A baicalein derivative and its preparation method and application

Abstract

The present invention discloses a baicalein derivative, its preparation method, and application. The baicalein derivative is 4'-methylamino baicalein. Experimental results demonstrate that the MAB described herein is not only low in toxicity but also exhibits a more significant inhibitory effect on excessive inflammatory responses induced by LPS or pathogenic bacteria infection than baicalein under the same conditions, and exhibits superior targeting ability to TLR4 protein. Therefore, it is expected that the MAB, or at least one of its pharmaceutically acceptable salts, tautomers, stereoisomers, and precursor compounds, may be used as an active ingredient in the preparation of a drug for the treatment of sepsis.

Description

Technical Field

[0001] The present invention relates to a baicalein derivative and a preparation method and application thereof, in particular to 4'-methylamino baicalein and a preparation method thereof and application thereof in preparing a drug for treating sepsis, belonging to the technical field of medicine. Background Art

[0002] Sepsis is a systemic inflammatory response syndrome (SIRS) caused by the invasion of bacteria and other pathogens into the human body, which can lead to shock, multiple organ dysfunction syndrome (MODS), and death. It is a common complication and the leading cause of death in critically ill patients. According to statistics, the annual incidence of sepsis worldwide has increased to approximately 48.9 million, of which 11 million die, accounting for 19.70% of the world's total mortality. Sepsis is common after severe burns, infection, shock, or major surgery. Its pathogenesis is complex and closely related to pathophysiological changes in multiple systems and organs. It involves systemic inflammatory network effects, immune dysfunction, and abnormal host responses to various infectious pathogens and microbial toxins. SIRS, a cardinal feature of sepsis, is caused by an excessive inflammatory response during the defense against infection and plays a key role in the pathogenesis of sepsis.

[0003] Existing studies have shown that carbapenem-resistant Klebsiella pneumoniae (CRKP) is one of the carbapenem-resistant Enterobacteriaceae (CRE) and has attracted attention due to its role in high morbidity and mortality worldwide; Gram-negative (G - ) bacteria are also the main pathogenic factor inducing sepsis. Bacterial death can release a large amount of G - Lipopolysaccharide (LPS), the main component of the bacterial wall, can serve as the main stimulus for triggering inflammatory responses in macrophages, dendritic cells, neutrophils, etc. During sepsis, the inflammatory cascade triggered by microbial infection can lead to excessive production of cytokines, called "cytokine storm", which is considered to be the main cause of organ damage.

[0004] In addition, clinical studies have found that TLR4 is one of the earliest Toll-like receptor subtypes discovered by humans. It is widely present in tissues such as the liver, macrophages, and lungs. It can mediate the occurrence of inflammatory responses by acting on the body's inflammatory response signal transduction pathway. By inhibiting the TLR4 signaling pathway, it can inhibit inflammation in the sepsis body and alleviate the disease.

[0005] Because the current antibiotics used to treat sepsis have poor therapeutic effects and drug resistance problems, there is an urgent need to find new drugs to treat sepsis.

[0006] Baicalein is a flavonoid compound with the following chemical structure:

[0007] Scutellaria baicalensis is the main active ingredient extracted from the dried root of Scutellaria baicalensis Georgi, a perennial herbaceous plant in the Lamiaceae family. It has multiple pharmacological activities, including anti-inflammatory, antioxidant, antibacterial, and anti-tumor effects. However, its direct clinical application is limited by its low bioavailability and poor solubility. Summary of the Invention

[0008] In view of the above problems and needs existing in the prior art, the purpose of the present invention is to provide a baicalein derivative and its preparation method and application, specifically to provide a new compound 4'-methylamine baicalein and its preparation method and application in the preparation of drugs for treating sepsis.

[0009] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0010] A baicalein derivative is 4'-methylamino baicalein (abbreviated as MAB), which has the following chemical structure:

[0011]

[0012] A preparation method of the 4'-methylamine baicalein is to adopt the following synthetic route:

[0013]

[0014] In one embodiment, the preparation method comprises the following steps:

[0015] a) dissolving the nitro-substituted cinnamic acid in a solvent and reacting it with oxalyl chloride at room temperature to obtain intermediate 2;

[0016] b) dissolving intermediate 2 and 3,4,5-trimethoxyphenol in a solvent, then adding BF3·Et2O and performing a reflux reaction to obtain intermediate 4;

[0017] c) dissolving intermediate 4 in a solvent, then adding elemental iodine, and reacting at 120-150° C. to obtain intermediate 5;

[0018] d) dissolving intermediate 5 in a solvent, then adding sodium metabisulfite and conducting a reflux reaction to obtain intermediate 6;

[0019] e) dissolving intermediate 6 with potassium carbonate and methyl iodide in a solvent and then reacting at room temperature to obtain intermediate 7;

[0020] f) The intermediate 7 is added to a HI / CH3COOH solution, and then reacted at 110-130°C to obtain the compound 4'-methylamine baicalein.

