Use of a class of sesquiterpene polyketide compounds as immunomodulators in preventing and treating immune diseases

Abstract

The present invention relates to a class of sesquiterpene polyketone compounds, pharmaceutical compositions and their use as immunomodulators and in preventing and treating immune diseases. Biological activity experiments show that the sesquiterpene polyketone compounds of the present invention can be used as immunomodulators for the prevention and / or treatment of immune-related diseases.

Description

Technical Field

[0001] The invention belongs to the field of natural medicines, and specifically relates to the use of a class of sesquiterpene polyketide compounds as immunomodulators in the preparation of medicines for preventing or treating immune-related diseases. Background Art

[0002] The balance of the immune system is essential to good health. When it is disrupted, it is easy to suffer from immune diseases. Tumors and infections are related to low immunity. Organ transplant rejection, allergies and autoimmune diseases are related to excessive immune response. [1] Among them, autoimmune diseases include more than 100 diseases such as inflammatory bowel disease (IBD), multiple sclerosis (MS) and rheumatoid arthritis (RA). They are severe, have a high incidence rate, a long disease cycle, and have no cure, which seriously endangers human health. Immunomodulators can be used to treat immune diseases caused by immune dysfunction by regulating the body's immune response (enhancement, inhibition or bidirectional regulation). For diseases related to low immunity such as tumors and infections, regulators that enhance the body's immunity can be used for intervention. For diseases related to excessive immune response of the body such as organ transplant rejection, allergies and autoimmune diseases, immunosuppressants are currently mainly used to improve disease symptoms and prognosis.

[0003] Chemical drugs such as corticosteroids, methotrexate, and cyclosporine A are non-specific immunosuppressants with serious toxic side effects when used for a long time; biological agents are expensive, the therapeutic effects vary greatly from person to person, and they increase the risk of infection and tumors in patients. Therefore, the development of new immunosuppressants to prevent and treat diseases related to excessive immune responses is still receiving attention from all over the world. In recent years, we have made important progress in the study of the pathogenic mechanisms of immune diseases. Inhibiting the expression of the pro-inflammatory factor IL17, promoting the expression of the anti-inflammatory factor IL10, and inhibiting the production of pro-inflammatory factors by DC cells have become effective strategies for inhibiting immune diseases related to excessive immune responses, which provides new ideas for the development of new immunosuppressants.

[0004] CD4 + T cells differentiate into Th17 cells with the participation of TGFβ and IL6. IL17 is a strong inflammatory cytokine mainly secreted by Th17 cells, which mediates a variety of inflammatory and autoimmune diseases. [2]. There is increasing evidence that IL17 plays a key role in the pathogenesis of inflammatory bowel disease, type 1 diabetes, multiple sclerosis, rheumatoid arthritis, psoriasis, systemic lupus erythematosus, asthma, Sjögren's syndrome and ankylosing spondylitis, and is associated with uveitis, autoimmune nephritis, autoimmune thyroiditis, Behcet's disease, lichen planus, respiratory diseases (including non-eosinophilic asthma, steroid-resistant asthma and cystic fibrosis), liver diseases (including drug-induced liver injury, alcoholic liver disease, non-alcoholic fatty liver disease, viral hepatitis, hepatocellular carcinoma, autoimmune hepatitis, primary biliary cirrhosis and primary sclerosing cholangitis) and cardiovascular diseases (including atherosclerosis, myocardial infarction, aneurysm and ischemic stroke). The clinical use of IL17 inhibitors for the treatment of various immune diseases such as psoriasis, arthritis and multiple sclerosis has shown certain efficacy. [3] .

[0005] IL10 is a type of cytokine with immunosuppressive and broad anti-inflammatory properties. IL10 can regulate the activity of a variety of myeloid or lymphoid cells, inhibiting their production of pro-inflammatory factors, thereby inhibiting organ transplant rejection, allergies, inflammation, and autoimmune diseases. [4] Type I regulatory T cells (Tr1) secrete a large amount of IL10 and are a type of cell with immunosuppressive effects. Currently, clinical trials of transferring Tr1 cells to prevent and treat autoimmune diseases or protect organ transplant immune rejection have shown efficacy. [5] .

[0006] Therefore, compounds that inhibit IL17 and / or promote IL10 can be used as immunomodulators to prevent and treat organ transplant rejection, allergies, inflammation and autoimmune diseases (inflammatory bowel disease, type 1 diabetes, multiple sclerosis, rheumatoid arthritis, psoriasis, systemic lupus erythematosus, asthma, Sjögren's syndrome and ankylosing spondylitis, uveitis, autoimmune nephritis, autoimmune thyroiditis, Behcet's disease, lichen planus, various respiratory diseases, various liver diseases and various cardiovascular diseases, etc.). In addition, compounds that inhibit IL17 and / or promote IL10 can also be used as immunomodulators for the treatment of inflammation-related diseases such as acne, herniated disc, and celiac disease. IL17 is also closely related to the occurrence of tumors, especially inflammation-related tumors. IL10 has a bidirectional regulatory effect on the occurrence and development of tumors. IL10 can inhibit the production of inflammatory factors that promote the occurrence and development of tumors and activate tumor-specific CD8 + T to exert anti-tumor effect [4] Therefore, compounds that inhibit IL17 and / or promote IL10 can also be used as immunomodulators for tumor treatment.

[0007] Dendritic cells (DC) are antigen presenting cells that can present antigens to T cells, provide a second signal for T cell activation, and can also induce T cell polarization by secreting cytokines IL6, TNFα, and IL12. However, when DC cell-mediated immune responses are overactivated, they can promote the occurrence of inflammatory cytokine storms and even sepsis, and mediate immune diseases such as organ transplant rejection, inflammation, and autoimmune diseases. [6] Therefore, under antigen stimulation, compounds that inhibit DC cells from secreting inflammatory factors such as IL6, TNFα, IL12, etc. can be used to treat inflammation and autoimmune diseases, sepsis, and organ transplant rejection. For pharmacodynamic experiments on inhibiting DC immune responses, mouse bone marrow-derived dendritic cells (BMDC) are often used.

[0008] Th2 cells mainly secrete cytokine IL4. IL4 has multiple biological functions and plays an important role in humoral immunity and acquired immunity. [7] IL4 can induce the activation of B cells and T cells and the differentiation of B cells into plasma cells. In addition, IL4 can inhibit lymphocyte apoptosis, promote the antigen presentation ability of macrophages and enhance the function of killing tumor cells. Therefore, compounds that promote IL4 can be used as immunomodulators for anti-tumor and anti-infection (especially anti-parasitic infection) related diseases. Summary of the invention

[0009] The purpose of the present invention is to provide a class of sesquiterpene polyketide compounds, pharmaceutical compositions and uses thereof. Specifically, the inventors discovered, isolated and identified a class of sesquiterpene polyketide compounds from a strain of Myrocera, which has been experimentally proven to have immunomodulatory activity and can be used to prevent and treat immune diseases, especially autoimmune diseases such as inflammatory bowel disease, multiple sclerosis, psoriasis and sepsis.

