Oleanolic acid amide derivative for treating inflammatory immune diseases, and preparation method therefor and use thereof

Abstract

Provided are an oleanolic acid amide derivative for treating inflammatory immune diseases, and a preparation method therefor. According to the method, natural oleanolic acid and methylamine are subjected to amide reaction under the presence of a catalyst to obtain an oleanolic acid amide derivative. Acute toxicity and pharmacokinetic experiments prove that the modified oleanolic acid derivative is a safe and effective drug, and has better water solubility and pharmacokinetic characteristics. Pathological experiments show that the oleanolic acid amide derivative can significantly improve the skin phenotype and severity index of psoriasis model mice, thereby effectively relieving the clinical symptoms of psoriasis. The oleanolic acid amide derivative can significantly improve the arthritis symptoms of adjuvant arthritis rats. The oleanolic acid amide derivative provided can be used for preparing a drug for treating inflammatory immune diseases such as psoriasis and rheumatoid arthritis.

Description

Oleanolic acid amide derivative for treating inflammatory immune diseases, preparation method and use thereof Technical Field The invention belongs to the field of medicine, and specifically relates to a method for preparing oleanolic acid amides and application of the oleanolic acid amides in preparing drugs for treating inflammatory diseases (taking psoriasis and rheumatoid arthritis as examples). Background Art Oleanolic acid, English name Oleanolic acid (OA), chemical formula 3β-hydroxyoleanolic acid-12-ene-17-carboxylic acid, molecular weight 456.700, CAS number 508-02-1, structural formula is as follows. Inflammation and immune response are basic pathological processes that occur when various immune cells of the body's immune system are stimulated by various damaging factors and are mainly defensive reactions. Inflammation and immune response play an important role in the host defense and the progression of immune-mediated diseases. Under normal circumstances, it is a normal physiological response of the immune system to injury, infection, and stress. This process acts as a signal to prompt the immune system to heal and repair damaged tissues to protect the body from infectious factors such as viruses and bacteria. However, excessive or persistent inflammatory immune responses can lead to and aggravate the occurrence of diseases. [1] Autoimmune diseases are a type of immune disease in which the immune system induces tissue damage and inflammatory response, ultimately leading to target organ damage. [2] Autoimmune diseases are accompanied by chronic inflammation, such as rheumatoid arthritis, inflammatory bowel disease and psoriasis, which bring pain and distress to thousands of patients. Natural medicines are considered to be an important resource for the treatment of inflammatory immune diseases. Oleanolic acid is a pentacyclic triterpenoid substance widely found in Oleaceae plants, and exists in daily food in the form of free acid or triterpenoid saponin aglycone. So far, a variety of pharmacological activities of oleanolic acid have been reported, including its antioxidant, anti-inflammatory, anti-asthmatic, anti-diabetic, anti-tumor, liver protection and immunomodulatory effects. [3] At present, most of the research on the pharmacological activity of oleanolic acid focuses on its anti-tumor and liver-protective effects. Studies have found that oleanolic acid can induce apoptosis and autophagy of tumor cells, inhibit their migration and invasion, and thus achieve the effect of inhibiting tumor development. [4] Recent studies have found that oleanolic acid has a certain therapeutic effect on inflammatory immune diseases. [5][6] . However, oleanolic acid has extremely poor water solubility, is difficult to absorb in the intestine, and has low solubility, which leads to low bioavailability and also makes it difficult to conduct mechanism studies in vitro. Based on this, researchers have modified natural oleanolic acid, attempting to improve water solubility and drugability by modification to increase bioavailability. Studies have found that the structural modification of OA has a great influence on its biological activity. The carboxyl group at the C-17 position can undergo esterification, amide and other reactions, connecting multiple groups to achieve the effect of modification without losing its pharmacological activity. The comprehensive drug database shows that more than 25% of known drugs have amide as a structural feature. The presence of amide bonds in drugs may enable them to better form hydrogen bonds with the amino acid fragments of their target proteins, thereby firmly binding. [7] ; The -COOH at the C-17 position causes high steric hindrance of OA, making it unable to effectively bind to the target protein. The amide reaction of -COOH may eliminate or reduce the high steric hindrance effect caused by the original -COOH, making it easier to form hydrogen bonds with the target protein and show better pharmacological activity. Therefore, the synthesis of oleanolic acid amide derivatives has broad application prospects. Psoriasis is an inflammatory, immune, papular, scaly skin disease that affects more than 60 million adults and children worldwide. [8] Psoriasis not only causes physical problems for people, but also seriously affects their mental health. Up to 30% of psoriasis patients suffer from anxiety and depression. [9] It can be seen that the physical and psychological risks brought by psoriasis are huge. The pathological characteristics of psoriasis show that epidermal keratinocytes, T cells, dendritic cells, etc. play an important role.

[0010] . In particular, the IL-23 / Th17 pathway plays an important role in psoriasis. Helper T cells 17 (Th17 cells) differentiate from precursors under the regulation of interleukin-1β (IL-1β), IL-6, transforming growth factor-β (TGF-β) and transcription factor RORγt. Th17 cell expansion and survival depend on IL-23 produced by bone marrow cells. Once activated, Th17 cells produce cytokines such as IL-17A, IL-17F and IL-22, which induce keratinocyte proliferation and participate in the course of psoriasis.

[0011] . Currently, the treatment drugs for psoriasis include steroids, retinoids, methotrexate, cyclosporine, biologics, and targeted small molecule drugs (TNF-α inhibitors, IL-17 and IL-23 inhibitors). However, these drugs have significant side effects or their effects do not meet expectations. Therefore, exploring and developing targeted drugs with definite efficacy and few adverse reactions is an urgent research goal for clinicians and pharmaceutical experts. Recent studies have found that SHP2 is a non-receptor protein tyrosine kinase that contains two SH2 domains and a catalytic protein tyrosinephosphatase (PTP) domain. It is encoded by PTPN11 and has key functions in cell proliferation, differentiation and survival. SHP2 is closely related to the onset of systemic lupus erythematosus. Studies have shown that in cultured human systemic lupus erythematosus T cells, inhibition of SHP2 can reduce the levels of IFN-γ and IL-17A, indicating that inhibition of SHP2 can be used to improve systemic lupus erythematosus.

[0012] . Similarly, SHP2 plays a key role in the proliferation and activation of T cells in the course of psoriasis. SHP2 enhances the activation of TLR7 / NF-κB, leading to further increase in the expression of psoriasis-related inflammatory cytokines IL-23A, TNF-α, IL-6, and IL-1β, thereby accelerating psoriasis-like skin inflammation.

[0013] . At the same time, the SHP2 allosteric inhibitor SHP099 suppressed skin inflammation caused by myeloid cells, especially macrophages. In addition, the optimized SHP2 inhibitor TK-453 significantly improved imiquimod-induced skin-like inflammation in mice by inhibiting the IL-23 / Th17 pathway

[0014] Therefore, exploring inhibitors targeting SHP2 from traditional Chinese medicines to treat psoriasis will have potential clinical value. Rheumatoid arthritis (RA) is a chronic, systemic, inflammatory, autoimmune disease with joints as the main target organs. It has a high disability rate and seriously affects the normal life and work of patients. In addition to joints, patients may also suffer damage to multiple organs such as lungs and blood vessels. According to statistics, about 1 in every 200 adults in the world is diagnosed with RA, and the incidence rate in women is 2 to 3 times that of men.

