A ppar multiple agonist and preparation method and use thereof

Abstract

The present application provides a kind of compound with PPAR multiple agonist activity and its preparation method and use, the compound is prepared by fermentation of Penicillium (Penicillium) Penicillium pasqualense ) NCPC0626, the preservation unit of the Penicillium is China General Microbiological Culture Collection Center, and the preservation number is CGMCC No.23277.The compound prepared by the present application has multiple PPAR α 、 β And gamma Agonist activity, can be used for preparing drug for treating or preventing PPARs mediated diseases.

Description

Technical Field

[0001] This invention belongs to the field of medicinal chemistry, specifically relating to a compound with PPAR multi-agonist activity, its preparation method and application, as well as the bacteria that produce this type of compound. Background Technology

[0002] With the global prevalence of obesity and related metabolic syndromes, non-alcoholic fatty liver disease (NAFLD) has become a significant cause of chronic liver disease in developed countries such as Europe and the United States, as well as in affluent regions of my country, with a prevalence of approximately 15% to 40%. Of these, 10% to 20% of NAFLD patients will develop non-alcoholic steatohepatitis (NASH). The global incidence of NASH is estimated at 3% to 5%, increasing to 22% in diabetic patients. Approximately 15% to 25% of NASH patients will develop cirrhosis, and it is estimated that by 2030, liver deaths related to NAFH will increase by 178%.

[0003] NASH is a chronic liver disease with an incompletely understood etiology. The widely accepted "two-hit" theory posits that the first hit primarily involves increased fat intake, increased hepatic synthesis, impaired fatty acid β-oxidation, and decreased very low-density lipoprotein (VLDL) secretion, leading to lipid accumulation. The second hit is oxidative stress, which further causes liver inflammation, necrosis, and fibrosis. The emerging multi-hit theory suggests that liver inflammation, rather than steatosis, is the primary cause of NASH progression to fibrosis. NASH is the result of multiple factors acting simultaneously, including genetic variations, abnormal lipid metabolism, oxidative stress, endoplasmic reticulum stress, mitochondrial dysfunction, altered immune responses, and gut microbiota imbalance. While the mechanisms by which obesity and NASH / NAFLD promote liver cancer development are not fully understood, various tumor microenvironment factors, such as elevated pro-inflammatory cytokines, immune cell responses, lipid metabolism disorders, and altered gut microbiota, can induce or synergistically promote the development and progression of liver cancer.

[0004] Peroxisome growth factor-activated receptors (PPARs), as a class of nuclear receptors, are major regulators of glucose and lipid metabolism, playing a crucial role in anti-steatolipin, anti-inflammation, and anti-fibrosis. PPARs include three subtypes: PPARα, PPARβ (or PPARδ), and PPARγ. PPARα enhances the β-oxidation capacity of fatty acids in mitochondria and peroxisomes, reduces the accumulation of triglycerides in the liver, and can directly control the progression of inflammatory responses. Unlike PPARα, PPARβ promotes β-oxidation of fatty acids in extrahepatic cells. PPARγ improves insulin sensitivity and the oxidation of free fatty acids, inhibits inflammatory responses, and prevents the progression of fibrosis.

[0005] Triple PPAR agonists play important roles in improving glucose and lipid metabolism, as well as in anti-inflammatory and anti-fibrotic effects, and can more effectively improve non-alcoholic fatty liver disease. Therefore, screening compounds with triple PPAR agonist activity is of great value for the prevention and treatment of NASH. Summary of the Invention

[0006] In view of the problems and needs of the existing technology, the purpose of this invention is to provide a compound with triple agonist activity of PPARs, a method for preparing such a compound and its pharmaceutical uses, and to provide a strain that produces such a compound.

[0007] To achieve the objectives of this invention, compounds with structures as shown in Formula I (hereinafter referred to as Formula I compounds) are provided:

[0008]

[0009] Where R1 is H or OH; R2 is H or OH.

[0010] Among the preferred compounds are: compound A, where R1 is H and R2 is OH; and compound B, where R1 is OH and R2 is OH.

[0011] This invention provides a strain for fermentation preparation of compound I, a Penicillium strain (Penicilliumpasqualense) NCPC0626, deposited at the China General Microbiological Culture Collection Center (CGMCC) on October 11, 2021, with accession number CGMCC No. 23277, and deposited at Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0012] This invention provides a method for preparing the compound of formula I, which involves fermentation extraction using Penicillium strain NCPC0626, and may include the following steps:

[0013] a. Preparation of seed culture for Penicillium strain NCPC0626;

[0014] b. Preparation of fermentation culture of Penicillium strain NCPC0626;

[0015] c. Extract the fermentation culture with an organic solvent, concentrate the extract under reduced pressure, remove the organic solvent, and obtain a crude extract;

[0016] d. Compound I was prepared by chromatographic separation of the crude extract.

