Small molecule compound TII-C2 and application thereof in preparation of medicine for treating TRADD related autoimmune diseases

Abstract

The invention discloses a small molecule compound TII-C2 and an application thereof in preparation of a medicine for treating TRADD related autoimmune diseases. According to the present invention, the combination of the small molecule compound TII-C2 and TRADD can destroy the combination of TRADD and TRAF2, significantly inhibit the activation of a TNFR1 downstream signal channel, and inhibit the abnormal proliferation of fibroblast-like synovial cells, such that the clinical manifestation of an arthritis mouse model is improved, and the important reference is provided for the treatment of rheumatoid arthritis and TRADD-related autoimmune diseases; good application prospects are realized.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a small molecule compound TII-C2 and an application thereof in preparing a drug for treating TRADD-related autoimmune diseases. Background Art

[0002] Tumor necrosis factor (TNF) receptor type 1-associated death domain protein (TRADD) is a unique adaptor protein that plays a central role in multiple downstream signaling events, including cell survival and apoptosis, in different cellular contexts. TRADD contains an N-terminal TNF receptor-associated factor 2 (TRAF2) binding domain and a C-terminal death domain (DD). TRADD is recruited to TNF receptor 1 (TNFR1) in a TNF-dependent manner, serving as a platform for recruiting other proteins. TRADD-N-terminus interacts with TRAF2 and recruits TRAF2 to TNFR1, thereby activating the nuclear factor-κB (NF-κB), c-Jun N-terminal kinase (JNK), and mitogen-activated protein kinase (MAPK) pathways, resulting in pro-inflammatory, pro-proliferative, and anti-apoptotic responses. On the other hand, when NF-κB activation is inhibited, TRADD binds to receptor-interacting protein kinase 1 (RIPK) and FAS-associated death domain protein (FADD), initiating the apoptotic cascade. Given the key role of TRADD in regulating cell survival, inflammation, and apoptosis, TRADD has been considered a promising drug target for the treatment of human diseases. However, the research on TRADD inhibitors is still at the basic research stage, and there is still a long way to go before practical application.

[0003] Autoimmune diseases are a type of disease caused by the body's immune response to self-antigens, resulting in damage to its own tissues. TNF-α is overproduced in some autoimmune diseases, such as rheumatoid arthritis (RA), psoriasis, and Crohn's disease (CD), leading to joint erosion, fibrosis, and structural formation. The sustained activation of TNF-α / TNFR1 signaling mainly promotes inflammation and tissue degeneration. TRADD is a core protein for downstream signaling of TNFR1. The binding of TRADD to TRAF2 to transmit downstream signals of TNFR1 may be one of the initiation mechanisms of autoimmune diseases. RA is a typical autoimmune disease characterized by synovitis, which leads to joint deformity and even loss of joint function. Fibroblast-like synoviocytes (FLSs) are one of the main cell types in the synovial tissue of RA patients and are key effector cells in the pathogenesis of RA. FLSs tumor-like abnormal proliferation, invasion, and migration lead to persistent synovial inflammation and destruction of cartilage and bone tissue, promoting joint deformity and loss of function. FLSs are one of the main cell types in the synovial tissue of RA patients and play a key role in the pathogenesis of RA. Therefore, how to effectively inhibit the abnormal proliferation of FLSs has important clinical significance for the treatment of RA and provides a feasible direction for the development of drugs for the treatment of RA.

[0004] Currently, biologics, such as TNF-α and interleukin (IL)-6 inhibitors, are used to treat a variety of autoimmune diseases, such as RA, ankylosing spondylitis, psoriasis, ulcerative colitis, and CD, and have changed the original treatments for some diseases. However, about 20-30% of patients do not respond to these biologic therapies, and long-term use of biologics can also bring many adverse reactions, including increased risk of serious infections and malignant tumors. In addition, biologics are expensive and some patients cannot afford them. Adverse reactions and cost issues limit the clinical application of anti-TNF-α biologics. In addition, due to the presence of multiple heterogeneous factors and complex immune-inflammatory pathological mechanism networks in autoimmune diseases, existing therapies have limited clinical efficacy in some patients. Therefore, new targets and treatment strategies for autoimmune diseases still need to be identified. Summary of the invention

[0005] In view of the deficiencies of the above-mentioned prior art, the present invention provides a small molecule compound TII-C2 and its use in the preparation of a drug for treating TRADD-related autoimmune diseases. The small molecule compound TII-C2 of the present invention can bind to TRADD, inhibit the binding of TRADD-TRAF2, inhibit the activation of the downstream signaling pathway of TNFR1 and the abnormal proliferation of FLSs. TII-C2 has a good inhibitory effect on the inflammatory immune response of the delayed-type hypersensitivity (DTH) model in mice. In addition, TII-C2 inhibits the abnormal proliferation of FLSs and restores immune homeostasis, improving the clinical manifestations of the collagen induced arthritis (CIA) model in mice.

