Azaphilone derivatives having activity against liver ischemia-reperfusion injury, and methods of making and using the same

Abstract

This invention belongs to the field of pharmaceutical compound technology, specifically relating to an azafibrone derivative with anti-hepatic ischemia-reperfusion injury activity, its preparation method, and its application. The structural formula of the azafibrone derivative is shown in Formula A: Among them, R 1 For CHO or COOH, R 2 The derivative is H or Cl. This application describes the extraction of an azafibrone derivative from the fermentation broth of *Cryptocaryas* fungi. Experiments have confirmed that this compound possesses anti-hepatic ischemia-reperfusion injury activity, indicating its potential to be developed into an anti-hepatic ischemia-reperfusion injury drug, providing a new option for the development of such drugs.

Description

Azafirone derivatives with anti-hepatic ischemia-reperfusion injury activity, their preparation methods and applications Technical Field

[0001] This invention belongs to the field of pharmaceutical compound technology, specifically relating to an azafiron derivative with anti-hepatic ischemia-reperfusion injury activity, its preparation method, and its application. Background Technology

[0002] Mangroves are mainly distributed in the intertidal zone of tropical and subtropical coasts. Their global distribution is strictly limited by environmental factors such as temperature, salinity, and tides. In my country, mangroves are only found in coastal provinces and are rare and protected species. Currently, my country has six national-level mangrove nature reserves (Dongzhai Port in Hainan, Beilun River Estuary in Guangxi, Shankou in Guangxi, Zhanjiang in Guangdong, Futian in Shenzhen, and Zhangjiang Estuary in Fujian), containing only 37 species of mangrove plants from 20 families. Of these, 23 species have medicinal value, and 13 are used in traditional medicine. The high temperature, high salinity, oxygen-deficient soil, and periodic seawater inundation create a unique ecological environment for mangroves, making them an important resource for drug development and the discovery of lead compounds.

[0003] Hepatic ischemia-reperfusion injury (HIRI) refers to the damage that occurs when blood flow to the liver is temporarily interrupted (ischemic) and then restored (reperfusion). This phenomenon is a common clinical challenge encountered in major liver surgery, liver transplantation, and liver trauma. HIRI usually leads to liver damage, increases the risk of early transplant failure, and may progress to liver failure, which in severe cases can lead to multiple organ failure and even death.

[0004] While a variety of preoperative and postoperative treatments for HIRI have been reported in the literature, these methods have only been effective in a small number of patients with prolonged ischemic periods and those undergoing minimal hepatectomy. Furthermore, to date, no drug treatments have been formally approved for the prevention or management of HIRI. Studies have shown that certain interventions, including calcium channel blockers, adenosine receptor agonists, energy metabolism regulators, and antioxidants, may have a protective effect against HIRI; however, due to potential adverse reactions, related research has been primarily limited to animal studies.

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

[0006] To address the aforementioned technical problems, the present invention first provides an azafibrone derivative, the structural formula of which is shown in formula (A):

[0007]

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

[0009] The second objective of this invention is to provide a method for preparing the azafifenone derivatives as described above, wherein the azafifenone derivatives are secondary metabolites of fungi of the genus *Dothiorella*.

[0010] The preparation method includes the following steps:

[0011] S1. Fungal culture: After activating the target fungus, it was inoculated into PDB medium and cultured in a shaker at 27°C for 4-5 days to obtain a seed culture. The seed culture was then transferred to rice liquid medium and cultured at room temperature for 27±3 days to obtain a fermentation broth. The target fungus was a strain of the genus *Cryptocoryne*, preserved at the China Marine Microbial Culture Collection Center with accession number MCCC 3J00072.

[0012] S2. Crude extraction: After fermentation, the rice liquid culture medium was soaked in methanol solution for 24 hours, then filtered and extracted 3 times. The filtrates were combined and concentrated to obtain crude extract. Then, it was extracted 3 times with ethyl acetate and concentrated under reduced pressure to obtain ethyl acetate extract.

[0013] S3. Separation and purification: The extract was mixed with silica gel and separated using a normal-phase silica gel column with gradient elution using ethyl acetate-petroleum ether as the initial eluent to obtain fractions Fr.1-Fr.10, wherein...