[0015] . Although RA manifests as a systemic autoimmune reaction, its clinical features are mainly synovial inflammation and joint damage. Fibroblast-like synovial cells (FLS) are a highly specific mesenchymal cell located in the synovial lining. As the main effector cell for RA synovial tissue proliferation and inflammation, FLS has the biological characteristics of abnormal proliferation, migration, invasion and secretion. In RA, FLS proliferates rapidly under the stimulation of platelet-derived growth factor, TGF-β, IL-1β, and TNF-α produced by various immune cells in the inflamed joints, transforming the synovial lining into invasive hyperplastic tissue. FLS also produces many inflammatory cytokines such as IL-1β, IL-6, TNF-α, etc., leading to synovial inflammation. More importantly, activated FLS will produce active substances such as matrix metalloproteinases (MMPs) to destroy collagen-rich joint tissue.

[0016] Therefore, inhibiting the abnormal function of FLS cells in RA can effectively control the proliferation of synovial inflammation, prevent the erosion of cartilage and ultimately alleviate the progression of RA. Targeting FLS therapy may become a promising treatment method. At present, the RA drugs used in clinical practice mainly include non-steroidal anti-inflammatory drugs, immunosuppressants, biological agents and glucocorticoids. This type of drug is mainly used to control and relieve the patient's condition and symptoms, but there are many adverse reactions or high treatment costs, so it is very important to find highly effective, low-toxic, economical and practical drugs. Studies have shown that oleanolic acid, as a plant extract, can not only significantly inhibit the swelling of the feet of RA rats and the release of cellular inflammatory factors, but also inhibit the expression of inflammatory factors such as FLS IL-6 and IL-1β induced by TNF-α in a concentration-dependent manner, and inhibit TNF-α-induced p38 mitogen activated protein kinase (Mitogen activated protein kinase, Therefore, further research on the mechanism of action of oleanolic acid and its modified molecules has broad clinical application value. The pro-inflammatory cytokine TNF-α is a key driver of inflammatory diseases such as RA. It can drive two independent receptors, tumor necrosis factor receptor 1 (TNFR1) and TNFR2, to control different signal transductions. The activation of TNFR1 leads to the activation of NF-κB and MAPK signal transduction, driving the inflammatory response, which is closely related to the occurrence and development of RA.

[0017] . Recent studies have found that TNF-α affects mitochondrial function and drives mtDNA release. Under inflammatory conditions, when the cytoplasmic DNA sensor cyclic GMP-AMP (Cyclic GMP-AMP synthase, cGAS) senses DNA that should not be present in the cytoplasm, it catalyzes ATP and GTP to synthesize a small molecule cyclic guanosine-adenosine (Cyclic GMP-AMP, cGAMP). cGAMP binds to the dimerized interferon stimulating gene (Interferon stimulating gene, STING), causing its conformation to change and transfer from the endoplasmic reticulum to the Golgi apparatus, where it recruits and phosphorylates TANK-binding kinase 1 (TBK1) protein and NF-κB inhibitor (Inhibitor of KappaB Kinase, IKK), which then activates IFN regulatory transcription factor 3 (IRF3) and NF-κB, respectively, thereby promoting the production of type I interferon (IFN-I). Studies have shown that damaged mitochondria increase in FLS of RA patients, and cGAS is highly expressed. At the same time, in TNF-α-stimulated FLS, overexpression of cGAS enhances the production of proinflammatory cytokines and matrix metalloproteinases (MMPs).

[0018] Therefore, it is of great clinical value to find natural drugs that target TNFR1 and inhibit the cGAS-STING axis in FLS to treat RA. References [1]PLACE DE,KANNEGANTI T D.The innate immune system and cell death in autoinflammatory and autoimmune disease[J].CurrOpin Immunol,2020,67:95-105. [2]DI FLORIO DN,SIN J,CORONADO MJ,et al.Sex differences in inflammation,redox biology,mitochondria and autoimmunity[J].Redox Biol,2020,31:101482. [3]LIN C,WEN X,SUN H.Oleanolic acid derivatives for pharmaceutical use:a patent review[J].Expert Opin Ther Pat,2016,26(6):643-55. [4]BORELLA R,FORTI L,GIBELLINI L,et al.Synthesis and Anticancer Activity of CDDO and CDDO-Me,Two Derivatives of Natural Triterpenoids[J].Molecules,2019,24(22). [5]AYELESO T B,MATUMBA M G,MUKWEVHO E.Oleanolic Acid and Its Derivatives:Biological Activities and Therapeutic Potential in Chronic Diseases[J].Molecules,2017,22(11). [6]CHOI J K,KIM S W,KIM D S,et al.Oleanolic acid acetate inhibits rheumatoid arthritis by modulating T cell immune responses and matrix-degrading enzymes[J].Toxicol Appl Pharmacol,2016,290:1-9. [7]TIAN T,LIU X,LEE E S,et al.Synthesis of novel oleanolic acid and ursolic acid in C-28position derivatives as potential anticancer agents[J].Arch Pharm Res,2017,40(4):458-68. [8]GRIFFITHS C E M,ARMSTRONG A W,GUDJONSSON J E,et al.Psoriasis[J].Lancet,2021,397(10281):1301-15. [9]MATTEIP L,COREY K C,KIMBALL A B.Psoriasis Area Severity Index(PASI)and the Dermatology Life Quality Index(DLQI):the correlation between disease severity and psychological burden in patients treated with biological therapies[J].J EurAcad Dermatol Venereol,2014,28(3):333-7.

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[0013] ZHU Y,WU Z,YAN W,et al.Allosteric inhibition of SHP2 uncovers aberrant TLR7 trafficking in aggravating psoriasis[J].EMBO Mol Med,2022,14(3):e14455.

[0014] WANG M, LI T, OUYANG Z, et al. SHP2 allosteric inhibitor TK-453 alleviates psoriasis-like skin inflammation in mice via inhibition of IL-23 / Th17 axis[J]. iScience, 2022, 25(4): 104009.

[0015] SMITH M H, BERMAN J R. What Is Rheumatoid Arthritis?[J]. Jama, 2022, 327(12): 1194.

[0016] NYGAARD G, FIRESTEIN G S. Restoring synovial homeostasis in rheumatoid arthritis by targeting fibroblast-like synoviocytes[J]. Nat Rev Rheumatol, 2020, 16(6): 316 - 33.

[0017] WILLEMSEN J, NEUHOFF M T, HOYLER T, et al. TNF leads to mtDNA release and cGAS / STING-dependent interferon responses that support inflammatory arthritis[J]. Cell Rep, 2021, 37(6): 109977.