[0006] The structure of the small molecule compound TII-C2 of the present invention is shown below:

[0007]

[0008] Application of the small molecule compound TII-C2 of the present invention in preparing medicine for treating TRADD-related autoimmune diseases.

[0009] The TRADD-related autoimmune diseases include but are not limited to RA, systemic lupus erythematosus, Syndrome, systemic sclerosis, psoriasis, pemphigus, inflammatory bowel disease, myasthenia gravis, Hashimoto's thyroiditis, primary antiphospholipid antibody syndrome, multiple sclerosis, idiopathic optic neuritis and neuromyelitis optica spectrum disease, any one or a combination of several thereof, preferably RA.

[0010] The small molecule compound TII-C2 of the present invention acts as a TRADD inhibitor to disrupt the binding of TRADD and TRAF2, selectively inhibits the activation of the downstream signaling pathway of TNFR1, inhibits the abnormal proliferation of RAFLSs, and exerts an anti-proliferative effect. There are currently no relevant reports. In the joints of RA patients, the abnormal inflammatory environment promotes the abnormal activation of the TNFR1 signaling pathway in FLSs, leading to tumor-like malignant proliferation of FLSs. The small molecule compound TII-C2 in the present invention acts as a TRADD inhibitor to inhibit the abnormal proliferation of RAFLSs. The specific mechanism is that TII-C2 and TRADD disrupt the mutual binding of TRADD and TRAF2, inhibit the activation of the downstream signaling pathway of TNFR1, and thus inhibit the abnormal proliferation of RAFLSs.

[0011] The small molecule compound TII-C2 of the present invention has a good inhibitory effect on the inflammatory immune response of the delayed hypersensitivity reaction model of mice. In addition, TII-C2 effectively inhibits the proliferation of fibroblast-like synoviocytes, restores immune balance, and improves the clinical manifestations of the arthritis mouse model.

[0012] Due to the adoption of the above technical solution, the present invention has the following advantages and beneficial effects:

[0013] The present invention first discovered that a small molecule compound TII-C2 binds to TRADD and exerts an immune anti-proliferative effect. The present invention verifies at the cellular level that TII-C2 destroys the mutual binding of TRADD and TRAF2, verifies at the cellular level that TII-C2 inhibits the proliferation and migration of MH7A cells, and verifies at the animal level that TII-C2 has an immunosuppressive ability.

[0014] The present invention first discovered that TII-C2 exerts anti-proliferative activity by inhibiting the downstream signaling pathway of TNFR1, and exhibits minimal toxicity in animals, and can be used as a combination drug in clinical practice. Therefore, TII-C2 is expected to become a new and effective means of treating autoimmune diseases.

[0015] In order to study the pharmacological effects of TII-C2, the present invention established a DTH model on BALB / c mice, and treated the mice with sodium carboxymethylcellulose, TII-C2 (25 mg / kg, 50 mg / kg, 75 mg / kg, 100 mg / kg) and prednisone group (5 mg / kg). The results showed that TII-C2 can effectively reduce ear swelling, spleen and thymus index of DTH.

[0016] The present invention further uses DBA / 1 mice to establish a CIA model, and uses sodium carboxymethylcellulose, TII-C2 (80 mg / kg) and etanercept (5 mg / kg) to treat mice. The results show that TII-C2 can inhibit lymphocyte proliferation under inflammatory conditions, regulate the ratio of T cell subsets (Th1 / Th2 and Th17 and Treg), inhibit the release of proinflammatory cytokines such as IL-1β, IL-17 and TNF-α, enhance the expression of anti-inflammatory cytokine IL-10, and improve the spleen and joint pathology of CIA mice, significantly restore the weight of CIA mice, reduce the overall score, arthritis index and arthritis swelling number and other overall indicators, and improve the clinical manifestations of CIA mice. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more clear.

[0018] Figure 1The synthetic route of TII-C2 in Example 1

[0019] Figure 2 NMR spectrum of TII-C2 in Example 1.

[0020] Figure 3 This is a schematic diagram of the results of detecting the affinity of TII-C2 and TRADD-N-terminal protein using surface plasmon resonance technology in Example 2 of the present application.

[0021] Figure 4 This is a schematic diagram of the effect of TII-C2 on MH7A cell viability detected by the CCK-8 method in Example 3 of the present application.

[0022] Figure 5 This is a schematic diagram of the results of detecting the effect of TII-C2 on MH7A cell proliferation using high-content cell imaging technology in Example 4 of the present application (AD).

[0023] Figure 6 This is a schematic diagram of the results of Transwell assay for detecting the effect of TII-C2 on MH7A cell migration in Example 5 of the present application (AC).

[0024] Figure 7 This is a schematic diagram of the results of immunoblotting to detect the effect of TII-C2 on the phosphorylation expression of NF-κB pathway members (IKKα / β, IκBα and p65) in Example 6 of the present application.