[0010] The first aspect of the present invention provides a class of sesquiterpene polyketide compounds or pharmaceutically acceptable salts thereof as immunomodulators, wherein the sesquiterpene polyketide compounds are represented by formula (I):

[0011]

[0012] Wherein, R1 is selected from: hydroxyl, acetoxy, or =O;

[0013] R2 is selected from: methyl, hydroxymethyl, acetylhydroxymethyl, aldehyde, or dimethoxymethyl;

[0014] R3 is selected from: methyl, hydroxymethyl, acetylhydroxymethyl, or aldehyde;

[0015] Or R2, R3 and the benzene ring form a 5-membered lactam ring or lactone ring;

[0016] R4, R5, R6, and R7 are carbon atoms, and any adjacent groups are selected from double bonds, and the remaining groups are selected from single bonds.

[0017] Furthermore, the pharmaceutically acceptable salt is a salt formed by the compound of formula (I) and an organic base or an inorganic base.

[0018] Furthermore, the formed salt is a sodium salt, a potassium salt, a calcium salt, a ferric salt, a magnesium salt, a zinc salt, an aluminum salt, a barium salt or an ammonium salt.

[0019] Preferably, the sesquiterpene polyketone compound is selected from the compounds of formula II to formula XII, and the specific structural formula is as follows:

[0020]

[0021] The second aspect of the present invention provides a method for preparing the above compound, comprising: fermenting the compound with a microorganism that produces a sesquiterpene polyketide compound as shown in formula (I), and then separating the compound by chromatography to obtain the compound.

[0022] Furthermore, the microorganism is a fungus of the genus Mycorrhiza.

[0023] Further optionally, the Mycorrhiza fungus is ZLW0801-19, and its preservation number is CGMCC No.19039.

[0024] The third aspect of the present invention provides a pharmaceutical composition for immunomodulation, comprising the above-mentioned sesquiterpene polyketide compound or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

[0025] Further optionally, the salt is any one of sodium salt, potassium salt, calcium salt, iron salt, magnesium salt, zinc salt, aluminum salt, barium salt and ammonium salt.

[0026] Further optionally, in one embodiment, the amount of the active ingredient (i.e., the compound of the present invention) contained in the pharmaceutical composition can be specifically used according to the patient's condition and the doctor's diagnosis, and the amount or concentration of the active compound can be adjusted within a wider range. The content of the compound of formula (I) or a pharmaceutically acceptable salt thereof is 1-90% by weight of the composition.

[0027] Further optionally, the pharmaceutically acceptable carrier includes diluents, lubricants, binders, disintegrants, stabilizers, solvents, etc. The diluents of the present invention include but are not limited to starch, microcrystalline cellulose, sucrose, dextrin, lactose, powdered sugar, glucose, etc.; the lubricants include but are not limited to magnesium stearate, stearic acid, sodium chloride, sodium oleate, sodium lauryl sulfate, poloxam, etc.; the binders include but are not limited to water, ethanol, starch slurry, syrup, hydroxypropyl methylcellulose, sodium carboxymethyl cellulose, sodium alginate, polyvinyl pyrrolidone, etc.; the disintegrants include but are not limited to starch effervescent mixtures, i.e. sodium bicarbonate and citric acid, tartaric acid, low-substituted hydroxypropyl cellulose, etc.; the stabilizers include but are not limited to polysaccharides such as acacia gum, agar, alginic acid, cellulose ethers and carboxymethyl chitosan, etc.; the solvents include but are not limited to water, balanced salt solutions, etc.

[0028] Further optionally, the pharmaceutical composition is an oral preparation or an injection; preferably, the oral preparation includes but is not limited to any one of ordinary tablets, dispersible tablets, enteric-coated tablets, granules, capsules, pills, powders, oral liquids or emulsions; preferably, the injection is selected from any one of small water injections, infusions or lyophilized powder injections.

[0029] The fourth aspect of the present invention also provides a compound of formula (I) or a pharmaceutically acceptable salt thereof for preparing an immunomodulator.

[0030] Furthermore, the immunomodulator is used for the prevention and / or treatment of immune-related diseases.

[0031] Furthermore, the immunomodulator is used to prevent and treat diseases or indications for which inhibition of IL17 and / or promotion of IL10 is beneficial.

[0032] Preferably, the disease or indication is selected from psoriasis, inflammatory bowel disease, type I diabetes, multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, asthma, Sjögren's syndrome, ankylosing spondylitis, organ transplant rejection, allergy, sepsis, uveitis, autoimmune nephritis, autoimmune thyroiditis, Behcet's disease, autoimmune liver disease, lichen planus, acne, herniated disc, and celiac disease.

[0033] Furthermore, the immunomodulator is used for diseases or indications where promoting IL4 is beneficial.

[0034] Preferably, the disease or indication is related to parasitic infection.

[0035] Furthermore, the immunomodulator is used to prevent and treat diseases or indications for which suppression of DC immune response is beneficial.

[0036] Preferably, the disease or indication is inflammatory and autoimmune disease, sepsis, organ transplant rejection.

[0037] Beneficial Effects

[0038] The sesquiterpene polyketide compounds of the present invention are derived from microbial fermentation, and are convenient for large-scale fermentation and industrial preparation. Through cell and animal activity tests, it is found for the first time that the compounds have significant immunomodulatory activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 It is shown that the compound of formula (III) significantly inhibits the expression of human IL17.

[0040] Figure 2 The compound of formula (III) inhibited the expression of IL12 / IL23 p40 in BMDC stimulated by LPS. + IL6 + and TNFα + The proportion of cells.

[0041] Figure 3 It is shown that the compound of formula (III) significantly inhibited the weight loss of enteritis mice ( Figure 3 A) Increased DAI score ( Figure 3 B) Shortened colon ( Figure 3 C); Colon tissue pathological analysis showed that the compound of formula (III) significantly inhibited the extent of colon inflammation, epithelial damage and intestinal structural lesions ( Figure 3 D and 3E).

[0042] Figure 4 It is shown that the compound of formula (III) significantly reduced the EAE score of EAE model mice ( Figure 4 A); Spinal cord tissue pathological analysis showed that treatment with the compound of formula (III) significantly inhibited the infiltration of inflammatory cells and the degree of demyelination in the spinal cord ( Figure 4 B).

[0043] Figure 5 As shown: Compared with the model group, the compound of formula (III) significantly inhibited the thickening of the ears of psoriasis-like mice ( Figure 5 A and 5B); The results of ear tissue pathological analysis showed that the treatment with the compound of formula (III) significantly inhibited the pathological damage of incomplete keratinization, hyperkeratosis, Munro body formation, acanthosis, inflammatory cell infiltration and dermal vascular dilation and congestion in the ear skin of psoriasis-like mice ( Figure 5 C and 5D).

[0044] Figure 6 It is shown that the compound of formula (III) significantly improved the survival rate of mice with LPS-induced sepsis.

[0045] Figure 7It is shown that the compound of formula (III) significantly inhibited the level of ALT in the serum of mice with liver damage. DETAILED DESCRIPTION

[0046] The present invention will be further illustrated below. It should be noted that the examples illustrate some preparation or use methods, however, it should be understood that these examples do not limit the present invention. The scope of protection of the present invention shall be subject to the contents recorded in the attached claims.

