New compound and application thereof in preparation of medicine for treating systemic lupus erythematosus

Abstract

The invention discloses a novel compound and application thereof in preparation of a medicine for treating systemic lupus erythematosus. In-vitro experiments show that the compound provided by the invention can further inhibit the secretion of anti-dsDNA IgG + B cell subtype mediated autoantibody by inhibiting the process that B cells are differentiated into plasmoblasts; in-vivo experiments show that the compound provided by the invention can effectively reduce the content of total IgG and anti-dsDNA-IgG in peripheral blood of a systemic lupus erythematosus model mouse, inhibit splenomegaly and peripheral lymph node hyperplasia of the mouse, slow down urine protein increment of the mouse, inhibit glomerular lesion and renal tubule lesion of the mouse and inhibit IFN-gamma generation of the mouse kidney; differentiation of spleen and peripheral lymph node plasmablasts of mice is inhibited, that is, differentiation and function development (antibody secretion) processes of B cells are directly acted. Therefore, the compound provided by the invention has a prospect of being developed into drugs for treating systemic lupus erythematosus.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical chemistry, and relates to new compounds and their applications. Specifically, it relates to a new compound and its application in the preparation of drugs for treating systemic lupus erythematosus. Background Art

[0002] Systemic lupus erythematosus (SLE) is a chronic diffuse connective tissue disease mainly caused by the abnormal activation of the immune system, which attacks its own tissues. The specific cause of this disease is not yet clear, but it may be related to genetics, environmental factors and estrogen. Long-term sun exposure, use of certain drugs, infections, and oral estrogen may all induce the exacerbation of SLE symptoms. This disease is prone to occur in women of childbearing age, especially in the population aged 10 to 40. The incidence rate of women is significantly higher than that of men, about 9:1. The symptoms of SLE are very diverse and often include fever, photosensitivity, rash, enlarged lymph nodes, muscle and joint pain, headache, fatigue, etc. This disease can damage various organs of the whole body, so it may cause various complications, including kidney damage, neuropsychiatric symptoms, etc.

[0003] Abnormal B cells are the core driving factors of SLE. B cells in SLE patients can spontaneously secrete immunoglobulins without external stimulation and have abnormal responses to exogenous stimuli, resulting in the generation of a large number of autoantibodies against autoantigens such as dsDNA and nuclear proteins. Plasmablasts are the direct executors of pathological damage. After B cells differentiate into plasmablasts, they further mature into long-lived plasma cells, continuously secreting pathogenic autoantibodies (such as anti-dsDNA antibodies), forming immune complexes that deposit in organs such as the kidneys and skin, triggering inflammatory reactions. It can be seen that the pathogenesis of SLE is closely related to the overactivation of B cells and the abnormal differentiation of plasmablasts. Targeting the regulatory pathways of these two types of cells is the core direction of current therapeutic research and development.

[0004] The treatment of systemic lupus erythematosus usually relies on multiple therapies, including drug treatment, phototherapy and lifestyle changes, etc. Commonly used drug treatments mainly include non-steroidal anti-inflammatory drugs, immunosuppressive drugs and corticosteroids, etc. Glucocorticoids and cyclophosphamide can non-specifically inhibit B cell proliferation and reduce antibody secretion, but they have extensive immunosuppressive side effects.

[0005] In order to develop drugs for treating SLE, the present invention is specifically proposed. Summary of the Invention

[0006] The object of the present invention is to provide a new compound and its application in the preparation of drugs for treating systemic lupus erythematosus.

[0007] The above object of the present invention is achieved by the following technical solutions:

[0008] A compound of the following structure or a pharmaceutically acceptable salt or solvate thereof:

[0009]

[0010] Use of the above compound or a pharmaceutically acceptable salt or solvate thereof in the preparation of a drug for treating systemic lupus erythematosus.

[0011] Furthermore, the above drug uses the above compound or a pharmaceutically acceptable salt or solvate thereof as an active ingredient and is formulated into a pharmaceutically acceptable dosage form through pharmaceutically acceptable excipients.

[0012] Even further, the excipients are solid, liquid or semi-solid.

[0013] Even further, the dosage forms include tablets, capsules, injections and pills.