[0014] 1) Collect the eluted fraction Fr.3 and further separate it by silica gel column chromatography. The eluent is dichloromethane-methanol with a volume ratio of 150:1. After spotting, select the fraction that absorbs at a wavelength of 265 nm and turns brownish-yellow after being developed with concentrated vanillin sulfuric acid. Combine and concentrate to obtain compound 1.

[0015] 2) Collect the eluted fraction Fr.5 and further separate it using a silica gel column with dichloromethane-methanol at a volume ratio of 50:1 to obtain subfractions Fr.5.1-5.3;

[0016] 2.1) Subfraction Fr.5.1 was first purified by Sephadex LH-20 gel chromatography column with dichloromethane-methanol at a volume ratio of 1:1. The purified product was further separated by silica gel chromatography column with dichloromethane-methanol at a volume ratio of 40:1. The fraction that absorbed at a wavelength of 265 nm and turned yellow after being developed with concentrated vanillin sulfate was selected, and the fractions were combined and concentrated to obtain compound 2.

[0017] 2.2) Subfraction Fr.5.3 was first purified by silica gel column chromatography with dichloromethane-methanol at a volume ratio of 35:1. The purified product was further separated by high performance liquid chromatography (HPLC) with a mobile phase of methanol-water mixture at a volume ratio of 75:25 and a flow rate of 2.0 mL / min. The fraction that absorbed at 265 nm and turned pale yellow after being developed with concentrated vanillin sulfate was selected, combined and concentrated to obtain compound 3.

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

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

[0020] Preferably, the rice liquid culture medium contains 100 g / L of rice and 80 mL / L of 0.3% w / v crude saline.

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

[0022] A third objective of this invention is to provide the application of the azafirone derivatives described above in the preparation of drugs for preventing hepatic ischemia-reperfusion injury.

[0023] The fourth objective of this invention is to provide a drug for preventing hepatic ischemia-reperfusion injury, characterized in that it comprises a pharmaceutically effective dose of the azafirone derivative as described above and a pharmaceutically acceptable carrier.

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

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

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

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

[0028] The beneficial effects of this application are as follows:

[0029] 1. This application describes the extraction of a novel class of azafibrone derivatives from the fermentation broth of *Synapticus* strains. This compound exhibits significant anti-inflammatory activity in in vitro cell experiments and shows significant liver injury-improving activity in a mouse model of liver ischemia-reperfusion injury, suggesting its potential application in the preparation of clinically effective drugs for treating liver ischemia-reperfusion injury. This application provides a new option for the development of drugs with anti-liver ischemia-reperfusion injury activity.

[0030] 2. The LPS-induced mouse macrophage RAW264.7 cell model showed that the azafibrone derivatives provided in this application can effectively inhibit NO release, with compound 1 showing the best inhibitory effect. In a mouse model of liver defect reperfusion injury, the efficacy of the azafibrone derivatives provided in this application was investigated, showing that they can significantly reduce the formation of vacuoles, neutrophil infiltration, and congestion in liver tissue caused by ischemia-reperfusion, significantly reduce the levels of important liver markers ALT and AST, significantly reduce the expression levels of inflammatory factors TNF-α, IL-1α, and IL-6 in liver serum tissue, and downregulate the protein expression level of AIM-2. This indicates that the azafibrone derivatives provided in this application can act as anti-inflammatory drugs, exerting their anti-HIRI effect by blocking the activation of the inflammatory response (inhibiting AIM2 inflammasome activation).

[0031] 3. The azafiron derivatives provided in this application are secondary metabolites of fungi in the genus *Cryptocoryne*. They can be prepared by microbial fermentation, which is simple, has a short cycle, mild culture conditions, few byproducts, and low cost. It can be obtained by unlimited fermentation, is not limited by resource shortages, is green and environmentally friendly, and is easy to industrialize. It not only meets the needs of modern green, low-carbon and environmentally friendly economy, but also provides a new way for the discovery of potential anti-HIRI drugs. Attached Figure Description

[0032] Figure 1 shows the HR-ESI-MS spectrum of the novel compound 3 provided in this application.

[0033] Figure 2 shows the novel compound 3 provided in this application. 1 H NMR spectrum.

[0034] Figure 3 shows the novel compound 3 provided in this application. 13 C NMR spectrum.

[0035] Figure 4 shows the novel compound 3 provided in this application. 1 H- 1 H COSY spectrum.

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

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

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

[0039] Figure 8 shows the toxicity screening of compounds 1-3 provided in this application on cells.