[0021] In a preferred embodiment, in step a), the solvent is dichloromethane, and the molar ratio of p-nitrocinnamic acid to oxalyl chloride is 1:(1.1-1.5).

[0022] In a preferred embodiment, in step b), the solvent is toluene, and the molar ratio of intermediate 2 to 3,4,5-trimethoxyphenol is (1-1.5):1, and the molar ratio of BF3·Et2O to 3,4,5-trimethoxyphenol is (1-3):1.

[0023] In a preferred embodiment, in step c), the solvent is dimethyl sulfoxide, and the molar ratio of intermediate 4 to elemental iodine is 1:(0.1-0.5).

[0024] In a preferred embodiment, in step d), the solvent is an ethanol aqueous solution with a volume fraction of 50% to 75%, and the molar ratio of the intermediate 5 to the sodium metabisulfite is 1:(2 to 5).

[0025] In a preferred embodiment, in step e), the solvent is acetone, and the molar ratio of intermediate 6 to potassium carbonate and iodomethane is 1:(2-5):(2-5).

[0026] One application of the 4'-methylamino baicalein is to use 4'-methylamino baicalein or at least one of its pharmaceutically acceptable salts, tautomers, stereoisomers, and precursor compounds as an active ingredient for preparing a drug for treating sepsis.

[0027] In one embodiment, the sepsis includes early sepsis, severe sepsis and septic shock.

[0028] The dosage form of the drug of the present invention is not limited and can be a unit dosage form suitable for sublingual administration, oral administration, transdermal administration, injection or pulmonary inhalation, as long as it is a dosage form that can effectively allow the active ingredient to reach the body. The unit dosage form can be a common dosage form such as tablets, capsules, powders, granules, injections, oral liquids, aerosols, lozenges, or sustained-release dosage forms such as nanoformulations.

[0029] The drug of the present invention, in addition to the main active ingredient MAB, may also contain a small amount of secondary ingredients that do not affect the effective active ingredients and / or pharmaceutically acceptable carriers, for example, sweeteners to improve taste, antioxidants to prevent oxidation, and various necessary excipients for preparations.

[0030] In addition, the definitions of terms in the present invention are as follows:

[0031] The term "pharmaceutically acceptable salt" refers to a salt formed by 4'-methylamino baicalein and a pharmaceutically acceptable inorganic acid or organic acid, wherein the inorganic acid is hydrochloric acid, hydrobromic acid, phosphoric acid, nitric acid or sulfuric acid; the organic acid is formic acid, acetic acid, propionic acid, succinic acid, 1,5-naphthalenedisulfonic acid, asiatic acid, oxalic acid, tartaric acid, lactic acid, salicylic acid, benzoic acid, valeric acid, diethylacetic acid, malonic acid, succinic acid, fumaric acid, pimelic acid, adipic acid, maleic acid, malic acid, sulfamic acid, phenylpropionic acid, gluconic acid, ascorbic acid, nicotinic acid, isonicotinic acid, methanesulfonic acid, p-toluenesulfonic acid, citric acid or amino acid; the "pharmaceutically acceptable" means that it is suitable for use in humans without excessive adverse side effects (such as toxicity, irritation and allergic reactions), that is, it has a reasonable benefit / risk ratio.

[0032] The term "stereoisomer" refers to isomers resulting from different spatial arrangements of atoms in a molecule, such as cis-trans isomers, enantiomers, conformers, and the like.

[0033] The term "precursor compound" refers to a compound that is inactive in vitro but can be converted into 4'-methylamino baicalein by metabolism or chemical reaction in vivo, thereby exerting its pharmacological effects.

