Azalophenanthrene ketone derivative with anti-hepatic ischemia-reperfusion injury activity and preparation method and application of azalophenanthrene ketone derivative

Abstract

The invention belongs to the technical field of pharmaceutical compounds, and particularly relates to an azaphenanthrone derivative with anti-hepatic ischemia-reperfusion injury activity as well as a preparation method and application of the azaphenanthrone derivative. The structural formula of the azaphenanthrone derivative is shown as a formula A, in the formula A, R1 is CHO or COOH, and R2 is H or Cl. According to the application, the azaphenanthrone derivative is extracted from an acupoint shell fungus fermentation solution, and experiments prove that the compound has the activity of resisting the hepatic ischemia-reperfusion injury, so that the compound has the potential to be developed into an active medicine for resisting the hepatic ischemia-reperfusion injury. And a new choice is provided for developing an active medicine for resisting the hepatic ischemia-reperfusion injury.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of pharmaceutical compounds, and particularly relates to an azapherones derivative with anti-liver ischemia-reperfusion injury activity, and a preparation method and application thereof. BACKGROUND

[0002] Mangrove forests are mainly distributed in the intertidal zone of tropical and subtropical coasts, and are strictly limited by environmental factors such as temperature, salinity and tides, and have a small global distribution range. In China, mangrove forests are only distributed in coastal provinces, and are rare protected species. At present, there are six national mangrove nature reserves in China (Dongzhai Port in Hainan, Beilun Estuary in Guangxi, Shankou in Guangxi, Zhanjiang in Guangdong, Futian in Shenzhen and Zhangjiang Estuary in Fujian), and there are only 20 families and 37 species of mangrove plants in the country, of which 23 species have medicinal value and 13 species are used as folk medicine. The high temperature, high salinity, soil hypoxia and periodic seawater immersion of mangrove forests create a unique ecological environment, and are one of the important resources for drug development and lead compound discovery.

[0003] Hepatic ischemia-reperfusion injury (HIRI) refers to the damage caused by the temporary interruption of blood flow to the liver (ischemia) and its subsequent restoration (reperfusion). This phenomenon is a common clinical challenge encountered in major liver surgery, liver transplantation surgery and liver trauma. HIRI often leads to liver damage, increases the risk of early graft failure, and can progress to liver failure, and in severe cases, can lead to multiple organ failure and even death.

[0004] Although various preoperative and postoperative treatment methods have been reported to alleviate HIRI, these methods are only effective in a small number of patients with long ischemia and small liver resection. In addition, so far, there is no officially approved drug treatment for the prevention or management of HIRI. Studies have shown that certain interventions, including calcium channel blockers, adenosine receptor agonists, energy metabolism modulators and antioxidants, may have a protective effect on HIRI, but due to adverse reactions, related research is mainly limited to animal studies.

[0005] Therefore, it is urgent to find and develop new drugs for the prevention and treatment of HIRI. SUMMARY

[0006] To solve the above technical problems, the present application first provides an azapherones derivative, the structural formula of which is shown as formula (A):

[0007]

[0008] wherein R1 is CHO or COOH, and R2 is H or Cl.

[0009] The second object of the present application is to provide a preparation method of the azapherones derivative, which is a secondary metabolite of Dothiorella sp.

[0010] The preparation method comprises the following steps:

[0011] S1. Fungus culture: after the target fungus is activated, it is inoculated into PDB culture medium, and cultured at 27℃ constant temperature shaking table for 4-5 days to obtain a seed liquid; the seed liquid is transferred into a rice liquid culture medium, and cultured at room temperature for 27±3 days to obtain a fermentation liquid; the target fungus is a Dothiorella sp. strain preserved in the China Marine Microbial and Fermentation Product Testing Center with the preservation number MCCC 3J00072;

[0012] S2. Crude extraction: after the fermentation is completed, methanol solution is added to the rice liquid culture medium, soaked for 24h, then filtered and extracted for 3 times, the filtrates are combined and concentrated to obtain a crude extract, then extracted with ethyl acetate for 3 times, and concentrated under reduced pressure to obtain an ethyl acetate part extract;

[0013] S3. Separation and purification: the extract is subjected to silica gel mixing, separated by normal phase silica gel column, gradient eluted with ethyl acetate-petroleum ether as the starting eluent, and components Fr.1-Fr.10 are obtained, wherein,

[0014] 1) the eluted component Fr.3 is collected, further separated by a silica gel column chromatography, the eluent is dichloromethane-methanol with a volume ratio of 150:1, after spotting, the fraction with absorption at 265nm and brown yellow color after vanillin-sulfuric acid coloration is selected, combined and concentrated to obtain compound 1;

[0015] 2) the eluted component Fr.5 is collected, further separated by a silica gel column chromatography, the eluent is dichloromethane-methanol with a volume ratio of 50:1, to obtain sub-components Fr.5.1-5.3;

