 95%, and the extraction method includes a cold immersion method, a percolation method, a microwave extraction method, an ultrasonic extraction method, and a reflux extraction method. method or continuous reflux extraction method.

Further, the number of extractions in step (1) is 1 to 3 times, the mass ratio of the input amount of ethanol solution to the medicinal material of lilac stems is (8 to 30):1, and in step (2) The number of times of dichloromethane and ethyl acetate extraction is 4 to 6 times, respectively, and the amount of dichloromethane and ethyl acetate used each time is 1/2 to 1/4 of the volume of the liquid to be extracted.

Furthermore, in step A, the silica gel for sample stirring is 100 to 200 mesh, the mass ratio of the ethyl acetate extraction part to the silica gel for sample stirring is 1:10, and the silica gel column is 200 to 200 mesh. It is a 300 mesh silica gel column, and the petroleum ether-acetone volume ratios in which gradient elution is performed are 5:1, 4:1, 3:1, 2:1, 1:1, and 0:1, respectively.

Furthermore, in step B, the methanol-water volume concentrations at which the gradient elution is performed are 5%, 15%, 30%, 50%, 70%, and 100%, respectively, and the chromatography is performed at the ODS medium pressure. The chromatography conditions are ODS-C18 chromatography column, chromatography column size of 800×25 mm, particle size of 20-45 μm, flow rate of 30 ml/min, column temperature of 25° C.

Furthermore, in step C, the chromatography conditions to create liquid phase chromatography are a YMC-Triart C18 chromatography column, a chromatography column size of 250×20 mm, a particle size of 5 μm, and a flow rate of 10 ml/min.
The present invention further provides the use of the above azulene compounds in the production of anti-inflammatory drugs.

The present invention provides oral preparations or injections by directly or indirectly adding the azulene compound to various pharmaceutically acceptable and commonly used auxiliary materials such as fillers, disintegrants, lubricants, and adhesives. An agent may also be prepared.

The oral preparations are tablets, capsules, granules, fat emulsions, microcapsules, and drops, and the injection preparations are injection solutions or powder injections.

Beneficial effect: The method for preparing azulene-based compounds according to the present invention is simple to operate, and pure compounds can be obtained by separation. According to experiments, the azulene-based compound according to the present invention had no obvious effect on the proliferation of RAW264.7 macrophages within 24 h, and inhibited LPS-induced NO release of RAW264.7 cells and IL-1β and IL in cells. -6 and TNF-α excessive secretion is significantly suppressed. The compounds according to the invention can therefore be used for the production of anti-inflammatory drugs.

FIG. 1 is a chemical plan view of an azulene compound according to the present invention. FIG. 2 shows ECD measured values and calculated value spectrograms of the azulene-based compound according to the present invention. FIG. 3 is a ¹ H-NMR spectrogram of Compound 1 according to the present invention. FIG. 4 is a ¹³ C-NMR spectrogram of Compound 1 according to the present invention. FIG. 5 is a mass spectrogram of Compound 1 according to the present invention. FIG. 6 is a DEPT carbon spectrogram of Compound 1 according to the present invention. FIG. 7 is an HMBC carbon spectrogram of Compound 1 according to the present invention. FIG. 8 is an HSQC carbon spectrogram of Compound 1 according to the present invention. FIG. 9 is a NOESY carbon spectrogram of Compound 1 according to the present invention. FIG. 10 is an HH-COSY carbon spectrogram of Compound 1 according to the present invention. FIG. 11 is a ¹ H-NMR spectrogram of Compound 2 according to the present invention. FIG. 12 is a ¹³ C-NMR spectrogram of compound 2 according to the present invention. FIG. 13 is a mass spectrogram of Compound 2 according to the present invention. FIG. 14 is a DEPT carbon spectrogram of Compound 2 according to the present invention. FIG. 15 is an HMBC carbon spectrogram of Compound 2 according to the present invention. FIG. 16 is an HSQC carbon spectrogram of Compound 2 according to the present invention. FIG. 17 is a NOESY carbon spectrogram of Compound 2 according to the present invention. FIG. 18 is an HH-COSY carbon spectrogram of Compound 2 according to the present invention. FIG. 19 is a schematic illustration of the effect of Compound 1 according to the invention on RAW264.7 cell activity. FIG. 20 is a schematic diagram of the effect of compound 2 according to the invention on RAW264.7 cell activity. FIG. 21 is a schematic representation of the effect of Compound 1 according to the present invention on LPS-induced RAW264.7 cell lipid peroxidation. FIG. 22 is a schematic representation of the effect of compound 2 according to the present invention on LPS-induced RAW264.7 cell lipid peroxidation.