[0025] Figure 8 This is a schematic diagram of the results of immunoblotting to detect the effect of TII-C2 on JNK and p38 phosphorylation expression in Example 6 of the present application.

[0026] Fig. 9 This is a schematic diagram of the results of immunoprecipitation detection of the effect of TII-C2 on the binding of TRADD and TRAF2 in Example 7 of the present application.

[0027] Fig.10 This is a schematic diagram of the therapeutic indicators of TII-C2 in Example 8 of the present application on body weight (A), spleen index (B) and thymus index (C) of DTH mice.

[0028] Fig.11 Schematic diagram of the therapeutic indicators of TII-C2 on the ears (A), ear swelling (B) and ED50 (C) of DTH mice in Example 9 of the present application.

[0029] Fig.12 This is a schematic diagram of the therapeutic effect of TII-C2 on the paws of CIA mice in Example 10 of the present application.

[0030] Fig.13This is a schematic diagram of the therapeutic indicators of Example 11 TII-C2 for CIA mice, including body weight (A), systemic score (B), arthritis index (C) and arthritis swelling number (D).

[0031] Fig.14 This is a schematic diagram of the treatment results of TII-C2 on spleen pathology (A) and pathological score diagram (B) of CIA mice in Example 12 of the present application.

[0032] Fig.15 This is a schematic diagram of the results of flow cytometry detection of helper T cell (T helper, Th) subsets in the spleen of each group of mice in Example 13 of the present application. Among them, Figure A is Th1 cells, Figure B is Th2 cells, Figure C is Th17 cells, and Figure D is Treg cells.

[0033] Fig.16 Schematic diagram of the treatment results of TII-C2 on knee joint pathology (A) and pathological score diagram (B) of CIA mice in Example 14 of the present application.

[0034] Fig.17 This is a schematic diagram of the results of enzyme-linked immunosorbent assay (ELISA) for detecting IL-1β (A), IL-17 (B), TNF-α (C) and IL-10 (D) in the serum of each group of mice in Example 15 of the present application. DETAILED DESCRIPTION

[0035] The present invention will be further described below in conjunction with the accompanying drawings.

[0036] GraphPad Prism 8.0.0 software was used for statistics and analysis. Statistical results are expressed as mean ± standard deviation. The t-test was used to compare the differences between two groups, and one-way analysis of variance was used to compare the differences between multiple groups. Two-way analysis of variance followed by Tukey's multiple comparison test was used to compare the differences between multiple groups with 2 variables. P < 0.05 was considered statistically significant.

[0037] Example 1: Preparation process of TII-C2

[0038] After mixing 2H-benzo[b][1,4]oxazin-3(4H)-one with methyl 2-bromoacetate, NaH (2.0 equivalents) and DMF (0.2 equivalents) were added, and the temperature was controlled at 80°C for overnight reaction to obtain methyl 2-(3-oxo-2,3-dihydro-4H-benzo[b][1,4]oxazin-4-yl)acetate. The product was added with 2-bromoacetyl chloride, AlCl3 (3.0 equivalents) and DCM (0.2 mol), and the temperature was controlled at 0°C for overnight reaction under reflux to obtain methyl 2-(6-(2-bromoacetyl)-3-oxo-2,3-dihydro-4H-benzo[b][1,4]oxazin-4-yl)acetate. The above product was added with 2-propylphenol (2.0 equivalents), NaHCO3 (2.0 equivalents) and DMF (0.2 equivalents) to react overnight to obtain methyl 2-(3-oxo-6-(2-(2-propylphenoxy)acetyl)-2,3-dihydro-4H-benzo[b][1,4]oxazin-4-yl)acetate. The above product was mixed with LiOH (3.0 equivalents) and MeOH (0.2 mol), refluxed for 6 hours, and the HCl solution was adjusted to pH = 2-3 to obtain 2-(3-oxo-6-(2-(2-propylphenoxy)acetyl)-2,3-dihydro-4H-benzo[b][1,4]oxazin-4-yl)acetic acid. The above product was mixed with SOCl2 (2.0 equivalents) and CH2Cl2 (0.2 equivalents), catalyzed by DMF, and reacted at 0°C to room temperature for 4 hours to obtain 2-(3-oxo-6-(2-(2-propylphenoxy)acetyl)-2,3-dihydro-4H-benzo[b][1,4]oxazin-4-yl)acetyl chloride. The above product was added with pyridin-4-ylmethylamine (1.1 equivalents), Et2N (2.0 equivalents) and DMF (0.2 mol), and reacted at 0°C to room temperature for 4 hours. The solvent was recovered under reduced pressure and evaporated to dryness, and the final product 2-(3-oxo-6-(2-(2-propylphenoxy)acetyl)-2,3-dihydro-4H-benzo[b][1,4]oxazin-4-yl)-N-(pyridin-4-ylmethyl)acetamide was obtained by column chromatography. 1 HNMR(400MHz,Chloroform-d)δ8.47(s,2H),7.77-7.74(m,2H),7.21-7.07(m,5H),6.93(td,J=7.4,1.1Hz,1H),6.73(dd,J=8.2,1.2Hz, 1H),5.22(s,2H),4.79(s,2H),4.70(s,2H),4.49(d,J=6.0Hz,2H),2.68-2.64(m,2H),1.64(d,J=7.6Hz,2H),0.94(t,J=7.4Hz,3H); Molecular formula C 27 H 27 N3O5.