[0016] 2.1) the sub-component Fr.5.1 is first purified by a Sephadex LH-20 gel column chromatography, the eluent is dichloromethane-methanol with a volume ratio of 1:1; the purified product is further separated by a silica gel column chromatography, the eluent is dichloromethane-methanol with a volume ratio of 40:1; the fraction with absorption at 265nm and yellow color after vanillin-sulfuric acid coloration is selected, combined and concentrated to obtain compound 2;

[0017] 2.2) Sub-fraction Fr.5.3 was purified by silica gel column chromatography with eluent of dichloromethane-methanol (35:1, by volume); the purified product was further separated by high performance liquid chromatography (HPLC) with mobile phase of methanol-water mixture (75:25, by volume) at a flow rate of 2.0 mL / min; the fraction with absorption at 265 nm and light yellow color after vanillin-sulfuric acid coloration was selected and combined to obtain compound 3 after concentration;

[0018] The compound 1 is an azafenidin derivative with R1 being CHO and R2 being H; the compound 2 is an azafenidin derivative with R1 being CHO and R2 being Cl; and the compound 3 is an azafenidin derivative with R1 being COOH and R2 being H.

[0019] Preferably, in the step S1, the strain is inoculated on a rice solid culture medium, and incubated at room temperature for 27±3 days, and then inoculated on a PDA culture medium, and incubated at room temperature for 5 days to obtain the activated Chaetomium strain; the rice solid culture medium comprises rice 100 g / L, NaBr 50 g / L and peptone 0.3 g / L.

[0020] Preferably, the rice liquid culture medium comprises rice 100 g / L and 0.3% w / v coarse salt water 80 mL / L.

[0021] Preferably, in the step S3, gradient elution is performed with a system of 0 / 1 ethyl acetate-petroleum ether: 1 / 0 ethyl acetate-petroleum ether; the third fraction Fr.1 is eluted at 3 / 7 ethyl acetate-petroleum ether; and the first fraction Fr.5 is eluted at 5 / 5 ethyl acetate-petroleum ether.

[0022] The third object of the present application is to provide use of the azafenidin derivative as described above in the preparation of a medicament for preventing liver ischemia-reperfusion injury.

[0023] The fourth object of the present application is to provide a medicament for preventing liver ischemia-reperfusion injury, characterized in that it comprises a pharmaceutically effective dose of the azafenidin derivative as described above and a pharmaceutically acceptable carrier.

[0024] Preferably, the azafenidin derivative accounts for 60-90% of the total mass or total volume of the medicament.

[0025] Preferably, the azafenidin derivative accounts for 62%, 65%, 70%, 72%, 75%, 78%, 80%, 85%, 88% of the total mass or total volume of the medicament.

[0026] Preferably, the pharmaceutically acceptable carrier comprises one or more of excipients, stabilizers, antioxidants, coloring agents, diluents, sustained-release agents, such as starch, lipids, waxes, dextrin, sucrose, lactose, microcrystalline cellulose, gelatin, citric acid, inorganic salts, hydroxypropyl methylcellulose, hydroxyethyl cellulose, and the like.

[0027] Preferably, the medicine is any one of an injection, a tablet, a granule, a pill, a capsule, a suspension, or an emulsion.

[0028] The beneficial effects of the present application are:

[0029] 1. The present application extracts a new azaphilone derivative from the fermentation broth of the small hole shell fungus strain, which shows significant anti-inflammatory activity in in vitro cell experiments and significantly improves liver damage in a mouse model of liver ischemia-reperfusion injury, suggesting that it can be used to prepare clinically active drugs for treating liver ischemia-reperfusion injury, and the present application provides a new choice for developing anti-liver ischemia-reperfusion injury active drugs.

[0030] 2. The LPS-induced mouse macrophage RAW264.7 cell model shows that the azaphilone derivative provided by the present application can effectively inhibit NO release, and compound 1 has the best inhibitory effect. The mouse model of liver ischemia-reperfusion injury shows that the azaphilone derivative provided by the present application can significantly reduce the formation of vacuoles in the liver tissue, neutrophil infiltration, and the production of hyperemia caused by ischemia-reperfusion, significantly reduce the levels of important liver indicators ALT and AST, significantly reduce the expression levels of inflammatory factors TNF-α, IL-1α, and IL-6 in the liver serum tissue, and down-regulate the protein expression level of AIM-2, indicating that the azaphilone derivative provided by the present application can be used as an anti-inflammatory drug, and plays an anti-HIRI role by blocking the activation of inflammatory response (inhibiting the activation of AIM2 inflammasome).