[0017] Preferably, step d includes the following steps:

[0018] 1) The crude extract obtained in step c was separated by silica gel column chromatography and eluted with a gradient of n-hexane / ethyl acetate to obtain PPARs active component a;

[0019] 2) Component a was separated by Sephadex LH-20 column chromatography and eluted with methanol to obtain component a1;

[0020] 3) Component a1 was separated using an ODS preparative chromatographic column and eluted with an acetonitrile / water gradient to obtain PPARs active components 3 and 5;

[0021] 4) Component 3 was separated by Sephadex LH20 column elution and eluted with methanol to give compound B;

[0022] 5) Component 5 was dried by evaporating the solvent under light-protected conditions to obtain compound A.

[0023] In step 1), the gradient elution process using n-hexane:ethyl acetate can be as follows: the elution solvents are: 1000 ml of n-hexane, 1000 ml of n-hexane-ethyl acetate (V / V = 10:1), 1000 ml of n-hexane-ethyl acetate (V / V = 4:1), 1000 ml of n-hexane-ethyl acetate (V / V = 1:1), 1000 ml of ethyl acetate, and 1000 ml of ethanol. The detection wavelength is 333 nm, and the samples are collected according to the peaks. The samples are evaporated to dryness under reduced pressure to obtain components a and b. The transcriptional activity of the compounds on PPARs is detected, and finally, component a is determined to be the active component.

[0024] In step 2), the sample was detected at 333 nm, and the eluent was collected according to the peak to obtain component a1;

[0025] In step 3), the acetonitrile / water gradient elution can be a linear concentration gradient elution of acetonitrile from 20% to 100% (0 min to 20 min); detection is performed at 333 nm, and the components are collected according to the peaks and numbered sequentially as components 1-9. The acetonitrile of components 1-9 is removed by vacuum distillation, and the remaining aqueous phase is extracted with ethyl acetate. The activity is measured separately to obtain PPARs active components 3 and 5.

[0026] In step 5), component 5 can be placed in an open brown bottle to evaporate the moisture, yielding compound A.

[0027] Preferably, step a may include the following steps: inoculating the slant culture or spore liquid of Penicillium strain NCPC0626 into a seed culture medium, culturing at a temperature of 22-30℃ and a rotation speed of 100-220 rpm for 48-72 h to obtain a seed liquid; wherein the seed culture medium is prepared by the following method: 10.0-40.0 g of corn starch, 10.0-40.0 g of glucose, 4.0-20.0 g of hot-pressed soybean meal, 1.0-6.0 g of malt powder, 1.0-3.0 g of yeast powder, 0.5-2.0 g of sodium chloride, 0.5-2.0 g of magnesium sulfate heptahydrate, adding tap water to make up to 1000 mL, pH 6.5-7.0; sterilizing at 121℃ for 30 min.

[0028] Step b may include the following steps: Inoculating the seed culture from step a at an inoculation rate of 3-10% into a solid fermentation medium, fermenting at a temperature of 22℃-30℃ for 7-20 days to obtain a fermented culture; the solid fermentation medium includes a solid granular matrix, a powdered matrix, inorganic salts, and trace elements, optionally added or not added; the solid granular matrix is ​​rice, millet, wheat bran, or corn grits, and the powdered matrix is ​​hot-pressed soybean meal, cottonseed meal, or corn gluten meal. The inorganic salt components may be phosphates or sodium nitrates, and the trace elements include cobalt chloride, zinc sulfate, and manganese chloride. The culture medium is sterilized at 121℃ for 30 minutes.

[0029] Preferably, the ratio of solid granular matrix (rice, millet, corn grits or bran) to water is 1:1 to 5:1:1; the powdered matrix (hot-pressed soybean meal) is added at 2%-10% of the total weight of the solid granular matrix after mixing with water; cottonseed meal is added at 0-5% of the total weight of the solid granular matrix after mixing with water; and corn gluten meal is added at 0-5% of the total weight of the solid granular matrix after mixing with water. The inorganic salts (phosphate, sodium nitrate, zinc sulfate, manganese chloride, and cobalt chloride) are added at 0-0.1% of the total weight of the solid granular matrix after mixing with water.

[0030] Step c may include the following steps: extracting 1-3 times, each time for 4-8 hours, and combining the extracts; wherein the organic solvent is ethyl acetate or acetone.

[0031] The present invention also provides a pharmaceutical use of the compound of formula I: using the compound to prepare a medicament for treating and / or preventing PPARs-mediated diseases. The PPARs-mediated diseases may be non-alcoholic fatty liver disease.