[0047] In the following examples, the mass spectrometer is the LCQ-Advantage mass spectrometer produced by Finnigan, USA. The superconducting nuclear magnetic resonance instrument is Bruker AV-400. The silica gel GF254 for thin layer chromatography and the silica gel for column chromatography (200-300 mesh) are both products of Qingdao Ocean Chemical Plant. The reversed phase ODS filler 50μm is a product of Japan YMC Company. The medium and low pressure liquid chromatograph is a product of Shanghai Lisui Electronic Technology Co., Ltd. The chromatographic column used for liquid phase separation is Phenomenex Gemini C18 column (10.0×250mm, 5μm). The methanol and acetonitrile used in liquid chromatography are chromatographically pure, the water is double distilled water, and the other reagents are analytically pure.

[0048] The experimental data at the in vitro cell level shown in the embodiment are expressed as mean ± standard deviation (Mean ± SD), and the animal experimental data are expressed as mean ± standard error (Mean ± SEM). For comparing the difference between two groups of data, Two-tailed Student's t test was used for analysis; for comparing the difference between more than 2 groups of data, One-way Anova was used for analysis; for each subject, the data with repeated measurements at different time points (such as body weight measurement, ear thickness measurement) were analyzed, and the repeated measures variance analysis in Two-way Anova was used to compare the differences between the groups. Statistical differences are indicated as follows: * represents P < 0.05, ** represents P < 0.01, *** represents P < 0.001, **** represents P < 0.0001, and ns represents no significant difference. Graphpad Prism 7.00 and FlowJo V10 (for flow) were used to analyze the acquired data, and all pathological sections were evaluated by pathologists.

[0049] Example 1 Large-scale fermentation of the fungus ZLW0801-19 and its sample pretreatment method

[0050] (1) The fungal strain ZLW0801-19 of the genus Mycorrhiza was cultured on a potato dextrose agar (PDA) slant at 25°C for 5 days. After activation on the PDA slant, it was inoculated into 4 conical flasks (250 mL) containing potato dextrose (PDB) medium to prepare seed solution. Each conical flask contained 100 mL of PDB medium and was cultured at 200 rpm at 25°C for 5 days to prepare seed solution. Fermentation was carried out in 24 conical flasks (500 mL), each containing 70 g of rice. First, distilled water (105 mL) was added to each conical flask, and the rice was soaked overnight, and then autoclaved at 120°C for 30 minutes. After cooling to room temperature, 5.0 mL of seed solution was inoculated into each conical flask and cultured at room temperature in the dark for 51 days.

[0051] (2) The fermented product was added to ethyl acetate for soaking extraction three times, and the extract was concentrated to dryness under reduced pressure to obtain a crude extract (81.7 g).

[0052] Example 2 Preparation of compounds of formula (II) to formula (XII)

[0053] In Example 1, the crude ethyl acetate extract (81.7 g) was subjected to silica gel column chromatography, and eluted with cyclohexane-ethyl acetate (100:0, 98:2, 95:5, 90:10, 80:20, 70:30, 50:50, 0:100, v / v) and methanol in sequence, with each gradient elution volume of 6 L, to obtain 7 fraction samples (F1-F7). Fraction F5 was subjected to medium-low pressure liquid phase ODS column chromatography, and eluted with methanol-water (70:30, 80:20, 90:10, and 100:0, v / v) in sequence, with each gradient elution volume of 2.5 L, to obtain 9 fraction samples (F5.1-F5.9). Fraction F5.2 was subjected to medium-low pressure liquid phase ODS column chromatography, and eluted with methanol-water (60:40, 70:30, 80:20, 90:10, v / v) in sequence, with each gradient elution volume of 0.7 L, to obtain 5 fraction samples (F5.2.1-F5.2.5). Fraction F5.2.4 (387 mg) was prepared by reverse phase preparative HPLC (Cosmosil Packed C18 column), and eluted with acetonitrile-water (50:50, v / v) at a flow rate of 3 mL / min to obtain the compound of formula (X) (t R :53.6min, 26.2mg). Fraction F5.2.5 (365mg) was prepared by reverse phase preparative HPLC (Phenomenex, Packed C18 column) and eluted with acetonitrile-water (55:45, v / v) at a flow rate of 8mL / min to obtain compound (XII) R:86.4min,7.0mg). Fraction F5.3 was chromatographed on a gel column LH-20 column and eluted with methanol to obtain 7 fractions (F5.3.1-F5.3.7). Fraction F5.3.7 (144mg) was prepared by reverse phase preparative HPLC (Cosmosil Packed C18 column) and eluted with methanol-water (75:25, v / v) at a flow rate of 3mL / min to obtain a compound of formula (II) (t R :63.7min,17.0mg) and compound of formula (V) (t R :69.9min, 17.5mg). Fraction F5.5 (1550mg) was prepared by reverse phase preparative HPLC (Phenomenex, Packed C18 column) and eluted with methanol-water (75:25, v / v) at a flow rate of 8mL / min to obtain compound (IV) R :73.5min,507.0mg). Fraction F5.7 was chromatographed on a silica gel column, eluted with cyclohexane-ethyl acetate (90:10,85:15,80:20,75:25,70:30,60:40,50:50,0:100,v / v), each gradient elution volume was 1.0L, and 8 fraction samples (F5.7.1-F5.7.8) were obtained. Fraction F5.7.4 (62.1mg) was prepared by reverse phase preparative HPLC (Cosmosil Packed C18column), eluted with methanol-water (82:18,v / v) at a flow rate of 3mL / min, and obtained a compound of formula (VI) (t R :21.3min,12.1mg). Fraction F6 was subjected to medium-low pressure liquid phase ODS column chromatography, and eluted with methanol-water (60:40,70:30,80:20,90:10,100:0,v / v) in sequence, with each gradient elution volume of 2.5L, to obtain 7 fraction samples (F6.1-F6.7). Fraction F6.3 was subjected to silica gel column chromatography, and eluted with cyclohexane-ethyl acetate (75:25,70:30,60:40,50:50,0:100v / v) in sequence, with each gradient elution volume of 0.3L, to obtain 6 fraction samples (F6.3.1-F6.3.6). Fraction F6.3.6 (349.7 mg) was prepared by reverse phase preparative HPLC (Cosmosil Packed C18 column) using acetonitrile-water (40:60, v / v) at a flow rate of 3 mL / min to obtain compound (IX) R:17.5min,7.7mg). Fraction F6.4 was subjected to medium-low pressure liquid phase ODS column chromatography, eluted with methanol-water (70:30, v / v), elution volume 0.7L, to obtain 5 fraction samples (F6.4.1-F6.4.5). Fraction F6.4.2 (1630mg) was prepared by reverse phase preparative HPLC (Phenomenex, Packed C18 column), eluted with methanol-water (70:30, v / v) at a flow rate of 8mL / min, to obtain compound of formula (III) (t R :35.2min,820mg) and compound of formula (XI) (t R :25.4min, 4.0mg). Fraction F6.4.3 (212.9mg) was prepared by reverse phase preparative HPLC (Cosmosil Packed C18 column) and eluted with acetonitrile-water (55:45, v / v) at a flow rate of 3mL / min to obtain compound (VII) (t R :63.3min,10.3mg) and compound of formula (VIII) (t R :25.0min,10.1mg).