Hereinafter, the technical solutions in the embodiments of the present invention will be clearly and completely explained, but it is clear that the described embodiments are only some embodiments of the present invention and not the entirety thereof. Those skilled in the art will understand that, based on the embodiments of the present invention, all other embodiments obtained without any creative effort fall within the protection scope of the present invention.

Unless otherwise specified, all reagents related to the Examples according to the present invention are commercially available products and can be purchased through commercial routes.
Example 1

This example is an azulene compound in lilac, comprising two compounds 1 and 2, which are isomers to each other, and whose chemical name is 8-(1,2-dihydroxypropan-2-yl)-2-hydroxy. -1,10-dimethyl-9-dihydroazulene-3,4-dione, and its structural formula is as follows.

Provided is an azulene compound.
The method for producing the azulene compound in lilac is as follows:

Take the medicinal material of dried lilac stems, add 8 times the amount of 70% (volume concentration) ethanol, perform heating and reflux extraction three times, for 2 hours, 2 hours, and 1 hour, respectively, and filter. step (1) of obtaining the extract as an extract by combining the extracts and concentrating under reduced pressure until the alcohol odor disappears;

Take the extract, disperse it in water, and extract it 5 times with dichloromethane and 5 times with ethyl acetate.The amount of dichloromethane and ethyl acetate used each time is 1/3 of the volume of the extracted liquid, and the two solvent extracts are extracted. A step (2) of collecting the water and drying the solvent under reduced pressure to obtain a dichloromethane extraction site, an ethyl acetate extraction site, and a water site;

Take the ethyl acetate extracted part obtained in step (2), completely dissolve it in methanol, add 100-200 mesh silica gel, perform dry sample stirring, and transfer it to a pre-packed 200-300 mesh silica gel column. Add the sample and perform separation by column chromatography and gradient elution with petroleum ether-acetone (volume ratios are 5:1, 4:1, 3:1, 2:1, 1:1, 0:1). was carried out, identified using silica gel GF254 and G thin plate, and 11 fractions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 were obtained by combining fractions containing similar spots. Fraction 4 was taken, dissolved in methanol, filtered, and the filtrate was subjected to chromatographic separation using ODS medium pressure with a 5%, 15%, 30%, 50%, 70%, 100% methanol-water gradient. Elution was performed, examined using silica gel GF254 and a thin plate, and fractions containing similar spots were combined, concentrated and dried to obtain 5 fractions A, B, C, D, and E, and fraction B was taken. Dissolve with 50% acetonitrile, filter, create liquid phase chromatographic separation, elute with 13% acetonitrile-water, collect fractions according to the peak time of each chromatographic peak, and concentrate each fraction. step (3) to obtain pure crystals of compounds 1 (200 mg) and 2 (450 mg), provided that the purity of both compounds detected by the HPLC area normalization method is 98% or higher; Be prepared.

However, the chromatography conditions for producing chromatography under ODS medium pressure are: ODS-C18 chromatography column, chromatography column size of 800 x 25 mm, particle size of 20 to 45 μm, flow rate of 30 ml/min, column temperature of 25 ° C. It is.

The chromatography conditions for making liquid phase chromatography are YMC-Triart C18 chromatography column, chromatography column size of 250×20 mm, particle size of 5 μm, flow rate of 10 ml/min.
Example 2
This example provides an azulene-based compound in lilac, and the preparation method thereof is as follows:

Take the medicinal material of dried lilac stems, add 15 times the volume of 80% ethanol, extract by cold soaking method, perform cold soak twice for 24 hours each time, filter, and combine the extracts. , a step (1) of obtaining the extract as an extract by vacuuming and concentrating until the alcohol smell disappears;