[0031] 3. The azaphilone derivative provided by the present application is a secondary metabolite of the small hole shell fungus, which can be prepared by microbial fermentation. The preparation method is simple, short in cycle, mild in culture conditions, low in by-products and cost, and can be obtained by infinite cycle fermentation, not limited by resource shortage, green and environmentally friendly, easy to realize industrialization, not only meets the demand of modern green low-carbon environmental protection economy, but also provides a new way for potential anti-HIRI drug discovery. BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1 The HR-ESI-MS spectrum of the new compound 3 provided by the present application is shown.

[0033] Figure 2 The HR-ESI-MS spectrum of the new compound 3 provided by the present application is shown. 1H NMR spectrum.

[0034] Figure 3 The new compound 3 provided in this application 13 C NMR spectrum.

[0035] Figure 4 The new compound 3 provided in this application 1 H- 1 H COSY spectrum.

[0036] Figure 5 HSQC spectrum of the novel compound 3 provided for this application.

[0037] Figure 6 HMBC spectrum of the novel compound 3 provided for this application.

[0038] Figure 7 The new compound 3 provided in this application 1 H- 1 H COSY and HMBC related spectra.

[0039] Figure 8 To screen the toxicity of compounds 1-3 provided in this application to cells.

[0040] Figure 9 The compounds 1-3 provided in this application were screened for inhibition of LPS-induced NO.

[0041] Figure 10 The effects of compounds 1-3 provided in this application on the expression of inflammatory factors TNF-α (Figure A), IL-6 (Figure B), and IL-1β (Figure C).

[0042] Figure 11 H&E staining of liver sections in a mouse liver ischemia-reperfusion model using compound 1 provided in this application.

[0043] Figure 12 The compound 1 provided in this application was used to detect aspartate aminotransferase (A) and alanine aminotransferase (B) in plasma of a mouse liver ischemia-reperfusion model.

[0044] Figure 13 The compound 1 provided in this application was used to detect the inflammatory factors IL-6 (A in the figure), TNF-α (B in the figure), and IL-1α (C in the figure) in the liver tissue of a mouse liver ischemia-reperfusion model.

[0045] Figure 14 The effect of compound 1 provided in this application on AIM2 inflammasome in tissue proteins of a mouse liver ischemia-reperfusion model. Detailed Implementation

[0046] The terms used herein have meanings commonly understood by those skilled in the art, unless otherwise specified.

[0047] The technical solutions of the present application will be described in more detail below in conjunction with the examples.

[0048] In this application, room temperature refers to 27±3℃, Dothiorella sp. (ZJQQYZ-1) is from China Marine Microbial and Fermentation Product Testing and Engineering Center, and the preservation number is MCCC 3J00072. The materials used in the culture medium are northeast rice, and the organic solvent extraction reagents petroleum ether, ethyl acetate, dichloromethane and methanol are all analytical pure reagents.

[0049] Example 1

[0050] 1. Extraction of azafenidin derivatives

[0051] The azafenidin derivatives have the following general structural formula:

[0052]

[0053] Among them, R1 is CHO or COOH, and R2 is H or Cl.

[0054] The azafenidin derivatives are secondary metabolites of Dothiorella sp. fungi. The preparation method comprises the following steps:

[0055] S1. Fungal culture

[0056] Activation: inoculate Dothiorella sp. (ZJQQYZ-1) on PDA medium and incubate at room temperature for 5 days to obtain activated strains. The composition of PDA medium is: potato powder 300.0 g / L, glucose 20.0 g / L, agar 15.0 g / L, chloramphenicol 0.1 g / L;

[0057] Primary culture: inoculate the activated strains on PDB medium and culture at 27℃ in a shaker (160 r / min) for 4-5 days to obtain about 500 mL of fungal seed liquid. The composition of PDB medium is: potato powder 300.0 g / L, glucose 20.0 g / L.

[0058] Fermentation culture: transfer about 10 ml of seed liquid into rice medium (100×1000 mL conical flask, each containing 100 g of rice, 50 g of NaBr and 0.3 g of proteose peptone), and grow at room temperature for 30 days to obtain fermentation broth.

[0059] S2. Crude extraction: 200 mL of methanol solution was added to the rice liquid medium after 30 days, soaked and extracted 3 times, the filtrate was concentrated to obtain the extract of the crude extract, then extracted with ethyl acetate 3 times, concentrated under reduced pressure to obtain the extract of the ethyl acetate fraction.