[0054] The physicochemical constants are as follows:

[0055] Compound of formula (II): colorless flaky crystals; (c 1.0,CH3OH); UV(CH3OH)λ max (logε)205(4.31),225(4.28),221(4.10),300(4.27)nm; ECD(c 3.4×10 –4 MCH3OH)λ max (Δε)221(+1.86),245(+1.71),334(–0.55),306(–0.74); IR(KBr)ν max 3399,3137,2974,2929,2864,1616,1430,1293,1262,1117,1045cm –1 ;ESI-MS(positive)m / z 373[M+H] + ,395[M+Na] + ;ESI-MS(negative)m / z 371[M–H] – ,743[2M–H] – ;HR-ESI-MS(positive)m / z 373.2379[M+H] + (calcd.for C 23 H33 O4,373.2379), the molecular formula of the compound is determined to be C 23 H 32 O4; 1 H and 13 C NMR see Table 1.

[0056] Compound of formula (III): pale yellow solid; (c 1.0,CH3OH); UV(CH3OH)λ max (logε)206(4.43),225(4.35),240(4.18),299(4.31)nm; IR(KBr)ν max 3146,2969,2929,2881,1621,1625,1448,1258cm -1 ;ESI-MS(positive)m / z 389[M+H] + ,411[M+Na] + ;ESI-MS(negative)m / z 387[M–H] – ,775[2M–H] – ;HR-ESI-MS(positive)m / z 389.2331[M+H] + (calcd.for C 23 H 33 O5,389.2328), the molecular formula of the compound is determined to be C 23 H 32 O5; 1 H and 13 C NMR see Table 1.

[0057] Compound of formula (IV): pale yellow needle-shaped crystals; (c 1.0,CH3OH); (CH3OH)λ max (logε)205(4.27),223(4.23),240(4.09),299(4.27)nm; IR(KBr)ν max 3399,3160,2973,2838,2868,1708,1619,1435,1268,1002cm –1 ;ESI-MS(positive)m / z 453[M+Na] + ,883[2M+Na] + ;ESI-MS(negative)m / z 429[M–H] – ,859[2M–H] –;HR-ESI-MS(positive)m / z 453.2261[M+Na] + (calcd.for C 25 H 34 O6Na,453.2253), the molecular formula of the compound is determined to be C 25 H 34 O6; 1 H and 13 C NMR see Table 1.

[0058] Compound of formula (V): pale yellow solid; (c 1.0,CH3OH); UV(CH3OH)λ max (logε)205(4.47),226(4.43),242(4.31),300(4.44)nm; IR(KBr)ν max 3430,2961,2931,2868,1719,1622,1435,1265,1039cm –1 ;ESI-MS(positive)m / z 453[M+Na] + ,883[2M+Na] + ;ESI-MS(negative)m / z 429[M–H] – ,859[2M–H] – ;HR-ESI-MS(positive)m / z 431.2433[M+H] + (calcd.for C 25 H 35 O6,431.2434), the molecular formula of the compound is determined to be C 25 H 34 O6; 1 H and 13 C NMR see Table 1.

[0059] Compound of formula (VI): light yellow oil; (c 1.0,CH3OH); UV(CH3OH)λ max (logε)205(4.52),226(4.47),242(4.31),298(4.45)nm; IR(KBr)ν max 3279,2934,2867,1708,1618,1434,1267,1032cm –1 ;ESI-MS(positive)m / z 495[M+Na] + ,967[2M+Na] +;ESI-MS(negative)m / z 471[M–H] – ,943[2M–H] – ;HR-ESI-MS(positive)m / z 495.2368[M+Na] + (calcd.forC 27 H 36 O7Na,495.2359), the molecular formula of the compound is determined to be C 27 H 36 O7; 1 H and 13 C NMR see Table 2.

[0060] Compound of formula (VII): light yellow oil; (c 1.0,CH3OH); UV(CH3OH)λ max (logε)205(4.37),225(4.35),239(4.18),300(4.35)nm; ECD(c 3.2×10 –4 MCH3OH)λ max (Δε)201(+1.34),235(+2.04),274(–0.80),324(+0.46); IR(KBr)ν max 3411,2929,2858,1618,1434,1386,1267,1240,1125,1050cm –1 ;ESI-MS(positive)m / z 387[M+H] + ,409[M+Na] + ;ESI-MS(negative)m / z 385[M–H] – ,771[2M–H] – ;HR-ESI-MS(positive)m / z 409.2009[M+Na] + (calcd.for C 23 H 30 O5Na, 409.2015), the molecular formula of the compound is determined to be C 23 H 30 O5; 1 H and 13 C NMR see Table 2.

[0061] Compound of formula (VIII): pale yellow solid; (c 1.0,CH3OH); UV(CH3OH)λ max(logε)206(4.36),226(4.30),240(4.16),299(4.33)nm; ECD(c 5.8×10 –4 MCH3OH)λ max (Δε)224(+0.99),249(+3.46),302(–0.76),361(–1.02); IR(KBr)ν max 3399,2934,2850,1617,1386,1267,1245,1125,1050,988cm –1 ;ESI-MS(positive)m / z 455[M+Na] + ,895[2M+Na] + ;ESI-MS(negative)m / z 431[M–H] – ,863[2M–H] – ;HR-ESI-MS(positive)m / z 455.2415[M+Na] + (calcd.for C 25 H 36 O6Na,455.2410), the molecular formula of the compound is determined to be C 25 H 36 O6; 1 H and 13 C NMR see Table 2.

[0062] Compound of formula (IX): light green solid; (c 1.0,CH3OH); UV(CH3OH)λ max (logε)215(4.57),262(4.06),305(3.27)nm; IR(KBr)ν max 3385,2929,2863,1675,1452,1386,1262cm –1 ;ESI-MS(positive)m / z 386[M+H] + ,793[2M+Na] + ;ESI-MS(negative)m / z 769[2M–H] – ;HR-ESI-MS(positive)m / z 386.2340[M+H] + (calcd.for C 23 H 32 NO4,386.2331), the molecular formula of the compound is determined to be C 23 H 31 NO4;1 H and 13 C NMR see Table 2.

[0063] Compound of formula (X): light green solid; (c 1.0,CH3OH); UV(CH3OH)λ max (logε)206(4.44),223(4.46),240(4.42),298(4.33)nm; ECD(c 6.0×10 –4 MCH3OH)λ max (Δε)204(+7.00),247(+18.5),330(+1.81); IR(KBr)ν max 3283,2970,2925,2867,1621,1618,1430,1267,1005cm –1 ;ESI-MS(positive)m / z 409[M+Na] + ,795[2M+Na] + ;ESI-MS(negative)m / z385[M–H] – ,771[2M–H] – ;HR-ESI-MS(positive)m / z 387.2176[M+H] + (calcd.for C 23 H 31 O5,387.2176), the molecular formula of the compound is determined to be C 23 H 30 O5; 1 H and 13 C NMR see Table 3.

[0064] Compound of formula (XI): white solid; (c 1.0,CH3OH); UV(CH3OH)λ max (logε)206(4.39),226(4.06),289(3.69)nm; ECD(c 6.4×10 –4 M CH3OH)λ max (Δε)220(+3.81),236(+3.09),246(+3.74),323(–0.54); IR(KBr)ν max 3301,2961,2872,1628,1462,1284,1125,1076cm –1 ;ESI-MS(positive)m / z 389[M+H] + ,411[M+Na]+ ;ESI-MS(negative)m / z 387[M–H] – ,775[2M–H] – ;HR-ESI-MS(positive)m / z389.2329[M+H] + (calcd.for C 23 H 33 O5,389.2328), the molecular formula of the compound is determined to be C 23 H 32 O5; 1 H and 13 C NMR see Table 3.