[0034] Compared with the prior art, the present invention has the following significant beneficial effects:

[0035] The experimental results of the present invention show that compared with baicalein, 4'-methylamino baicalein (MAB) can play a more significant role in protecting body temperature and improving the survival rate of severe sepsis in a mouse septic shock model induced by a lethal dose of LPS; in the inflammatory response of mouse RAW264.7 cells stimulated by LPS or heat-killed CRKP (HK-CRKP) and mouse primary peritoneal macrophages stimulated by LPS, compared with baicalein, at the same concentration, MAB can significantly inhibit the expression of cytokines such as tumor necrosis factor α (TNF-α), interleukin 6 (IL-6), interleukin 10 (IL-10), interleukin 1β (IL-1β), chemokines (such as MCP-1, MIP-2), interferon β (IFN-β) and interleukin 10 (IL-10); and, by CETSA (Cellular Thermal Shift A cellular heat shift assay (CTSA) demonstrated that the MAB's binding ability to the TLR4 protein was significantly superior to that of baicalein. This demonstrates that the MAB described herein can inhibit excessive inflammatory responses induced by LPS or pathogenic bacteria, thereby reducing damage and even death caused by excessive inflammatory responses. Furthermore, the experimental results of the present invention showed that in Raw264.7 cells, a 30 μM MAB exposure for 24 hours was non-cytotoxic and did not affect normal cell proliferation. In primary mouse peritoneal macrophages, a 100 μM MAB exposure for 24 hours was non-cytotoxic. This demonstrates that the MAB described herein, or at least one of its pharmaceutically acceptable salts, tautomers, stereoisomers, or precursor compounds, as an active ingredient can be used to prepare a drug for the treatment of sepsis. The MAB described herein offers significant improvements and unexpected technical benefits over baicalein. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 The effect of MAB on the survival rate (1A) and body temperature (1B and 1C) of mice with LPS-induced septic shock is shown; in the figure: * indicates p < 0.05 compared with the model group; ** indicates p < 0.01 compared with the model group; *** indicates p < 0.001 compared with the model group;

[0037] Figure 2 The cytotoxicity of different concentrations of MAB after co-incubation with mouse Raw264.7 cells (2A) and mouse primary peritoneal macrophages (2B) for 24 hours is shown; in the figure: ** indicates p < 0.01 compared with the blank group; *** indicates p < 0.001 compared with the blank group;

[0038] Figure 3 shows the effects of different concentrations of Baicalein or MAB on the expression of TNF-α in LPS-stimulated mouse RAW264.7 cells ( Figure 3A), IL-6( Figure 3B )、IL-10( Figure 3C )、MCP-1( Figure 3D )、MIP-2( Figure 3E ), IFN-β( Figure 3F ) and NO( Figure 3G ) regulatory effect; in the figure: * indicates p < 0.05 compared with the control group; ** indicates p < 0.01 compared with the control group; *** indicates p < 0.001 compared with the control group; ns indicates no significant difference compared with the control group;

[0039] Figure 4 shows the effects of different concentrations of Baicalein or MAB on the expression of IL-1β in LPS-stimulated mouse RAW264.7 cells ( Figure 4A )、CXCL 10( Figure 4B )、iNOS( Figure 4C ) and COX-2mRNA( Figure 4D ) regulatory effect; in the figure: * indicates p < 0.05 compared with the control group; ** indicates p < 0.01 compared with the control group; *** indicates p < 0.001 compared with the control group; ns indicates no significant difference compared with the control group;

[0040] Figure 5 shows the effects of different concentrations of Baicalein or MAB on TNF-α ( Figure 5A ), IL-6( Figure 5B )、IL-10( Figure 5C )、MCP-1( Figure 5D )、MIP-2( Figure 5E ), IFN-β( Figure 5F ) and NO( Figure 5G ) regulatory effect; in the figure: * indicates p < 0.05 compared with the control group; ** indicates p < 0.01 compared with the control group; *** indicates p < 0.001 compared with the control group; ns indicates no significant difference compared with the control group;

[0041] Figure 6 shows the effects of different concentrations of Baicalein or MAB on the expression of IL-1β in primary mouse peritoneal macrophages stimulated by LPS. Figure 6A )、CXCL 10( Figure 6B )、iNOS( Figure 6C ) and COX-2mRNA( Figure 6D ) regulatory effect; in the figure: * indicates p < 0.05 compared with the control group; ** indicates p < 0.01 compared with the control group; *** indicates p < 0.001 compared with the control group; ns indicates no significant difference compared with the control group;

[0042] Figure 7 shows the effects of different concentrations of Baicalein or MAB on TNF-α ( Figure 7A ), IL-6( Figure 7B )、IL-10( Figure 7C )、MCP-1( Figure 7D )、MIP-2( Figure 7E ), IFN-β( Figure 7F ) and NO( Figure 7G ) regulatory effect; in the figure: * indicates p < 0.05 compared with the control group; ** indicates p < 0.01 compared with the control group; *** indicates p < 0.001 compared with the control group; ns indicates no significant difference compared with the control group;