[0040] Figure 9 shows the screening of compounds 1-3 provided in this application for inhibiting LPS-induced NO production.

[0041] Figure 10 shows 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 shows the H&E staining of liver sections in a mouse liver ischemia-reperfusion model induced by compound 1 provided in this application.

[0043] Figure 12 shows the detection of aspartate aminotransferase (A) and alanine aminotransferase (B) in the plasma of a mouse liver ischemia-reperfusion model provided by Compound 1 of this application.

[0044] Figure 13 shows the detection of inflammatory factors IL-6 (A), TNF-α (B), and IL-1α (C) in liver tissue of a mouse liver ischemia-reperfusion model by compound 1 provided in this application.

[0045] Figure 14 shows 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] Unless otherwise stated, the terms used herein have the meanings commonly understood by those skilled in the art.

[0047] The technical solution of the present invention will be described in more detail below with reference to the embodiments.

[0048] In this application, room temperature refers to 27±3℃. The fungus *Dothiorella* sp. ZJQQYZ-1 was obtained from the China Marine Microbial Culture Collection Center, with accession number MCCC 3J00072. The culture medium was made from Northeast China rice. The organic solvent extraction reagents, petroleum ether, ethyl acetate, dichloromethane, and methanol, were all analytical grade reagents.

[0049] Example 1

[0050] 1. Extraction of azafiron derivatives

[0051] Azafifenone derivatives have the following general structural formula:

[0052]

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

[0054] This azafifenone derivative is a secondary metabolite of the fungus *Dothiorella* sp. The preparation method includes the following steps:

[0055] S1. Fungal culture

[0056] Activation: Dothiorella sp. ZJQQYZ-1 was inoculated onto PDA medium and incubated at room temperature for 5 days to obtain the activated strain. The PDA medium consisted of: potato extract powder 300.0 g / L, glucose 20.0 g / L, agar 15.0 g / L, and chloramphenicol 0.1 g / L.

[0057] Primary culture: The activated strain was inoculated onto PDB medium and cultured in a shaker (160 r / min) at 27°C for 4-5 days to obtain approximately 500 mL of fungal seed culture. The PDB medium consisted of 300.0 g / L potato extract and 20.0 g / L glucose.

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

[0059] S2. Crude extraction: After 30 days, 200 mL of methanol solution was added to the rice liquid culture medium, and the mixture was soaked and filtered three times. The filtrates were combined and concentrated to obtain a crude extract. Then, it was extracted three times with ethyl acetate and concentrated under reduced pressure to obtain an ethyl acetate fraction extract.

[0060] S3. Separation and purification: The extract was mixed with silica gel and separated using a normal-phase silica gel column with gradient elution using ethyl acetate-petroleum ether as the initial eluent to obtain fractions Fr.1-Fr.10, wherein...

[0061] 1) The third fraction Fr.3 was obtained by elution in a 3 / 7 ethyl acetate-petroleum ether system. Fr.3 was further separated by silica gel column chromatography with dichloromethane-methanol in a volume ratio of 150:1. After spotting, the fraction that absorbed at a wavelength of 265 nm and turned brownish-yellow after being developed with concentrated sulfuric acid vanillin was selected, and the fractions were combined and concentrated to obtain compound 1.

[0062] 2) The fifth fraction Fr.5 was obtained by elution in a 5 / 5 ethyl acetate-petroleum ether system. Fr.5 was further separated by silica gel column chromatography with dichloromethane-methanol in a volume ratio of 50:1 to obtain subfractions Fr.5.1-5.3.

[0063] 2.1) Subfraction Fr.5.1 was first purified by Sephadex LH-20 gel chromatography column with dichloromethane-methanol at a volume ratio of 1:1. The purified product was further separated by silica gel chromatography column with dichloromethane-methanol at a volume ratio of 40:1. The fraction that absorbed at a wavelength of 265 nm and turned yellow after being developed with concentrated vanillin sulfate was selected, and the fractions were combined and concentrated to obtain compound 2.

[0064] 2.2) Subfraction Fr.5.3 was first purified by silica gel column chromatography with dichloromethane-methanol at a volume ratio of 35:1. The purified product was further separated by high performance liquid chromatography (HPLC) with a mobile phase of methanol-water mixture at a volume ratio of 75:25 and a flow rate of 2.0 mL / min. The fraction that absorbed at 265 nm and turned pale yellow after being developed with concentrated vanillin sulfate was selected, combined and concentrated to obtain compound 3.