[0065] Compound of formula (XII): white solid; (c 1.0,CH3OH); UV(CH3OH)λ max (logε)206(4.41),227(4.09),290(3.76)nm; ECD(c 2.9×10 –4 M CH3OH)λ max (Δε)220(+5.47),235(+4.19),246(+5.58),320(–1.30); IR(KBr)ν max 3479,29743,2881,1740,1623,1489,1270,1045cm –1 ;ESI-MS(positive)m / z 453[M+Na] + ;ESI-MS(negative)m / z 429[M–H] – ,HR-ESI-MS(positive)m / z 453.2246[M+Na] + (calcd.for C 25 H 34 O6Na,453.2353), the molecular formula of the compound is determined to be C 25 H 34 O6; 1 H and 13 C NMR see Table 3.

[0066] Table 1 Compounds of formula (II) to (V) 13 C NMR and 1 H NMR data and assignments

[0067]

[0068]

[0069] a The data recorded in DMSO-d6( 1 H NMR for 300MHz, 13 C NMR for 75MHz)

[0070] b The data recorded in DMSO-d6( 1 H NMR for 400MHz, 13 C NMR for 100MHz)

[0071] Table 2 Compounds of formula (VI) to (IX) 13 C NMR and 1 H NMR data and assignments

[0072]

[0073]

[0074] a The data recorded in DMSO-d6( 1 H NMR for 300 MHz, 13 C NMR for 75 MHz)

[0075] b The data recorded in DMSO-d6( 1 H NMR for 400MHz, 13 C NMR for 100MHz)

[0076] Table 3 Compounds of formula (X) to (XII) 13 C NMR and 1 H NMR data and assignments

[0077]

[0078] a The data recorded in DMSO-d6( 1 H NMR for 300MHz, 13 C NMR for 75MHz)

[0079] Example 3 Effects of Sesquiterpene Polyketide Compounds of Formula (II) to Formula (XII) on IL17 Expression

[0080] After T cells differentiate, they produce specific cytokines, such as Th17 producing cytokine IL17, Th2 producing cytokine IL4, and Tr1 producing cytokine IL10. IL17-GFP mice, IL4-eGFP mice, and IL10-eGFP mice (all from JAX) are transgenic mice with a green fluorescent reporter gene (GFP), which have a GFP fluorescent protein coding sequence connected to the downstream of the gene of the specific cytokine produced after T cells differentiate. Therefore, the expression level of cytokines can be measured by detecting the expression of GFP using a fluorescence detection system.

[0081] Primary spleen lymphocytes of fluorescent reporter mice IL17-GFP mice were extracted and co-cultured with the compound under Th17 differentiation conditions. The IL17-GFP level was detected by flow cytometry to evaluate the effect of the compound on IL17 expression (this method uses primary lymphocytes to better simulate complex physiological processes; the use of GFP mice and flow cytometry makes the experiment simple, reproducible and reliable). The specific experimental steps are as follows:

[0082] Anti-mouse CD3 Ab (antibody) coating: Add anti-mouse CD3 Ab diluted in IMDM medium to a final concentration of 10 μg / mL into a 96-well plate and incubate at 37°C for 2 hours.

[0083] Preparation of single cell suspension: IL17-GFP mice raised in a specific pathogen-free (SPF) environment were killed by cervical dislocation, and the spleen was taken out and placed in IMDM culture medium. The spleen was ground with a sterilized pathological slide in a clean bench, and the ground cell suspension was filtered into a centrifuge tube with a 40 μm cell strainer; centrifuged (4°C, 1400 rpm, 7 minutes), the supernatant was discarded, red blood cell lysis buffer was added, mixed, and allowed to stand at room temperature for 5 minutes, and then IMDM was added to stop the lysis of red blood cells; filtered with a 40 μm cell strainer and centrifuged again (4°C, 1400 rpm, 7 minutes), the supernatant was discarded, and the cells were resuspended in IMDM containing 10% FBS; the cells were diluted 100 times, and the cells were counted under a microscope. According to the counting results, 1×10 6 / mL (final concentration) of cell suspension.

[0084] Th17 cell differentiation inducing factors were added: anti-mouse CD28 Ab (1 μg / mL), rhTGFb1.2 (0.25 ng / mL), rmIL6 (40 ng / mL), anti-mouse IL4 (5 μg / mL), anti-mouse IFNγ (5 μg / mL) (the cytokines here are all final concentrations).

[0085] Dilution of samples to be tested: dilute the compound to 10 μM (final concentration) with IMDM containing 10% FBS; dilute the positive compound SR2211 that inhibits IL17 to 1 μM (final concentration).

[0086] Plating: remove the antibody coated on the plate, wash once with IMDM, add 100 μL of cell suspension and 100 μL of final concentration sample solution to each well for co-culture (e.g. 100 μL 4×10 6 The cell suspension with a concentration of 100 μL / mL was co-cultured with 100 μL of 20 μM compound, and the final cell concentration in the culture system was 2×10 6 / mL, compound concentration was 10 μM).

[0087] Cell culture: The sample solution and cells were cultured in a humidified incubator at 37°C and 5% CO2 for 72 hours.

[0088] Cell surface staining and flow cytometry: Collect the cultured cells in a flow tube, add 3 mL of pre-cooled PBS, centrifuge (4°C, 1400 rpm, 7 minutes), discard the supernatant, then add 0.25 μL of APC anti-mouse CD4 antibody to 50 μL of cell suspension for surface staining, and place at 4°C in the dark for 20 minutes. Resuspend the cells with 1 mL of pre-cooled PBS, add 2 mL of PBS, centrifuge again (4°C, 1400 rpm, 7 minutes), discard the supernatant, add 200 μL of PBS to each tube, and analyze GFP by flow cytometry. + CD4 + The ratio of cells.

[0089] IMDM culture medium and fetal bovine serum (FBS) were purchased from Gibco; anti-mouse CD28 Ab, anti-mouse CD3 Ab, anti-mouse IL4, anti-mouse IFNγ, APC anti-mouse CD4 were purchased from Sungene; rmIL6 was purchased from Peprotech; rhTGFb1.2 was purchased from R&D; red blood cell lysate was purchased from Tiangen; and PBS was purchased from Solarbio.

[0090] The formula used is: (DMSO group GFP + CD4 + Ratio of -compound group to GFP + CD4 + ratio) / DMSO group GFP + CD4 + The relative inhibitory activity of the compound on IL17 was calculated by the ratio of × 100%.

[0091] The results are shown in Table 4: Under the condition of differentiation of spleen lymphocytes into Th17 cells, the relative inhibitory activity of the compounds of formula (II), formula (III), formula (V), formula (XI) and formula (XII) of the present invention on IL17 was significantly enhanced. This shows that the compounds of formula (II), formula (III), formula (V), formula (XI) and formula (XII) of the present invention have the effect of inhibiting the expression of IL17 and can be used to prevent and treat immune-related diseases, especially IL17-mediated immune diseases.