[0039] The results are shown in the attached figure. The synthetic route of TII-C2 ( Figure 1 ) and NMR spectra ( Figure 2 ).

[0040] Example 2: Surface Plasmon Resonance Technology to Detect the Affinity of TII-C2 and TRADD-N-Terminal Proteins

[0041] 1. Reagent preparation

[0042] PBS (10×), pH 7.4 was diluted 10 times with deionized water to prepare running buffer 1: containing 2 mM potassium dihydrogen phosphate (KH2PO4), 137 mM sodium chloride (NaCl), 10 mM sodium dihydrogen phosphate (Na2HPO4), 2.7 mM potassium chloride (KCl). Filter through a 0.22 μM filter membrane before use.

[0043] PBS (10×), pH 7.4 was diluted with deionized water and DMSO was added to prepare running buffer 2: containing 2 mM KH2PO4, 137 mM NaCl, 10 mM Na2HPO4, 2.7 mM KCl, 5% DMSO, and filtered through a 0.22 μM filter membrane before use.

[0044] Dilute 50 mM NaOH 100 times with deionized water to prepare 0.5 mM NaOH for later use.

[0045] 2. Chip preparation

[0046] SPR detection was performed on a Biacore T200 instrument. The ligand (TRADD-N-terminal protein) was diluted 20 times with a fixation reagent (10mM sodium acetate, pH 4.5). First, the surface of the CM5 chip was activated with 400mM EDC and 100mM NHS at a flow rate of 10μL / min for 900s. Secondly, the diluted ligand was injected into the experimental channel (Fc4) at a flow rate of 30μL / min, with a fixed amount of approximately 9000RU. Finally, the chip was blocked with 1M ethanolamine at 10μL / min for 900s. The reference channel (Fc3) was only activated and blocked, and no ligand was coupled. Ligand coupling was performed in running buffer 1.

[0047] 3. Multi-cycle analysis of analytes

[0048] The analyte (compound TII-C2) was diluted to 500 μM with PBS (10×), DMSO and deionized water, and then diluted with running buffer 2 at different concentrations. The diluted analytes were injected into the reference channel (Fc3) and the experimental channel (Fc4) at a flow rate of 30 μL / min, with a binding time of 60 s and a dissociation time of 60 s. The binding and dissociation steps were all performed in running buffer 2. After each concentration analysis, the chip was regenerated with 0.5 mM NaOH at a flow rate of 30 μL / min for 5 s to clean the undissociated analyte. The temperature of the chip compartment and the sample compartment was selected to be 25°C, and the affinity between the analyte and the ligand was measured.

[0049] The results are as follows Figure 3 As shown, the dissociation parameter of TII-C2 and TRADD-N-terminal protein is 17.4 μM, which is a medium binding strength.

[0050] Example 3: CCK-8 method to detect the effect of TII-C2 on MH7A cell viability

[0051] The fibroblast-like synoviocyte cell line MH7A from patients with rheumatoid arthritis was diluted to a cell suspension of 3-4×10 4 100 μL of cell suspension was added to each well of a 96-well plate. The initial number of cells per well was 3-4×10 3 The cells were divided into a normal group and a TII-C2 group and cultured for 24 hours. TII-C2 was given MH7A in 5 concentration gradients (100nM, 1μM, 10μM, 50μM, 100μM). After 24 hours of culture, CCK-8 solution (10μL) was added to each well and incubated for 0.5-4 hours. The absorbance of the plate at 450nm was detected using an ELISA reader.

[0052] The results are as follows Figure 4 As shown, the IC50 value of TII-C2 against MH7A cells was 57.89 μM.

[0053] Example 4: Detection of the effect of TII-C2 on MH7A cell proliferation using high-content cell imaging technology

[0054] After MH7A cell digestion, 100 μL of cell suspension was transferred to each well of a 96-well plate. The number of cells was approximately 3 × 10 3 / each well. Incubate overnight in a 37°C, 5% CO2 incubator. Treat with TNF-α (20 ng / mL) and compound TII-C2 (100 nM, 1 μM, 10 μM) for 24 h. Wash the plate twice with PBS and fix each well with 4% paraformaldehyde for 20 min. Wash three times with PBS and stain with DAPI for 5 min. After washing with PBS, take pictures of the cells using a high-content cell imaging system and count the number of cells in each well.