[0018] WANG Y, SU G H, ZHANG F, et al. Cyclic GMP-AMP Synthase Is Required for Cell Proliferation and Inflammatory Responses in Rheumatoid Arthritis Synoviocytes[J]. Mediators Inflamm, 2015, 2015: 192329. Summary of the Invention The present invention modifies the C-17 position of OA by amide reaction, prepares a novel oleanolic acid derivative code-named OA-17, studies its toxicology, pharmacokinetics and pharmacodynamics, and compares it with the parent OA. The results show that OA-17 has better water solubility than OA and stronger drug potential. Most importantly, OA-17 shows better efficacy than OA in the pharmacodynamic study of psoriasis, and effectively reduces the adjuvant arthritis process in rats by inhibiting FLS. The present invention also conducts a mechanism study on OA-17. In psoriasis, OA-17 can significantly inhibit the expression of T cell SHP2 and PI3K / mTOR, thereby inhibiting the skin inflammation of psoriasis mice induced by imiquimod in pathological experiments; in RA, OA-17 can inhibit the TNF-α receptor TNFR1 in FLS, thereby significantly inhibiting the expression of its downstream cGAS and STING, thereby alleviating the inflammatory changes of FLS, and finally alleviating the progression of RA. Based on this, the present invention provides an oleanolic acid derivative that can be used to prepare a method for treating psoriasis and RA, which has good social and economic benefits. Therefore, the purpose of the present invention is to provide an oleanolic acid amide derivative having the effect of treating psoriasis and a preparation method thereof. The method uses natural oleanolic acid as a lead compound to design an oleanolic acid amide derivative OA-17 (modified at its C-17 position). OA-17 can significantly inhibit the expression of T cell SHP2, thereby inhibiting the skin inflammation of psoriasis mice induced by imiquimod in pathological experiments. The oleanolic acid derivative has the following molecular structure: The present invention thus provides an oleanolic acid amide derivative having the effect of treating psoriasis, and its molecular structure is shown in formula (II). The present invention provides stereoisomers, solvent compounds, prodrugs, metabolites, pharmaceutically acceptable salts or co-crystals of the oleanolic acid amide derivatives having the effect of treating psoriasis. The present invention further provides a pharmaceutical composition containing the oleanolic acid amide derivatives, or stereoisomers, solvent compounds, prodrugs, metabolites, pharmaceutically acceptable salts or cocrystals thereof. Specifically, a pharmaceutically acceptable carrier is also included. The present invention also provides the use of the oleanolic acid amide derivatives, or their stereoisomers, solvent compounds, prodrugs, metabolites, pharmaceutically acceptable salts or co-crystals in the preparation of drugs for treating or preventing psoriasis. The present invention also provides a method for preparing the oleanolic acid amide derivatives, which comprises the following steps: (1) Take the raw material oleanolic acid, the reactant methylamine hydrochloride, add the catalyst 2-(7-azobenzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate, and the catalyst N,N-diisopropylethylamine at 20-25° C., and stir at a constant speed for 6-15 hours; (2) dissolving the mixture of step 1 in an organic solvent, adding saturated aqueous NaCl solution and saturated aqueous potassium hydrogen sulfate solution, and extracting with ethyl acetate 2-3 times; (3) Collect the organic phase to obtain the target product. Preferably, in step (1), the equivalent ratios of oleanolic acid to methylamine hydrochloride, 2-(7-azobenzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate, and N,N-diisopropylethylamine are 1:1, 1:1.3, and 1:2, respectively. Specifically, in step (2), the organic solvent is ethyl acetate. Specifically, in step (3), the organic phase is collected, concentrated using a rotary evaporator, and silica gel powder is added and rotary evaporated to obtain a solid powder. More specifically, the obtained solid powder is purified by silica gel column chromatography; preferably, gradient elution is performed using a mixture of petroleum ether and ethyl acetate as the eluent to obtain a purified solution of the target product, which is concentrated and vacuum dried to obtain the target product; more preferably, the volume ratio of the eluent petroleum ether and ethyl acetate in the silica gel purification is 3:1 and 1:1. Compared with the prior art, the beneficial effects of the present invention are as follows: 1. Toxicology and pharmacokinetic experiments show that OA-17 is safe and effective, has no toxic damage to any organ, and has better water solubility and pharmacokinetic characteristics than precursor OA; 2. Pharmacodynamic experiments have shown that OA-17 is superior to OA in the treatment of psoriasis. It can significantly improve the skin inflammatory response of mice with psoriasis and significantly reduce the levels of psoriasis-related inflammatory factors such as IL 17, IFN-γ, TNF-α, IL-23a, etc. 3. Molecular docking showed that OA-17 can bind to SHP2, a key drug target in the course of psoriasis, and its binding ability is higher than that of precursor OA. Molecular biology experiments showed that OA-17 can significantly inhibit the expression of SHP2 in T cells in terms of mechanism, thereby inhibiting skin inflammation in psoriasis mice induced by imiquimod in pathological experiments. 4. The present invention provides a new medication option for the treatment of psoriasis patients, and the parent drug has a wide source, simple synthesis steps, low cost and few by-products, clear ingredients and dosages, safe medication without obvious side effects, and has good social and economic benefits. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1. HE staining of heart, liver, spleen and kidney of mice in each group. Figure 2. Standard curve of OA-17 in rat plasma. Figure 3. Average blood drug concentration-time curve of rats after intragastric administration of 100 mg / kg OA-17. Figure 4. Changes in skin phenotype of mice in each group. Figure 5. Effects of OA-17 and OA on PASI scores of mice in each group, including erythema, scales, thickness and cumulative score (n=4, Mean±SD) (*P<0.05 and **P<0.01OA-17 (25 mg / kg) vs. Model, #P<0.05 and ##P<0.01OA-17 (25 mg / kg) vs. OA (25 mg / kg)). Figure 6. Changes in spleen and spleen index of mice in each group (n=4, Mean±SD). Among them, **P<0.01 vs. Model, #P<0.05 OA-17 (25 mg / kg, 50 mg / kg, 100 mg / kg) vs. OA (25 mg / kg, 50 mg / kg, 100 mg / kg), &&P<0.01 vs. Normal). Figure 7. Pathological changes in the skin of mice in each group Figure 8. Expression of PCNA in the skin tissues of mice in each group. Figure 9. Effects of OA-17 and OA on IMQ-induced serum IL-17A levels in mice (n=4, Mean±SD). Among them, **P<0.01 and ***P<0.0001 vs. Model, ##P<0.01 and ###P<0.001 OA-17 (25 mg / kg, 50 mg / kg, 100 mg / kg) vs. OA (25 mg / kg, 50 mg / kg, 100 mg / kg), &&&P<0.001 Model vs. Normal. Figure 10. Effects of OA and OA-17 on the expression of il-17a and il-23 mRNA in IMQ-induced mouse skin tissue (n=4, Mean±SD). Among them, ***P<0.001 vs. Model, ###P<0.001 vs. OA (25 mg / kg, 50 mg / kg, 100 mg / kg), &&P<0.01 and &&&P<0.001 vs. Normal. Fig. 11. Molecular docking results of OA, OA-17 and SHP2. FIG. 12 . Effects of OA-17 and SHP2 inhibitor on the activity of mouse spleen T cells (n=4, Mean±SD) (**P<0.01 vs. ConA). Figure 13. Effects of OA-17 on the expression of SHP2 and PI3K / mTOR pathway-related proteins in skin tissue Figure 14. Effects of OA-17 on clinical manifestations of AA rats (n=6, Mean±SD). (A) Body weight; (B) Paw swelling; (C) Arthritis index; (D) Number of swollen joints; (E) Whole body score. (#P<0.05, ##P<0.01vs Normal; *P<0.05, **P<0.01vs AA model) Figure 15. Effects of OA-17 on spleen and thymus indexes of AA rats (n=6, Mean±SD). Among them, #P<0.05, ##P<0.01 vs Normal; **P<0.01 vs AA model. Figure 16. Effects of OA-17 on the expression of TNF-α and IL-1β in the serum of AA rats (n=6, Mean±SD), where **P<0.01 vs Normal; ##P<0.01 vs AA model. Figure 17. Effects of OA-17 on the expression of CRP, CCP and RF in AA rats (n=6, Mean±SD). **P<0.01 vs Normal; ##P<0.01 vs AA model. Figure 18. Th17 / Treg cell ratio in rat spleen (n=6, Mean±SD). **P<0.01 vs Normal; ##P<0.01 vs AA model). Figure 19. X-ray changes of the ankle joint and paw on the secondary side of AA rats. Figure 20. OA-17 inhibits MH7A cell migration and invasion, wherein (A) cell migration; (B) cell invasion. Figure 21. OA-17 regulates the expression of mitochondrial damage-related genes and related inflammatory factors mRNA in MH7A cells (n=6, Mean±SD). Among them, **p<0.01, ***p<0.001 vs TNF-α group. FIG. 22 OA-17 regulates the expression of mitochondrial damage-related proteins cGAS and STING in MH7A cells. Figure 23. Molecular docking results of OA-17 and TNFR1. DETAILED DESCRIPTION The technical solution of the present invention will be clearly described below in conjunction with the accompanying drawings. Obviously, the described embodiments are not all embodiments of the present invention, and all other embodiments obtained by ordinary technicians in the field without making creative work are within the protection scope of the present invention. Example 1: Preparation method of OA-17 (1) Take 200 mg of oleanolic acid, add 216.46 mg of the catalyst 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate and 152.56 μL of N,N-diisopropylethylamine, respectively, and