[0060] S3. Separation and purification: the extract was mixed with silica gel, separated by normal phase silica gel column, gradient eluted with ethyl acetate-petroleum ether as the starting eluent, to obtain components Fr.1-Fr.10, wherein,

[0061] 1) the third component Fr.3 eluted in 3 / 7 ethyl acetate-petroleum ether system, Fr.3 was further separated by silica gel column chromatography, the eluent was dichloromethane-methanol with a volume ratio of 150:1, after spotting, the fraction with absorption at 265 nm wavelength and brownish yellow color after vanillin with concentrated sulfuric acid was selected, and then combined and concentrated to obtain compound 1;

[0062] 2), the fifth component Fr.5 eluted in 5 / 5 ethyl acetate-petroleum ether system, Fr.5 was further separated by silica gel column chromatography, the eluent was dichloromethane-methanol with a volume ratio of 50:1, to obtain sub-components Fr.5.1-5.3;

[0063] 2.1) the sub-component Fr.5.1 was first purified by Sephadex LH-20 gel column chromatography, the eluent was dichloromethane-methanol with a volume ratio of 1:1; the purified product was further separated by silica gel column chromatography, the eluent was dichloromethane-methanol with a volume ratio of 40:1; the fraction with absorption at 265 nm wavelength and yellow color after vanillin with concentrated sulfuric acid was selected, and then combined and concentrated to obtain compound 2;

[0064] 2.2) the sub-component Fr.5.3 was first purified by silica gel column chromatography, the eluent was dichloromethane-methanol with a volume ratio of 35:1; the purified product was further separated by high performance liquid chromatography (HPLC), the mobile phase was methanol-water mixture with a volume ratio of 75:25, and the flow rate was 2.0 mL / min; the fraction with absorption at 265 nm wavelength and light yellow color after vanillin with concentrated sulfuric acid was selected, and then combined and concentrated to obtain compound 3;

[0065] The above compound 1 is an azaphilone derivative with R1 being CHO and R2 being H; compound 2 is an azaphilone derivative with R1 being CHO and R2 being Cl, and compound 3 is an azaphilone derivative with R1 being COOH and R2 being H.

[0066] 2. Characterization of azaphilone derivatives

[0067] The structure of the compound was determined by NMR nuclear magnetic resonance, high resolution mass spectrometry (HRESIMS) and ECD calculation,

[0068] Compound 1 (R1 is CHO, R2 is H): white solid; IR (KBr) υ max : 3381, 2963, 2920, 1680, 1625, 1598, 1580 cm -1 ; UV (MeOH) λ max : 290 (1.2) nm; HRESIMS: m / z 316.1672 [M + H] + (calcd for C 19 H 24 O4, 316.1675). 1 Hand 13 C NMR (DMSO-d6) data are listed in Table 1. Nomenclature: 2,4-dihydroxy-6-(5,7-dimethyl-2-oxo-trans-3-trans-5-nonadienyl)-3-methylbenzaldehyde.

[0069] Compound 2 (R1 is CHO, R2 is CI): white solid; IR (KBr) υ max : 3380, 2965, 2923, 1678, 1626, 1600, 1581 cm -1 ; UV (MeOH) λ max : 291 (1.3) nm; HRESIMS: m / z 350.1360 [M + H] + (calcd for C 19 H 23 CI04, 316.1361). 1 H and 13 C NMR (CDC13) data are listed in Table 1. Nomenclature: 6-((1E,3E)-3,5-dimethylhepta-l,3-dien-l-yl)-2,4-dihydroxy-3-methylbenzaldehyde.

[0070] Compound 3 (R1 is COOH, R2 is H): white solid; IR (KBr) υ max : 3380, 2965, 2922, 1681, 1630, 1588, 1568 cm -1 ; UV (MeOH) λ max : 290 (1.2) nm; HRESIMS: m / z 355.1512 [M + Na] + (calcd for C 19 H24 O5,355.1510); 1 Hand 13 C NMR(CD3OD) data were listed in Table 1. Compound 1 was named as dothioretone A.

[0071] Represented by compound 3, see Figures 1-7 , respectively, HR-ESI-MS spectrum of compound 3, 1 H NMR spectrum, 13 C NMR spectrum, 1 H- 1 H COSY spectrum, HSQC spectrum, HMBC spectrum and main 1 H- 1 H COSY and HMBC correlation charts.

[0072] Table 1 NMR nuclear magnetic resonance data of compounds 1-3 (125MHz / 500MHz, TMS, ppm)

[0073]

[0074]

[0075] Example 2

[0076] Study on anti-HIRI activity of compounds 1-3

[0077] Experimental materials: mouse macrophage cell strain RAW 264.7 cells, C57BL / 6 mice, fetal bovine serum (FBS), penicillin-streptomycin mixture, DMEM high-sugar culture medium, carbon dioxide incubator, centrifuge, electrophoresis instrument, inverted microscope, cell culture plate, lipopolysaccharide, PBS buffer, cell lysis solution, enzyme-linked immunoassay kit (TNF-α, IL-1β, IL-6, ALT, AST), TRIzol lysis solution, anhydrous ethanol, isopropanol, TBST, fixing solution, hematoxylin-eosin, CCK8 kit, NO kit, BCA protein quantification kit, protease phosphatase inhibitor, PMSF protease inhibitor, skimmed milk powder, primary antibody (AIM-2, β-actin), goat anti-mouse / rabbit HRP labeled secondary antibody, etc.