[0055] The results are as follows Figure 5 As shown in the figure, compared with the Control, the number of cells induced by TNF-α increased significantly and the proliferation ability was strong. Compared with the TNF-α group, TII-C2 (1μM and 10μM) significantly inhibited the proliferation ability of MH7A cells ( n=5). **P<0.01, ***P<0.001.

[0056] Example 5: Transwell assay to detect the effect of TII-C2 on MH7A cell migration

[0057] The migration ability of MH7A cells was detected by Transwell plate method. MH7A cells were trypsinized to prepare cell suspension. 3 ) were transferred to the upper chamber of the Transwell chamber. 500 μL of 10% FBS and TNF-α (20 ng / mL) or compound TII-C2 (100 nM, 1 μM, 10 μM) were added to the lower chamber (i.e., the 24-well plate) and cultured for 24 h. The chamber was washed twice with PBS and 0.25% crystal violet solution was added for 20-30 min. The chamber was washed 3 times with PBS and 5 fields of view were randomly selected for photography under an inverted microscope. Image J software was used to count the number of cells and calculate the migration rate. Migration rate = number of cells in the treatment group / number of cells in the blank control group.

[0058] The results are as follows Figure 6 As shown in the figure, compared with the Control, the number of cell migration induced by TNF-α was significantly increased. Compared with the TNF-α group, TII-C2 (1μM and 10μM) significantly inhibited the number of cell migration ( n=5). **P<0.01, ***P<0.001.

[0059] Example 6: Immunoblotting to detect the effect of TII-C2 on the activation of NF-κB, JNK and MAPK pathways

[0060] MH7A cells were seeded into 6-well plates and cultured for 284 hours before adding TII-C2 (10 μM) and culturing for 12 hours. TNF-α (20 ng / mL) was added for stimulation for different time periods (0 min, 5 min, 15 min, 30 min, 60 min). The supernatant was discarded and washed 2-3 times with PBS. 100-200 μL of lysis buffer (RIPA: PMSF: phosphatase inhibitor = 99:1:1) was added to each well and lysed for 30 minutes at 4°C. The lysate was transferred to an EP tube. Centrifuged at 4°C, 12000 g for 10 minutes, and the supernatant was retained. The BCA kit was used for protein quantification, and the supernatant was used for immunoblotting experiments to detect the expression of phosphorylation of NF-κB, JNK and MAPK pathway members.

[0061] The results are as follows Figure 7 As shown in the figure, in the Vehicle group cells stimulated by TNF-α, the phosphorylation levels of IKKα / β, IκBα and p65 increased significantly over time. In the cells treated with TII-C2, the phosphorylation levels of IKKα / β, IκBα and p65 changed with the TNF-α stimulation time, but the changes were significantly lower than those in the Vehicle group (15 and 30 min) ( n=3). *P<0.05, **P<0.01.

[0062] The results are as follows Figure 8 As shown in Figure 2, in the Vehicle group stimulated by TNF-α, the phosphorylation levels of JNK and p38 in cells increased significantly over time. However, the phosphorylation levels of JNK and p38 in cells treated with TII-C2 changed with the TNF-α stimulation time, but the changes were significantly lower than those in the Vehicle group. *P<0.05, **P<0.01.

[0063] Example 7: Immunoprecipitation to detect the effect of TII-C2 on the binding of TRADD and TRAF2

[0064] 1. Human embryonic kidney cell line HEK-293T was plated in a 10 cm culture dish. When the cell confluence reached 60-70%, TII-C2 (10 μM) was added and cultured for 12 h, then TNF-α (20 ng / mL) was added for different stimulation times (0 min, 5 min, 15 min, 30 min). After stimulation, the cells were washed 1-2 times with PBS, 1 mL of Co-IP lysis buffer was added, and the cells were lysed on a shaker at 4 ° C for 30 min, and the lysate was collected. The protein lysate was then broken in an ultrasonic cell disruptor, centrifuged at 4 ° C, 12000 g for 15 min, and the supernatant was aspirated. The protein concentration was determined by the BCA kit.

[0065] 2. Mix IgG and ProteinA / G plus-agarose and incubate at 4℃ for 2h. Wash ProteinA / G plus-agarose with IP wash solution 3 times, discard the supernatant each time; incubate ProteinA / G plus-agarose and protein sample at 4℃ for 2h. Centrifuge at 4℃, 2000rpm for 5min, and keep the supernatant for subsequent use.

[0066] 3. Mix TRADD antibody with Protein A / G plus-agarose and incubate on a shaker at 4°C for 2 hours. Wash Protein A / G plus-agarose 3 times with IP wash solution, discard the supernatant each time and keep Protein A / G agarose;

[0067] 4. Mix the supernatant from step 2 and the ProteinA / G plus-agarose from step 3 and incubate overnight at 4°C on a shaker. Wash three times with IP wash buffer and centrifuge at 2000 rpm for 5 min at 4°C.

[0068] 5. Add 32 μL IP lysis buffer and 8 μL protein loading buffer (5×) to the precipitated complex and boil at 100°C to denature the protein. Then perform immunoblotting.