[0065] Compound 1 is an azafibrone derivative with R1 being CHO and R2 being H; compound 2 is an azafibrone derivative with R1 being CHO and R2 being Cl; and compound 3 is an azafibrone derivative with R1 being COOH and R2 being H.

[0066] 2. Characterization of azafiron derivatives

[0067] The structure of the compound was determined by NMR, HRESIMS, and ECD calculations.

[0068] Compound 1 (R1 is CHO, R2 is H): white solid; IR(KBr)υ max :3381,2963,2920,1680,1625,1598,1580cm -1 ;UV(MeOH)λmax :290(1.2)nm; HRESIMS:m / z316.1672[M+H] + (calcd for C 19 H 24 O4,316.1675); 1 Hand 13 The C10 NMR (DMSO-d6) data are shown in Table 1. It is named 2,4-dihydroxy-6-(5,7-dimethyl-2-oxo-trans-3-trans-5-nonadienyl)-3-methylbenzaldehyde.

[0069] Compound 2 (R1 is CHO, R2 is Cl): white solid; IR(KBr)υ max :3380,2965,2923,1678,1626,1600,1581cm -1 ;UV(MeOH)λ max :291(1.3)nm; HRESIMS:m / z350.1360[M+H] + (calcd for C 19 H 23 ClO4, 316.1361); 1 H and 13 The C10 NMR (CDCl3) data are shown in Table 1. It is named 6-((1E,3E)-3,5-dimethylhepta-1,3-dien-1-yl)-2,4-dihy-droxy-3-methylbenzaldehyde.

[0070] Compound 3 (R1 is COOH, R2 is H): white solid; IR(KBr)υ max :3380,2965,2922,1681,1630,1588,1568cm -1 ;UV(MeOH)λ max :290(1.2)nm; HRESIMS:m / z355.1512[M+Na] + (calcd for C 19 H 24 O5,355.1510); 1 Hand 13 The C NMR (CD3OD) data are shown in Table 1. It is named dothioretone A.

[0071] Taking compound 3 as an example, see Figures 1-7, which show the HR-ESI-MS spectra 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 related diagram.

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

[0073]

[0074]

[0075] Example 2

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

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

[0078] 1. Cell experiments

[0079] Cell culture: Mouse macrophage cell line RAW 264.7 was cultured in DMEM high glucose medium containing 10% fetal bovine serum (FBS), 1% penicillin (100 U / mL) and streptomycin (100 μg / mL) at 37°C in a 5% CO2 incubator. When the cells reached 70%-80% confluence, they were passaged at a ratio of 1:3, and cells in the logarithmic growth phase were used for experiments.

[0080] 1) Cell viability assay: When cells reached logarithmic growth, they were seeded in 96-well plates at 2 × 10⁴ cells per well. After 24 hours, the original culture medium was aspirated, and basal medium containing the drug and simple basal medium were added. Five concentration gradients of the drug were set up: 50, 25, 12.5, 6.25, and 3.125 μM. After another 24 hours, the CCK8 assay was performed: 110 μl of a mixture of DMEM medium and CCK8 solution (10:1) was added to each well, and the mixture was incubated at 37°C for 1 hour. The absorbance was measured at 450 nm using a microplate reader. This was to assess the effect of the drug on the viability of RAW 264.7 macrophages.

[0081] The results are shown in Figure 8. Compounds 1-3 had no toxic effect on RAW264.7 macrophages at a concentration of 6.25 μM.

[0082] 2) Nitric Oxide (NO) Content Determination: The nitric oxide content in cell supernatant was determined using the nitrate reduction method. Logarithmic growth phase mouse RAW 264.7 cells were seeded into 24-well plates and cultured for 24 hours. Then, compounds with different concentration gradients or LPS at a concentration of 1 μg / mL were added. The experiment was divided into three groups: a blank control group (fresh culture medium only), an LPS model group (fresh culture medium + 1 μg / mL LPS), and a test compound group (compound diluted in fresh culture medium + 1 μg / mL LPS). After 24 hours of LPS stimulation, the cell supernatant was collected and centrifuged. The NO concentration in the cell supernatant was detected and calculated using a nitric oxide assay kit.

