Natural heteroterpenoids and their uses in pharmaceuticals

Abstract

This invention belongs to the field of pharmaceutical technology and relates to heteroterpenoid compounds of formulas (I) and (II) isolated from plants of the Rhododendron genus and their medicinal uses. These compounds exhibit significant NF-κB transcriptional repressive activity, effectively inhibiting macrophage inflammatory responses and demonstrating significant protective effects against lipopolysaccharide and ischemia-reperfusion-induced acute kidney injury in mice. They can be used as drugs or lead compounds for the preparation of new drugs for the prevention and treatment of inflammation-related diseases.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology and relates to a pair of natural heteroterpene enantiomers isolated from plants of the Rhododendron genus and their use in the preparation of drugs for the prevention and treatment of inflammation-related diseases. Background Technology

[0002] Studies have shown that inflammatory diseases are ubiquitous in human life, posing serious threats to health and attracting increasing attention. Inflammation is a defensive response of the body to stimuli, often triggering various immune diseases such as cardiovascular, brain, respiratory, and digestive disorders. Metabolic diseases such as obesity, insulin resistance, type 2 diabetes, non-alcoholic fatty liver disease, and atherosclerosis can lead to persistent chronic inflammation. Furthermore, tissue damage caused by various factors can trigger intense inflammatory responses, such as acute kidney injury, acute liver injury, and acute lung injury.

[0003] In recent years, with the increasing global aging trend, accelerated industrialization leading to environmental degradation, the rapid spread of chronic diseases such as obesity and diabetes, and the rapid development of medical imaging, the incidence of acute kidney injury (AKI) has risen sharply and has quickly developed into a global public health problem. According to statistics, of the approximately 13 million people diagnosed with AKI globally each year, about 1.7 million die from AKI and its complications. Studies have shown that AKI is not only about damage to kidney tissue; it involves changes in renal homeostasis, with inflammatory responses occurring throughout the development of AKI, and various immune cells participating in kidney damage and repair. When renal tubular epithelial cells and endothelial cells secrete inflammatory factors, they can promote the activation of immune cells, including macrophages, thereby inducing an inflammatory response. In recent years, strategies for preventing and treating AKI by regulating the inflammatory response have received increasing attention, and clinical practice and animal experimental data have shown that anti-inflammatory drugs can effectively alleviate AKI.

[0004] Studies have shown that macrophages are the primary immune cells mediating inflammatory responses. Under different environmental stimuli, they can regulate the expression and secretion of inflammatory genes by altering their own number and phenotype or interacting with other cells in tissues. Macrophages are closely related to the development of autoimmune diseases, metabolic diseases, septic tissue damage, and acute kidney injury caused by various factors. In recent years, macrophages have become one of the important target cells for the prevention and treatment of inflammation-related diseases. Transcription factor NF-κB is an important component of the inflammatory signaling pathway. Sustained activation of NF-κB is closely related to many inflammatory diseases, and inhibiting NF-κB activation can effectively alleviate various inflammation-related diseases. Some studies suggest that downregulating NF-κB expression may be a potential strategy for treating septic tissue damage. In summary, inflammation is a fundamental pathological process in the body, a defensive response to external infection. Organ and tissue damage caused by various factors is accompanied by the occurrence and development of inflammation, and anti-inflammatory drugs can effectively prevent and treat these diseases. However, to date, no naturally derived small-molecule anti-inflammatory drugs with a specific disease as their primary indication have been marketed. Existing anti-inflammatory drugs are not suitable for treating multiple diseases and may even cause significant side effects. Therefore, developing safe, effective, and highly selective drugs to inhibit inflammatory responses is an important strategy for treating immune diseases, improving metabolic diseases, and treating tissue and organ damage. In recent years, it has been found that many natural drug active ingredients have shown varying degrees of anti-inflammatory effects in multiple inflammatory models, possessing advantages such as abundant sources, few adverse reactions, and long-lasting effects, demonstrating excellent prospects for application development.

[0005] Rhododendron is the largest genus in the Ericaceae family, and its species resources are very abundant in my country, with about 571 varieties, accounting for more than 50% of the world's total. Therefore, my country is an important distribution center of Rhododendron plants worldwide. Rhododendron plants have important medicinal value, and many have the effects of clearing heat and detoxifying, relieving cough and asthma. Heterpenoids are an important class of components in Rhododendron plants, and have activities such as inhibiting histamine release (Journal of Natural Products 2010, 73 (7): 1203), anti-HIV virus (Tetrahedron, 2001, 57 (8): 1559), and inhibiting protein tyrosine phosphatase 1B (Journal of Natural Products 2018, 81 (8): 1810).