[0069] The results are as follows Fig. 9 As shown, in Vehicle cells, as the time of TNF-α stimulation was prolonged (0-15min), the co-expression of TRADD and TRAF2 was gradually upregulated. At around 15-30min, the co-expression was gradually weakened. In TII-C2-treated cells, as the time of TNF-α stimulation was prolonged (0-15min), the co-expression of TRADD and TRAF2 was gradually upregulated, but its binding was significantly lower than that of the Vehicle group. TII-C2 inhibited the binding of TRADD and TRAF2 ( n=3). *P<0.05, **P<0.01.

[0070] Example 8: Therapeutic effect of TII-C2 on mouse DTH model

[0071] The abdominal skin of 8-10 week SPF inbred BALB / c mice was shaved in an area of ​​about 2cm×2cm. On the first and second days, 100μL of 0.5% DNFB (dissolved in acetone: olive oil = 4:1) solution was pipetted and evenly dripped on the depilated area for sensitization. The corresponding drug treatment was started on the day of sensitization. On the 6th and 7th days, 10μL of 0.2% DNFB solution was administered to the left ear and evenly dripped on the front and back sides of the left ear of the mouse for sensitization (the normal group mice were also dripped with DNFB solution). Compound TII-C2 (25mg / kg, 50mg / kg, 75mg / kg, 200mg / kg) and prednisone group (5mg / kg) were gavaged at the corresponding doses, once a day. The normal group and model group were gavaged with 0.5% sodium carboxymethylcellulose for a total of 7 days. The weight changes of mice in each group were recorded every day. After the mice were anesthetized and killed, the spleen and thymus were removed and weighed. Organ index = organ weight / body weight.

[0072] The results are as follows Fig.10As shown, compared with the normal group, the body weight of DTT mice decreased, and treatment with 50, 75 or 100 mg / kg of TII-C2 significantly restored the body weight of DTH mice (Figure A). In addition, the effect of TII-C2 on the spleen (B) and thymus index (C) of DTH mice was detected. Compared with the normal group, the spleen and thymus of DTH mice were significantly swollen. As the dose of TII-C2 increased, the spleen index and thymus index of DTH mice showed a downward trend, and there was a significant difference compared with the DTH group (BC, n=8). *P<0.05, **P<0.01, ***P<0.001.

[0073] Example 9: Calculation of ear swelling

[0074] After the mice were anesthetized and killed, a 6 mm ear punch was used to punch round ear pieces at the same position on both ears and weighed. The difference in weight between the left and right ears was the degree of swelling.

[0075] The results are as follows Fig.11 The results showed that compared with the normal group, the model group had significantly swollen ears and unclear blood vessels in the ears. With the gradual increase in the dose of TII-C2, the swelling of the mice ears was suppressed more and more, and the blood vessels in the ears were clearly visible. The dose of 100 mg / kg was equivalent to the therapeutic effect of the positive drug (prednisone) (AB). In addition, based on the results of different doses of TII-C2 on the inhibition of ear swelling in DTH mice, we calculated that the ED50 value of TII-C2 was 47.92 mg / kg (C, n=8). **P<0.01,***P<0.001.

[0076] Example 10: Establishment of mouse CIA model

[0077] 1. Dissolve 10 mg chicken CCII collagen in 5 mL of 0.01 mol / L acetic acid (30 μL glacial acetic acid + 10 mL sterile double distilled water) to a final concentration of 2 mg / mL and dissolve in a 4°C refrigerator overnight;

[0078] 2. Transfer the dissolved collagen to a mortar and grind on ice for 2 hours. In addition, inactivate 50 mg of BCG (80°C water bath for 1 hour), add it to 12.5 mL of sterile liquid paraffin, transfer it to a mortar and grind it for 2 hours, and the final concentration is 4 mg / mL of complete Freund's adjuvant;

[0079] 3. Mix equal volumes of complete Freund's adjuvant and collagen, and continue grinding on ice until completely mixed and the emulsion is milky white;

[0080] 4. Inject 0.15 mL of the emulsion intradermally at multiple points on the tail base and back of the mouse. On the 21st day after immunization, inject 0.1 mL intradermally at multiple points on the back of the mouse again to strengthen the induction.

[0081] 5. After CCII was injected again on the 21st day, the paw swelling of mice was observed and scored every three days. Mice with paw swelling on the 28th day were randomly divided into three groups, namely CIA+ sodium carboxymethylcellulose (CMC-Na, acting as a carrier of the drug) group, CIA+ compound TII-C2 (80 mg / kg / d) group, and CIA+ etanercept (5 mg / kg / d) group. The normal group consisted of mice that were not injected with collagen, with 6-10 mice in each group, and the drug administration lasted for 14 days. The posterior paws of mice in each group were photographed on the last day.

[0082] 8-10 week old SPF inbred DBA / 1 mice were used as experimental mice.