react at 20-25°C for 1 hour. Then, add 29.56 mg of methylamine hydrochloride in a 1:1 ratio and stir at a constant speed for 6-15 hours. (2) The mixture of step 1 is dissolved in an organic solvent, saturated aqueous NaCl solution and saturated aqueous potassium hydrogen sulfate solution are added, and the mixture is extracted with ethyl acetate for 2-3 times. The organic phase is collected, concentrated by a rotary evaporator, and silica gel powder is added for rotary evaporation. The obtained solid powder is subjected to silica gel column chromatography. A mixture of petroleum ether and ethyl acetate in a volume ratio of 3:1 and 1:1 is used as an eluent for gradient elution to obtain a purified solution of the target product, which is concentrated and vacuum dried to obtain the target product. The yield is 82%. Example 2: Acute toxicity study of OA-17 1. Experimental Methods 7-8 weeks old C57BL / 6 mice weighing 20±2g were selected and randomly divided into 3 groups, namely CMC-Na group, OA-17 (1g / kg) group, and OA-17 (10g / kg) group, with 10 mice in each group. OA-17 / CMC-NA was gavaged twice within 24 hours, 0.2mL each time, and observed for 7 days. The behavior, hair, mental state, etc. of the mice were observed twice a day. The amount of water and food consumed by the mice in each group was recorded daily, and the weight of the mice was weighed. On the 4th day, the obtained heart, liver, kidney, and spleen were washed in PBS buffer, dehydrated on filter paper, and the spleen index was calculated. It was placed in 4% formalin solution and soaked for 24 hours, and then sliced ​​for HE staining and observed under a microscope. Peripheral blood (50μL) was collected for biochemical index detection, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), and urea nitrogen (BUN), and the effects of OA-17 on the liver and kidney were observed. 2. Experimental results During the observation period, the oral administration group had no erect hair, no changes in body color, normal movement, no spasms, ataxia, no convulsions, normal body reflexes, smooth fur, no abnormal secretions in the eyes, nose, and mouth, and the feces and urine were normal compared with the control group. There was no obvious shrinkage or dilation of the pupils, and the eye examination signs such as protrusion were all normal. No mice died in all groups within 7 days of observation. The weight of mice in each group changed to varying degrees with the increase in feeding time, but no obvious abnormalities were observed. After the observation period, the mice were killed, and the results of macroscopic observation and recording showed that there were no obvious abnormalities in the main tissues and organs of the mice in the oral administration group, such as the heart, liver, spleen, lung, and kidney. As shown in Figure 1, the main histopathological staining results showed that the cell structure and morphology of the liver lobules in the drug-treated group were normal, without lesions or abnormalities, and the germinal center, marginal zone and red and white pulp junction in the spleen were normal. At the same time, the spleen tissue structure of the drug-treated group was basically the same as that of the control group, the lung tissue structure was clear, without edema and effusion, the alveolar structure was normal, the alveolar septa were slender and not widened, and the glomerular and renal unit cell structures were normal. Biochemical index detection showed that the ALT, AST, and BUN indicators of the mice in the drug-treated group were normal, and OA-17 did not affect the liver and kidney functions (Table 1). Table 1. Effects of OA-17 on blood biochemical parameters of mice Example 3: Pharmacokinetic study of single intragastric administration of OA and OA-17 1. Experimental Methods (1) Construction of standard curve Take a healthy male SD rat, take blood from the ocular venous plexus and place it in a 1.5mL centrifuge tube pre-filled with 20μL heparin sodium solution, centrifuge at 3500rpm and 4℃ for 10min, accurately draw 50μL of the upper plasma, add OA-17 standard reference solution with concentrations of 160, 320, 640, 1280, and 2560ng / mL, respectively, and mix for 1min. Then add 5μL of 100ng / mL internal standard reference solution, mix for 1min, then add 200μL acetonitrile, mix for 2min, centrifuge at 14000rpm for 10min, take 200μL of supernatant in a 1.5mL centrifuge tube, add 800μL acetonitrile to dilute, filter, and take 2μL for injection analysis. (2) Detection of blood drug concentration and calculation of pharmacokinetic parameters after single oral administration of OA-17 in rats Three healthy male SD rats were fasted for 12 hours and then intragastrically administered with OA-17 at a dose of 100 mg / kg. Blood was collected from the eye sockets at 10, 20, 40, 60, 120, 180, 240, 360, 480, 600, 720, and 1440 min after administration. About 200 μL of blood was collected from each rat at each time point and placed in a 1.5 mL centrifuge tube pre-filled with 20 μL of heparin sodium solution. The blood was centrifuged at 3500 rpm and 4°C for 10 min. 50 μL of the upper plasma was accurately drawn and placed in a 1.5 mL centrifuge tube and mixed for 1 min. 5 μL of 100 ng / mL internal standard control solution was added and mixed for 1 min. Then 200 μL of acetonitrile was added and mixed for 2 min. The supernatant was centrifuged at 14000 rpm for 10 min, 200 μL was placed in a 1.5 mL centrifuge tube, 800 μL of acetonitrile was added for dilution, and 2 μL was taken for injection analysis. 2. Experimental results (1) Construction of standard curve The concentration of OA-17 in the sample (ng / mL) was used as the horizontal axis, and the peak area ratio of OA-17 to the internal standard was used as the vertical axis. The weighted least square method (W = 1 / C 2 ) to obtain a standard curve, and the relative standard deviation (RSD) should be less than 20%. The results show that this method has good linearity in the range of 1-1000ng / mL, and Figure 2 is the standard curve. (2) Detection of blood drug concentration and calculation of pharmacokinetic parameters after single oral administration of OA-17 in rats The average blood drug concentration-time curve of rats given 100 mg / kg by oral gavage is shown in Figure 3. The measured data were processed by two-compartment model analysis using DAS 3.0 pharmacokinetic software to obtain the main pharmacokinetic parameters of OA-17 in rats under extravascular administration. The results are shown in Table 2. Table 2 Pharmacokinetic parameters of OA-17 in rats after administration Example 4: Effects of OA and OA-17 on Imiquimod-induced Psoriasis Model in Mice 1. Experimental Methods After one week of adaptive feeding, BALB / c mice weighing 20±2g were anesthetized by inhalation of 1.5% isoflurane, and the hair on the back of the mice was removed with a depilatory cream to form a 2cm×3cm bare area. After depilation, the mice were randomly divided into 9 groups, namely the normal group, the model group, the OA low, medium and high dose group (25mg / kg, 50mg / kg, 100mg / kg), the OA-17 low, medium and high dose group (25mg / kg, 50mg / kg, 100mg / kg), and the positive drug MTX (2mg / kg) group, with 3 mice in each group. First, the mice in each drug group were pre-medicated, and the OA low, medium and high dose group, the OA-17 low, medium and high dose group and the MTX group were given the pre-prepared drug suspension, among which the OA and OA-17 groups were given oral gavage once a day, and the MTX group was given oral gavage once every 2 days, and 0.2mL was given each time. The normal group and the model group were given an equal amount of CMC-Na by oral gavage. On the 6th day after intragastric administration, 5% Imiquimod (IMQ) cream 62.5 mg was applied to the exposed skin of mice in the model group, OA low, medium and high dose groups (25 mg / kg, 50 mg / kg, 100 mg / kg), OA-17 low, medium and high dose groups (25 mg / kg, 50 mg / kg, 100 mg / kg), and positive drug MTX (2 mg / kg) groups, and the same amount of vaseline cream was applied to the back of mice in the normal group at a fixed time every day. Until the 12th day, the mice in each group were weighed and killed by dislocation. 2. Evaluation indicators (1) Psoriasis Area and Severity Index (PASI): The skin lesions were scored, and the changes in the skin lesions were recorded with a camera every day. The mice were given a score of 0-4 for the degree of erythema, scaling, and thickening of the skin lesions, and the total score was obtained by adding the three scores. The PASI scoring standard is as follows: none = 0 points; mild = 1 point; moderate = 2 points; severe = 3 points; extremely severe = 4 points; total score = scaling + thickening + erythema (0-12 points). (2) Spleen index = mouse spleen weight (mg) / mouse body weight (g) (3) Pathological observation of mouse skin tissue and detection of epidermal thickness: The skin on the back of the mouse was cut with clean scissors and divided into four pieces. One piece of skin was placed in 4% paraformaldehyde for dehydration, paraffin embedding, sectioning, and HE staining. The pathological changes of mouse skin tissue were observed under a microscope. HE-stained sections were selected for observation and photography under a microscope to analyze the thickening of the epidermis. (4) Immunohistochemical observation of epidermal cell proliferation: A piece of skin tissue from the back of each mouse was fixed in pre-prepared 4% formaldehyde, embedded in paraffin, and sectioned. After dewaxing and antigen retrieval, it was labeled with immunohistochemical marker antibody PCNA. After sealing, the slices were observed under a microscope to analyze the proliferation of epidermal cells in the skin of each group. (5) ELISA method for detecting IL-17A levels in mouse serum: After killing mice by cervical dislocation, blood was collected from the eyeballs and placed in EP tubes. The tubes were placed in an ice box for 2 h and centrifuged at 4°C (2000 rpm, 10 min). The supernatant was taken as mouse serum. The ELISA method was used for detection according to the instructions of the kit. (6) qPCR detection of IL-17a and IL-23 mRNA expression in mouse skin tissue: RNA from the skin tissue of each group of mice was extracted using the Trizol method. -ΔΔCT The relative quantitative analysis of il-17a and il-23 mRNA in the skin tissue of each group of mice was performed by the method. 