[0006] Based on the current state of the technology, the inventors of this application intend to provide new natural heteroterpenoid compounds and their uses in pharmaceuticals, specifically involving the isolation of heteroterpenoid enantiomers from plants of the Rhododendron genus such as Rhododendron anthopogonoides Maxim. and their uses in pharmaceuticals. Summary of the Invention

[0007] The purpose of this invention is to provide new natural heteroterpenoid compounds and their uses in pharmaceuticals based on the current state of the prior art, specifically involving a pair of new heteroterpenoid enantiomers isolated from plants of the Rhododendron genus, particularly involving compounds with structures as shown in formula (I) (S,E)-2-(8-hydroxy-4,8-dimethyl-3-nonenyl)-2,7-dimethyl-2H-chromogenen-5-ol and compounds with structures as shown in formula (II) (R,E)-2-(8-hydroxy-4,8-dimethyl-3-nonenyl)-2,7-dimethyl-2H-chromogenen-5-ol.

[0008] A further object of the present invention is to provide pharmaceutical uses for the compounds. Experiments have demonstrated that the compounds of formulas (I) and (II) possess significant NF-κB transcriptional repressive activity, effectively inhibiting the inflammatory response of the RAW264.7 macrophage cell line; the compound of formula (I) can inhibit the transcriptional expression and secretion of inflammatory factors in mouse bone marrow-derived primary macrophages; and formula (I) has a significant protective effect against lipopolysaccharide (LPS) and ischemia / reperfusion (I / R)-induced acute kidney injury in mice. The compounds of formulas (I) and (II) can be used as drugs or lead compounds for the preparation of novel anti-inflammatory drugs for related diseases.

[0009]

[0010] The compound of the present invention is prepared by the following method:

[0011] The dried branches and leaves of Rhododendron species were used as raw materials. After pulverization, they were extracted with organic solvents and / or water, and concentrated under reduced pressure to obtain the total extract. The organic solvent used could be alcohols, such as ethanol or methanol, preferably 95% (v / v) ethanol. The total extract was dispersed in water and extracted with ethyl acetate. After solvent recovery under reduced pressure, the ethyl acetate extract was obtained and purified by macroporous resin HP-20 column chromatography, silica gel column chromatography, and ODS reversed-phase silica gel column chromatography. The racemic compound was then prepared by high performance liquid chromatography. Finally, the enantiomers (I) and (II) were separated by chiral chromatography. The structure of compound (I) was identified as (S,E)-2-(8-hydroxy-4,8-dimethyl-3-nonenyl)-2,7-dimethyl-2H-chromogenen-5-ol, and the structure of compound (II) was identified as (R,E)-2-(8-hydroxy-4,8-dimethyl-3-nonenyl)-2,7-dimethyl-2H-chromogenen-5-ol.

[0012] This invention tested the NF-κB transcriptional repressive activity of the compounds shown in formulas (I) and (II) and investigated their effects on macrophage inflammatory responses. The results showed that the compounds in formulas (I) and (II) exhibited significant NF-κB transcriptional repressive activity, effectively inhibiting the expression of inflammatory genes in the LPS-induced RAW264.7 macrophage cell line. Compound (I) inhibited the transcriptional expression and secretion of BMDM inflammatory genes in mouse bone marrow-derived monocytes / macrophages co-induced by LPS and interferon (IFNγ). Furthermore, compound (I) showed significant protective effects against LPS and I / R-induced acute kidney injury in mice. The compounds in formulas (I) and (II) of this invention can be used as drugs or lead compounds for the preparation of novel anti-inflammatory drugs for related diseases. Attached Figure Description

[0013] Figure 1 The effects of compounds (I) and (II) of the present invention in Example 3 on LPS-induced expression of inflammatory genes in RAW264.7 macrophages are shown, wherein a. compound (I) inhibits inflammatory gene expression; b. compound (II) inhibits inflammatory gene expression; and compared with the LPS-treated group, *P < 0.05, **P < 0.01, ***P < 0.001, &&& indicates that compared with the DMSO group, P < 0.001.

[0014] Figure 2 The effect of compound (I) of the present invention in Example 4 on the expression of inflammatory genes in BMDM macrophages co-induced by LPS and IFNγ is shown in the figure. Compared with the LPS+IFNγ treatment group, *P < 0.05, **P < 0.01, &&& indicates that compared with the DMSO group, P < 0.001.

[0015] Figure 3 The effect of compound (I) of the present invention in Example 4 on the secretion of inflammatory factors in BMDM macrophages induced by LPS and IFNγ, wherein, compared with the LPS+IFNγ treatment group, *P < 0.05, ***P < 0.001.

[0016] Figure 4 To protect LPS-induced acute kidney injury in mice with sepsis, compound (I) of the present invention in Example 5 was used. The following were measured: 1. Plasma LDH level (a), liver function indicators AST and ALT (b), and kidney function indicators Crea and BUN (c); 2. RT-QPCR detection of the expression of kidney injury molecules NGAL and KIM1 genes; 3. H&E staining of kidney tissue; 4. Immunohistochemistry detection of the expression of kidney injury marker NGAL (f), macrophage marker F4 / 80 (g), and neutrophil marker Ly6G (h). Compared with the model group, *P < 0.05, **P < 0.01, ***P < 0.001, & indicates P < 0.05 compared with the control group, &&& indicates P < 0.001.