[0092] Table 4 Effects of compounds of formula (II) to (XII) on IL17 expression (10 μM)

[0093]

[0094]

[0095] Example 4 Effects of Sesquiterpene Polyketide Compounds of Formula (II) to Formula (XII) on IL4 Expression

[0096] Primary spleen lymphocytes from fluorescent reporter mice IL4-eGFP were extracted and co-cultured with the compounds under Th2 differentiation conditions. The IL4-GFP level was detected by flow cytometry to evaluate the effect of the compounds on IL4 expression. The culture medium used for Th2 was RPMI-1640; the cell density was 2×10 6 The differentiation conditions used were: anti-mouse CD28 Ab (1 μg / mL), rmIL2 (2 ng / mL), rmIL4 (40 ng / mL), and Anti-mouse IFNγ (10 μg / mL). Other experimental steps for Th2 differentiation were consistent with those for Th17 differentiation in Example 3.

[0097] rmIL4 and rmIL2 were purchased from Peprotech; RPMI-1640 culture medium was purchased from Sigma.

[0098] When evaluating the effect of compounds on IL4 expression, the formula was used: Compound group GFP + CD4 + ratio of DMSO group to GFP + CD4 + The relative activity of the compound on IL4 was calculated by the ratio of 1:1 × 100%.

[0099] The specific results are shown in Table 5: Under the condition of spleen lymphocyte differentiation into Th2 cells, the relative activity of the compounds of formula (II), formula (III), formula (X), formula (XI) and formula (XII) of the present invention on IL4 is significantly enhanced. This shows that the compounds of formula (II), formula (III), formula (X), formula (XI) and formula (XII) of the present invention have the effect of promoting the expression of IL4 and can be used to resist parasitic infections.

[0100] Table 5 Effects of compounds of formula (II) to (XII) on IL4 expression (10 μM)

[0101] Compounds Relative Activity(%) (10μM) P-value Formula II 169.77±4.65 **** Formula III 165.12±33.48 ** Formula IV 97.91±7.68 ns Formula V 101.63±5.82 ns Formula VI 89.77±2.79 ns Formula VII 88.61±2.56 ns Formula VIII 87.68±1.63 ns Formula IX 87.68±2.56 ns Formula X 247.21±1.16 **** Formula XI 216.75±3.26 **** Formula XII 346.98±2.33 ****

[0102] Example 5 Effects of Sesquiterpene Polyketide Compounds of Formula (II) to Formula (XII) on IL10 Expression

[0103] Primary spleen lymphocytes from fluorescent reporter mice IL10-eGFP were extracted and sorted, and co-cultured with the compounds under Tr1 differentiation conditions. The IL10-eGFP level was detected by flow cytometry to evaluate the effect of the compounds on IL10 expression. The culture medium used for Tr1 was RPMI-1640; the cell density was 1×10 6 The differentiation conditions used were: anti-mouseCD28 Ab (1 μg / mL), rmIL27 (50 ng / mL), rhTGFb1.2 (0.5 ng / mL). Other experimental steps of Tr1 differentiation were consistent with those of Th17 differentiation in Example 3.

[0104] rmIL27 was purchased from Biolegend Company; RPMI-1640 culture medium was purchased from Sigma Company.

[0105] When evaluating the effect of compounds on IL10, the formula was used: Compound group GFP + CD4 + ratio of DMSO group to GFP + CD4 + The relative activity of the compound on IL10 was calculated by the ratio of 1:1 × 100%.

[0106] The specific results are shown in Table 6: Under the condition of differentiation of spleen lymphocytes into Tr1 cells, the relative activity of the compounds of formula (II), formula (III), formula (IV), formula (VI), formula (VII), formula (VIII), formula (IX), formula (X), formula (XI) and formula (XII) of the present invention on IL10 is significantly enhanced. This shows that the above compounds have the effect of promoting the expression of IL10, can exert a broad-spectrum anti-inflammatory effect, and are used for anti-inflammatory and autoimmune diseases.

[0107] Table 6 Effects of compounds of formula (II) to (XII) on IL10 expression (20 μM)

[0108] Compounds Relative Activity(%) (20μM) P-value Formula II 265.43±7.25 **** Formula III 285.40±9.68 **** Formula IV 118.74±1.09 ** Formula V 112.78±4.28 ns Formula VI 117.79±5.93 * Formula VII 114.74±1.95 * Formula VIII 115.47±3.49 * Formula IX 116.12±5.19 * Formula X 274.15±4.40 **** Formula XI 236.75±10.91 **** Formula XII 332.97±3.83 ****

[0109] Based on the results of Examples 3, 4 and 5 above, it can be seen that sesquiterpene polyketide compounds have the activity of inhibiting IL17, promoting IL4 and promoting IL10 expression, and can be used for the prevention and treatment of immune diseases.

[0110] Example 6 Compounds of formula (III) inhibit human IL17 expression

[0111] Human peripheral blood mononuclear cells (PBMCs) were isolated from healthy human peripheral blood samples using EasySep TM Human CD4 + T Cell Isolation Kit II (purchased from Stem Cell) was used to isolate human naive CD4 + T cells for subsequent human Th17 differentiation.

[0112] The sorted human naive CD4 + T cells were diluted to 5 × 10 5 / mL, add anti-human CD28 Ab (1μg / mL); rhIL6 (40ng / mL); rhTGFβ1.2 (0.5ng / mL); anti-human IL4 (10μg / mL); anti-human IFNγ (10μg / mL); add DMSO and 2.5μM compound of formula (III) to the above cell suspension, inoculate on a culture plate coated with anti-human CD3 Ab (5μg / mL) for 2 hours, and culture in a cell culture incubator; add rhIL6 on the third day, wash off anti-human CD23 Ab and anti-human CD28 Ab on the fifth day, transfer to a new culture medium containing rhIL6, rhTGFβ1.2, anti-human IL4, anti-human IFNγ, and continue to culture for 2 days; divide the wells with a new culture medium containing rhIL6, rhTGFβ1.2, anti-human IL4, anti-human IFNγ, and culture for another 2 days.

[0113] After human Th17 cells were differentiated, they were stimulated with 50 ng / mL PMA, 1 μg / mL Ionomycin and Golgi-Plug for 5 hours. Flow cytometry antibodies BV421 anti-human IL17A and BV500 anti-human CD4 were added and stained according to the experimental steps of the intracellular staining kit.

[0114] Among them, anti-human CD3, anti-human CD28, anti-human IFNγ, and anti-human IL4 were purchased from Tonbo Biosciences; rhIL6 was purchased from Peprotech; BV421 anti-human IL17A was purchased from BioLegend, and BV500 anti-human CD4 was purchased from BD.

[0115] The experimental results are as follows Figure 1 Shown: Human naive CD4 + Under the condition of T cell differentiation into human Th17, the compound of formula (III) significantly inhibited the proliferation of human CD4 + IL17 in T cells + This indicates that the compound of formula (III) has the effect of inhibiting the expression of human IL17.