3. Experimental results (1) Psoriasis Skin Phenotype and Severity Index (PASI) After modeling, the mice were photographed and scored every day. As shown in Figure 4, the skin of normal mice was smooth and delicate on d6-12; IMQ-induced mice showed slight scaling and slight redness on d6, and the skin lesions gradually worsened on d7-12, and the scaling was densely distributed and thickened, and the erythema color deepened. Compared with the model group mice, the distribution of dandruff on the back of the OA (25 mg / kg, 50 mg / kg) medication group was slightly improved without significant improvement. OA (100 mg / kg), OA-17 (25 mg / kg, 50 mg / kg, 100 mg / kg) and MTX (2 mg / kg) can significantly improve the erythema, scaling and thickening of the mouse skin. As shown in Figure 5, the scaling, erythema, thickening and total scores of the normal group mice on d6-12 were all 0; the scaling, thickening, erythema and total scores of the skin of the IMQ-induced model group mice continued to increase on d6-12; there was no significant difference between the OA (25 mg / kg, 50 mg / kg) group and the model group, and the scaling and thickening of the back skin of the mice in the OA (100 mg / kg) group were slightly improved compared with the model group, and the scaling, erythema, thickening and total scores were reduced on d12. The skin scaling, thickening, erythema and total scores of mice in the OA-17 (25 mg / kg, 50 mg / kg, 100 mg / kg) and MTX (2 mg / kg) groups continued to increase on d6-11, but the values ​​were lower than those in the model group. On d12, OA-17 (25 mg / kg, 50 mg / kg, 100 mg / kg) and MTX (2 mg / kg) could significantly reduce the IMQ-induced skin scaling, thickening, erythema and total scores of mice. (2) Spleen index measurement results As shown in Figure 6, compared with the normal group mice, the spleen of the model group mice was significantly enlarged, and the spleen index was significantly increased, indicating that the model was successfully established; compared with the model group mice, the spleen enlargement of the OA-17 (25mg / kg, 50mg / kg, 100mg / kg) and MTX (2mg / kg) groups was significantly alleviated, and the spleen index was significantly reduced, and the larger the dose of OA-17 group, the smaller the spleen index. However, compared with the model group, the spleen of the OA (25mg / kg, 50mg / kg) group did not show a significant decrease, but the spleen of the OA (100mg / kg) group was significantly reduced compared with the model group, and the spleen index was also significantly reduced. It shows that OA (100mg / kg), OA-17 (25mg / kg, 50mg / kg, 100mg / kg) and MTX (2mg / kg) all have therapeutic effects on IMQ-induced psoriasis mice, but OA-17 (100mg / kg) has the best therapeutic effect, second only to the positive control drug MTX. (3) Observation of mouse skin tissue pathology and detection of epidermal thickness The skin of each group of mice was removed and stained with HE. The pathological changes of skin tissue were observed under a microscope. As shown in Figure 7, the skin tissue structure of the normal group of mice was normal, and there were only two to three layers of epidermal cells. Compared with the normal group of mice, the epidermis of the IMQ-induced model group mice was significantly thickened, capillary hyperplasia and inflammatory cell infiltration were observed in the superficial epidermis, and parakeratosis was observed in the stratum corneum. The acanthosis was thickened with epidermal protrusions extending downward. Compared with the model group, the epidermal thickness, parakeratosis, capillary hyperplasia and inflammatory cell infiltration of the OA (100 mg / kg), OA-17 (25 mg / kg, 50 mg / kg, 100 mg / kg) and MTX (2 mg / kg) groups were significantly reduced, while there was no significant difference between the OA (25 mg / kg) and OA (50 mg / kg) groups and the model group. (4) Immunohistochemical observation of epidermal cell proliferation Proliferating Cell Nuclear Antigen (PCNA) can reflect the proliferation of keratinocytes. Immunohistochemical positive expression is located in the cell nucleus and is brown-yellow. As shown in Figure 8, PCNA in the normal group of mice is only arranged linearly in the basal layer of the epidermis; compared with the normal group, the number of PCNA-positive cells in the model group of mice increased significantly; compared with the model group, the number of PCNA-positive cells in the OA (100 mg / kg), OA-17 (25 mg / kg, 50 mg / kg, 100 mg / kg) and MTX (2 mg / kg) groups was significantly reduced, while there was no significant difference between the OA (25 mg / kg) and OA (50 mg / kg) groups and the model group. (5) ELISA method to detect mouse serum IL-17A levels IL-17A is an important inflammatory cytokine in patients with psoriasis and is involved in the occurrence of psoriasis. The level of IL-17A in mouse serum was detected using an ELISA kit. As shown in Figure 9, compared with the normal group of mice, the expression of IL-17A in the serum of the model group mice was significantly increased; compared with the model group, the serum IL-17A levels of mice in the OA (100 mg / kg) and OA-17 (25 mg / kg, 50 mg / kg, 100 mg / kg) and MTX (2 mg / kg) groups were significantly downregulated, and the treatment effect of the OA-17 (100 mg / kg) group was the most obvious among the OA-17 administration groups, second only to the positive control drug MTX group. (6) Detection of IL-17a and IL-23 mRNA expression in mouse skin tissue by qPCR The expression of il-17a and il-23 mRNA in mouse skin tissue was detected by qPCR. As shown in Figure 10, compared with normal mice, the expression of il-17a and il-23 mRNA in the skin tissue of the model group mice was significantly increased; compared with the model group mice, there was no significant change in the expression of il-17a and il-23 mRNA in the OA (25 mg / kg, 50 mg / kg) group; compared with the model group mice, OA (100 mg / kg) and OA-17 (25 mg / kg, 50 mg / kg, 100 mg / kg) and MTX (2 mg / kg) can significantly reduce the expression of il-17a and il-23 mRNA, and the effect of OA-17 (100 mg / kg) group in the OA-17 administration group is the most significant. This can all show that OA-17 can relieve psoriasis symptoms by inhibiting the expression of various inflammatory factors and cytokine mRNA. Example 5: Study on the mechanism of OA-17 in treating psoriasis 1. Experimental Methods (1) Discovery Studio molecular docking technology was used to explore the binding ability of OA and OA-17 with SHP2: 4OHL human non-receptor type 11 protein tyrosine phosphatase protein subunits were retrieved from the RCSB PDB website and imported into the Discovery Studio software for processing, including removal of water molecules, side chain repair, main chain end processing, and determination of active pockets. The chemical structure of OA-17 was drawn using ChemDraw software and imported into the Discovery Studio small molecule window to prepare ligand molecules. After successful preparation, CDOCKER in the Discovery Studio software was used to perform simulated molecular docking experiments between small molecules and proteins, and the docking results of OA and OA-17 with proteins were analyzed respectively. (2) Effects of OA-17 and SHP2 inhibitors on mouse splenic T cell activity: Normal C57 BL / 6 mouse spleens were isolated in a clean bench, washed with 75% ethanol, and half of them were placed in a culture dish. 5 mL of mouse lymphocyte separation solution was added, and the spleen was wrapped with sterile gauze and fully ground with a homogenizer to prepare a splenic lymphocyte suspension. 