[0032] Compound I was found to significantly enhance the transcriptional activity of PPARα, β, and γ, exhibiting multiple agonist activity for PPARα, β, and γ, and thus serving as an effective component of a multiple PPAR agonist. In a mouse primary hepatocyte model, compound I demonstrated significant anti-fibrotic and anti-inflammatory activity. Furthermore, using a carbon tetrachloride-induced liver fibrosis model mouse as the research subject, and with hepatic steatosis and fibrosis as indicators, this invention verified that compound I, as a multiple PPARα, β, and γ agonist, can effectively reduce the degree of hepatic steatosis, fibrosis, and ballooning degeneration in the model mice.

[0033] When it comes to the above-mentioned pharmaceutical uses, according to common knowledge in the art, not only the compound of formula (I) described in this invention itself, but also its pharmaceutically acceptable salts, tautomers, stereoisomers, precursor compounds, hydrates or solvates can have the same or similar pharmaceutical uses.

[0034] The term "pharmaceutically acceptable salt" refers to a salt formed by the compound and a pharmaceutically acceptable inorganic or organic acid, including but not limited to: hydrochloric acid, hydrobromic acid, phosphoric acid, nitric acid, and sulfuric acid; and the organic acid including but not limited to: formic acid, acetic acid, propionic acid, succinic acid, 1,5-naphthalenedisulfonic acid, linalool, 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, and amino acids; "pharmaceutical acceptable" means a substance suitable for human use without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), i.e., a reasonable benefit / risk ratio.

[0035] The present invention also provides a pharmaceutical composition comprising a compound of formula I or a pharmaceutically acceptable salt, isomer, precursor compound, hydrate or solvate thereof, and a pharmaceutically acceptable carrier, excipient, or excipient. The pharmaceutical composition may also comprise another therapeutic agent. The pharmaceutical composition is suitable for preparing medicaments for treating and / or preventing PPARs-mediated diseases.

[0036] The drug described in this invention can be administered to patients via various routes of administration, including but not limited to oral, transdermal, intramuscular, subcutaneous, and intravenous injection.

[0037] The dosage form of the drug described in this invention is not limited, as long as it enables the active ingredient to effectively reach the body, including: tablets, sugar-coated tablets, film-coated tablets, enteric-coated tablets, capsules, hard capsules, soft capsules, oral liquids, lozenges, granules, powders, pills, powders, ointments, pills, suspensions, powders, solutions, injections, suppositories, ointments, plasters, creams, sprays, drops, patches, etc.; oral dosage forms are preferred, such as: capsules, tablets, oral liquids, granules, pills, powders, pills, ointments, ointments, etc.

[0038] The medicaments described in this invention, in addition to containing the main active ingredient, may also contain small amounts of minor components that do not affect the effective ingredient and / or pharmaceutically acceptable carriers, as well as various excipients necessary for formulation. For example, when the medicament is an oral dosage form, it may contain commonly used excipients, such as binders, fillers, diluents, tableting agents, lubricants, disintegrants, colorants, flavoring agents, and humectants; if necessary, the tablets may be coated. Suitable fillers include cellulose, mannitol, lactose, and other similar fillers; suitable disintegrants include starch, polyvinylpyrrolidone, and starch derivatives, such as sodium glycolate starch; suitable lubricants include, for example, magnesium stearate; suitable pharmaceutically acceptable humectants include sodium dodecyl sulfate.

[0039] Compared with the prior art, the present invention has the following significant advantages:

[0040] The research results of this invention show that the compound of formula I can significantly enhance the transcriptional activity of PPARα, β, and γ, exhibiting multiple agonist activities of PPARα, β, and γ, and can serve as an effective component of a multiple PPARs agonist. In a mouse primary hepatocyte model, the compound of formula I demonstrated significant anti-fibrotic and anti-inflammatory activities. Moreover, using a carbon tetrachloride-induced liver fibrosis model mouse as the research subject, and using the degree of hepatic steatosis and fibrosis as indicators, this invention verified that the compound of formula I, as a multiple PPARα, β, and γ agonist, can effectively reduce the degree of hepatic steatosis, fibrosis, and ballooning degeneration in model mice. It can be used to prepare drugs for the treatment and / or prevention of non-alcoholic fatty liver disease, and has broad application prospects and significant pharmaceutical value. Attached Figure Description

[0041] Figure 1 Liver pathological sections of mice with liver fibrosis model

[0042] Wherein, F: normal group; M: model group; L3: 1 mg / kg for 3 weeks; H3: 3 mg / kg for 3 weeks; L4: 1 mg / kg for 4 weeks; H4: 3 mg / kg for 4 weeks. Detailed Implementation

[0043] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto. Unless otherwise specified, the methods used in the following embodiments are conventional methods.