[0014] Beneficial effects:

[0015] Those skilled in the art know that the onset of systemic lupus erythematosus is closely related to the overactivation of B cells and the abnormal differentiation of plasmablasts. Targeting the regulatory pathways of these two types of cells is the core direction of current therapeutic research and development. In vitro experiments show that the compound provided by the present invention can further inhibit the secretion of autoantibodies mediated by the anti-dsDNA IgG+ B cell subtype by inhibiting the process of B cell differentiation into plasmablasts; in vivo experiments show that the compound provided by the present invention can effectively reduce the contents of total IgG and anti-dsDNA-IgG in the peripheral blood of systemic lupus erythematosus model mice, inhibit splenomegaly and peripheral lymph node hyperplasia in mice, slow down the increase in urinary protein in mice, inhibit glomerular and tubular lesions in mice, inhibit the generation of renal IFN-gamma in mice, and inhibit the differentiation of plasmablasts in the spleen and peripheral lymph nodes of mice, that is, directly act on the process of B cell differentiation and functional development (antibody secretion). Therefore, the compound provided by the present invention has the prospect of being developed into a drug for treating systemic lupus erythematosus. Description of the drawings

[0016] Figure 1 Shows the inhibitory effect of compound L3011 on the activities of GM12878 and peripheral blood PBMC at a drug concentration of 10 μM;

[0017] Figure 2 Shows the inhibition of the formation of ELISpot spots of specific anti-dsDNA IgG in primary B cells of SLE patients when compound L3011 targets the B cell differentiation stage;

[0018] Figure 3 Shows the effects of different addition times of compound L3011 on the production of specific anti-dsDNA IgG and total IgG in primary B cells of SLE patients;

[0019] Figure 4 Inhibitory effect of compound L3011 at different concentrations on the differentiation of primary B cells from SLE patients into plasmablasts PB

[0020] Figure 5 Compound L3011 reduces the levels of total IgG and anti-dsDNA-IgG in the peripheral blood of mice

[0021] Figure 6 Digital photographs of the spleens of mice in different groups and determination of spleen indices

[0022] Figure 7 Digital photographs of the peripheral lymph nodes of mice in different groups

[0023] Figure 8 Curves of changes in urinary protein content in mice in different groups

[0024] Figure 9 In A, pathological anatomy of mouse kidneys and PAS staining; in B, scoring of glomerular and tubular lesions. It can be seen that the degrees of glomerular and tubular lesions in the L3011 intervention group are significantly reduced, *vs. model control group, P<0.05

[0025] Figure 10 Compound L3011 reduces the production level of lupus-related marker IFN-γ in the kidneys of MRL / lpr mice. After continuous intervention with compound L3011 at doses of 2 mg / kg and 10 mg / kg for 6 weeks, the production of renal IFN-γ in lupus model mice is inhibited in vivo, *vs. model control group, P<0.05

[0026] Figure 11 Statistics of plasmablasts PB accounting for total splenic B cells. Compound L3011 significantly inhibits the differentiation of splenic plasmablasts in MRL / lpr mice, *vs. model control group, P<0.05

[0027] Figure 12 Statistics of plasmablasts PB accounting for total lymph node B cells. Compound L3011 inhibits the differentiation of plasmablasts in the peripheral lymph nodes of MRL / lpr mice, *vs. model control group, P<0.05 Detailed implementation methods

[0028] The following specifically introduces the substantial content of the present invention in combination with examples, but does not limit the protection scope of the present invention hereby

[0029] Example 1: Preparation of compound L3011

[0030]

[0031] The synthesis route is as follows

[0032]

[0033] (i) DIPEA, IPA, 40 °C; (ii) TsOH, IPA, 90 °C

[0034] (i) Dissolve 2,4-dichloropyrrolo[3,2-d]pyrimidine (2.05 g, 10 mmol) in isopropanol (IPA) (30 mL), add isopropylamine (1.29 mL, 15 mmol) and N,N-diisopropylethylamine (DIPEA) (4.35 mL, 25 mmol), and react with stirring at 40 °C for 4 h. Monitor by TLC hexane:ethyl acetate 2:1. When the substrate has completely reacted, dilute with water (100 mL), extract with ethyl acetate (3 × 100 mL), wash with saturated brine, dry over anhydrous sodium sulfate, distill under reduced pressure on a rotary evaporator, and purify the mixture by silica gel column chromatography (hexane / ethyl acetate = 10 / 1 to 2 / 1) to obtain the product (1.8 g, 79.3%).