[0017] Figure 5 To protect I / R-induced acute kidney injury in mice, compound (I) of the present invention in Example 6 was used for the following methods: a. Detection of plasma renal function indicators Crea and BUN using a biochemical analyzer; b. Detection of renal injury molecules NGAL and KIM1 gene expression by RT-QPCR; c. H&E staining of kidney tissue; df. Detection of renal injury marker NGAL (d), macrophage marker F4 / 80 (e), and neutrophil marker Ly6G (f) protein expression by immunohistochemistry; g. Detection of renal tissue cell apoptosis using a TUNEL assay kit. ** P < 0.01 compared to the model group, &&& indicates P < 0.01 compared to the control group. Detailed Implementation

[0018] The present invention will be further described below with reference to specific implementation examples, but this does not limit the present invention.

[0019] Example 1: Extraction of the compound of the present invention from Rhododendron simsii.

[0020] (1) Extraction: 10 kg of crushed dried branches and leaves of Rhododendron simsii were percolated with 100 L of 95% ethanol at room temperature and concentrated under reduced pressure to obtain 2.2 kg of extract. The extract was dispersed in water, extracted with ethyl acetate, and concentrated under reduced pressure to obtain 1.2 kg of extract.

[0021] (2) Separation: The ethyl acetate extract was separated by macroporous HP-20 column chromatography, eluting with a gradient of ethanol-water system (10:90→30:70→70:30→95:5), and the fractions were collected. The ethanol-water (70:30) fraction (350 g) was separated by silica gel column chromatography (100~200 mesh), eluting with a gradient of petroleum ether-ethyl acetate system (50:1→30:1→20:1→10:1→5:1), and the fractions were collected. The petroleum ether-ethyl acetate (10:1) fraction (10 g) was separated by ODS reversed-phase silica gel column chromatography, eluting with a gradient of methanol-water system (60:40→70:30→80:20→90:10). The methanol-water (70:30) fraction (1 g) was prepared by semi-preparative high performance liquid chromatography to obtain 250 mg of racemic compound, which was then separated by chiral chromatography to obtain 150 mg of compound (I), namely (S,E)-2-(8-hydroxy-4,8-dimethyl-3-nonenyl)-2,7-dimethyl-2H-chromogenen-5-ol, and 80 mg of compound (II), namely (R,E)-2-(8-hydroxy-4,8-dimethyl-3-nonenyl)-2,7-dimethyl-2H-chromogenen-5-ol.

[0022] The high-performance liquid chromatograph (HPLC) was a Shimadzu Essentia LC-16, with a Kromasil C18 column (150 × 10 mm, 5 μm), methanol-water (78:22) as the eluent, and a retention time of 15.0 min. The chiral column was a DAICELCHIRALPAK IF (250 mm × 10 mm, 5 μm), with n-hexane-isopropanol (92:8) as the eluent, and a retention time of 10.0 min. Detection wavelengths were 210 nm and 254 nm, and the flow rate was 3.0 mL·min⁻¹.

[0023] Compounds of formula (I) and (II) share the following common physicochemical properties and spectral data: Appearance: colorless oily substance; Molecular formula: C22H32O3; Molecular weight: 344; Maximum absorption wavelength in ultraviolet (methanol): 230 (4.35), 283 (3.87) nm; Maximum absorption frequency in infrared (potassium bromide): 3387, 2973, 2933, 2856, 1624, 1582, 1455, 1377, 1332, 1205, 1143, 1096, 1078, 991, 906, 826 cm⁻¹; High-resolution electrospray ionization mass spectrometry (mass-to-charge ratio): negative ion mode 343.2282 [MH]⁻ (indicating molecular formula C22H32O3); ¹H and ¹³C NMR spectra are shown in Table 1.

[0024] Furthermore, the specific rotation of compound (I) is: [α]25D +68.0 (c 0.15, methanol); the maximum absorption wavelength in circular dichroism (EMD) (methanol) is: 205 (-4.23), 221 (+2.07), 240 (-0.43), 280 (+3.27) nm; the specific rotation of compound (II) is: [α]25D -68.9 (c 0.09, methanol); the maximum absorption wavelength in EMD (methanol) is: 205 (+3.81), 220 (-2.38), 241 (+0.44), 280 (-3.29) nm.

[0025] Table 1 shows the 1H NMR data (400 MHz; chemical shift: ppm; coupling constant: Hz; solvent: deuterated chloroform) and 1C NMR data (150 MHz; chemical shift: ppm; solvent: deuterated chloroform) of compounds (I) and (II).

[0026] Table 1

[0027] serial number Hydrogen spectrum data Carbon spectrum data 2 78.3 3 5.48 (d, J = 10.0) 127.2 4 6.62 (d, J = 10.0) 117.0 4a 5 6 6.12 (br s) 7 8 6.22 (br s) 8a 9 1.64 (m) 1.74 (m) 10 2.11 (m) 11 5.11 (t, J = 6.8) 124.5 12 13 1.94 (m) 14 1.42 (m) 15 1.42 (m) 16 17 1.21 (s) 18 1.21 (s) 19 1.56 (s) 20 1.37 (s) 21 2.19 (s)

[0028] Example 2: Assay of the transcriptional repressive activity of compounds (I) and (II) of the present invention against NF-κB

[0029] 1. Construction of stable transgenic NF-κB luciferase reporter gene cells

[0030] (1) The HEK 293 cell line was obtained from Cell Bank and cultured in a carbon dioxide constant temperature incubator in High GlucoseDulbecco's modified Eagle's medium (HG-DMEM) complete medium containing 10% fetal bovine serum (FBS). When the cells reached 90% confluence, they were digested with trypsin and counted (2×105 / mL), then plated and allowed to adhere for 24 hours.