[0044] The specifications and models of some of the raw materials involved in the following embodiments are as follows:

[0045] Nuclear magnetic resonance spectrometer: Bruker 500MHz (TMS is an internal standard).

[0046] Low-resolution mass spectrometer: Waters LC-MS ZQ 2000 (ESI mode).

[0047] Medium-pressure preparative chromatography system: Buchi Corporation, 2 Buchi C-601 pumps.

[0048] Analytical high-pressure liquid chromatography system: Waters Corporation, 2 Model 515 pumps, 996 detector.

[0049] Analytical chromatographic column: Chromasil ODS column (4.6×250mm, 10μm).

[0050] Preparative high-pressure liquid chromatography system: Waters Prep LC System, 2487 detector.

[0051] Preparative chromatography column: Nano-Micro Unisil ODS column (21.2×250mm, 10μm).

[0052] Chromatographic silica gel: 300-400 mesh, Qingdao Marine Chemical Plant.

[0053] Hydroxypropyl dextran gel Sephadex LH20 chromatography medium: Pharmacia.

[0054] Microplate reader: Perkin Elmer Victor21420 Multilabel Counter

[0055] Chromatographic grade methanol and acetonitrile were purchased from Honeywell, Inc., USA; other reagents were of analytical grade and purchased from Tianjin Yongda Chemical Reagent Co., Ltd.

[0056] Example 1. Preparation of fermentation culture of bacteria producing compound I

[0057] (a) Preparation of seed culture of Penicillium pasqualense NCPC0626

[0058] The *Penicillium pasqualense* strain NCPC0626 was inoculated into seed culture medium and cultured at 26°C and 200 rpm for 72 h to obtain seed culture. The seed culture medium was prepared as follows: 30.0 g corn starch, 10.0 g glucose, 4.0 g hot-pressed soybean meal, 5.0 g malt powder, 3.0 g yeast powder, 2.0 g sodium chloride, and 1.0 g magnesium sulfate heptahydrate were dissolved in tap water and brought to a final volume of 1000 ml. The pH was adjusted to 6.5, and the medium was sterilized at 121°C for 30 min.

[0059] (b) Preparation of solid-state fermentation culture of Penicillium pasqualense NCPC0626

[0060] The seed culture was inoculated at an 8% inoculation rate into 750 mL Erlenmeyer flasks containing 100 g of solid rice fermentation medium and incubated at 26 °C for 7 days. The fermentation medium was prepared as follows: rice and water were mixed evenly at a 4:1 ratio and soaked for 3 hours. Then, 5% hot-pressed soybean meal, 1% cottonseed meal, 0.2% potassium dihydrogen phosphate, 0.05% cobalt chloride, 0.01% manganese chloride, and 0.01% zinc sulfate were added. After thorough mixing, the mixture was dispensed into 750 mL Erlenmeyer flasks, 100 g per flask, sealed with rubber stoppers, and sterilized at 121 °C for 30 min.

[0061] Example 2. Preparation of fermentation culture of compound I producing bacteria

[0062] (a) Preparation of seed culture for Penicillium pasqualense strain NCPC0626:

[0063] The fungus *Penicillium asqualense* strain NCPC0626 was inoculated into seed culture medium and cultured at 22°C and 100 rpm for 72 h to obtain seed culture. The seed culture medium was prepared as follows: 10.0 g corn starch, 40.0 g glucose, 14.0 g hot-pressed soybean meal, 6.0 g malt powder, 1.0 g yeast powder, 1.0 g sodium chloride, and 0.5 g magnesium sulfate heptahydrate were dissolved in tap water and brought to a final volume of 1000 ml. The pH was adjusted to 7.0, and the medium was sterilized at 121°C for 30 min.

[0064] (b) Preparation of solid-state fermentation culture of Penicillium pasqualense NCPC0626

[0065] The seed culture was inoculated at a rate of 5% into a 750mL Erlenmeyer flask containing 100g of solid corn residue fermentation medium, and incubated statically at 30℃ for 14 days. The fermentation medium was prepared by mixing corn residue and water in a 3:1 ratio, adding 0.2% sodium nitrate by weight, and soaking for 4 hours. The mixture was then dispensed into 750mL Erlenmeyer flasks, 100g per flask, sealed with rubber stoppers, and sterilized at 121℃ for 30 minutes.