[0083] result Fig.12 As shown in the figure, this experiment successfully established a CIA mouse model. Compared with normal mice, CIA mice had joint swelling and secondary lesions in the hind paws. Compared with CIA mice, TII-C2 and etanercept reduced paw swelling.

[0084] Example 11: Evaluation of CIA Model

[0085] 1. Mouse weight: After CIA mice develop joint swelling, their body weight is measured and recorded every three days.

[0086] 2. Systemic performance score: After the CIA mice develop joint swelling, the score is recorded every three days, and the highest systemic score is 8 points. The scoring rules are as follows Table 1:

[0087] Table 1 Scoring criteria for systemic manifestations

[0088]

[0089] 3. Arthritis index score: After the CIA mice developed joint swelling, the arthritis index was recorded every three days, and the highest score was 16. The scoring criteria were as follows: 0 = normal; 1 = erythema and slight swelling of the ankle joint; 2 = erythema and slight swelling from the ankle joint to the metatarsal joint or the palm joint; 3 = erythema and moderate swelling from the ankle joint to the metatarsophalangeal joint or the palm joint; 4 = erythema and severe swelling from the ankle joint to the toe joint.

[0090] 4. Joint swelling score: After the CIA mice develop joint swelling, the score is recorded every three days, with the highest score being 24. Each paw of the mouse counts one ankle joint and five toe joints.

[0091] The results are as follows Fig.13As shown, the mice lost a significant amount of body weight after inflammation, and TII-C2 treatment significantly restored the weight of mice on d33-d42 (A). Both TII-C2 and etanercept significantly reduced the systemic score (B), arthritis index (C), and arthritis swelling number (D) of CIA mice on d36-d42 ( n=8~10). **P<0.001,***P<0.001 vs Normal; # p<0.05, ## p<0.01, ### p<0.001vs CIA-Veh(TII-C2); & p<0.05, && p<0.01, &&& p<0.001vs CIA-Veh(Ecep).

[0092] Example 12: Spleen pathology scoring

[0093] Paraffin tissues of spleens of mice in each group were sliced ​​and stained with hematoxylin-eosin (H&E). The lesions were divided into three grades from mild to severe based on the density of lymphoid sheath cells, hyperplasia of lymphoid nodules (germinal centers), hyperplasia of marginal zones, and congestion of red pulp. The rating criteria are shown in Table 2 below.

[0094] Table 2 Spleen pathology scoring criteria

[0095]

[0096] The results are as follows Fig.14 As shown, compared with the mice in the Normal group, the spleen of CIA mice was significantly enlarged, the surface was rough and uneven, and the cell density of the periarterial lymphoid sheath, the germinal center and the red pulp congestion were significantly aggravated. Compared with CIA mice, TII-C2 and etanercept treatment significantly improved spleen pathology, reduced the number of germinal centers, and reduced the number of marginal zones (A, B) ( n=5). *P<0.05, **P<0.01, ***P<0.001.

[0097] Example 13: Detection of the percentage of Th cell subsets in mouse spleen by flow cytometry

[0098] 1. Isolation of spleen lymphocytes: After the mice were killed, the spleen was removed from the sterile clean bench and placed in a culture dish containing 2% FBS. The spleen was wrapped with gauze and ground evenly with a grinding rod to prepare a spleen cell suspension. The spleen cell suspension was transferred to a 15mL centrifuge tube containing mouse lymphocyte separation solution (the volume ratio of 1640 culture medium and mouse lymphocyte separation solution = 1:1). Centrifuge at 800g for 30min at room temperature. The middle layer of mononuclear cells (lymphocytes and monocytes) in a cloudy state was aspirated into another centrifuge tube. Wash twice with 10mL sterile PBS solution and mix by inversion. Centrifuge at 800g for 10min at room temperature. Resuspend the fine precipitate, transfer to a 96-well plate, add 10% FBS containing T cell stimulator, and continue to culture for 4-6h.

[0099] 2. Flow cytometry detection of T cell subsets: Transfer the stimulated cell suspension in the 96-well plate to the EP tube, centrifuge at 800g for 10min, and discard the supernatant. Add 100μL PBS buffer (containing 2% fetal bovine serum, the same below) to each tube and mix well to make a cell suspension. Add surface antibodies: BV-510anti-Mouse CD3, FITC anti-Mouse CD4 and APC anti-MouseCD25, and incubate in the dark for 30min. Add 1mL PBS buffer to each tube, centrifuge at 800g for 10min, and discard the supernatant. Add 100μL fixative to each tube and incubate in the dark for 30min. Add 1mL PBS buffer, centrifuge at 800g for 10min, and discard the supernatant. Add 100μL permeabilization solution to each tube and incubate in the dark for 10min. During the whole process of permeabilization, the EP tube should be placed steadily without shaking. Add intracellular antibodies: APC anti-Mouse IL-4, BV-421anti-Mouse IFN-γ, PEanti-MouseIL-17A and PE anti-MouseFoxp3, and incubate in the dark for 30 minutes. Add 200 μL PBS buffer to each tube and filter out large particles that may remain through a gauze mesh. Flow cytometry was used to detect the percentage of Th cell subsets expressed in mouse spleen.