[0043] Figure 8 shows the effects of different concentrations of Baicalein or MAB on the expression of IL-1β ( Figure 8A )、CXCL 10( Figure 8B )、iNOS( Figure 8C ) and COX-2mRNA( Figure 8D ) regulatory effect; in the figure: * indicates p < 0.05 compared with the control group; ** indicates p < 0.01 compared with the control group; *** indicates p < 0.001 compared with the control group; ns indicates no significant difference compared with the control group;

[0044] Figure 9 The targeting binding ability of MAB to TLR4 protein at different temperatures is shown; in the figure: * indicates p < 0.05 compared with the DMSO group; ** indicates p < 0.01 compared with the DMSO group; *** indicates p < 0.001 compared with the DMSO group; # indicates p < 0.05 compared with MAB and Baicalein at the same temperature. DETAILED DESCRIPTION

[0045] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally performed under conventional conditions or as recommended by the manufacturer.

[0046] Example 1: Preparation of 4'-methylamine baicalein (MAB)

[0047] The following synthetic route is adopted:

[0048]

[0049] The preparation method comprises the following steps:

[0050] a) Nitro-substituted cinnamic acid 1 (6.7 g, 34 mmol) was dissolved in 100 mL of dichloromethane (DCM) solvent, and oxalyl chloride (6.6 g, 51 mmol) was added dropwise in an ice bath. After the addition was complete, the ice bath was removed and the mixture was reacted at room temperature. After the reaction was complete as determined by TLC (approximately 3 hours), the solvent was concentrated to dryness to obtain an off-white solid, which was intermediate 2;

[0051] b) The obtained intermediate 2 and 3,4,5-trimethoxyphenol 3 (6 g, 33 mmol) were dissolved in 100 mL of toluene, and then 20 mL of BF3·Et2O was added and the reaction was stirred at reflux temperature. When the reaction was completed by TLC monitoring (about 5 hours), the reaction solution was slowly added to an ice-water mixture and stirred for 30 min. The mixture was extracted three times with ethyl acetate (EA) to obtain an organic phase, which was then washed twice with a saturated NaCl solution, dried over anhydrous Na2SO4, filtered, and dried under reduced pressure to obtain intermediate 4;

[0052] c) The obtained intermediate 4 (5.6 mmol) was dissolved in 15 mL of dimethyl sulfoxide (DMSO), and then iodine (142 mg, 0.56 mmol) was added and reacted at 130° C. After the reaction was completed by TLC (about 3 hours), the reaction solution was cooled to room temperature, and then 300 mL of water was added. A large amount of solid precipitated. After the solid precipitated, sodium thiosulfate aqueous solution was added to remove excess iodine. The mixture was stirred at room temperature for half an hour and then filtered. The filter cake was washed with water, dried, and then chromatographed on a silica gel column to obtain intermediate 5;

[0053] d) Intermediate 5 (2 g, 5.6 mmol) was dissolved in 50 mL of 65% aqueous ethanol, and sodium metabisulfite (3.2 g, 16.8 mmol) was added and refluxed. After reflux for 2 hours, 4 mL of concentrated hydrochloric acid was added and the reaction was continued for 1 hour. Water was then added to terminate the reaction, and the pH was adjusted to 8-9 with aqueous ammonia. The organic phase was extracted three times with dichloromethane to obtain an organic phase, which was then washed twice with saturated NaCl solution, dried over anhydrous Na2SO4, filtered, and dried under reduced pressure to obtain intermediate 6;

[0054] e) Intermediate 6 (1 g, 3 mmol) was dissolved in 10 mL of acetone with potassium carbonate (412 mg, 3 mmol) and iodomethane (426 mg, 3 mmol), and the mixture was reacted at room temperature for 8 hours. Water was then added to terminate the reaction, and the organic phase was extracted with dichloromethane (DCM). The organic phase was then washed with brine, dried over anhydrous sodium sulfate, filtered, and spin-dried to give a crude product, which was then purified by column chromatography to give intermediate 7;

[0055] f) Intermediate 7 (500 mg, 1.47 mmol) was added to 10 mL of HI / CH3COOH solution and reacted at 110°C. After the reaction was completed by TLC (approximately 18 hours), the mixture was cooled to room temperature and carefully poured into crushed ice. 20% sodium thiosulfate (5 mL) was then added and stirred for 30 minutes. The precipitate was collected by filtration and recrystallized from ethanol to obtain the target compound 4'-methylamine baicalein.