[0066] Example 3. Preparation of fermentation culture of bacteria producing compound I

[0067] (a) Preparation of seed culture for Penicillium pasqualense strain NCPC0626:

[0068] The fungus *Penicillium pasqualense* strain NCPC0626 was inoculated into seed culture medium and cultured at 30°C and 220 rpm for 48 hours to obtain seed culture. The seed culture medium was prepared as follows: 40.0 g corn starch, 30.0 g glucose, 20.0 g hot-pressed soybean meal, 1.0 g malt powder, 2.0 g yeast powder, 0.5 g sodium chloride, and 2.0 g magnesium sulfate heptahydrate were dissolved in tap water and brought to a final volume of 1000 ml. The pH was adjusted to 6.7, and the medium was sterilized at 121°C for 30 min.

[0069] (b) Preparation of solid-state fermentation culture of Penicillium pasqualense NCPC0626

[0070] The seed culture was inoculated at a rate of 3% into 750ml Erlenmeyer flasks containing 100g of solid millet fermentation medium, and incubated statically at 30℃ for 10 days. The fermentation medium was prepared as follows: millet and water were mixed evenly at a ratio of 8:3 and soaked for 10 hours. Then, corn gluten powder and 0.2% potassium dihydrogen phosphate were added at 2.5% of the total weight, mixed evenly, and dispensed into 750ml Erlenmeyer flasks, 100g per flask, sealed with rubber stoppers, and sterilized at 121℃ for 30 minutes.

[0071] Example 4. Preparation of Compound A and Compound B

[0072] 1) Preparation of fermentation culture of compound I producing bacteria

[0073] (a) Preparation of seed culture for Penicillium pasqualense strain NCPC0626:

[0074] The fungus *Penicillium pasqualense* strain NCPC0626 was inoculated into seed culture medium and cultured at 26°C and 200 rpm for 72 h to obtain seed culture. The seed culture medium was prepared as follows: 20.0 g corn starch, 20.0 g glucose, 8.0 g hot-pressed soybean meal, 3.0 g malt powder, 2.5 g yeast powder, 1.5 g sodium chloride, and 1.5 g magnesium sulfate heptahydrate were dissolved in tap water and brought to a final volume of 1000 ml. The pH was adjusted to 6.8, and the medium was sterilized at 121°C for 30 min.

[0075] (b) Preparation of solid-state fermentation culture of Penicillium pasqualense NCPC0626

[0076] The seed culture was inoculated at a rate of 10% into a 750ml Erlenmeyer flask containing 100g of a fermentation medium made from a mixture of wheat bran and hot soybean flour. The flask was then incubated statically at 26℃ for 20 days. The fermentation medium was prepared as follows: wheat bran, hot soybean flour, and water were mixed in a 1:1:2 ratio, then dispensed into 750ml Erlenmeyer flasks (100g per flask), sealed with gauze, and sterilized at 121℃ for 30 minutes.

[0077] (ii) Extraction and preparation of compounds A and B

[0078] Approximately 6 kg of the fermentation product (solid-state fermentation) from step 1) was soaked in 12 L of ethyl acetate and stirred. After 4 hours, the mixture was filtered and the filtrate was evaporated under reduced pressure to obtain 25 g of a dark brown paste.

[0079] 25g of a dark brown paste was dissolved in a small amount of ethyl acetate, mixed with silica gel, and separated by silica gel column chromatography using a Buchi medium-pressure preparative chromatography system with a silica gel column bed of Φ3.6×40cm. The elution solvents were, in sequence: 1000ml of n-hexane, 1000ml of n-hexane-ethyl acetate (V / V = 10:1), 1000ml of n-hexane-ethyl acetate (V / V = 4:1), 1000ml of n-hexane-ethyl acetate (V / V = 1:1), 1000ml of ethyl acetate, and 1000ml of ethanol. Peaks were collected, and the detection wavelength was 333nm. The eluents were collected separately and evaporated to dryness under reduced pressure to obtain components a and b, weighing 4g and 21g respectively. The transcriptional activity of the compounds on PPARs was detected using mammalian cell one-hybrid assay. Component a was determined to be the active component, and component b was discarded.

[0080] Component a was separated by Sephadex LH20 column (eluting with pure methanol) and detected by analytical high-performance liquid chromatography system at a detection wavelength of 333 nm. The eluent was collected according to the characteristic ultraviolet absorption peak and concentrated to dryness under reduced pressure to obtain component a1 (2.8 g).

[0081] Component a1 was separated using an ODS preparative column (21.2 × 250 mm) and a Waters preparative high-performance liquid chromatography system. Acetonitrile and water were used as the mobile phase, with elution of acetonitrile at a linear concentration gradient of 20%–100% (0 min–20 min) at a flow rate of 18 mL / min. Detection was performed at 333 nm. Peaks were collected and numbered sequentially from the first peak to 9. Acetonitrile was removed under reduced pressure, and the remaining aqueous phase was extracted with ethyl acetate. The mixture was separated using a separatory funnel, and the upper ethyl acetate phase was concentrated to obtain 9 components. Quantitative analysis was performed on each component, with components 3 and 5 identified as the active components.