[0031] (2) Dilute the NF-κB luciferase reporter plasmid pcELAM-L with serum-free medium OPTI-MEMI to a concentration of 1.0 µg DNA / 50 µL and mix gently.

[0032] (3) Dilute 2 µL of Lipofectamine 2000 transfection reagent with 50 µL of OPTI-MEMI medium and mix gently.

[0033] (4) Mix the diluted DNA (step 2) and diluted Lipofectamine 2000 (step 3) and incubate at room temperature for 20 minutes.

[0034] (5) Aspirate the old culture medium from the 24-well plate, wash twice with phosphate-buffered saline (PBS), and add 0.5 mL of OPTI-MEMI medium.

[0035] (6) Add the complex directly to each well, shake the culture plate, and mix gently.

[0036] (7) After transfection at 37℃ and 5% CO2 for 48 h, the cells were passaged into fresh culture medium. After 48 h, 0.5 mg / mL of G418 was added for selection, and finally stable transgenic HEK 293 / NF-kB cells with high expression of NF-kB luciferase reporter gene were obtained.

[0037] 2. Assay of the transcriptional repressive activity of compounds (I) and (II) against NF-κB

[0038] HEK 293 / NF-κB cells (1×10⁴ / well / 50 µL) were seeded in 96-well cell culture plates and cultured for 24 h. Each well was stimulated with 50 µL of HG-DMEM medium containing tumor necrosis factor-α (TNFα) (20 ng / mL). Simultaneously, a 2 mM stock solution of the compound was prepared using dimethyl sulfoxide (DMSO), and the compound was serially diluted 1:3. 1 µL of each solution was added to the cell-containing culture plate. After 6 h of incubation, luciferase substrate was added, and after 30 min of incubation, the fluorescence signal intensity was detected using a microplate reader, and the IC50 was calculated. The IC50 of compound (I) was 11.94 ± 2.12 μM, and the IC50 of compound (II) was 12.72 ± 0.83 μM.

[0039] Example 3 Effects of compounds (I) and (II) of the present invention on the inflammatory response of RAW 264.7 macrophages

[0040] 1. Culture and compound treatment of RAW 264.7 macrophage cell line

[0041] RAW 264.7 macrophages were obtained from Cell Bank and cultured in HG-DMEM complete medium containing 10% FBS and 0.2% penicillin-streptomycin (PS) in a CO2 incubator. When the RAW264.7 cells reached 80%-90% confluence, they were passaged at a 1:5 ratio. RAW264.7 cells were seeded at 1.2 × 10⁵ cells / well in 48-well plates and cultured overnight. Treatment with LPS (100 ng / mL) alone or with LPS (100 ng / mL) and the compound for 24 h was performed, with a DMSO solvent control group included.

[0042] 2. Real-time quantitative PCR (RT-QPCR) experiment

[0043] (1) Total RNA extraction and cDNA reversal: Discard the culture medium and rinse with PBS. Add 200 µL of TRIzol reagent to each well and lyse on ice for 10-15 min. Transfer the lysate from the culture plate to a pre-chilled EP tube on ice, add 40 µL of RNAase-free chloroform, vortex for about 15 s to mix thoroughly, and then the layers will quickly separate. After standing for 2-3 min, centrifuge (conditions: 4°C, 12000 rpm, 15 min), carefully aspirate the clear supernatant to a new pre-chilled EP tube, then add 100 µL of RNAase-free isopropanol and mix thoroughly. Incubate at -20°C for 2 h or overnight to precipitate. Remove the precipitated RNA sample and centrifuge (conditions: 4°C, 12000 rpm, 15 min). After centrifugation, discard the liquid in the sample tube, add pre-chilled 70% ethanol to wash the obtained white precipitate and centrifuge (conditions:

[0044] (4°C, 7500 rpm, 5 min). After discarding the ice-cold ethanol, invert the sample tube on the table to allow the ethanol to evaporate, then add a certain volume of RNAase-free H2O to dissolve the RNA. Determine the RNA concentration using absorbance spectrometry. Construct the reverse transcription system according to the instructions provided in the reverse transcription kit. Take 1 µg of RNA sample to construct a 20 µL reverse transcription system, incubate at 37°C for 15 min, then inactivate by heating in an 85°C metal bath for 15 s. Dilute to 200 µL with double-distilled water (dd H2O) to obtain the cDNA sample for subsequent experiments.