[0056] 1 H NMR (600MHz, DMSO-d6) δ7.81 (d, J = 8.5 Hz, 2H), 6.87 (s, 1H), 6.71 (d, J = 8.6 Hz, 2H), 6.67 (s, 1H), 3.91 (s, 3H).

[0057] 13 C NMR (101MHz, DMSO-d6) δ182.38,164.99,154.53,152.72,149.92,146.74,130.20,128.54,117.59,114.26,105.32,101.06,91.45,56.69.

[0058] ESI-MS: M=299.08; found m / z=300.0862.

[0059] Example 2: Investigating the effects of MAB on the survival rate and body temperature of mice with LPS-induced septic shock

[0060] 1) Sample preparation

[0061] The required amounts of baicalein and 4'-methylaminobaicalein (MAB) were weighed and dissolved in 3% dimethyl sulfoxide (DMSO) and diluted to 4 mg / mL with sterile saline. The positive control drug, dexamethasone sodium phosphate injection (DXM), was diluted to 0.5 mg / mL with PBS. A septic shock mouse model was induced using 12.5 mg / kg of lipopolysaccharide (LPS).

[0062] 2) Experimental methods

[0063] Construction of mouse septic shock model: 7-week-old female C57BL / 6J mice that had been adaptively raised for 1 week were evenly distributed into different groups according to their body weight and recorded with ear tags.

[0064] Grouping: LPS group, LPS+Baicalein (20 mg / kg), LPS+Baicalein (40 mg / kg), LPS+MAB (20 mg / kg), LPS+MAB (40 mg / kg) and LPS+DXM (5 mg / kg) groups, a total of 6 groups, 11 rats in each group.

[0065] On the day of modeling, LPS was diluted with sterile PBS to the experimental concentration. According to the preliminary experiment, the optimal lethal concentration was 12.5 mg / kg. It was injected intraperitoneally to establish a septic shock model in mice, and drugs were injected at the same time. The living conditions and survival of the mice were observed continuously for 120 hours.

[0066] 3) Measurement and statistical methods

[0067] The survival status and survival conditions of mice in each group were recorded for 5 consecutive days, and the survival curves were drawn using Graphpad Prism 9 software. The Log-Rank test was used for survival analysis.

[0068] 4) Experimental results

[0069] The experimental results are as follows Figure 1 As shown by Figure 1 The results showed that: ① A 12.5 mg / kg dose of LPS successfully induced a septic shock mouse model, with 80% of the mice in the model group dying within 40 hours. The mice also developed symptoms such as lethargy, tremors, piloerection, and chills during the experiment. ② After treatment with 20 mg / kg and 40 mg / kg of Baicalein, the survival rates of mice with septic shock were 0% and 20%, respectively. ③ MAB at 20 mg / kg and 40 mg / kg effectively protected mice from septic shock, with survival rates reaching 30% and 65%, respectively, both of which were statistically significant compared with the LPS model group. ④ The positive drug DXM effectively protected mice from septic shock, with a survival rate of 90%, which was statistically significant compared with the LPS model group. ⑤ Compared with 20 mg / kg of Baicalein, 20 mg / kg of MAB was more effective in protecting mice from septic shock. ⑥ 40 mg / kg of MAB lowered the body temperature of mice in the LPS-induced septic shock model, helping them maintain or restore normal body temperature.

[0070] The above experimental results show that MAB has a protective effect on LPS-induced septic shock model mice, can prolong the survival time of mice and maintain the body temperature of mice, and its effect is significant compared with baicalein under the same conditions.

[0071] Example 3: Investigating the regulatory effect of MAB on LPS-induced inflammatory responses in Raw264.7 cells and primary mouse peritoneal macrophages

[0072] 1) Sample preparation

[0073] Baicalein or MAB was dissolved in dimethyl sulfoxide (DMSO) to a 50 mmol / L stock solution and further diluted with serum-free DMEM cell culture medium to form sample solutions with gradient concentrations of 0, 3, 10, and 30 μmol / L. Lipopolysaccharide (LPS) at 100 ng / mL was used as a stimulator for cell-level experiments.

[0074] 2) Experimental methods

[0075] ①Cell culture of mouse macrophage cell line Raw264.7

[0076] Culture the cells in DMEM high-glucose medium containing 10% fetal bovine serum (FBS) at 37°C in a cell culture incubator with 5% CO2. When the cell confluence reaches 80%, passage them at a ratio of 1:4-1:5. Gently scrape the cells with a cell scraper to disperse cell clumps, or count and plate the cells.