3 × 10 6 The spleen lymphocyte suspension collected in vitro was divided into control group, ConA (5ng / mL) stimulation group, OA-17 (25mg / kg, 50mg / kg, 100mg / kg group), and SHP2 inhibitor group. The CCK8 method was used to detect the lymphocyte activity of each group after 24h of culture. (3) Effect of OA-17 on the expression of SHP2 and PI3K / mTOR pathway-related proteins in skin tissue: 1% protease inhibitor (PMSF) and 1% phosphatase inhibitor were added to the RIPA lysis buffer and mixed well. 50 mg of mouse skin from the normal group, MTX group, and OA-17 (100 mg / kg, 50 mg / kg, and 25 mg / kg) group was weighed and added to 500 μL of the prepared lysis buffer for thorough grinding. After centrifugation at 12000 g for 15 min at 4°C, the supernatant was aspirated. After BCA quantification and addition of loading buffer to denature it, the expression of SHP2, PI3K, mTOR, and p-mTOR proteins in the skin of each group of mice was detected by Western blot. 2. Experimental results (1) Molecular docking technology showed that OA-17 had a stronger affinity with SHP2 than OA Figure 11 shows the molecular docking results of OA and OA-17 with SHP2, where the green bonds are hydrogen bonds, which are a type of intermolecular force and a force between permanent dipoles. Hydrogen bonds are particularly important for biopolymers, as they are part of the reason why the secondary, tertiary and quaternary structures of proteins and nucleic acids are stabilized. Therefore, the more hydrogen bonds there are in the molecular docking results, the stronger the affinity between the two molecules. As shown in Figure 11, there are three hydrogen bonds between OA-17 and SHP2, while there is only one hydrogen bond between OA and SHP2. This may be able to preliminarily explain that the affinity of OA, i.e. OA-17, after being modified by the present invention, to the target SHP2 is stronger. (2) OA-17 and SHP2 inhibitors inhibit abnormal proliferation of mouse splenic T cells As shown in Figure 12, compared with the Control group, the T cell activity in the ConA stimulation group was significantly enhanced, and compared with the ConA stimulation group, the spleen T cell activity in the OA-17 (10 μM) and SHP2 inhibitor groups was significantly reduced. This indicates that OA-17 and SHP2 inhibitors can inhibit the abnormal proliferation of T cells. (3) OA-17 inhibits the expression of SHP2 and PI3K / mTOR pathway-related proteins in skin tissue As shown in Figure 13, compared with the normal group of mice, the expression of PI3K / AKT related proteins and SHP2 proteins in the skin of the model group mice was significantly increased; compared with the model group mice, the expression of PI3K / mTOR related proteins and SHP2 proteins in the skin of the OA-17 (25mg / kg, 50mg / kg, 100mg / kg) and MTX (2mg / kg) groups mice was significantly reduced. This shows that OA-17 can alleviate psoriasis by inhibiting the expression of SHP2 and PI3K / mTOR. Example 6: Effects of OA and OA-17 on AA rat model 1. Experimental Methods Use 0.1 mol / L glacial acetic acid solution to dissolve chicken type II collagen to a final concentration of 2 mg / mL, pipette and blow to fully dissolve it, and place it in a refrigerator at 4°C overnight. The next day, take out the prepared collagen solution, mix it with an equal volume of complete Freund's adjuvant, and continue to grind. You can use a 1 mL syringe without a needle to repeatedly blow and beat instead of grinding, so that the mixed liquid is fully emulsified to a milky white color. The rat's right hind paw was disinfected with a 75% ethanol cotton ball, 0.1 mL of the mixture was drawn with a 1 mL syringe, and the needle was inserted from bottom to top into the rat's paw for intradermal injection. A bulge was formed after injection, and pressure was applied to prevent outflow. The same amount of saline was injected into the paw of the normal group of rats as a control; the day of modeling was d0, and inflammatory symptoms began to appear after d14-d16. According to the overall evaluation after modeling, the rats were randomly divided into four groups: model group, OA-17 (25 mg / kg) group, OA-17 (50 mg / kg) group, OA-17 (100 mg / kg) group, and a normal control group, with 8 rats in each group. The rats were weighed before administration, and the daily dosage of each mouse was calculated according to the dosage. The administration started from the 17th day to the 30th day. The normal group was given the corresponding saline as a control. After the administration cycle, the rats were anesthetized and killed, and the relevant indicators were observed and recorded. 2. Evaluation indicators (1) Rat weight After modeling (from day 0), the same experimenter weighed and recorded the weight of rats in each group every 3 days. (2) Number of swollen joints and arthritis index score After modeling (from day 0), the same experimenter scored the paw lesions of rats in each group every 3 days according to the scoring criteria, and counted the number of swollen joints. Each rat's toe counted 5 finger joints and 1 ankle joint, with a maximum of 24 swollen joints, and a maximum score of 24 points. At the same time, the same experimenter evaluated the arthritis index of the ankle joints of rats in each group every 3 days according to the scoring criteria, with a maximum score of 16 points for each rat. The scoring details are as follows: Table 2. Arthritis index scoring criteria (maximum 16 points per rat) Table 3. Joint swelling number scoring criteria (maximum 24 points per rat) (3) Measurement of the secondary paw volume Before modeling, the volume of the left hind paw (non-inflammatory side) of the rats was measured and recorded as the initial value. Starting from the 16th day, the same experimenter measured the volume of the left hind paw of each group of rats every 3 days. Each rat was measured 3 times in a row using a toe volume meter. The average value was the joint volume, and the swelling degree was obtained by subtracting the corresponding initial value when calculating. (4) Calculation of rat spleen and thymus index The rats were deprived of food the night before being killed, and the weight of each rat was weighed and recorded before the experiment. The rats were killed after anesthesia, and the thymus and spleen tissues of the rats were removed in a clean bench, peeled clean and placed in pre-labeled sterile centrifuge tubes (the centrifuge tubes need to be weighed in advance), and the total weight was weighed on an electronic balance. The thymus or spleen index of each rat was the ratio of the net weight of the thymus or spleen to the corresponding body weight. (5) Detection of inflammatory factors and related reaction proteins in rat serum by enzyme-linked immunosorbent assay Femoral artery blood was collected from each rat, allowed to stand for 2 hours, centrifuged at 2500 r / min for 10 minutes, serum samples were collected, and frozen at -80°C for later use. FLS cell culture supernatants of rats in each group were collected, centrifuged at 2500 r / min for 10 minutes, supernatants were collected, and frozen at -80°C for later use. The levels of inflammatory factors and related reaction proteins in serum were detected according to the ELISA kit steps. (6) Effect of flow cytometry on Th17 / Treg immune balance in rats Place the spleen tissue on a gauze containing lymphocyte separation fluid, grind it evenly until the spleen cells are fully detached, rinse the spleen cells adhering to the filter with PBS, and collect the washed spleen cells. Centrifuge the prepared single-cell suspension at 500g for 5 minutes, remove the supernatant, and take the precipitate. Resuspend the cell pellet with 2mL of hemolysin and react at room temperature for 10 minutes to lyse the red blood cells. Centrifuge at 500g for 5 minutes, remove the supernatant, and take the precipitate. Add 2mL of PBS to wash the cell pellet, resuspend it and centrifuge it at 500g for 5 minutes, remove the supernatant, and take the precipitate. Use 1mL of RPMI culture medium to mix and resuspend the cells by blowing, dilute the cells at 1:10 or 1:20 times, and count the cells. Take 5×10 6 Each lymphocyte was stimulated, T cell co-stimulator was added, and cultured at 37℃ for 4h. After stimulation, centrifuge at 500g for 5min, discard the supernatant, add 5mL PBS to resuspend the cells, centrifuge at 500g for 5min, and discard the supernatant. Add 100μL PBS to resuspend the cells, add 1μL FITC-CD4 antibody, vortex to mix, and react at room temperature and away from light for 20min. Add 2mL cell fixative to each tube, incubate at room temperature and away from light for 20min, centrifuge at 600g for 5min, and discard the supernatant. Add 2mL cell permeabilization solution to each tube, incubate at room temperature and away from light for 10min, centrifuge at 600g for 5min, and discard the supernatant. Add 2mL cell permeabilization solution to each tube, centrifuge at 600g for 5min, and discard the supernatant. Add 1μL PE-IL17A antibody, vortex to mix, incubate at room temperature and away from light for 30min, add 2mL cell permeabilization solution to each tube, centrifuge at 600g for 5min, and discard the supernatant. Finally, add 300 μL PBS to each tube, vortex to mix, and then test on the instrument. (7) X-ray scanning of rat ankle joints to observe bone destruction X-ray imaging system (Leica, Germany) was used to perform X-ray imaging of the secondary ankle joints of AA rats. The rats in the experimental group were anesthetized by inhalation of isoflurane and placed on the imaging board. The GTI-2000 imaging system was used to photograph the secondary inflamed ankle joints. 