[0045] (2) RT-QPCR experiment: 5 µL of cDNA sample was used as a template, and 0.5 µL of a pre-prepared 5 µM forward and reverse primer mixture and 4.5 µL of 2×SYBR Green QPCR Master Mix (Biotool) were added to a 384-well PCR plate to obtain a 10 µL reaction mixture. PCR amplification was then performed using a Stratagene Mx3005 (Agilent Technologies) instrument. The reaction conditions were: pre-denaturation (95°C, 5 min), denaturation reaction (95°C, 30 s), annealing (60°C, 30 s), extension (72°C, 30 s), for 40 cycles.

[0046] 3. Data Processing

[0047] All experimental data are expressed as mean ± standard deviation (mean ± SEM), and the difference between the two groups was tested using the two-waystudents' t-test.

[0048] like Figure 1 As shown in Figure a, LPS can stimulate the inflammatory response in RAW264.7 cells, and compound (I) can significantly reduce the transcriptional expression of pro-inflammatory factors such as IL-1β, IL-6, TNF-α, and COX-2 induced by LPS; Figure 1 As shown in b, compound (II) can reduce the transcriptional expression of pro-inflammatory factors such as IL 1β and COX 2.

[0049] Example 4 Effect of compound (I) of the present invention on the inflammatory response of BMDM macrophages

[0050] 1. Isolation and culture of primary mouse bone marrow-derived macrophages

[0051] Mouse bone marrow-derived mononuclear macrophages (BMDMs) were isolated and obtained from male C57BL / 6J mice aged 10-16 weeks. Mice were euthanized by cervical dislocation and immersed in 75% ethanol for 5 min. The hind leg bones were then immediately separated and transferred to a clean bench. The bones were washed sequentially with PBS-PBS-complete culture medium. The leg bones were separated from the joint using scissors and ophthalmic forceps. The bone marrow was expelled using a 2 mL sterile syringe and dispersed into single cells in a culture dish. The single-cell suspension was collected and centrifuged (500 g, 5 min). The culture medium was discarded, and the cells were resuspended in erythrocyte lysis buffer. After lysis for 5 min, culture medium was added to stop the lysis, and the cells were centrifuged again (500 g, 5 min). The liquid was discarded, and the cells were resuspended in fresh culture medium and counted. Cells were seeded into 24-well plates at a density of 1 × 10^6 / mL. The culture medium and conditions used were the same as for RAW 264.7 cells. In addition, 20 ng / mL of macrophage colony-stimulating factor (M-CSF) was added during the culture process, and the culture medium was changed every 2-3 days. Mature M0 macrophages were obtained on the seventh day. Subsequently, the cells were treated with LPS (50 ng / mL) + INF-γ (125 ng / mL) alone or with LPS (50 ng / mL) + INF-γ (125 ng / mL) and the compound for 24 h. A DMSO solvent control group was included.

[0052] 2. Detection of inflammation-related cytokine levels in cell culture medium

[0053] Cell culture supernatants from different treatment conditions were collected and used directly for detection or frozen at -80°C for later use. The levels of IL-6 and TNF-α in the culture supernatants were detected using homogeneous time-resolved fluorescence (HTRF) technology. After centrifugation (5000 rpm, 3 min) of the culture supernatants, the levels of IL-6 and TNF-α in the culture supernatants were detected using IL-6 and TNF-α kits, respectively. The procedures were performed strictly according to the instructions provided with each kit. Taking TNF-α as an example, the specific experimental method is as follows:

[0054] (1) Preparation of working solution: Remove the kit from the -80°C container, thaw on ice, and centrifuge rapidly. Dilute the 20×TNF-α Eu Cryptate antibody (hereinafter referred to as antibody 1) and TNF-α d2 antibody (hereinafter referred to as antibody 2) in the kit with the detection solution. Then mix the two antibodies and aliquot them to prepare the working solution for later use. Add 300 µL of distilled water to the TNF-α standard to obtain the standard stock solution (concentration of 18 ng / mL) for later use.

[0055] (2) Preparation of standard curve: Take eight 1.5 mL EP tubes and label them 0-7#. Take 60 µL of stock solution and add it to tube #7, then add 120 µL of diluent and mix well. Take 100 µL of liquid from tube #7 and add 130 µL of diluent and mix well to obtain standard #6. Dilute the standard #5, #4, #3, #2, and #1 in the same way. Take 130 µL of diluent as standard #0, which is the blank control.

[0056] (3) Add 10 µL of standard and sample to the 384-well white plate in sequence, followed by 2 µL of premixed antibody working solution, centrifuge quickly, cover with sealing film, incubate at room temperature for 1 h, discard sealing film, read the microplate reader (detection wavelengths are 665 nm and 620 nm) and calculate the sample concentration.

[0057] 3. Real-time quantitative PCR (RT-QPCR) experiment

[0058] RNA was extracted with an appropriate amount of TRIzol, and the expression of inflammation-related genes was detected by RT-qPCR. The specific experimental methods and data processing methods are the same as those in parts 2 and 3 of "Example 3".

[0059] like Figure 2 As shown, LPS + INF-γ can stimulate the inflammatory response of BMDM cells, and compound (I) can significantly reduce the transcriptional expression of pro-inflammatory factors such as IL-1β, IL-6, TNF-α, CD14 and COX2 induced by LPS + INF-γ.