Take the extract, disperse it in water, and extract it 4 times with dichloromethane and 4 times with ethyl acetate in order.The amount of dichloromethane and ethyl acetate used each time is 1/2 of the volume of the extracted liquid, and the two solvent extracts are extracted. A step (2) of collecting the water and drying the solvent under reduced pressure to obtain a dichloromethane extraction site, an ethyl acetate extraction site, and a water site;

Take the ethyl acetate extracted part obtained in step (2), completely dissolve it in methanol, add 100-200 mesh silica gel, perform dry sample stirring, and transfer it to a pre-packed 200-300 mesh silica gel column. Add the sample and perform separation by column chromatography and gradient elution with petroleum ether-acetone (volume ratios are 5:1, 4:1, 3:1, 2:1, 1:1, 0:1). was carried out, identified using silica gel GF254 and G thin plate, and 11 fractions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 were obtained by combining fractions containing similar spots. Fraction 4 was taken, dissolved in methanol, filtered, and the filtrate was subjected to chromatographic separation using ODS medium pressure with a 5%, 15%, 30%, 50%, 70%, 100% methanol-water gradient. Elution was performed, examined using silica gel GF254 and a thin plate, and fractions containing similar spots were combined, concentrated and dried to obtain 5 fractions A, B, C, D, and E, and fraction B was taken. Dissolve with 50% acetonitrile, filter, create liquid phase chromatographic separation, elute with 13% acetonitrile-water, collect fractions according to the peak time of each chromatographic peak, and concentrate each fraction. and step (3) to obtain pure crystals of Compounds 1 and 2, provided that the purity of both compounds detected by HPLC area normalization method is 98% or more.

The rest is the same as in Example 1.
Example 3
This example provides an azulene-based compound in lilac, and its preparation method is as follows:

Take the medicinal material of dried lilac stems, add 30 times the volume of 95% ethanol, extract by osmosis method, let it permeate for a week, filter, combine the extracts, and reduce the pressure until the alcohol smell disappears.・Step (1) of obtaining the extract as an extract by concentrating;

Take the extract, disperse it in water, and extract it 6 times with dichloromethane and 6 times with ethyl acetate.The amount of dichloromethane and ethyl acetate used each time is 1/4 of the volume of the extracted liquid, and the two solvent extracts are extracted. A step (2) of collecting the water and drying the solvent under reduced pressure to obtain a dichloromethane extraction site, an ethyl acetate extraction site, and a water site;

Take the ethyl acetate extracted part obtained in step (2), completely dissolve it in methanol, add 100-200 mesh silica gel, perform dry sample stirring, and transfer it to a pre-packed 200-300 mesh silica gel column. Add the sample and perform separation by column chromatography and gradient elution with petroleum ether-acetone (volume ratios are 5:1, 4:1, 3:1, 2:1, 1:1, 0:1). was carried out, identified using silica gel GF254 and G thin plate, and 11 fractions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 were obtained by combining fractions containing similar spots. Fraction 4 was taken, dissolved in methanol, filtered, and the filtrate was subjected to chromatographic separation using ODS medium pressure with a 5%, 15%, 30%, 50%, 70%, 100% methanol-water gradient. Elution was performed, examined using silica gel GF254 and a thin plate, and fractions containing similar spots were combined, concentrated and dried to obtain 5 fractions A, B, C, D, and E, and fraction B was taken. Dissolve with 50% acetonitrile, filter, create liquid phase chromatographic separation, elute with 13% acetonitrile-water, collect fractions according to the peak time of each chromatographic peak, and concentrate each fraction. and step (3) to obtain pure crystals of Compounds 1 and 2, provided that the purity of both compounds detected by HPLC area normalization method is 98% or more.
The rest is the same as in Example 1.
Example 4
This example provides an azulene-based compound in lilac, and step (1) in the preparation method is

Take the medicinal material of dried lilac stems, add 20 times the amount of 5% (volume concentration) ethanol, perform heating and reflux extraction three times, for 3 hours, 2 hours, and 1 hour, respectively, and filter. The extract is obtained as an extract by combining the extracts and concentrating under reduced pressure until the alcohol smell disappears.
The rest is the same as in Example 1.
1. Structural analysis of compounds according to the present invention

Its structure is mainly identified using spectroscopic techniques such as ultraviolet, infrared, mass spectra, and nuclear magnetic resonance ( ¹ H-NMR, ¹³ C-NMR, 2D-NMR), and specific spectrograms are shown in Figures 2 to 3. 18, and its spectral data and analysis process are as follows.