[0078] 1. Cell experiment

[0079] Cell culture: mouse macrophage cell strain RAW 264.7 cells were cultured in DMEM high-sugar culture medium containing 10% fetal bovine serum (FBS), 1% penicillin (100 U / mL) and streptomycin (100 μg / mL), and placed in a 37℃, 5% CO2 incubator for culture. When the cell fusion reached 70%-80%, it was subcultured at a ratio of 1:3, and the logarithmic phase growth cells were used for experiment.

[0080] 1) Cell viability assay: When the cells were in logarithmic growth, 96-well plates were seeded with 2 x 104 cells per well. After 24 hours, the original culture medium was aspirated, and the basic culture medium containing drugs and the basic culture medium alone were added. The drug groups were set at five concentration gradients, 50, 25, 12.5, 6.25, and 3.125 μM, respectively. After another 24 hours, the CCK8 kit was used to detect the absorbance at 450 nm after incubation at 37°C for 1 hour. The effect of the drugs on the viability of RAW 264.7 macrophages was detected.

[0081] Results are shown in Figure 8 , and compounds 1-3 had no toxic effects on macrophages RAW264.7 at a concentration of 6.25 μM.

[0082] 2) Nitric oxide (NO) content determination: The nitric oxide content of the cell supernatant was determined using the nitrate reduction method. The logarithmically growing mouse RAW 264.7 cells were inoculated in a 24-well plate, and after 24 hours of culture, different concentrations of compounds or LPS at a concentration of 1 μg / mL were added. The experiment was divided into three groups: a blank control group (only fresh culture medium was added), an LPS modeling group (fresh culture medium + LPS 1 μg / mL), and a test compound group (fresh culture medium diluted with compounds + LPS 1 μg / mL). After 24 hours of LPS stimulation, the cell supernatant was collected and centrifuged, and the nitric oxide detection kit was used to detect and calculate the concentration of NO in the cell supernatant.

[0083] The detection steps are as follows according to the reagent instructions: take out Griess Reagent I and II and the cell supernatant and restore to room temperature. (1) Prepare the standard curve: dilute the standard concentration to 1, 5, 10, 20, 40, 60, and 100 μM with fresh culture medium. (2) Add 50 μL per well of the standard and cell supernatant to a 96-well plate. (3) Restore to room temperature and add 50 μL of Griess Reagent I and 50 μL of Griess Reagent II, respectively. (4) Measure the absorbance at 540 nm using a microplate reader. (5) Calculate the concentration of NO in the cell supernatant according to the standard curve.

[0084] Results are shown in Figure 9 At the experimental concentration, compounds 1-3 and L-NMMA (positive control, 12.5 μM) had significant inhibitory activity on the release of NO from LPS-induced macrophages RAW264.7. Compared with the positive control drug, compound 1 showed strong inhibitory activity, with an IC 50 of 1.13 μM, and compounds 2 and 3 had moderate inhibitory activity, with IC50 The concentrations were 23.8 and 9.6 μM, respectively (positive control L-NMMA: 17.7 μM).

[0085] Furthermore, the effect of compound 1 on the expression of inflammatory factors in LPS-induced RAW264.7 macrophages was also examined. See [link / reference]. Figure 10 Compared with the blank group, LPS stimulation significantly increased the expression levels of inflammatory factors TNF-α, IL-6, and IL-1β in RAW264.7 cells, but compound 1 significantly reduced the expression levels of inflammatory factors.

[0086] 2. Mouse experiment

[0087] Establishment of a mouse ischemia-reperfusion model: Fifty-four male C57BL / 6 mice aged 8-12 weeks (20-30g) were divided into six groups: sham-operated group, model group, and drug-treated group (compound 1). The mice received intraperitoneal injections of the drug (10mg / kg) for three days, while the sham-operated group received only the same dose of saline. Mice were fasted for 12 hours prior to surgery but allowed free access to water. Mice were anesthetized with isoflurane inhalation. The abdominal area was shaved and disinfected with 75% alcohol before being fixed to a sterile drape lined with a heated blanket. A 1.5-2cm incision was made along the midline of the abdomen. The abdominal mucosa was gently dissected, and the intestines were opened with a saline-soaked cotton swab to expose the liver. The left / middle lobe hepatic pedicle was isolated and blocked using a non-invasive microvascular clamp, blocking approximately 70% of the total liver volume. After confirming that the left / middle lobe liver had darkened in color, the wound was covered with moist sterile gauze to prevent fluid loss. One hour after ischemia, the vascular clamps were removed, and the blood supply to the ischemic area was observed to be restored. The abdominal cavity was moistened with physiological saline and sutured layer by layer. The mice were placed in a 37°C incubator until they awoke and their vital signs stabilized, and then transferred to clean cages. In the sham surgery group, only laparotomy was performed to expose the liver and close the cavity; no vascular clamps were used.