[0060] like Figure 3 As shown, LPS + INF-γ can stimulate the inflammatory response of BMDM cells, and compound (I) can significantly reduce the secretion of inflammatory factors TNF-α and IL-6 induced by LPS + INF-γ.

[0061] In summary, compounds (I) and (II) of the present invention have significant anti-inflammatory effects and can be used as new drugs or lead compounds for the prevention or treatment of inflammation-related diseases.

[0062] Example 5: Compound (I) of the present invention protects against LPS-induced septic kidney injury.

[0063] 1. Preparation of compounds

[0064] Compound (I) was dissolved in DMSO to obtain a 60 mg / mL stock solution of compound (I), which was then aliquoted for later use. Before the experiment, the stock solution of compound (I) was removed, and an appropriate volume of 0.5% hydroxypropyl methylcellulose (HPMC) was added to prepare a dosage concentration of 30 mg / kg for subsequent animal experiments.

[0065] 2. Establishment of an LPS-induced sepsis-induced kidney injury model

[0066] Ten-week-old C57BL / 6J mice were randomly divided into a control group (Sham), an LPS (10 mg / kg) group, and a drug treatment group (30 mg / kg), with eight mice in each group. The drug treatment group was intraperitoneally injected with pre-administered compound (I) for 3 days, while the control group and LPS group were intraperitoneally injected with the solvent HPMC. One hour after drug administration on the fourth day, the LPS group and the drug treatment group were intraperitoneally injected with LPS, while the control group was intraperitoneally injected with physiological saline. Blood was collected from the orbital cavity 24 hours later, and the kidneys were fixed and cryopreserved.

[0067] 3. Evaluation of plasma liver and kidney function

[0068] Blood was collected from the orbital cavity into anticoagulant tubes containing EDTA. After mixing, the blood was allowed to stand at room temperature for 20 min, then centrifuged (4℃, 1000 rpm, 5 min). The supernatant was collected and diluted 4 times with physiological saline for later use. Plasma lactate dehydrogenase (LDH) levels were measured using an automated biochemical analyzer to evaluate LPS-induced tissue damage; aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels were measured to evaluate liver function; and creatinine (Crea) and blood urea nitrogen (BUN) levels were measured to evaluate kidney function.

[0069] 4. The transcriptional expression of neutrophil gelatinase-associated lipocalin (NGAL) and kidney injury molecule (KIM1), markers of kidney injury, was detected using RT-QPCR. The specific experimental methods are as follows:

[0070] (1) Total RNA extraction and cDNA reversal: Weigh an appropriate amount of kidney tissue (30-50 mg) and place it into a pre-chilled EP tube on ice. Add 3-4 tissue homogenizing beads (3 mm in diameter) to each tube, add 500 µL of TRIzol reagent, and homogenize using a tissue homogenizer (homogenization conditions: 75 Hz, 3 min, 5 s interval). Repeat homogenization twice. After homogenization, add 500 µL of TRIzol and lyse on ice for 10-15 min. Then add 200 µL of RNAase-free chloroform and vortex for about 15 s to mix thoroughly. After standing for 2-3 min, centrifuge (conditions: 4°C, 12000 rpm, 15 min). Carefully aspirate the supernatant to a new pre-chilled EP tube, then add 500 µL of RNAase-free isopropanol and mix thoroughly. Incubate at -20°C for 2 h or overnight to precipitate. Remove the precipitated RNA sample and centrifuge (4°C, 12000 rpm, 15 min). After centrifugation, discard the liquid in the sample tube, add pre-cooled 70% ethanol to wash the resulting white precipitate, and centrifuge again (4°C, 7500 rpm, 5 min). Discard the ice-cold ethanol, invert the sample tube on a table to allow the ethanol to dry, then add a certain volume of RNAase-free H2O to dissolve the RNA. Determine the RNA concentration using absorbance spectrophotometry, and construct the reverse transcription system according to the instructions provided in the reverse transcription kit for subsequent operations. Take 1 µg of RNA sample to construct a 20 µL reverse transcription system, incubate at 37°C for 15 min, then inactivate by heating in an 85°C metal bath for 15 s, and dilute to 200 µL with ddH2O to obtain the cDNA sample for subsequent experiments.

[0071] (2) RT-QPCR experiment: The specific experimental method is the same as part 2(2) in “Example 3”.

[0072] 5. Hematoxylin-eosin (H&E) staining was used to examine the pathological damage to the kidneys.

[0073] After blood was collected from the mouse orbital cavity, kidney tissue was quickly freed and fixed with 4% paraformaldehyde (PFA) for 24 hours. The fixation was then replaced with 70% ethanol the next day for long-term preservation. Subsequent paraffin block preparation, embedding, sectioning, and H&E staining were performed. All kidney pathology images were captured and archived under identical settings using a Leica DM6B microscope equipped with an sCMOS camera.

[0074] 6. Immunohistochemical staining was used to investigate the expression of the kidney injury marker NGAL, the macrophage marker F4 / 80, and the neutrophil marker Ly6G protein.