[0083] The detection steps were performed according to the reagent instructions: Griess Reagent I and II and cell supernatant were removed and allowed to return to room temperature. (1) Prepare a standard curve: Dilute the standard concentrations to 1, 5, 10, 20, 40, 60, and 100 μM with fresh culture medium. (2) Add 50 μL of the standard and cell supernatant to each well of a 96-well plate. (3) After returning to room temperature, add 50 μL of Griess Reagent I and 50 μL of Griess Reagent II sequentially. (4) Measure the absorbance at 540 nm using a microplate reader. (5) Calculate the NO concentration in the cell supernatant based on the standard curve.

[0084] Referring to Figure 9, at the experimental concentrations, compounds 1-3 and L-NMMA (positive control, 12.5 μM) significantly inhibited the release of NO from LPS-induced RAW264.7 macrophages. Compared with the positive control, compound 1 showed strong inhibitory activity, with an IC50 value of [missing value]. 50 Compounds 2 and 3 exhibited moderate inhibitory activity at a concentration of 1.13 μM, with an IC50 value of 1.13 μM. 50 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 macrophages RAW264.7 was examined. As shown in Figure 10, compared to the control 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 administration significantly reduced these expression levels.

[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 liver tissue was removed, it was internally fixed with 4% paraformaldehyde overnight. It was then dehydrated by soaking in different concentrations of ethanol (70% ethanol, 1 h; 80% ethanol, 1 h; 90% ethanol, 1 h; 95% ethanol, 1 h; anhydrous ethanol, 1 h), and cleared (xylene). The liver tissue was then embedded in paraffin and sectioned using a microtome. Dewaxing: 1) Complete soaking in xylene for 20 min; 2) Complete soaking in xylene for 30 min; 3) Complete soaking in anhydrous ethanol for 6 min; 4) Complete soaking in anhydrous ethanol for 6 min; 5) Complete soaking in 95% ethanol for 6 min; 6) Complete soaking in 85% ethanol for 6 min; 7) Complete soaking in 70% ethanol for 6 min; 8) Complete soaking in pure water for 6 min. Staining: 1) Hematoxylin for 4 min; 2) Rinse with tap water for 10 min; 3) Differentiation with hydrochloric acid ethanol for 3 s; 4) Rinse with tap water for 10 min (blue return); 5) Eosin for 1 min. Dehydration: 1) Soak in anhydrous ethanol for 5 min; 2) Soak in anhydrous ethanol for 5 min; 3) Soak in anhydrous ethanol for 5 min. Permeation: 1) Soak in xylene for 10 min; 2) Soak in xylene for 10 min; 3) Soak in xylene for 10 min. Mounting: After mounting with neutral resin, allow to air dry at room temperature.

[0090] The slides were photographed and observed under a microscope. Referring to Figure 11, HE staining revealed that the hepatocytes in the normal control group mice had normal morphology and size; in the model group mice, some areas of hepatocytes showed edema, fatty degeneration, patchy necrosis, necrosis of liver lobule structures, inflammatory cell infiltration, and concentration in the liver lumen; in the drug group mice, the pathological damage to liver tissue was reduced, with mild hepatocyte edema, no patchy necrosis, and a small amount of neutrophil infiltration. This indicates that azafiron derivatives have a significant ameliorative effect on liver tissue damage in mice.

[0091] 2) Aspartate aminotransferase (AST / GOT) and alanine aminotransferase (ALT / GPT) activity assay: Preheat reagent one to 37°C for 20 min. Dilute the 10 μmol / mL standard solution with reagent one to prepare standard solutions of 5, 2.5, 1.25, 0.625, 0.3125, 0.15625, and 0.078 μmol / mL for later use. Set up control / standard wells (without sample) and sample wells, adding 20 μl of reagent one (matrix solution) to each well. Add 5 μl of sample to the sample wells and gently shake the plate. Mix well and react at 37°C for 30 minutes. Add 20 μl of reagent II (2,4-dinitrophenylhydrazine solution) to each well, gently shake the plate to mix, and react at 37°C for 20 minutes. Add 200 μl of reagent III (0.4 mol / L sodium hydroxide solution) to each well, gently shake the plate to mix, and incubate at room temperature for 15 minutes. Measure the OD value of each well using a microplate reader at a wavelength of 505 / 510 nm. Substitute the absolute OD value (OD value of the measured well minus the OD value of the control well) into the standard curve to obtain the corresponding AST / GOT and ALT / GPT activity values.