[0082] Fraction 3 was separated by Sephadex LH20 column (eluting with pure methanol), detected by analytical high-performance liquid chromatography system, and the eluent was collected according to peak and concentrated to dryness to obtain compound B (12 mg).

[0083] Component 5 was placed in an open brown bottle and allowed to evaporate the water to obtain compound A (253 mg).

[0084] Compounds A and B were identified as having the following structures by LC-MS and NMR data analysis, respectively; their chemical structures have not been reported.

[0085] Compound A: Yellow solid powder, readily soluble in ethyl acetate, methanol, and DMSO. UV λmax (CH3CN) = 249, 333 nm. ESI-MS m / z 340 [M+H] + 338 [MH] - Molecular formula C 20 H 21 NO4.

[0086] Compound B: Yellow solid powder, readily soluble in ethyl acetate, methanol, and DMSO. UV λmax (CH3CN) = 252, 335 nm. ESI-MS m / z 356 [M+H] + 354 [MH] - Molecular formula C 20 H 21 NO5.

[0087]

[0088] Compound A: R1 = H, R2 = -OH; Compound B: R1 = R2 = -OH;

[0089] Chemical structures of compounds A and B

[0090] The NMR data of compounds A and B are shown in Table 1.

[0091] Table 1. NMR data of compounds A and B

[0092]

[0093]

[0094] a 500MHz, DMSO-d6, TMS=0; b 125MHz, DMSO-d6

[0095] Example 5. Effects of compounds on the transcriptional activity of PPARs

[0096] The effect of the compound on PPAR transcriptional activity was detected using mammalian cell one-hybrid assay. HEK293 cells were transfected with two plasmids: pBIND carrying the PPAR ligand-binding domain and pGL3-Gal4 carrying the Gal4 DNA-binding domain. After treatment with the compound for 24 hours, luciferase activity was detected after cell lysis.

[0097] (1) HEK293 cell culture: HEK293 cells were cultured in DMEM high-glucose medium (containing 1% penicillin and streptomycin) with 10% fetal bovine serum in a 5% CO2, 37°C incubator. The HEK293 cell density was 2×10⁶ cells / year. 4 96-hole plate laying is performed using individual holes.

[0098] (2) Plasmid transfection: Overnight HEK293 cells were transfected with 5 μl serum-free 1640 medium diluted with 0.2 μl Lipofectamine 3000 reagent, 5 μl serum-free 1640 medium diluted with 0.5 μg target plasmid DNA (reporter plasmid to expression plasmid ratio of 4:1), and 0.2 μl P3000 reagent. The mixture was thoroughly mixed and allowed to stand for 15 min. 10 μl of the mixture was added to each well of a 96-well plate and gently shaken. Serially diluted compounds A and B were added, and the cells were incubated at 37°C and 5% CO2 for 24 hours.

[0099] (3) Reporter gene detection: Remove the 96-well plate from the incubator, discard the culture medium, wash once with PBS, discard the PBS, add 50 μl / well Lysis buffer, lyse for 10-15 minutes, add 50 μl / well reaction buffer, and detect with an ELISA reader.

[0100] (4) Experimental results: As shown in Table 2, compounds A and B of formula (I) have efficient partial agonist effects on PPARα, PPARβ and PPARγ.

[0101] Table 2. Agonistaltic activity of compounds A and B on PPARs

[0102]

[0103] Example 6. Effects of compound A on fibrosis and inflammatory gene expression in mouse primary hepatocytes

[0104] Mouse primary hepatocytes were treated with compounds, and changes in the expression levels of intracellular fibrosis markers monocyte chemoattractant protein-1 (MCP1), type I collagen α1 (COL1A1), matrix metalloproteinase inhibitor 1 (TIMP1), and inflammatory factors tumor necrosis factor α (TNFα) and interleukin-6 (IL-6) were detected using Real-time PCR.

[0105] (1) Extraction of primary mouse hepatocytes: Kunming mice were anesthetized with sodium barbital and their livers were perfused. First, the livers were perfused with solution A (HBSS, 0.1 mM EGTA, 10 mM Hepes, 50 ml), and then with solution B (Leibovitz's L-15, 0.02 mg / ml Liberase™, 50 ml). The livers were removed and placed in petri dishes with DMEM / F12 medium. The livers were minced in a clean bench, filtered three times, and centrifuged at 500 rpm for 3 min at 4°C for 3 times. The DMEM / F12 medium was changed each time. The cells were collected and suspended in Hepato ZYMESFM, plated in 24-well plates, and cultured in a 5% CO2, 37°C incubator.