[0035] (ii) Dissolve 2-chloro-N-(1-methylethyl)pyrrolo[3,2-d]pyrimidin-4-amine (1.14 g, 5 mmol) in isopropanol (IPA) (20 mL), add 1-methyl-1H-pyrazol-4-amine (0.62 mL, 6.5 mmol) and p-toluenesulfonic acid (TsOH) (1.12 g, 6.5 mmol), and react with stirring at 90 °C for 8 h. Monitor by TLC dichloromethane:methanol 20:1. When the substrate has completely reacted, quench with saturated sodium bicarbonate solution (30 mL), extract with ethyl acetate (3 × 50 mL), wash with saturated brine, dry over anhydrous sodium sulfate, distill under reduced pressure on a rotary evaporator, and purify the mixture by silica gel column chromatography (dichloromethane / methanol = 200 / 1 to 20 / 1) to obtain the target product (1.1 g, 76.4%), and recrystallize to a purity of not less than 95%. 1 H NMR (400 MHz, MeOD) δ 7.81 (s, 1H), 7.56 (s, 1H), 7.19 (d, J = 3.0 Hz, 1H), 6.14 (d, J = 3.0 Hz, 1H), 4.40 (hept, J = 6.5 Hz, 1H), 3.84 (s, 3H), 1.33 (d, J = 6.5 Hz, 6H). 13 C NMR (100 MHz, MeOD) δ 157.00, 151.43, 148.89, 131.86, 127.79, 126.54, 122.44, 111.17, 100.95, 43.21, 38.93, 23.15.

[0036] Example 2: Cytotoxicity of Compound L3011

[0037] I. Experimental Materials

[0038] Biological safety cabinet, Ficoll (Cytiva), pipettes, 10 mL pipettes, CD40L (MCE), RPMI-1640 medium, fetal bovine serum, CCK8 detection reagent, 96-well plates, multi-spectrum microplate reader (450 nm filter), tabletop centrifuge, cell counting chamber, cell counter, CO2 incubator, inverted microscope, 10 μL, 100 - 200 μL multi-channel pipettes.

[0039] The GM12878 human B lymphocytes were purchased from Hunan Fenghui Biotechnology Co., Ltd., and the compound L3011 was prepared in Example 1.

[0040] II. Experimental methods

[0041] 1. GM12878 cell line

[0042] For its suspension growth characteristics, it was cultured in RPMI-1640 medium containing 10% FBS.

[0043] 2. Extraction of human peripheral blood mononuclear cells (PBMC)

[0044] Human peripheral blood mononuclear cells were extracted from the peripheral blood of healthy individuals or patients with systemic lupus erythematosus. The subjects were informed of the content of participation and signed the informed consent form. All patients' clinical information was collected. The trial was approved by the Clinical Trial Ethics Committee of West China Hospital, Sichuan University, filing number: 2024(1315). 10 mL of peripheral blood was drawn from each subject using a 10 mL EDTA anticoagulant blood collection tube. In the biological safety cabinet, peripheral blood mononuclear cells (PBMC) were separated by Ficoll density gradient centrifugation: The volume of peripheral blood was diluted to 20 mL with sterile PBS buffer and carefully added to the upper layer of 10 mL Ficoll reagent to ensure a clear layering interface; centrifuged at 400 g, acceleration 1, deceleration 1, 20 °C for 30 min; carefully aspirate the middle white membrane layer (PBMC) and dilute it with twice the volume of sterile PBS buffer; centrifuge at 400 g for 10 min. The collected PBMC were resuspended in the prepared complete 1640 medium (containing 10% fetal bovine serum, 1% streptomycin-penicillin, 10 nM CD40L) for subsequent experiments.

[0045] 3. CCK-8 incubation

[0046] The cells to be detected were GM12878 and primary PBMC from healthy individuals. In the biological safety cabinet, the cells were diluted to 1×10 6 cells / mL using 1640 complete medium (the medium for PBMC contains CD40L, 10 μg / μL). Take a sterile 96-well plate and add 100 μL of the GM12878 or PBMC cell suspension to each well, approximately 1×105 cells / well, and culture overnight under the conditions of 37 °C, 5% CO2, and 90% humidity.