[0077] ② Culture of primary mouse peritoneal macrophages

[0078] SPF-grade C57BL / 6J female mice (6-8 weeks old) were intraperitoneally injected with a sterile 3.5% sodium thioglycolate solution, 1 mL per mouse, in each abdomen. 3.5 days later, the mice were sacrificed by cervical dislocation and immersed in 75% alcohol for 5 minutes. The abdominal skin of the mice was cut open, and the peritoneal cavity was flushed twice with a 20 mL syringe filled with pre-warmed sterile DMEM. The flushing fluid was collected in a sterile 50 mL centrifuge tube and repeatedly aspirated and pipetted. The tube was centrifuged at 1000 rpm for 5 minutes, the supernatant discarded, and the tube was resuspended in 10% FBS DMEM medium. The tube was diluted, counted, and plated. After routine culture for 2 hours, the medium was changed. The adherent cells are primary peritoneal macrophages.

[0079] ③Cell proliferation / toxicity assay

[0080] After incubation with mouse Raw264.7 cells or primary mouse peritoneal macrophages with different concentrations of MAB, 10 μL of CCK8 Enhanced Solution was added to each well according to the CCK-8 kit instructions. After addition of the reagent, the culture plate was gently shaken to facilitate mixing. The cells were incubated in an incubator for 1 hour, and the absorbance at 450 nm was measured using a microplate reader. Cell viability was calculated as follows:

[0081] Cell survival rate = [(As-Ab) / (Ac-Ab)] × 100%

[0082] Where As is the absorbance of the drug test well; Ac is the absorbance of the negative control well; Ab is the absorbance of the blank well.

[0083] ④ Enzyme-linked immunosorbent assay (ELISA)

[0084] Refer to the operating steps in the kit instructions:

[0085] The capture antibody was diluted to the working concentration with PBS, coated on the ELISA plate, 100 μL / well, and allowed to stand at room temperature overnight; the capture antibody was discarded, and 300 μL of wash buffer (PBS containing 0.05% Tween 20, pH 7.2-7.4) was added to each well with a dispenser. After 1 minute, the wash buffer was patted dry on a paper, and the plate was washed three times. After patting dry, 300 μL / well of reagent diluent (1% BSA in PBS, pH 7.2-7.4) was added, and the plate was blocked at room temperature for 1 hour; the reagent diluent was discarded, and the plate was washed three times with the above-mentioned wash buffer (the same below), and 100 μL / well of the standard and the sample to be tested were added, and the plate was incubated at room temperature for 2 hours; the sample to be tested was discarded, the plate was washed three times, and 100 μL / well of the detection antibody was added, and the plate was incubated at room temperature for 2 hours; the detection antibody was discarded, the plate was washed three times, and HRP was added. 100 μL / well, incubate at room temperature in the dark for 20 min; discard HRP and wash the plate three times, add AB solution, and incubate at room temperature in the dark for 20 min; add 50 μL / well of stop solution, and measure the OD value at 450 nm (detection wavelength) / 570 nm (correction wavelength) with a microplate reader. Calculate the corresponding protein concentration according to the standard curve.

[0086] ⑤ Determination of nitric oxide (NO) content by Griess method

[0087] Follow the steps in the kit instructions: a) Prepare all reagents as required and preheat at 37°C for 5 min. In a 96-well plate, add reagent 1 (5 μL), reagent 2 (10 μL), and reagent 3 (5 μL) to the sample wells, standard wells, and blank wells to be tested. Add sample (cell culture supernatant), standard, and distilled water at 60 μL / well, mix well, and react at 37°C for 60 min. b) Add reagent 4 to each well at 20 μL / well and react at 37°C for 30 min. c) Add reaction mix to each well at 100 μL / well and react at 37°C in the dark for 15 min. Measure the NO content in the cell supernatant with a microplate reader at 530 nm.

[0088] ⑥RNA extraction and real-time quantitative PCR analysis (qRT-PCR)

[0089] Total RNA was extracted from cells according to the kit instructions, and 0.5 μg of total RNA sample was reverse transcribed into complementary DNA (cDNA) in a total volume of 10 μL, including 5 μL SYBR, 2.2 μL RNase-free ddH2O, 0.4 μL forward primer, 0.4 μL reverse primer and 2 μL cDNA. The PCR was performed on a LightCycler fluorescent quantitative PCR system. β-Actin was used as an internal control, and 2 μL cDNA was used.-ΔΔCt Methods Data analysis was performed.

[0090] 3) Measurement and statistical methods

[0091] The experimental results of each group were statistically analyzed by one-way analysis of variance (ANOVA) using SPSS27.0 software, and the data were expressed as mean ± standard error (SEM).