3. Experimental results (1) Effects of OA-17 on clinical manifestations in rats On the 16th day after modeling, the body weight, arthritis index, number of paw swelling and degree of paw swelling of the rats were measured and recorded every 3 days. The results are shown in the figure below (Figure 14). From the 14th to 16th day after modeling, the paws of the rats began to swell and reached a peak around the 27th day. The body weight of AA model rats was significantly reduced compared with normal rats (P<0.01). Daily oral administration began on the 18th day after modeling. Compared with the model group, the arthritis index, number of paw swelling and degree of paw swelling in the OA-17 (50 mg / kg / day) group and the OA-17 (100 mg / kg / day) group were significantly reduced compared with the AA rat group on the 10th day after administration (the 26th day of modeling) (P<0.05). In summary, OA-17 can significantly improve the arthritis index, number of paw swelling and degree of paw swelling of AA rats. The results are shown in Figure 14 below, which shows the weight changes (Figure 14A), paw swelling (Figure 14B), arthritis index (Figure 14C), number of swollen joints (Figure 14D) and systemic score (Figure 14E) of AA rats. (2) Effects of OA-17 on spleen and thymus indexes in rats The results are shown in Figure 15. Compared with normal rats, the thymus and spleen of AA model rats were significantly enlarged and the index was increased; compared with the model group, the OA-17 administration group significantly reduced the spleen and thymus index of AA rats (P<0.01). (3) Detection of TNF-α and IL-6 levels in rat serum by enzyme-linked immunosorbent assay The ELISA method was used to detect the changes in the levels of inflammatory factors TNF-α and IL-6 in the serum of rats in each group. The results showed (Figure 16) that compared with normal rats, the serum IL-6 level of AA model rats increased significantly (P<0.01); compared with the AA group rats, the OA-17 administration group significantly reduced the serum IL-6 level (P<0.01). (4) Detection of CRP, CCP and RF levels in rats by ELISA ELISA was used to detect the changes in the levels of C-reactive protein (CRP), CCP (anti-cyclic citrullinated peptide antibody) and rheumatoid factor (RF) in rats of each group. The results showed (Figure 17) that compared with normal rats, the levels of CRP, CCP and RF in AA model rats were significantly increased (P<0.01); compared with AA group rats, the high-dose OA-17 group significantly reduced the levels of CRP, CCP and RF in rats (P<0.01). (5) Flow cytometry detection of the effect on Th17 / Treg immune balance in rat spleen As shown in Figure 18, the Th17 / Treg ratio in the OA-17 medium and high dose groups was significantly lower than that in the AA model group (P < 0.05), while there was no significant change in the low dose group compared with the AA group. (6) X-ray scanning of rat ankle joints to observe bone destruction AA model rats have swelling of soft tissues in the secondary joints, osteoporosis, decreased bone density, obvious bone erosion lesions, and narrowing or even disappearance of joint spaces. As shown in Figure 19, the OA-17 administration group was compared with the model group, and the symptoms of soft tissue swelling, bone erosion, and narrowing of joint spaces were alleviated, especially the medium and high dose administration groups were significantly different from the model group. Experimental Example 7: Study on the mechanism of OA-17 in treating arthritis 1. Experimental Methods (1) Transwell assay to evaluate the effect of OA-17 on the invasion ability of MH7A cells Matrigel glue (0.1%) was evenly applied on the bottom membrane of the upper chamber of Transwell, dried at 37°C for 30 min, and washed three times with PBS; cells were collected by trypsin digestion and blow-through, washed 1-2 times with PBS, resuspended in serum-free medium, and diluted with serum-free medium according to the required density; 600 μL of DMEM medium containing 20% ​​FBS was added to the lower chamber of the 24-well plate; 100 μL of cell suspension (1×10 5 The cells were co-incubated with TNF-α (20 ng / mL) and OA-17 (1 μM, 5 μM) for 24 hours; the upper chamber was carefully removed with tweezers and washed with PBS, fixed with 4% paraformaldehyde for 10 minutes, washed three times with PBS, stained with crystal violet dye for 5 minutes, soaked and washed with clean water several times, and the excess cells on the bottom membrane surface of the upper chamber were carefully wiped off with a wet cotton swab; 9 fields of view were randomly selected under an optical microscope for photography, and the invading cells were statistically analyzed. (2) Scratch assay to determine the effect of OA-17 on the migration ability of MH7A cells MH7A cells (5×10 5 / well) were inoculated in a 6-well plate. When the cells grew to cover the entire bottom surface, the serum-free medium was continued to be cultured for 12 hours to eliminate the influence of normal cell growth on the scratch test results; a 200 μL sterile pipette tip was used to form a scratch on the bottom of the well plate, and the scratched cells were gently washed 3 times with PBS to wash away the scratched cells; the cells were stimulated with TNF-α (20 ng / mL) and treated with different concentrations of OA-17 (1 μM, 5 μM) for 24 hours; the culture medium was aspirated, washed 3 times with PBS, and fixed with 4% paraformaldehyde for 20 minutes; the fixative was aspirated, washed 3 times with PBS, the cells were stained with crystal violet, washed with water, dried and photographed, and then the distance change of the scratch area was measured and analyzed. (3) OA-17 regulates the expression of cgas, sting and various inflammatory factors mRNA in MH7A cells MH7A cells (5×10 5 / well) were evenly inoculated in a six-well plate, and a blank control group (Control group), TNF-α stimulation group, and OA-17 (0.1 μM, 1 μM, 10 μM) groups were set up and cultured at 37°C in a 5% CO2 incubator for 48 h. Trizol was added to each well for lysis and RNA was extracted using 2 -ΔΔCT The method was used to quantitatively analyze the mRNA of cgas, sting and various inflammatory factors in MH7A. (4) OA-17 regulates the expression of mitochondrial damage-related proteins cGAS and STING in MH7A cells MH7A cells (5×10 5 / well) was added to a six-well plate and cultured for 12 hours, then TNF-α (20ng / mL) and OA-17 (1μM, 5μM) were added and co-cultured for 24 hours; the supernatant was removed and washed 3 times with pre-cooled PBS; RIPA lysis buffer and protease inhibitors were mixed in proportion, and an appropriate amount of the mixture was added to the well plate, and the plate was lysed on crushed ice for 30 minutes; the lysate was collected on ice, the centrifuge temperature was set to 4°C, and the centrifuge was centrifuged at 12000rpm for 10 minutes. After centrifugation, the supernatant was retained as the total protein sample. The total protein concentration of each group was detected using the microplate method in the BCA protein quantification kit, and a BSA standard curve was established. The total protein concentration was calculated according to the absorbance of the sample to be tested, and the total protein content was adjusted to the same concentration with PBS; the sample was mixed with 5×Loding buffer in a ratio of 4:1, boiled in boiling water for 5 minutes to denature the protein, and stored at -80°C. 2. Experimental results (1) Effects of OA-17 on the migration and invasion of MH7A cells The migration and invasion of MH7A cells were detected by scratch assay and Transwell chamber, and the results showed that OA-17 (1μM, 5μM) could inhibit the migration and invasion of MH7A cells in a concentration-dependent manner. In the scratch assay (Figure 20), the scratches prepared with the pipette tip in the normal group and the TNF-α group were almost filled with MH7A cells, while the migration ability of the OA-17 treatment group decreased. The scratches made by the pipette tip in the normal group and the TNF-α group were almost filled with MH7A cells, while the migration ability of the cells in the OA-17 (1μM, 5μM) treatment group decreased. At the same time, the Transwell results showed that OA-17 (1μM, 5μM) had a significant inhibitory effect on MH7A invasive cells compared with the TNF-α group (p<0.001) (Figure 20B). (2) Real-time fluorescence quantitative PCR (qPCR) was used to detect the content of cGAS, STING and related inflammatory factors. As shown in Figure 21, compared with normal MH7A cells, all tested inflammatory factors such as IL-1β and IL-6 were significantly increased under TNF-α stimulation (p<0.001), while OA-17 (0.1μM, 1μM, 10μM) significantly reduced the increased expression of cgas, sting and related inflammatory factors (p<0.01). This indicates that OA-17 can significantly inhibit the expression of cgas, sting and related inflammatory factor mRNA in MH7A. (3) OA-17 regulates the expression of mitochondrial damage-related proteins cGAS and STING in MH7A cells As shown in Figure 22, compared with the TNF-α group, the OA-17 treatment groups (0.1 μM, 1 μM, 10 μM) led to down-regulation of cGAS and STING expression, and the results of qPCR showed consistent expression of mRNA levels of related factors, confirming the regulatory effect of OA-17 on MH7A mitochondrial damage-related proteins. (4) Discovery Studio molecular docking technology analysis showed that OA-17 has a strong affinity with TNFR1 Figure 23 shows the molecular docking results of OA-17 and TNFR1, where the green bond is a hydrogen bond, which is a type of intermolecular force and a force between permanent dipoles. Hydrogen bonds are particularly important for biopolymers, and are part of the reason why the secondary, tertiary and quaternary structures of proteins and nucleic acids are stabilized. As shown in Figure 23, there is an intermolecular hydrogen bond between OA-17 and TNFR1, and the affinity is strong. This also proves that OA-17 can directly interact with TNFR1, thereby inhibiting the expression of downstream cGAS / STING related to mitochondrial damage by inhibiting TNFR1, and ultimately inhibiting the inflammatory changes of MH7A. The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. An oleanolic acid amide derivative having the effect of treating psoriasis, characterized in that: Its molecular structure is shown in formula (II):