(1) Compounds 1 and 2 obtained in Example 1 were taken for structural analysis. However, compounds 1 and 2 were pale yellow amorphous powders, and high-resolution mass spectra ESI-TOF-MSm/z is 301.1046[M+Na] ₊ .

Compound 1 and Compound 2 both have a molecular formula of C ₁₅ H ₁₈ O ₅ , an exact molecular weight of 278.1154, and a degree of unsaturation of 7. Moreover, ^{the 1} H NMR, ¹³ C NMR, HMBC, DEPT, and HSQC spectrograms of both compounds almost match.

As can be seen from comprehensive analysis of ¹ H NMR and ¹³ C NMR, the structure of the compound contains three methyl groups [δC24.2, 23.5 and 11.0 (respectively C-11, C-15 and C-12), δH2.06 (s, Me-12), δH1.38 (s, Me-15) and δH1.37 (s, Me-11)], and two methylene groups [δC69 .1 and 38.6 (respectively C-14 and C-9), δH 3.65 (d, J = 11.3 Hz, H-14a) and δH 3.53 (d, J = 11.3 Hz, H -14b), δH3.02 (d, J = 17.7Hz, H-9a) and 2.00 (dd, J = 2.0, 17.6Hz, H-9b)] and two methines [δC126. 2 and 125.5 (corresponding to C-5 and C-7, respectively), δH6.78 (s, H-5), δH6.49 (d, J = 2.4Hz, H-7)], Eight quaternary carbons [δC189.3, 181.8, 171.2, 160.0, 147.0, 135.9, 76.6 and 45.7 (respectively C-3, C-4, C- 8, C-6, C-2, C-1, C-13 and C-10)].

By combining two-dimensional nuclear magnetic resonance spectrum data such as HMBC, DEPT, and HSQC spectra, the planar structure of the compound was determined, and its chemical planar structural formula is shown in Figure 1, and its chemical name is 8-(1,2-dihydroxy propan-2-yl)-2-hydroxy-1,10-dimethyl-9-dihydroazulene-3,4-dione. Since this compound has two chiral centers, namely C-10 and C-13, the planar structure includes (10R,13S), (10S,13R), (10R,13R), (10S,13S ) There are four steric configurations. The absolute configuration of the compound is (10R,13R ) and (10R, 13S), and a specific spectrogram is shown in FIG. As can be seen from the NOESY spectrum data, there are C-11, C-12, and C-15 methyl hydrogen-related signals for compound 1, but only C-11 and C-12 methyl hydrogen-related signals are generated for compound 2. Therefore, it can be determined that the absolute configurations of Compound 1 and Compound 2 are (10R, 13R) and (10R, 13S), respectively, and the structural formulas thereof are shown in Example 1.
(2) The spectral data of compounds 1 and 2 are summarized as follows.

Compound 1: ^1H NMR (600MHz, MeOD) δ6.79 (s, 1H, H-5), 6.49 (d, J = 2.4Hz, 1H, H-7), 3.70 (d, J = 11.3Hz, 1H, H-14a), 3.62 (d, J = 11.3Hz, 1H, H-14b), 3.14 (d, J = 17.7Hz, 1H, H-9a), 2.43 (dd, J=17.6, 2.0Hz, 1H, H-9b), 2.07 (s, 3H, H-12), 1.38 (s, 3H, H-15), 1 .36 (s, 3H, H-11).

^13C NMR (150MHz, MeOD) δ 187.9 (C-3), 180.5 (C-4), 169.7 (C-8), 158.7 (C-6), 145.6 (C- 2), 134.6 (C-1), 124.7 (C-5), 124.1 (C-7), 74.9 (C-13), 68.1 (C-14), 44. 2 (C-10), 37.0 (C-9), 22.9 (C-11), 22.1 (C-15), 9.6 (C-12).