[0100] The results are as follows Fig.15 As shown, compared with the Normal group, the percentages of Th1 and Th17 cells in the spleen lymphocytes of CIA mice were upregulated, and the percentages of Th2 and Treg cells were downregulated. Compared with the CIA group, TII-C2 or etanercept treatment significantly inhibited the percentages of Th1 and Th17 cells (A, C) and restored the Treg ratio (D). TII-C2 treatment increased the proportion of Th2, while etanercept treatment had no significant effect on the increase of Th2 (B). TII-C2 can restore the balance of the immune system of CIA mice by regulating the proportion of T cell subsets and exert its immune regulatory function ( n=5). *P<0.05, **P<0.01.

[0101] Example 14: Knee joint pathology scoring

[0102] After the mice were killed by cervical dislocation, the knee joints of the mice were taken and the soft tissues around the joints were removed. After fixation with 4% polyformaldehyde, decalcification, dehydration, paraffin embedding, sectioning, and H&E staining. The pathological morphology of the knee joints was observed and recorded, and sub-item scores were given according to synovial cell hyperplasia, inflammation, pannus, bone erosion, and cell erosion. According to the severity of the lesions, they were divided into four grades, and the rating standards are shown in Table 3 below.

[0103] Table 3 Knee joint pathology scoring criteria

[0104]

[0105]

[0106] The results are as follows Fig.16 As shown in the figure, compared with the Normal group, the synovial cells in the CIA group proliferated significantly and were arranged in disorder, resulting in narrowing of the joint cavity. In addition, a large number of inflammatory cells infiltrated into the joint cavity, accompanied by pannus formation, erosion of the cartilage surface, and a large number of leukocytes. Compared with the CIA group, TII-C2 and etanercept improved synovial cell proliferation, joint tissue inflammation, pannus formation, inflammatory cell infiltration, and cartilage tissue damage to varying degrees (A, B) ( n=5). *P<0.05, **P<0.01, ***P<0.001.

[0107] Example 15: The ELISA method was used to detect the expression of IL-1β (A), IL-17 (B), TNF-α (C) and IL-10 (D) in the serum of each group of mice.

[0108] Blood was collected from the mouse eyeball and collected in a 1.5 mL centrifuge tube. The tube was left to stand at room temperature for 1 hour and centrifuged at 3000 rpm for 15 minutes. The supernatant was transferred to a new centrifuge tube. The expression of IL-1β, IL-17, TNF-α and IL-10 in mouse serum was subsequently detected using an ELISA kit.

[0109] The results are as follows Fig.17 As shown in Figure 2, the levels of IL-1β, IL-17, and TNF-α in mouse serum were significantly increased, and the anti-inflammatory cytokine IL-10 was significantly downregulated. Compared with the CIA-Veh group, TII-C2 can significantly inhibit the expression of IL-1β, IL-17, and TNF-α in mouse serum (Figure AC), and restore the expression of IL-10 (Figure D). In addition, TII-C2 is more effective than etanercept in increasing the expression of IL-10 (Figure 2). n=5). *P<0.05, **P<0.01, ***P<0.001.

[0110] The specific embodiments of the present invention are described in detail above, but they are only examples. There are many methods and ways to implement the technical solution. For those skilled in the art, the present invention can be modified and varied in various ways. Without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be considered as the protection scope of the present invention.

Claims

1. A small molecule compound TII-C2, characterized in that Its structure is as follows:

2. Use of the small molecule compound TII-C2 according to claim 1 in the preparation of a drug for treating TRADD-related autoimmune diseases.

3. The use according to claim 2, characterized in that: The TRADD-related autoimmune diseases include rheumatoid arthritis, systemic lupus erythematosus, Any one or a combination of syndrome, systemic sclerosis, psoriasis, pemphigus, inflammatory bowel disease, myasthenia gravis, Hashimoto's thyroiditis, primary antiphospholipid antibody syndrome, multiple sclerosis, idiopathic optic neuritis, and neuromyelitis optica spectrum disorder.

4. The use according to claim 3, characterized in that: The TRADD-related autoimmune disease is rheumatoid arthritis.

5. The use according to claim 2, characterized in that: The drug can destroy the binding of TRADD and TRAF2, selectively inhibit the activation of the downstream signaling pathway of TNFR1, and inhibit the abnormal proliferation of RAFLSs.

6. The use according to claim 2, characterized in that: The drug has an inhibitory effect on the inflammatory immune response of the delayed hypersensitivity reaction model.

7. The use according to claim 2, characterized in that: The drug can effectively inhibit the proliferation of fibroblast-like synoviocytes and restore immune balance.

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