[0116] Example 7 The compound of formula (III) inhibits BMDC from secreting inflammatory cytokines

[0117] Bone marrow cells were extracted from the leg bones of C57BL / 6 mice and cultured in bone marrow-derived dendritic cell (BMDC) culture medium (1640 medium containing 10% FBS pre-warmed with 50 ng / mL GM-CSF and 20 ng / mL IL4) for 2 days. The supernatant was transferred to a new dish and supplemented with BMDC culture medium for another 2 days. The medium was half-changed and cultured for another 2 days. The cells were collected, BMDCs were enriched with magnetic beads, and the cells were diluted to 2×10 5 / mL, 1mL per tube was inoculated into a flow tube and cultured overnight; DMSO and 5μM compound of formula (III) were added to the BMDC cultured overnight, and after culturing for 18 hours under LPS (100ng / mL) stimulation, Golgi-Plug was added for 6 hours; Purified Rat Anti-Mouse CD16 / CD32 was added for blocking for 15 minutes, and flow antibodies Percepcy5.5anti-mouse CD11c, eF450 anti-mouse MHCⅡ, FITC anti-mouse TNFα, PE anti-mouseIL6 and APC anti-mouse IL12 / IL23 p40 were added, and staining was performed according to the experimental steps of the intracellular staining kit.

[0118] Among them, Percepcy5.5 anti-mouse CD11c, FITC anti-mouse TNFα, PE anti-mouseIL6 and APC anti-mouse IL12 / IL23 p40 were purchased from Biolegend; eF450 anti-mouse MHCⅡ was purchased from Invitrogen; Purified Rat Anti-Mouse CD16 / CD32 was purchased from BD.

[0119] The experimental results are as follows Figure 2 As shown: The compound of formula (III) inhibited the LPS-stimulated BMDC (CD11c + MHCII + IL12 / IL23 p40 in cells + IL6 + and TNFα + This indicates that the compound of formula (III) has the effect of inhibiting the production of inflammatory cytokines IL12 / IL23, IL6 and TNFα by BMDC.

[0120] Example 8 The compound of formula (III) has the effect of inhibiting DSS-induced acute enteritis

[0121] Dextran sulfate sodium (DSS)-induced enteritis in mice is a classic inflammatory bowel disease (IBD) model. The modeling steps are as follows: from day 0 to day 4, C57BL / 6 mice freely drink 3% DSS (purchased from MP Company); on day 5, 3% DSS is withdrawn and replaced with water, and the mice are kept for another 3 days.

[0122] Ten-week-old male C57BL / 6 mice were divided into four groups, with eight mice in each group. The treatments of each group were as follows: blank control group (Control+Vehicle): free drinking of pure water, intraperitoneal injection of solvent twice a day; DSS model group (DSS+Vehicle): free drinking of 3% DSS, intraperitoneal injection of solvent twice a day; high-dose group (DSS+Formula III(H)): free drinking of 3% DSS, intraperitoneal injection of 40 mg / kg of the compound of formula (III) twice a day; low-dose group (DSS+Formula III(L)): free drinking of 3% DSS, intraperitoneal injection of 20 mg / kg of the compound of formula (III) twice a day. Vehicle refers to the solvent for dissolving the compound of formula (III), which is composed of 5% DMSO+5% Solutol HS 15 (polyethylene glycol-15 hydroxystearate, purchased from BASF)+90% water for injection.

[0123] During the experiment, the mice were weighed every day, the degree of fecal formation and occult blood in the feces were monitored, and scores were given according to the severity of the disease (0-4 points for each item). The clinical disease activity index (DAI) is an indicator used clinically to evaluate the degree of disease activity. The DAI score = (weight loss score + stool formation score + blood in the stool score) / 3.

[0124] On the 8th day of the experiment, the mice were killed and the complete colon was removed to measure the length of the mouse colon. The colon was flushed with PBS and the distal colon was dissected for pathological analysis.

[0125] The experimental results showed that the compound of formula (III) significantly inhibited the weight loss of enteritis mice ( Figure 3 A) Increased DAI score ( Figure 3 B) Shortened colon ( Figure 3 C) phenomenon (n=8); the results of pathological analysis showed that the compound of formula (III) significantly inhibited the degree of colon inflammation, epithelial damage and intestinal structural lesions ( Figure 3 D and 3E) (n=8). These results indicate that the compound of formula (III) has the effect of inhibiting DSS-induced acute intestinal inflammation and can be used as a treatment for inflammatory bowel disease.

[0126] Example 9 The compound of formula (III) has the effect of treating EAE in mice

[0127] Experimental autoimmune meningitis (EAE) induced by MOG35-55 is a classic mouse model that simulates human multiple sclerosis (MS). The modeling steps are as follows: Take MOG35-55 (Shanghai Jier Biochemical Company) and adjust the concentration to 2 mg / mL; Take incomplete Freund's adjuvant (Sigma Company), add inactivated Mycobacterium tuberculosis H37 RA (DIFCO Company), and adjust the concentration to 5 mg / mL; Mix MOG35-55 solution with Freund's adjuvant in a volume ratio of 1:1, and emulsify on ice for 2 hours to an oil-in-water state; After the mice are anesthetized, immunize subcutaneously behind the ears and thighs of the mice, inject 50 μL at each site, a total of 200 μL / mouse, and this is set as day 0; On the 0th and 2nd days of immunization, each mouse is intraperitoneally injected with 300 ng pertussis toxin (List labs company). The mice immunized in this experiment were 10-week-old female C57BL / 6 mice.

[0128] The EAE score is an indicator used to evaluate the degree of EAE disease activity, and can be scored from 0 to 5 points according to the severity of the disease: 0 points - no obvious abnormalities; 0.5 points - tail tip weakness, no abnormality in movement; 1 point - the entire tail is weak, and there is no abnormality in movement; 1.5 points - the entire tail is weak, the two hind limbs can be separated, and occasional stepping on air during movement; 2 points - the entire tail is weak, the two hind limbs are combined, and it is common to step on air during movement; 2.5 points - one hind limb drags; 3 points - both hind limbs drag, and the mouse can turn over by itself after being placed on its back; 3.5 points - both hind limbs drag, and the mouse cannot turn over by itself after being placed on its back, and the forelimbs can normally grasp the metal bars of the cage; 4 points - the mouse drags its two hind limbs, cannot turn over by itself after being placed on its back, one forelimb is weak, and cannot normally grasp the metal bars of the cage; 4.5 points - the mouse is quadriplegic; 5 points - the mouse dies or the score exceeds 4 for 2 consecutive days.

[0129] Ten days after modeling, 23 mice with initial disease (EAE score 0.5-1 points) were randomly divided into two groups, and the treatment of each group was as follows: EAE model group (EAE+Vehicle): intraperitoneal injection of solvent twice a day for 5 consecutive days, n=11; Formula (III) compound treatment group (EAE+Formula III): intraperitoneal injection of 40 mg / kg Formula (III) compound twice a day for 5 consecutive days, n=12.

[0130] The EAE scores of mice were scored every day during the experiment. On the 5th day after the intervention of the compound of formula (III), the mice were killed and the tissues of the spinal cord enlargement area were taken for pathological analysis.

[0131] The experimental results showed that the compound of formula (III) significantly reduced the EAE score of EAE model mice ( Figure 4 A); Spinal cord pathological analysis results showed that treatment with the compound of formula (III) significantly inhibited the infiltration of inflammatory cells and the degree of demyelination in the spinal cord ( Figure 4 B). These results indicate that the compound of formula (III) has the effect of treating EAE in mice and can be used as a treatment for multiple sclerosis.