Compound 2: ¹ HNMR (600MHz, MeOD) δ6.79 (s, 1H, H-5), 6.51 (d, J = 2.4Hz, 1H, H-7), 3.68 (d, J = 11.3Hz, 1H, H-14a), 3.56 (d, J = 11.3Hz, 1H, H-14b), 3.05 (d, J = 17.7Hz, 1H, H-9a), 2 .47 (dd, J=17.6, 2.0Hz, 1H, H-9b), 2.08 (s, 3H, H-12), 1.40 (s, 3H, H-15), 1. 38 (s, 3H, H-11).

^13C NMR (150MHz, MeOD) δ 187.9 (C-3), 180.4 (C-4), 169.8 (C-8), 158.6 (C-6), 145.6 (C- 2), 134.5 (C-1), 124.8 (C-5), 123.6 (C-7), 75.2 (C-13), 67.7 (C-14), 44. 2 (C-10), 37.2 (C-9), 22.8 (C-11), 22.1 (C-15), 9.6 (C-12).

2. Research on the anti-inflammatory effect of compounds according to the present invention (1) Experimental materials and reagents 1. drugs and reagents

Two compounds 1 and 2 prepared in Example 1 (self-extracted, purity 98% or higher); Mouse peritoneal macrophage RAW264.7 (Shanghai Hengke Biotechnology Co., Ltd.); Fetal bovine serum (FBS) (Thermo Fisher/Gibco company). CCK-8 reagent kit, IL-1β reagent kit, IL-6 reagent kit, TNF-α reagent kit, DMEM medium, penicillin and streptomycin mixture (100x) (Beijing Soraibao Technology Co., Ltd.), DMSO, LPS ( (American sigma company); Nitric oxide detection reagent kit (Shanghai Biyuntian Biotechnology Co., Ltd.).
2. Laboratory instrument

BT 125D electronic balance (Beijing Sartorius Scientific Instruments Co., Ltd.); SPECTRO star Nano full wavelength microplate reader (BMGLABTECH); Form 311 carbon dioxide incubator (Thermo Scientific Company).
3. Test drug and treatment method

The compound concentration was determined by accurately weighing 127.82 mg of the compound and 227.79 mg of the compound, placing them in a 5 ml volumetric flask, dissolving them in a small amount of DMSO, and adjusting the amount to the marked line by diluting with DMEM complete medium. DMEM medium mother liquors containing drugs of 20 mmol/L and 19.98 mmol/L are obtained, respectively, and diluted to the required concentration with DMEM medium during the experiment. The volume ratio of DMSO is controlled within three thousandths.
(2) Experimental method

1. Cell culture: RAW264.7 cells are introduced into a DMEM medium containing a mixture of 10% FBS, 1% penicillin and streptomycin, subcultured in an incubator containing 5% _CO2 at 37°C, and subcultured every other day.
2. Evaluation of the effects of both compounds on RAW264.7 cell activity

RAW264.7 cells in the logarithmic growth phase were taken, the cell density was adjusted to 1 × 10 ⁴ ml ^-1 with DMEM medium containing 10% FBS culture medium, and the cells were seeded on a 96-well plate and treated with compounds containing different drug concentrations. Cell culture medium was placed in a 96-well plate at 100 μL/hole, and drug supply groups were designed with drug concentrations of 12.5, 25, 50, 100, 200, and 400 μmol/L, respectively, and a normal cell control group was set separately. Then, cell culture medium without drug is added to the normal group, and each group has 6 multiple holes. After culturing for 24 h at 37°C and 5% _CO2 , the supernatant was discarded, 100 μL of 10% CCK-8 solution was added to each well, and the absorbance (OD value) was measured at 450 nm using a microplate reader. Calculate survival rate.
Evaluation of the effects of both compounds on LPS-induced RAW264.7 cell lipid peroxidation

(1) How to draw a standard curve: Dilute the standard product into solutions with concentrations of 0, 1.56, 3.125, 6.25, 12.5, 25, 50, and 100 mM, and add an equal amount of Griess reagent. The absorbance was measured at a wavelength of 550 nm using a microplate reader, and a standard curve was drawn.