[0088] Six hours later, blood was collected from the orbital cavity of the mice, and liver tissue was extracted for later use. After the blood was kept at room temperature for 2 hours, it was centrifuged at 7000 RPM for 10 minutes at 4°C. The upper serum layer was aspirated using a 200 μl pipette, avoiding the lower plasma layer. The serum was then stored at -80°C. Liver tissue extraction: After successful blood collection, the mice were placed flat on the operating table, and their limbs were secured with rubber bands. The original incision was extended vertically to approximately 4 cm to enter the abdominal cavity. An abdominal retractor was used to expose the surgical field. A sterile, saline-moistened cotton ball was used to gently push the liver and gastrointestinal tract upwards and downwards to expose the liver pedicle. A 1.5 cm section of the left lobe of the liver was removed, fixed in 4% paraformaldehyde solution for 24 hours, dehydrated, embedded, and sectioned in paraffin for H&E staining. The remaining liver tissue was retained in a 1.5 ml centrifuge tube and stored at -80°C.

[0089] 1) Staining experiment: After the liver tissue was taken out, it was fixed with 4% paraformaldehyde overnight. Different concentrations of ethanol were used for dehydration (70% ethanol, 1 h, 80% ethanol, 1 h, 90% ethanol, 1 h, 95% ethanol, 1 h, anhydrous ethanol, 1 h), transparency (xylene), and then the liver tissue was paraffin-embedded and sliced using a microtome. De-waxing: 1) complete immersion in xylene for 20 min; 2) complete immersion in xylene for 30 min; 3) complete immersion in anhydrous ethanol for 6 min; 4) immersion in anhydrous ethanol for 6 min; 5) complete immersion in 95% ethanol for 6 min; 6) complete immersion in 85% ethanol for 6 min; 7) complete immersion in 70% ethanol for 6 min; 8) complete immersion in pure water for 6 min. Staining: 1) hematoxylin for 4 min; 2) tap water rinse for 10 min; 3) hydrochloric acid ethanol differentiation for 3 s; 4) tap water rinse for 10 min (reblue); 5) eosin for 1 min. Dehydration: 1) anhydrous ethanol immersion for 5 min; 2) anhydrous ethanol immersion for 5 min; 3) anhydrous ethanol immersion for 5 min. Permeation: 1) xylene for 10 min; 2) xylene for 10 min; 3) xylene for 10 min. Mounting: after mounting with neutral resin, it was left to dry at room temperature.

[0090] Photographing under microscope. See Figure 11 HE staining found that the normal control group of mice had normal liver cell morphology; the model group of mice had partial area of liver cell edema, fatty degeneration, flaky necrosis, liver lobule structure necrosis, inflammatory cell infiltration, and liver tube cavity concentration; the drug group of mice had reduced liver tissue pathological damage, mild liver cell edema, no flaky necrosis, and a small amount of neutrophil infiltration. It showed that azafenidin derivatives had a significant improvement effect on mouse liver tissue damage.

[0091] 2) Alanine aminotransferase (AST / GOT) and aspartate aminotransferase (ALT / GPT) activity detection: reagent one was preheated at 37° for 20 min, 10 μmol / mL standard solution was diluted with reagent one to 5, 2.5, 1.25, 0.625, 0.3125, 0.15625, 0.078 μmol / mL standard solution for standby, control hole / standard hole (no sample) and sample hole were set, 20 μl of reagent one (matrix liquid) was added to each hole, 5 μl of sample was added to the sample hole, the hole plate was gently shaken and mixed, 37°C reaction for 30 min, 20 μl of reagent two (2, 4-dinitrophenylhydrazine solution) was added to each hole, the hole plate was gently shaken and mixed, 37°C reaction for 20 min, 200 μl of reagent three (0.4 mol / L sodium hydroxide solution) was added to each hole, the hole plate was gently shaken and mixed, and it was placed at room temperature for 15 min, wavelength 505 / 510 nm, and the OD value of each hole was determined by enzyme label instrument. The absolute OD value (determination hole OD value minus control hole OD value) was substituted into the standard curve to obtain the corresponding AST / GOT and ALT / GPT activity value.

[0092] Results are shown in Figure 12 Compared with the normal control group, the model group had increased AST / GOT and ALT / GPT levels in the plasma (P<0.05); compared with the model group, the drug group significantly reduced the AST / GOT and ALT / GPT levels in the plasma, indicating that azapheton derivatives improved liver tissue damage.