[0106] After overnight cell culture, the cells were treated with fatty acids (65 μM sodium oleate, 45 μM palmitic acid), insulin (100 nM), glucose (4.5 mg / ml), TNF-α (50 ng / ml), IL-1β (25 ng / ml), and TGF-β1 (8 ng / ml). Compound A was added, while the control group was treated with an equal volume of DMSO. The cells were cultured for another 24 hours.

[0107] (2) RNA extraction: Discard the culture medium in the cell culture plate, add 200 μl of TRIzon lysis buffer to each well of the culture plate and repeatedly pipette to lyse the cells. Let it stand at room temperature for 5 minutes, add 40 μl of chloroform, cap the tube, and vortex vigorously for 15 seconds. Let it stand at room temperature for 2 minutes, centrifuge at 12000 rpm and 4°C for 10 minutes. Carefully aspirate the colorless aqueous phase into a new centrifuge tube, add the same volume of 70% ethanol, invert and mix well, and add the mixture to the Spin Columns RM adsorption column that has been loaded into the collection tube. Centrifuge at 12000 rpm for 20 seconds, discard the waste liquid, and return the adsorption column to the collection tube. Add 700 μl of Buffer RW1 to the adsorption column, centrifuge at 12000 rpm for 20 seconds, discard the waste liquid in the collection tube, and return the adsorption column to the collection tube again. Add 500 μl of Buffer RW2 (with anhydrous ethanol added), centrifuge at 12000 rpm for 20 seconds, discard the waste liquid in the collection tube, and repeat this step. Centrifuge at 12000 rpm for 2 minutes, discard the waste liquid, open the cap of the adsorption column and let it air dry at room temperature for 5 minutes. Place the adsorption column in a new RNase-free centrifuge tube, add 50 μl of RNase-Free Water to the middle of the adsorption column, let it stand at room temperature for 1 minute, centrifuge at 12000 rpm for 1 minute, and collect the RNA solution. Repeat this step again to detect the concentration of extracted RNA. The extracted RNA can be stored at -80℃ for a long time.

[0108] (3) RNA reverse transcription: RNA reverse transcription was performed using ReverTra Ace qPCR RT Master Mix. The RNA was denatured in a 65°C water bath for 5 minutes and then rapidly cooled on ice, as shown in Table 3.

[0109] Table 3. Genomic DNA Removal Reaction System

[0110] composition volume gDNA Remover (already added) 2μl RNA 0.5μg Nuclease-free Water Add to 8 μl Total 8μl

[0111] Mix all liquids thoroughly and let stand at 37°C for 5 minutes. The reverse transcription reaction system and procedure are shown in Tables 4 and 5.

[0112] Table 4 Reverse transcription reaction system

[0113] composition volume The above reaction mixture 8μl 5×RT Master Mix II 2μl Total 10μl

[0114] Table 5 PCR reaction procedure

[0115] Temperature (°C) Time (minutes) 37 15 50 5 98 5 4 Hold

[0116] After the reaction is complete, cDNA can be used as a template for real-time quantitative PCR detection experiments.

[0117] (4) Real-time fluorescence quantitative PCR, the reaction system and procedures are shown in Tables 6 and 7.

[0118] Table 6 PCR reaction system

[0119] Components volume cDNA 1μl Sybr Green Mixture (2×) 10μl Primer-F (10μM) 1μl Primer-R (10μM) 1μl RNase-free water 7μl Total 20μl

[0120] Table 7 Reaction procedures

[0121] <00​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​(2) Experimental Methods: The experiment included groups F, M, H3, L3, H4, and L4. Except for group F, which consisted of 5 males and 5 females, the other mice were subcutaneously injected with 20% CCl4 corn oil solution (10 ml / kg, twice a week) in their backs for 2 weeks to establish a mouse model of liver fibrosis. Fifty mice (25 males and 25 females) were randomly divided into groups M, H3, L3, H4, and L4, with 10 mice in each group (half males and half females). Intraperitoneal administration began in week 3. The experimental groups received compound A intraperitoneally, while the blank control group and model group received the excipient, once daily at 0.1 ml / 10 g. Groups L3 and H3 received the compound for 3 consecutive weeks, while the other groups received it for 4 consecutive weeks. The effects of the compound on liver pathology in the mouse model of liver fibrosis were investigated.

[0132] At the end of the experiment, mice in each group were sacrificed, and liver tissue of approximately 1cm × 0.5cm was cut off, fixed with 4% buffered paraformaldehyde, and pathological sections were prepared. HE and Masson staining were used to observe the pathological changes in the liver tissue of the mice.