(2) The inhibitory effect of RAW264.7 on NO production induced by LPS is measured by the Griess method. RAW264.7 cells in the logarithmic growth phase were seeded in a 96-well plate (5 x ¹⁰⁴ cells/well) and cultured for 24 hours at 37°C and 5% _CO2 . Add 100 μL of DMEM medium containing (low dose 50 μmol/L, medium dose 100 μmol/L, high dose 200 μmol/L), add 100 μL of blank medium to each hole of the model group and blank group, and culture for 1 h. 100 μL of LPS (2 μg/ml) solution was added to each remaining group except for the blank group, and after continued culturing for 24 h, 50 μL of supernatant was absorbed from each well into a new 96-well plate, and 50 μL of Griess I solution and Sequentially add 50 μL of Griess II solution, measure the OD value at 550 nm using a microplate reader, and calculate the NO suppression rate.
4. Detect the content of inflammatory factors IL-1β, IL-6 and TNF-α by ELISA method

The test group division, modeling and drug feeding process were the same as in step 3 (2), and the cell supernatant was collected and divided into each test group at 3, 6, 12, and 24 h according to the operating instructions of the cell factor detection reagent kit. The secreted amounts of IL-1β, IL-6, and TNF-α in the supernatant of RAW264.7 cells are measured.
5. Data processing and analysis

The data were subjected to t-test analysis using Excel software, and the results are expressed as "x±s". Histograms are drawn with Graph Pad Prism 8.0.2 software.
(3) Experimental results 1. Evaluation results of the effects of both compounds on RAW264.7 cell activity

As shown in FIGS. 19 and 20, the detection results by the CCK-8 method show that at 400 μmol/L, both compounds clearly have no effect on the proliferation of RAW264.7 macrophages within 24 hours.
2. Evaluation of the effects of both compounds on LPS-induced RAW264.7 cell lipid peroxidation The results of the effects of both compounds on LPS-induced RAW264.7 cell NO release are shown in Table 1 below and Figures 21 and 22. .

As a result of the analysis, as can be seen by comparing the model group of compounds 1 and 2 with the control group, there was a very significant difference between the two (P<0.01), and NO production in RAW264.7 cells was increased by LPS stimulation. The modeling success was markedly increased. As can be seen by comparing the drug supply group and the model group, there is a very significant difference between the high and medium dose groups of compounds 1 and 2 (P<0.01), and a significant difference in the low dose group. (P<0.05), and Compounds 1 and 2 have a clear inhibitory effect on NO production in RAW264.7 cells stimulated by LPS.

Table 1 Effects of both compounds on LPS-induced RAW264.7 cell lipid peroxidation

3. Detect the content of inflammatory factors IL-1β, IL-6 and TNF-α by ELISA method

As shown in Tables 2 and 3, the experimental results show that the contents of IL-1β, IL-6, and TNF-α in the cell supernatant of the model group after LPS stimulation were as seen by comparing the model group and the blank group. The modeling was successful as both increased significantly. As can be seen by comparing the drug supply group and the model group, the IL-1β content in the high-dose groups of compounds 1 and 2 and the medium-dose group of compound 2 showed a very significant downward trend at all four time points. In addition, IL-1β in the middle dose group of compound 1 also expressed a certain degree of downward trend, and both compounds expressed a dose-dependent relationship at the same time point, and IL-1β in the high dose group of compound 1 also expressed a certain degree of downward trend. The content of 6 decreased very significantly at all four time points, and the middle dose group of compound 1 and the high and middle dose groups of compound 2 also exhibited a downward trend. The content of TNF-α in the high-dose groups of compounds 1 and 2 both decreased significantly, whereas the content of TNF-α in the low-dose groups of compounds 1 and 2 was suppressed to some extent. As can be seen, compounds 1 and 2 can clearly suppress the excessive secretion of IL-1β, IL-6 and TNF-α by RAW264.7 cells under LPS-induced conditions to a certain extent. It was found that compounds 1 and 2 have clear anti-inflammatory effects.

Table 2 Effect of Compound 1 on LPS-induced secretion of inflammatory factors IL-1β, IL-6 and TNF-α in RAW264.7 cells

Table 3 Effect of compound 2 on LPS-induced secretion of inflammatory factors IL-1β, IL-6 and TNF-α in RAW264.7 cells

Note: In Tables 1, 2, 3 and Figures 21 and 22 above, ^# means a comparison between the model group and the blank group, ^# represents P<0.05, ^## represents P<0.01 ^* means comparison between compound (high, medium, low) dose group and model group; ^* represents P<0.05; ^** represents P<0.01.