2. The stereoisomer, solvate, prodrug, metabolite, pharmaceutically acceptable salt or co-crystal of the oleanolic acid amide derivative having the effect of treating psoriasis according to claim 1.

3. A pharmaceutical composition comprising the oleanolic acid amide derivative according to claim 1, or a stereoisomer, solvate, prodrug, metabolite, pharmaceutically acceptable salt or cocrystal thereof.

4. The pharmaceutical composition of claim 4, further comprising a pharmaceutically acceptable carrier.

5. Use of the oleanolic acid amide derivatives according to claim 1, or their stereoisomers, solvates, prodrugs, metabolites, pharmaceutically acceptable salts or co-crystals in the preparation of drugs for treating or preventing psoriasis.

6. The method for preparing the oleanolic acid amide derivatives according to claim 1, comprising the following steps: (1) Take the raw material oleanolic acid, the reactant methylamine hydrochloride, add the catalyst 2-(7-azobenzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate, and the catalyst N,N-diisopropylethylamine at 20-25° C., and stir at a constant speed for 6-15 hours; (2) dissolving the mixture in step 1 in an organic solvent, adding saturated aqueous NaCl solution and saturated aqueous potassium hydrogen sulfate solution, and extracting with ethyl acetate 2-3 times; (3) Collect the organic phase to obtain the target product.

7. The preparation method according to claim 6, characterized in that: In step (1), the equivalent ratios of oleanolic acid to methylamine hydrochloride, 2-(7-azobenzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate, and N,N-diisopropylethylamine are 1:1, 1:1.3, and 1:2, respectively.

8. The preparation method according to claim 6, characterized in that: In step (2), the organic solvent is ethyl acetate.

9. The preparation method according to claim 6, characterized in that: In step (3), the organic phase is collected, concentrated using a rotary evaporator, and silica gel powder is added and rotary evaporated to obtain a solid powder.

10. The preparation method according to claim 9, characterized in that: The obtained solid powder is purified by silica gel column chromatography; preferably, gradient elution is performed using a mixture of petroleum ether and ethyl acetate as the eluent to obtain a purified solution of the target product, which is concentrated and vacuum dried to obtain the target product; more preferably, the volume ratio of the eluent petroleum ether and ethyl acetate in the silica gel purification is 3:1 and 1:1.

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