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

[0093] 3) Enzyme-linked immunosorbent assay (ELISA) for detecting inflammatory factor levels: Logarithmic growth phase mouse RAW 264.7 cells were counted using a cell counting chamber and seeded into 96-well plates. After culturing for 24 hours, different concentration gradients of compounds or LPS at a concentration of 1 μg / mL were added. The experiment was divided into three groups: blank control group (fresh culture medium only), LPS model group (fresh culture medium + 1 μg / mL LPS), and test compound group (compound diluted in fresh culture medium + 1 μg / mL LPS). After 24 hours of LPS stimulation, the cell supernatant was collected and centrifuged. The cell culture medium was centrifuged at 1500 rpm for 10 minutes at 4°C, and the supernatant was collected. The procedure was performed according to the instructions of the ELISA kit specific to the measured factor. First, the standard and sample were diluted. Then, blank wells and sample wells were prepared, samples were added, and the mixture was incubated at 25°C for 2 hours. After washing, 50 μL of enzyme dilution buffer was added, and the mixture was incubated for 1 hour. After washing, the chromogenic reagent was added, and the mixture was incubated in the dark for 25 minutes. Finally, stop solution was added, and absorbance was measured at 450 nm. The levels of TNF-α, IL-6, and IL-1α were determined.

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

[0095] 4) Western Blot analysis of protein expression in related signaling pathways: Three different treatment groups were set up: sham operation group, model group, and drug treatment group (1 mg / kg). After 24 h of treatment in each group, protein extraction was performed using the following method.

[0096] Total protein extraction: Remove frozen liver tissue from the freezer at -80°C and thaw on ice. Cut 0.030g of liver tissue (avoiding non-liver tissue) and mince it. Place the minced tissue into a 1.5mL enzyme-free EP tube, pushing the tissue to the bottom. Add 1mL of protein lysis buffer (containing 10μL PMSF, freshly prepared) to each tube and lyse on ice for 20 minutes. Place 3-4 small zirconia grinding beads into the tube, balance it, and place it in a tissue homogenizer. Adjust the vibration intensity and time parameters, and start the instrument. After homogenization, homogenize the liver tissue and centrifuge at 12000rpm at 4°C for 0.5h. Carefully aspirate the supernatant; this is the total protein from the tissue.

[0097] Protein quantification was performed using a BCA protein concentration assay kit. BCA working solution was prepared at a ratio of BCA:copper reagent = 50:1. The sample was diluted with PBS by the corresponding factor, and 200 μL of the prepared BCA working solution was added. The sample was incubated at 37°C for 30 min, and the absorbance value was measured at 562 nm.

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

[0099] As shown in Figure 14, the azafirone derivatives provided in this 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 exacerbation of hepatic ischemia-reperfusion injury (HIRI), the most critical factor is the outbreak of inflammation triggering a cascade of inflammatory responses, which in turn induces hepatocyte apoptosis and severe tissue damage. The above experiments show that compounds 1-3 exhibit significant anti-inflammatory activity in in vitro cells. In in vitro mouse model experiments, compound 1 improved HIRI activity, and its mechanism of action may involve inhibiting the expression of the AIM2 inflammasome, thereby reducing inflammatory factors such as TNF-α and IL-6. The azafiron derivatives provided in this application hold promise for development into novel drugs against hepatic ischemia-reperfusion injury.

[0101] The above are merely preferred embodiments of the present invention and are not intended to limit the scope of the invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. The application of azafibrone derivatives in the preparation of drugs for preventing hepatic ischemia-reperfusion injury, wherein the structural formula of the azafibrone derivative is shown in formula (A): (A), where, R1 is CHO, and R2 is H.

2. The application as described in claim 1, characterized in that, The drug comprises a pharmaceutically effective dose of the azafibrone derivative and a pharmaceutically acceptable carrier.

3. The application as described in claim 2, characterized in that, The azafiron derivatives account for 60-90% of the total mass or volume of the drug.

4. The application as described in claim 2, characterized in that, The pharmaceutically acceptable carriers include carriers that function as one or more of the following: excipients, stabilizers, antioxidants, colorants, diluents, and sustained-release agents.

5. The application as described in claim 4, characterized in that, The drug is any one of the following: injection, tablet, granule, pill, capsule, suspension, or emulsion.

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