[0093] 3) ELISA method for detecting inflammatory factor levels: the logarithmic growth period of mice RAW 264.7 cells was taken, and the cells were counted by a cell counting plate, then inoculated in a 96-well plate, and cultured for 24 h, then different concentration gradients of compounds or lμg / mL of LPS were added. The experiment was divided into 3 groups in total, a blank control group (only fresh culture medium was added), an LPS modeling group (fresh culture medium + LPS lμg / mL), and a compound to be tested group (fresh culture medium diluted compound + LPS lμg / mL). After LPS stimulation for 24 h, the cell supernatant was collected and centrifuged. The cell culture medium was centrifuged at 1500r / min for 10 min at 4℃, and the supernatant was collected. According to the ELISA kit instructions specific to the measured factor, the standard and sample were diluted, then the blank hole and the sample to be tested were set, the sample was added, incubated at 25℃, washed after 2 h, then enzyme diluent 50μL was added, incubated for 1 h, then washed, then color developing agent was added, developed in the dark for 25 min, then stop solution was added, and the absorbance was measured at 450 nm wavelength. The TNF-α, IL-6 and IL-1α contents were determined.

[0094] The effect of azapheton derivatives on the expression level of inflammatory factors in liver tissue induced by LRG1 is shown in Figure 13 As shown, azapheton derivatives can inhibit the expression level of TNF-α, IL-6, IL-1α inflammatory factors in tissue serum.

[0095] 4) Western Blot method for detecting related signal pathway protein expression: 3 different treatment groups were set: sham operation group, modeling group, and drug treatment group (1mg / kg). After 24 h of treatment in different treatment groups, the following method was used for protein extraction.

[0096] Total protein extraction: The frozen liver tissue was taken out from the -80°C refrigerator, placed on ice to thaw, and then 0.030 g of liver tissue (note to avoid non-liver tissue parts) was cut and placed into an enzyme-free 1.5 mL EP tube. The tissue was pushed to the bottom of the tube, 1 mL of protein lysis solution (10 μL of PMSF was added, and it was prepared immediately before use) was added to each tube, and the lysis was performed on ice for 20 min. 3-4 small-sized zirconium oxide grinding beads were placed in the tube, and the tissue homogenizer was balanced. The vibration intensity and time parameters were adjusted, and the instrument was started. After the homogenization was completed, the liver tissue homogenate was centrifuged at 12000 rpm and 4°C for 0.5 h. The supernatant was carefully aspirated, and the total protein of the tissue was obtained.

[0097] BCA protein concentration determination kit was used for protein quantification. BCA working solution was prepared at a ratio of BCA: copper reagent = 50: 1. The sample was diluted by PBS by the corresponding multiple, 200 μL of prepared BCA working solution was added, and the absorbance value was measured at 562 nm after incubation at 37°C for 30 min.

[0098] Electrophoresis, membrane transfer, and color development: 5xLoading Buffer (4:1) was added to the sample, and it was boiled at 100°C for 10 min. The same amount of protein sample (30 μg) was separated on a 10% SDS polyacrylamide gel under the following electrophoresis conditions: 150V, 60 min. The PVDF membrane was activated by soaking in methanol for 1 min, and was placed in the membrane transfer clamp according to the sandwich structure (positive electrode-sponge-filter paper-PVDF membrane-gel-filter paper-sponge-negative electrode). The membrane was transferred (300 min, 90 min) to the PVDF membrane (0.45 μm) by electrophoresis. The membrane was blocked with 5% skim milk powder for 2 h. The corresponding primary antibody was incubated at 4°C overnight. After TBST washing, the membrane was transferred to the corresponding horseradish peroxidase-labeled secondary antibody, and was incubated at room temperature for 1 h. After washing, the hypersensitive luminescence solution was used for development on the multicolor fluorescence, chemiluminescence, and visible light imager, and the analysis was performed. The band gray value was quantitatively analyzed by Image software, and β-Actin was used as an internal reference.

[0099] As shown in Figure 14 The azafenidin derivative provided by the application can effectively inhibit the expression of AIM2 protein, indicating that compound 1 can improve HIRI by inhibiting the expression of inflammasome AIM2.

[0100] In the aggravation of HIRI, the most critical factor is that the outbreak of inflammation triggers a cascade of inflammatory responses, thereby inducing hepatocyte apoptosis and severe tissue damage. The above experiments show that compounds 1-3 exhibit significant anti-inflammatory activity in in vitro cells, and compound 1 has improved HIRI activity in in vivo mouse model experiments, and the mechanism of action may be to reduce inflammatory factors such as TNF-α and IL-6 by inhibiting the expression of AIM2 inflammasome to play an anti-inflammatory role. The azapherones derivatives provided by the application are expected to be developed as a new anti-liver ischemia-reperfusion injury drug.

[0101] The above is only a preferred embodiment of the present application, and is not intended to limit the present application; although the present application has been described in detail with reference to the foregoing embodiments, it should be understood by those skilled in the art that any modification, equivalent replacement and improvement made within the spirit and principle of the present application should be included in the protection scope of the present application.