[0132] Example 10 The compound of formula (III) has the effect of treating psoriasis in mice

[0133] The mouse psoriasis induced by topical application of imiquimod (IMQ) is very similar to human psoriasis in phenotype and histological characteristics. The modeling steps are as follows: 25 mg of IMQ (Sichuan Mingxin Lidi Company) was applied to the mouse ears every day from day 0 to day 7 of the experiment, and the disease reached its peak on day 7; IMQ was applied every other day thereafter to maintain the disease.

[0134] Eight-week-old male Babl / c mice were divided into three groups, and the treatment of each group was as follows: blank control group (Control+Vehicle): no IMQ was applied during the whole experiment, and the solvent was intraperitoneally injected twice a day after the 7th day, n=6; model group (IMQ+Vehicle): IMQ was applied for 7 consecutive days, and then IMQ was applied every other day to maintain the condition, and the solvent was intraperitoneally injected twice a day after the 7th day, n=6; formula (III) compound treatment group (EAE+Formula III): IMQ was applied for 7 consecutive days, and then IMQ was applied every other day to maintain the condition, and 20 mg / kg of formula (III) compound was intraperitoneally injected every day after the 7th day, n=8.

[0135] The thickness of the ears was measured every day during the experiment. The mice were killed on the 21st day of the experiment, and the ears of the mice were photographed and the ear tissues were taken for pathological analysis.

[0136] The experimental results showed that compared with the model group, the compound of formula (III) significantly inhibited the thickening of the ears of psoriasis-like mice ( Figure 5 A and 5B); H&E staining scores of ear pathological sections showed that treatment with the compound of formula (III) significantly inhibited the parakeratosis, hyperkeratosis (squamous epithelial hyperplasia), Munro body formation, acanthosis, inflammatory cell infiltration and dermal vascular dilation and congestion in the ear skin of psoriasis-like mice ( Figure 5 C and 5D). These results indicate that the compound of formula (III) has the effect of treating psoriasis in mice and can be used for the treatment of psoriasis.

[0137] Example 11 The compound of formula (III) has a protective effect on LPS-induced sepsis in mice

[0138] The mouse sepsis model induced by lipopolysaccharide (LPS) is a classic animal model of sepsis. In the experiment, a lethal dose of 35 mg / kg LPS (purchased from Sigma) was injected intraperitoneally to induce sepsis.

[0139] Male C57BL / 6 mice were divided into 2 groups, and the treatments of each group were as follows: Model group (LPS+Vehicle): single intraperitoneal injection of solvent, n=11; Formula (III) compound treatment group (LPS+Formula III): single intraperitoneal injection of 20 mg / kg Formula (III) compound, n=13. Mice were pretreated with solvent or Formula (III) compound for 2 hours, and then intraperitoneally injected with a lethal dose of 35 mg / kg LPS to induce sepsis.

[0140] The survival of mice was observed and the time of death of mice was recorded until the 6th day.

[0141] Graphpad 7.0 was used to draw the survival curve and perform significance analysis. Figure 6 It is shown that the compound of formula (III) significantly increased the survival rate of mice. These results indicate that the compound of formula (III) has a protective effect on LPS-induced sepsis in mice and can be used for the prevention and treatment of sepsis.

[0142] Example 12 The compound of formula (III) has a protective effect on APAP-induced liver damage

[0143] Acetaminophen (APAP)-induced liver injury simulates clinical drug-induced liver injury. The mice were fasted but not watered one day before the experiment, and 250 mg / kg APAP (purchased from Sigma) was intraperitoneally injected to induce drug-induced liver injury in mice.

[0144] Male C57BL / 6 mice were divided into 2 groups, and the treatments of each group were as follows: Model group (APAP+Vehicle): single intraperitoneal injection of solvent, n=6; Formula (III) compound treatment group (APAP+Formula III): single intraperitoneal injection of 20 mg / kg Formula (III) compound, n=6. After the mice were pretreated with solvent or Formula (III) compound for 0.5 hours, 250 mg / kg APAP was intraperitoneally injected to induce drug-induced liver injury in mice.

[0145] Blood samples were collected from the eye sockets of mice before APAP injection and 3 hours, 6 hours, 12 hours, and 24 hours after injection, and heparin sodium was used for anticoagulation. After blood samples were collected, the ALT kit (purchased from Shanghai Rongsheng Biopharmaceutical Co., Ltd.) was used to detect the level of alanine aminotransferase (ALT) in mouse serum.

[0146] Specific results such as Figure 7 It is shown that the compound of formula (III) significantly inhibited the level of ALT in the serum of mice with liver injury. These results indicate that the compound of formula (III) has a protective effect on APAP-induced liver injury and can be used for the prevention and treatment of drug-induced liver injury.

[0147] The content of the present invention merely illustrates some specific implementation plans for which protection is sought, wherein the technical features recorded in one or more technical solutions may be combined with any one or more technical solutions, and these technical solutions obtained by combination are also within the protection scope of the present application, just as these technical solutions obtained by combination have been specifically recorded in the disclosed content of the present invention.

[0148] References:

[0149] [1]Horwitz DA, Fahmy TM, Piccirillo CA, et al. Rebalancing immunehomeostasis to treat autoimmune diseases. Trends Immunol, 2019, 40(10):888-908.

[0150] [2]Maddur MS, Miossec P, Kaveri SV, et al. Th17 cells. The American Journal of Pathology, 2012, 181(1):8-18.

[0151] [3]Patel DD, Kuchroo V K. Th17 cell pathway in human immunity: Lessons from genetics and therapeutic interventions. Immunity, 2015, 43(6): 1040-1051.

[0152] [4]Bedke T, Muscate F, Soukou S, et al. Il-10-producing t cells and their dual functions. Semin Immunol, 2019, 44: 101335.

[0153] [5]Roncarolo MG,Gregori S,Bacchetta R,et al.The biology of tregulatory type 1cells and their therapeutic application in immune-mediateddiseases.Immunity,2018,49(6):1004-1019.

[0154] [6]Reizis B.Plasmacytoid dendritic cells:development,regulation,andfunction.Immunity,2019,50(1):37-50.

[0155] [7]Ho I C,Miaw S C.Regulation of il-4 expression in immunity anddiseases.Advances in Experimental Medicine and Biology,2016,941:31-77.

Claims

1. Use of a sesquiterpene polyketide compound or a pharmaceutically acceptable salt thereof in the preparation of a medicament for preventing or treating immune-related diseases, characterized in that: The immune-related disease is selected from psoriasis, inflammatory bowel disease, multiple sclerosis, sepsis or drug-induced liver injury, and the sesquiterpene polyketide compound is represented by formula (III): (III) Among them, R1=OH, R2=CH2OH.

2. The use according to claim 1, characterized in that The pharmaceutically acceptable salt is a salt formed by the sesquiterpene polyketone compound and an organic base or an inorganic base.

3. The use according to claim 2, characterized in that: The formed salt is a sodium salt, a potassium salt, a calcium salt, a ferric salt, a magnesium salt, a zinc salt, an aluminum salt, a barium salt or an ammonium salt.

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