[0092] 4) Experimental results

[0093] Figure 2 The cytotoxicity of different concentrations of MAB after incubation with mouse Raw264.7 cells (2A) and mouse primary peritoneal macrophages (2B) for 24 h is shown; Figure 2 As shown, in Raw264.7 cells, MAB had no cytotoxicity at 0-30 μM within 24 hours and did not affect the normal proliferation of cells; in primary mouse peritoneal macrophages, MAB had no cytotoxicity at 0-100 μM within 24 hours.

[0094] Figure 3 shows the regulatory effects of different concentrations of Baicalein or MAB on TNF-α (3A), IL-6 (3B), IL-10 (3C), MCP-1 (3D), MIP-2 (3E), IFN-β (3F) and NO (3G) in LPS-stimulated mouse RAW264.7 cells; As shown in Figure 3, it can be seen that in Raw264.7 cells, compared with baicalein, at the same concentration, MAB intervention can better inhibit the secretion of TNF-α, IL-6, IL-10, MCP-1, MIP-2, IFN-β and NO stimulated by LPS, and shows a clear dose-dependent relationship.

[0095] Figure 4 shows the regulatory effects of different concentrations of Baicalein or MAB on IL-1β (4A), CXCL10 (4B), iNOS (4C) and COX-2 mRNA (4D) in LPS-stimulated mouse RAW264.7 cells. As shown in Figure 4, in Raw264.7 cells, compared with baicalein, at the same concentration, MAB can significantly inhibit the expression of IL-1β, CXCL10, iNOS and COX-2 mRNA produced by LPS stimulation.

[0096] Figure 5 shows the regulatory effects of different concentrations of Baicalein or MAB on TNF-α (5A), IL-6 (5B), IL-10 (5C), MCP-1 (5D), MIP-2 (5E), IFN-β (5F) and NO (5G) in mouse primary peritoneal macrophages stimulated by LPS; As shown in Figure 5, it can be seen that in mouse primary peritoneal macrophages, compared with baicalein, at the same concentration, MAB intervention can better inhibit the secretion of TNF-α, IL-6, IL-10, MCP-1, MIP-2, IFN-β and NO stimulated by LPS, and shows an obvious dose-dependent relationship.

[0097] Figure 6 shows the regulatory effects of different concentrations of Baicalein or MAB on IL-1β (6A), CXCL 10 (6B), iNOS (6C) and COX-2 mRNA (6D) in mouse primary peritoneal macrophages stimulated by LPS; As shown in Figure 6, in mouse primary peritoneal macrophages, compared with baicalein, at the same concentration, MAB can significantly inhibit the expression of IL-1β, CXCL 10, iNOS and COX-2 mRNA stimulated by LPS.

[0098] The above experimental results show that compared with baicalein, MAB can more significantly inhibit the levels of inflammatory factors and chemokines in LPS-induced Raw264.7 cells and primary mouse peritoneal macrophages, and has a significant anti-inflammatory regulatory effect.

[0099] Example 4: Investigating the regulatory effect of MAB on the inflammatory response of mouse Raw264.7 cells induced by heat-killed CRKP (HK-CRKP)

[0100] 1) Sample preparation

[0101] Baicalein or MAB was dissolved in dimethyl sulfoxide (DMSO) to a 50 mmol / L stock solution, and further diluted with serum-free DMEM cell culture medium to prepare sample solutions with gradient concentrations of 0, 3, 10, and 30 μmol / L.

[0102] 2) Strain recovery and cultivation

[0103] Take out a CRKP (HS11286) glycerol tube from the -80℃ freezer, pick up a small amount of bacterial liquid with a sterile inoculating loop and inoculate it on the LB agar plate according to the three-zone streak method, then place it in a 37℃ constant temperature incubator and invert it for 14 hours, and then store it in a 4℃ refrigerator for use.

[0104] 3) Preparation of HK-CRKP bacterial suspension

[0105] A single colony was picked and placed in a shaker tube containing 2 mL of LB liquid medium. The tube was incubated at 250 rpm at 37°C for 12 hours, and the absorbance at 600 nm was measured to determine the bacterial concentration. After the measurement, the bacterial solution was heat-inactivated in a water bath at 65°C for 45 minutes. After inactivation, the solution was centrifuged at 4400 rpm for 15 minutes, the supernatant was discarded, and 1 mL of DMEM was added to resuspend the solution. The bacterial concentration at this point was the measured bacterial concentration. The seeding density of the Raw264.7 cell plate was 2.5 × 10 5 cells / well, and the required bacterial concentration was calculated based on the number of infected cells at MOI=10 (i.e., one cell was infected with 10 bacteria).