Finally, although the above is only a preferred embodiment of the present invention and is not intended to limit the present invention, and the present invention has been described in detail with reference to the foregoing embodiment, it is still clear to those skilled in the art that the foregoing implementation The technical proposals described in the examples may be modified or some of their technical features may be replaced with equivalents. Any modification, equivalent substitution, improvement, etc. made within the spirit and principle of the present invention shall fall within the protection scope of the present invention.

Claims (6)

An azulene compound characterized by being Compound 1 or Compound 2 shown by the following structural formula.
A method for producing an azulene compound according to claim 1, comprising:
A step (1) of introducing an ethanol solution into lilac stems, which is a medicinal material, for extraction, collecting the extract after filtration, and concentrating the extract as an extract;
The extract is taken out, dispersed in water, extracted sequentially with dichloromethane and ethyl acetate, the two solvent extracts and water are collected, and the solvent is concentrated under reduced pressure and dried to obtain a dichloromethane extracted portion and an ethyl acetate extracted portion. and step (2) of obtaining a water portion;
A step (3) of taking out the ethyl acetate extract obtained in step (2) and performing separation and purification by chromatography to obtain an azulene compound,
Separation and purification by chromatography in step (3)
The ethyl acetate extract obtained in step (2) was dissolved in methanol, silica gel was added and stirred, the resulting sample was placed in a silica gel column and separated by column chromatography, and petroleum ether with different volume ratios were dissolved. step A of performing gradient elution with /acetone to obtain 11 fractions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and 11;
Fraction 4 is taken and separated by medium pressure chromatography using an ODS column, and gradient elution is performed with methanol/water in different volume ratios to obtain 5 fractions A, B, C, D and E. ,
Fraction B was taken and separated by liquid phase chromatography, eluted with acetonitrile/water with a volume concentration of 13%, fractions were collected according to the peak time of each chromatographic peak, and each fraction was concentrated to make it pure. Step C of obtaining crystals of compounds 1 and 2,
In step A, the silica gel for stirring is 100 to 200 mesh, the mass ratio of the ethyl acetate extraction part to the silica gel for stirring is 1:10, and the silica gel in the silica gel column is 200 to 300 mesh. and the volume ratios of petroleum ether and acetone for gradient elution are 5:1, 4:1, 3:1, 2:1, 1:1, and 0:1, respectively,
In step B, the volume concentrations of the methanol aqueous solution used for gradient elution are 5%, 15%, 30%, 50%, 70%, and 100%, respectively, and the medium pressure chromatography using the ODS column is performed using the ODS column. - Using a C18 chromatography column, the chromatography column size is 800 x 25 mm, the particle size is 20-45 μm, the flow rate is 30 ml/min, and the column temperature is 25 ° C.
The liquid phase chromatography in step C uses an azulene chromatography column of YMC-Triart C18, the chromatography column size is 250 x 20 mm, the particle size is 5 μm, and the flow rate is 10 ml/min. Method for producing system compounds. The ethanol solution in step (1) is an aqueous ethanol solution with a volume concentration of 5 to 95%, and the extraction is performed by a cold immersion method, an osmosis method, a microwave extraction method, an ultrasonic extraction method, a reflux extraction method or a continuous reflux extraction method. 3. The method for producing an azulene compound according to claim 2, wherein the method is extraction by. The number of times of extraction in step (1) is 1 to 3 times, the mass ratio of the input amount of ethanol solution to the medicinal material lilac stem is (8 to 30):1, and the dichloromethane in step (2) is The number of times of extraction with , and ethyl acetate is 4 to 6 times, respectively, and the amount of dichloromethane and ethyl acetate used each time is 1/2 to 1/4 of the volume of the liquid to be extracted. 3. The method for producing an azulene compound according to claim 2. Use of the azulene compound according to claim 1 in the preparation of an anti-inflammatory drug. The anti-inflammatory drug is an oral preparation or an injection prepared by adding the azulene compound directly or indirectly to a pharmaceutically acceptable carrier or excipient, and the oral preparation is in the form of a tablet or a capsule. 6. The use of an azulene compound according to claim 5, wherein the injectable preparation is an injection solution or a powder injection preparation.