[0075] The obtained tissue sections were stained. The antibody dilution ratios used in the experiment were F4 / 80 antibody (1:200), Ly6G antibody (1:200), and NGAL antibody (1:1000). The secondary antibody dilution ratio linked to DyLight 488 or DyLight 555 was (1:500). All section images were scanned using a Pannoramic MIDI (3D HISTECH, Hungary) scanner. The expression of NGAL, F4 / 80, and Ly6G in the images was quantitatively analyzed using Image-Pro Plus software or counting methods. The specific operation steps are as follows:

[0076] (1) Dewaxing and hydration: The tissue was placed in xylene I and II for 15 min each, ethanol I and II for 5 min each, and then washed with PBS once for 5 min each time.

[0077] (2) Antigen retrieval: Boil the antigen retrieval solution in a microwave oven on high heat, immerse the tissue slices in the solution, then microwave on low heat for 15 minutes. Remove the slices and allow them to cool naturally to room temperature (to prevent the tissue from drying out). Wash twice with PBS for 5 minutes each time.

[0078] (3) After washing with PBS, the tissue was immersed in 2% Sudan Black dye (solvent is 70% ethanol aqueous solution) for 10 min.

[0079] (4) Rinse with running water for 10 minutes, wipe the liquid around the tissue dry with paper, and circle the tissue with an immunohistochemistry pen.

[0080] (5) Blocking: Block with 5% bovine serum albumin solution (BSA) at room temperature for 1 hour.

[0081] (6) Primary antibody incubation: Add 50 µL of diluted primary antibody to the tissue in the circle and incubate at room temperature for 1 h. Wash 3 times with PBS, 5 min each time.

[0082] (7) Secondary antibody incubation: Add 50 µL of diluted secondary antibody to the tissue in the circle, incubate at room temperature for 1 h, and rinse 3 times with PBS for 5 min each time.

[0083] (8) Hoechst incubation: Add 50 µL of diluted Hoechst staining solution to the tissue in the circle, incubate in the dark for 10 min, and rinse once with PBS for 5 min each time.

[0084] (9) Mounting: Cover the tissue with an anti-fluorescence quencher, cover with a glass slide, moisten the cover slide with 50% glycerin to prevent air bubbles, and seal the edges with nail polish. Scan and photograph the images, and evaluate and quantify them.

[0085] 7. Data Processing

[0086] All experimental data are expressed as mean ± standard deviation (mean ± SEM), and the difference between the two groups was tested using the two-ways students' t-test.

[0087] like Figure 4 As shown, compound (I) can reduce LPS-induced plasma LDH levels. Figure 4 a) Reduces plasma AST and ALT levels, thereby improving LPS-induced liver function damage ( Figure 4 b); LPS significantly increased plasma levels of Crea and BUN, indicating a marked abnormality in renal function and a decrease in glomerular filtration rate. Administration of compound (I) significantly inhibited the levels of Crea and BUN. Figure 4 c), indicating that the compound can effectively improve renal function; compound (1) can significantly inhibit the transcriptional expression of NGAL and KIM1, suggesting that compound (I) has a protective effect against renal injury. Figure 4 d) H&E staining was used to observe the pathological damage of kidney tissue. In septic mice, inflammatory cell infiltration, a small number of tubular casts and cell necrosis were observed in the kidney tissue, but no obvious damage was seen to the glomeruli and Bowman's capsule structures. Compound (I) can alleviate the pathological damage to the kidneys caused by LPS. Figure 4 e); Immunohistochemistry was used to detect the expression of NGAL in kidney tissue. Compound (I) could alleviate LPS-induced high expression of NGAL. Figure 4 f); Immunohistochemical results of F4 / 80 showed that administration of compound (I) significantly reduced the proportion of F4 / 80 positive cells. Figure 4 g), indicating that compound (I) can resist LPS-induced recruitment and infiltration of macrophages by the kidneys; Ly6G is a marker protein for neutrophil infiltration, and immunohistochemical results showed that compound (I) could significantly reduce the number of Ly6G-positive cells (g). Figure 4 h), indicating that compound (I) can inhibit the infiltration of neutrophils in kidney tissue.

[0088] Therefore, the compound (I) of this invention has a significant protective effect against LPS-induced septic kidney injury and can be used as a new drug or lead compound for the prevention or treatment of inflammatory tissue damage.

[0089] Example 6: Compound (I) of the present invention protects against ischemia-reperfusion (I / R) induced acute kidney injury.

[0090] 1. Establishment of an I / R-induced kidney injury model

[0091] Ten-week-old C57BL / 6J mice were randomly divided into a control group (Sham), an I / R group, and a treatment group (30 mg / kg), with eight mice in each group. The treatment group received intraperitoneal injection of pre-administered compound (I) for 3 days, while the control group and the I / R group received intraperitoneal injection of the solvent HPMC. On the fourth day, one hour after the intraperitoneal injection of compound (I), the treatment group and the I / R group underwent renal ischemia-reperfusion surgery. The specific procedures are as follows:

[0092] (1) After anesthetizing the animal with sodium pentobarbital, make 1.5-2.0 cm incisions on both sides of the back. After cutting the outer skin with ophthalmic scissors, separate the skin layer by layer with ophthalmic forceps. After finding the renal pedicle, quickly block the blood flow of the left and right renal pedicles with experimental miniature arterial clamps. The kidneys will turn from bright red to purplish-black, indicating that the clamping was successful.