[0106] 4) Measurement and statistical methods

[0107] The experimental results of each group were statistically analyzed by one-way analysis of variance (ANOVA) using SPSS27.0 software, and the data were expressed as mean ± standard error (SEM).

[0108] 5) Experimental results

[0109] Figure 7 shows the regulatory effects of different concentrations of Baicalein or MAB on TNF-α (7A), IL-6 (7B), IL-10 (7C), MCP-1 (7D), MIP-2 (7E), IFN-β (7F) and NO (7G) in HK-CRKP-stimulated mouse Raw264.7 cells; As shown in Figure 7, in Raw264.7 cells, compared with baicalein, at the same concentration, MAB intervention can better inhibit the secretion of TNF-α, IL-6, IL-10, MCP-1, MIP-2, IFN-β and NO stimulated by HK-CRKP, and shows a significant dose-dependent relationship.

[0110] Figure 8 shows the regulatory effects of different concentrations of Baicalein or MAB on IL-1β (8A), CXCL 10 (8B), iNOS (8C), and COX-2 mRNA (8D) in HK-CRKP-stimulated mouse RAW264.7 cells. As shown in Figure 8 , in Raw264.7 cells, compared with baicalein, at the same concentration, MAB can significantly inhibit the expression of IL-1β, CXCL 10, iNOS, and COX-2 mRNA produced by HK-CRKP stimulation.

[0111] The above experimental results show that compared with baicalein, MAB can more significantly inhibit the levels of inflammatory factors and chemokines in Raw264.7 cells induced by HK-CRKP, and has a significant anti-inflammatory regulatory effect.

[0112] Example 5: Cellular Thermal Shift Assay (CETSA)

[0113] 1) Wash a whole dish of RAW264.7 cells three times with pre-chilled PBS, scrape the cells with a cell scraper (or gently blow them off), and centrifuge at 1000 rpm for 5 minutes;

[0114] 2) Discard the supernatant and add 1 ml of M-PER lysis buffer (containing PMSF and protease inhibitors) to the cell pellet, resuspend, and incubate on ice for 30 minutes;

[0115] 3) Centrifuge at 20,000 g for 20 min at 4°C, collect the supernatant, and add PMSF again;

[0116] 4) Divide the supernatant into 3 equal parts;

[0117] 5) Solvent (DMSO) and MAB / Baicalein (200 μM) were added to two tubes respectively, with equal volumes of drug-dissolving solvent and drug, and both were rotated at room temperature for 30 min;

[0118] 6) Divide each tube into 12 equal portions and heat in a metal bath at a gradient of 38, 42, 46, 50, 54, 58, 62, 66, 70, 74, 78, and 82°C. Place the mixture in a metal bath for 3 minutes, then remove and immediately place on ice.

[0119] 7) Centrifuge at 20,000 g for 20 min at 4°C;

[0120] 8) Aspirate the supernatant, add loading medium and freeze at -20℃. Do not denature it again.

[0121] Figure 9 The targeting ability of MAB to TLR4 protein at different temperatures was shown. Figure 9 As shown, the targeting binding ability of MAB to TLR4 protein is significantly better than that of baicalein.

[0122] In summary, it can be seen that the MAB described in the present invention is not only low in toxicity, but also has a more significant inhibitory effect on excessive inflammatory responses induced by LPS or pathogen infection than baicalein under the same conditions, and has better targeted binding ability to the TLR4 protein. Therefore, it is expected that the MAB described in the present invention or at least one of its pharmaceutically acceptable salts, tautomers, stereoisomers, and precursor compounds can be used as an active ingredient to prepare a drug for the treatment of sepsis. The MAB described in the present invention has achieved significant progress and unexpected technical effects compared to baicalein.

[0123] Finally, it should be pointed out that the above are only some preferred embodiments of the present invention and should not be understood as limiting the scope of protection of the present invention. Some non-essential improvements and adjustments made by those skilled in the art based on the above contents of the present invention fall within the scope of protection of the present invention.

Claims

1. A use of a baicalein derivative, wherein the baicalein derivative is 4'-methylamino baicalein, having the following chemical structure: ; It is characterized by: At least one of 4'-methylamine baicalein or a pharmaceutically acceptable salt thereof is used as an active ingredient in preparing a drug for treating sepsis.

2. The use according to claim 1, characterized in that: The sepsis includes early sepsis, severe sepsis and septic shock.

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