Claims

1. An azafibrone derivative, characterized in that, The structural formula of the azaphilone derivative is shown as formula (A): Wherein, R1 is CHO or COOH, and R2 is H or Cl.

2. A process for the preparation of azafenifen derivatives according to claim 1, characterized in that, The azaphilone derivative is a secondary metabolite of Dothiorella sp. fungi. The preparation method comprises the following steps: S1. Fungus culture: after the target fungus is activated, it is inoculated into PDB culture medium, and cultured at 27 DEG C in a constant-temperature shaker for 4-5 days to obtain a seed liquid; the seed liquid is transferred into a rice liquid culture medium, and cultured at room temperature for 27±3 days to obtain a fermentation liquid; the target fungus is a Dothiorella sp. strain preserved in the China Marine Microbial and Fermentation Product Testing and Engineering Research Center with a preservation number of MCCC 3J00072; S2. Crude extraction: after the fermentation is completed, methanol solution is added to the rice liquid culture medium, soaked for 24 h, then filtered and extracted for 3 times, the filtrates are combined and concentrated to obtain a crude extract, then extracted with ethyl acetate for 3 times, and concentrated under reduced pressure to obtain an ethyl acetate part extract; S3. Separation and purification: the extract is subjected to silica gel mixing, separated by normal phase silica gel column, and gradient eluted with ethyl acetate-petroleum ether as the starting eluent to obtain components Fr.1-Fr.10, wherein, 1) the eluted component Fr.3 is collected, further separated by a silica gel column, the eluent is dichloromethane-methanol with a volume ratio of 150:1, after spotting, the fraction with absorption at a wavelength of 265 nm and brown yellow color after vanillin-sulfuric acid coloration is selected, and combined and concentrated to obtain compound 1; 2) the eluted component Fr.5 is collected, further separated by a silica gel column, the eluent is dichloromethane-methanol with a volume ratio of 50:1, to obtain sub-components Fr.5.1-5.3; 2.1) the sub-component Fr.5.1 is first purified by a Sephadex LH-20 gel column, the eluent is dichloromethane-methanol with a volume ratio of 1:1; the purified product is further separated by a silica gel column, the eluent is dichloromethane-methanol with a volume ratio of 40:1; the fraction with absorption at a wavelength of 265 nm and yellow color after vanillin-sulfuric acid coloration is selected, and combined and concentrated to obtain compound 2; 2.2) the sub-component Fr.5.3 is first purified by a silica gel column, the eluent is dichloromethane-methanol with a volume ratio of 35:1; the purified product is further separated by high performance liquid chromatography (HPLC), the mobile phase is methanol-water mixture with a volume ratio of 75:25, and the flow rate is 2.0 mL / min; the fraction with absorption at a wavelength of 265 nm and light yellow color after vanillin-sulfuric acid coloration is selected, and combined and concentrated to obtain compound 3; The compound 1 is an azaphilone derivative with R1 being CHO and R2 being H; the compound 2 is an azaphilone derivative with R1 being CHO and R2 being Cl; and the compound 3 is an azaphilone derivative with R1 being COOH and R2 being H.

3. The method for preparing azafiron derivatives as described in claim 2, characterized in that, In the step S1, the strain is inoculated on a rice solid culture medium, and fermented at room temperature for 27±3 days, then inoculated on a PDA culture medium, and incubated at room temperature for 5 days to obtain the activated Chalara strain; the rice solid culture medium comprises rice 100 g / L, NaBr 50 g / L and peptone 0.3 g / L.

4. The method for preparing azafiron derivatives as described in claim 2, characterized in that, The rice liquid culture medium comprises rice 100 g / L and 0.3% w / v coarse salt water 80 mL / L.

5. The method for preparing azafiron derivatives as described in claim 2, characterized in that, In the step S3, gradient elution is performed with a system of 0 / 1 ethyl acetate-petroleum ether: 1 / 0 ethyl acetate-petroleum ether, the third component Fr.1 is eluted with a system of 3 / 7 ethyl acetate-petroleum ether, and the first component Fr.5 is eluted with a system of 5 / 5 ethyl acetate-petroleum ether.

6. Use of the azafenizon derivative of claim 1 in the preparation of a medicament for preventing liver ischemia-reperfusion injury.

7. A medicament for preventing liver ischemia-reperfusion injury, characterized by, The azafenizon derivative of claim 1 in a pharmaceutically effective dose and a pharmaceutically acceptable carrier.

8. The medicament according to claim 7, wherein The azafenizon derivative accounts for 60-90% of the total mass or total volume of the medicament.

9. The medicament according to claim 7, wherein The pharmaceutically acceptable carrier comprises one or several functional carriers of excipients, stabilizers, antioxidants, colorants, diluents, and sustained-release agents.

10. The medicament according to claim 9, wherein The medicament is any one of an injection, a tablet, a granule, a pill, a capsule, a suspension, or an emulsion.

Images