[0093] (2) After clamping for 30 minutes, the clamps were removed, and blood perfusion was restored. The kidneys were observed to rapidly change from purplish-black to bright red, returning to their original color. The abdominal cavity was then closed by layered sutures. Postoperatively, the mice were kept warm at 24-29℃ and given water and food. Physiological saline was used during the operation to keep the mice adequately hydrated.

[0094] (3) Blood was collected from the orbital cavity 24 hours after reperfusion, and the kidney was cryopreserved and fixed.

[0095] 2. TUNEL staining of kidney tissue

[0096] (1) Dewaxing and hydration: The tissue was placed in xylene I and II for 15 min each, ethanol I and II for 5 min each, and then washed with PBS once for 5 min each time.

[0097] (2) Permeability: Accurately weigh and prepare Proteinase K stock solution with a concentration of 200 µg / mL; dilute the stock solution with PBS at a ratio of 1:9 to prepare a working solution with a final concentration of 20 µg / mL. Then add 100 µL of the above Proteinase K working solution to each sample to completely cover the tissue, incubate at 37°C for 20 min, and wash three times with PBS for 5 min each time.

[0098] (3) Equilibration: Add 50 µL of Equilibration Buffer to each sample to completely cover the tissue, and incubate at room temperature for 20 min.

[0099] (4) Staining and labeling: Prepare TdT incubation buffer according to the ratio of Recombinant TdT enzyme (1 µL): FITC-12-dUTP Labeling Mix (5 µL): Equilibration Buffer (50 µL); absorb the Equilibration Buffer with absorbent paper; add 56 µL of TdT incubation buffer to each tissue sample and incubate at 37℃ for 1 h, being careful not to let the slide dry during incubation; wash 3 times with PBS for 5 min each time.

[0100] (5) Hoechst incubation: Add 50 µL of diluted Hoechst staining solution to the tissue (area 1cm×1cm), incubate in the dark for 10 min, and rinse once with PBS for 5 min each time.

[0101] (6) Covering the slide: Cover the tissue with an anti-fluorescence quencher, cover with a glass slide, moisten the cover with 50% glycerin to prevent air bubbles, and seal the edges with nail polish.

[0102] 3. The preparation of compounds, evaluation of renal function, RT-QPCR experiments, H&E staining, immunohistochemistry experiments, and data processing were all the same as in Example 5.

[0103] like Figure 5 As shown, compound (I) can inhibit the levels of Crea and BUN in plasma. Figure 5 (a) indicates that the compound can effectively improve renal function; compound (I) can significantly inhibit the transcriptional expression of NGAL and KIM1, suggesting that compound (I) has a protective effect against renal injury. Figure 5 b) H&E staining was used to observe the pathological damage of kidney tissue. I / R caused a large amount of renal tubular cell shedding. After administration of compound (I), the pathological damage caused by I / R was improved, and cell apoptosis and shedding were reduced. Figure 5 c); Immunohistochemistry was used to detect the expression of NGAL in kidney tissue. Compound (I) could reduce the high expression of NGAL induced by I / R. Figure 5 d); Immunohistochemical results of F4 / 80 cells showed that administration of compound (I) significantly reduced the proportion of F4 / 80 positive cells. Figure 5 e), indicating that compound (I) can resist I / R-induced recruitment and infiltration of macrophages by the kidney; immunohistochemical results showed that compound (I) could significantly reduce the number of Ly6G positive cells ( Figure 5 f), indicating that compound (I) can inhibit the infiltration of neutrophils in kidney tissue. Furthermore, TUNEL staining was used to examine kidney tissue cell apoptosis; compound (1) significantly reduced the number of TUNEL-positive cells. Figure 5g), further suggesting that compound (I) has a protective effect against kidney damage.

[0104] The compound (I) of this invention has a significant protective effect against I / R-induced acute kidney injury and can be used as a new drug or lead compound for the prevention or treatment of kidney injury.

Claims

1. Natural heteroterpenoids with structures of formula (I) and formula (II), ； The compound is a pair of heteroterpene enantiomers, wherein... The compound of formula (I) is (S,E)-2-(8-hydroxy-4,8-dimethyl-3-nonenyl)-2,7-dimethyl-2H-chromogenen-5-ol; the compound of formula (II) is (R,E)-2-(8-hydroxy-4,8-dimethyl-3-nonenyl)-2,7-dimethyl-2H-chromogenen-5-ol; both have the molecular formula C 22 H 32 O3; molecular weight is 344.

2. Use of the compounds of formula (I) and formula (II) according to claim 1 in the preparation of anti-inflammatory drugs.

3. Use of the compounds of formula (I) and formula (II) according to claim 1 in the preparation of medicaments for the prevention or treatment of acute kidney injury.

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