[0133] (3) Experimental results: as attached Figure 1 As shown, compound A can improve the degree of liver fibrosis, steatosis, and ballooning degeneration in model mice, with more significant effects with increasing dose and longer administration time. Specifically, F: normal group; M: model group; L3: 1 mg / kg for 3 weeks; H3: 3 mg / kg for 3 weeks; L4: 1 mg / kg for 4 weeks; H4: 3 mg / kg for 4 weeks.

Claims

1. Compounds with structures as shown in Formula I: Formula I in, Compound A: R1 is H, R2 is OH; Compound B: R1 is OH, R2 is OH.

2. A Penicillium strain Penicillium pasqualense NCPC0626, with accession number CGMCCNo.23277.

3. The method for preparing the compound according to claim 1, characterized in that... The compound was derived from a Penicillium strain. Penicillium pasqualense Prepared by fermentation and extraction of NCPC0626 strain, the strain's preservation number is CGMCCNo.23277.

4. The preparation method according to claim 3, characterized in that... Includes the following steps: a. Preparation of seed culture for Penicillium strain NCPC0626; b. Preparation of fermentation culture of Penicillium strain NCPC0626; c. Extract the fermentation culture with an organic solvent, concentrate the extract under reduced pressure, remove the organic solvent, and obtain a crude extract; d. The crude extract is separated by chromatography to prepare compound I, including the following steps: 1) The crude extract obtained in step c was separated by silica gel column chromatography and eluted with a gradient of n-hexane / ethyl acetate to obtain PPARs active component a; 2) Component a was separated by Sephadex LH-20 column chromatography and eluted with methanol to obtain component a1; 3) Component a1 was separated using an ODS preparative chromatographic column and eluted with an acetonitrile / water gradient to obtain PPARs active components 3 and 5; 4) Component 3 was separated by Sephadex LH20 column elution and eluted with methanol to give compound B; 5) Component 5 was dried in the dark to obtain compound A.

5. The method for preparing the compound according to claim 4, characterized in that: In step d, 1), the solvents used for gradient elution are, in sequence: 1000 ml of n-hexane, 1000 ml of n-hexane-ethyl acetate (volume ratio 10:1), 1000 ml of n-hexane-ethyl acetate (volume ratio 4:1), 1000 ml of n-hexane-ethyl acetate (volume ratio 1:1), 1000 ml of ethyl acetate, and 1000 ml of ethanol. The detection wavelength is 333 nm, and the samples are collected according to peaks. The samples are evaporated to dryness under reduced pressure to obtain components a and b. The transcriptional activity of the compounds on PPARs is then detected, and component a is finally determined to be the active component. In step d, 2), the sample is detected at 333 nm, and the eluent is collected according to the peak to obtain component a1; In step d, 3), the acetonitrile / water gradient elution is performed by eluting with a linear concentration gradient of 20% to 100% acetonitrile over a period of 0 min to 20 min, detecting at 333 nm, collecting the components according to the peaks, and numbering them sequentially as components 1 to 9. The activity of each component is then measured to obtain PPARs active components 3 and 5.

6. The preparation method according to any one of claims 4-5, characterized in that: Step a is as follows: The slant culture or spore solution of Penicillium strain NCPC0626 is inoculated into a seed culture medium at a temperature of 22-30 ℃ and a rotation speed of 100-220 rpm for 48-72 h to obtain a seed solution. The seed culture medium formula is: 10.0-40.0 g corn starch, 10.0-40.0 g glucose, 4.0-20.0 g hot-pressed soybean meal, 1.0-6.0 g malt powder, 1.0-3.0 g yeast powder, 0.5-2.0 g sodium chloride, 0.5-2.0 g magnesium sulfate heptahydrate, and water is added to a final volume of 1000 mL, with a pH of 6.5-7.

0. Step b is as follows: the seed liquid obtained in step a is inoculated into a solid fermentation medium at an inoculation rate of 3-10%, the fermentation temperature is 22℃-30℃, and the fermentation time is 7-20 days to obtain a fermented culture; the solid fermentation medium includes a solid granular matrix, a powdered matrix, inorganic salts, and trace elements, which may or may not be added; the solid granular matrix is ​​rice, millet, wheat bran, and corn grits, and the powdered matrix is ​​hot-pressed soybean meal, cottonseed meal, and corn gluten meal; Step c is as follows: extract 1-3 times, 4-8 hours each time, and combine the extracts; the organic solvent is ethyl acetate or acetone.

7. The use of the compound of claim 1 in the preparation of a medicament for treating and / or preventing PPARs-mediated diseases, wherein the disease is non-alcoholic fatty liver disease.

8. A pharmaceutical composition comprising the compound of claim 1 and a pharmaceutically acceptable carrier, excipient, or excipient.

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