[0047] Observe the cell growth status and density under a microscope, and select wells with good growth status and uniform cell distribution and density for experiments. Prepare the drug sample solution to a final concentration of 20 μM, 100 μL / well, and add it to the 96-well plate. Three replicate wells for each compound are used as experimental wells. The negative control wells are only inoculated with cells of the same density without adding the drug solution, and the blank control wells are not inoculated with cells and only contain the same 1640 complete medium as the experimental wells. Culture for 24 h under the conditions of 37 °C, 5% CO2, and 90% humidity. The CCK-8 detection reagent is rewarmed and centrifuged at room temperature before use, and the CCK-8 working solution is prepared by diluting it 9:1 with freshly prepared 1640 medium containing 10% FBS. Centrifuge the 96-well plate at 500 g, acceleration speed 4, deceleration speed 1, for 5 min, and carefully discard the culture supernatant. Add 100 μL of the CCK-8 working solution to each well and transfer it to the cell culture incubator for 4 h.

[0048] 4. Determine the IC50 value

[0049] The cells to be detected are GM12878 and healthy human primary PBMC. In the biosafety cabinet, dilute the cells to 1×10 6 cells / mL using 1640 complete medium (the medium for PBMC contains CD40L, 10 μg / μL). Take a sterile 96-well plate and add 100 μL of the GM12878 or PBMC cell suspension to each well, approximately 1×10 5 cells / well, and culture overnight under the conditions of 37 °C, 5% CO2, and 90% humidity.

[0050] Observe the cell growth status and density under a microscope, and select wells with good growth status and uniform cell distribution and density for experiments. Prepare the drug sample solution to final concentrations of 2000.00, 666.67, 222.22, 74.07, 24.69, 8.23, 2.74, 0.91, 0.30, 0 μM, 100 μL / well (two-fold dilution), and add it to the 96-well plate. Three replicate wells for each compound are used as experimental wells. The negative control wells are only inoculated with cells of the same density without adding the drug solution, and the blank control wells are not inoculated with cells and only contain the same 1640 complete medium as the experimental wells. Culture for 24 h under the conditions of 37 °C, 5% CO2, and 90% humidity. The CCK-8 detection reagent is rewarmed and centrifuged at room temperature before use, and the CCK-8 working solution is prepared by diluting it 9:1 with freshly prepared 1640 medium containing 10% FBS. Centrifuge the 96-well plate at 500 g, acceleration speed 4, deceleration speed 1, for 5 min, and carefully discard the culture supernatant. Add 100 μL of the CCK-8 working solution to each well and transfer it to the cell culture incubator for 4 h.

[0051] 5. Detection and calculation: Measure the absorbance at 450 nm using a microplate reader. Process and analyze the results using Excel and Graphpad Prism. The calculation formula for cell viability detection is as follows:

[0052]

[0053] III. Experimental Results

[0054] As Figure 1 shown, compound L3011 showed no obvious inhibitory effect on the activities of GM12878 and peripheral blood PBMCs at a drug concentration of 10 μM. The IC50 value of compound L3011 for inhibiting GM12878 was 330.1 μM, and the IC50 value for inhibiting peripheral blood PBMCs was 151.5 μM, indicating no obvious inhibitory effect. In summary, compound L3011 has high safety.

[0055] Example 3: In vitro inhibition of the secretion of autoantibody IgG by B cells from SLE patients by compound L3011

[0056] I. Experimental materials

[0057] Biological safety cabinet, Ficoll (Cytiva), pipettes, 10 mL pipettes, RPMI-1640 medium, fetal bovine serum, EasySep TM Release Human CD19 Positive Selection Kit (STEMCELL), 96-well plates (MSIPS4510), human IgG B cell ELISPOT kit for ELISpot experiments (Catalog#CT780-PR5), CTL enzyme-linked spot analyzer, tabletop centrifuge, cell counting chamber, cell counter, CO2 incubator, 10 μL, 100 - 200 μL multi-channel pipettes.

[0058] Compound L3011 was prepared in Example 1.

[0059] II. Experimental methods

[0060] 1. Positive sorting of primary B cells from peripheral blood

[0061] In this experiment, a human CD19 positive sorting kit was used. Prepare a dilution buffer: sterile PBS buffer containing 2% FBS. Dilute the Release Buffer 1:40 with the buffer to obtain a final concentration of "1×" Release Buffer, and prepare it freshly before use. Resuspend the test PBMCs with 1 mL of dilution buffer to make the final cell concentration 1×10 8Cells / mL to obtain a sorted sample, which was transferred to a 5 mL polystyrene flow tube. Gently mix the anti-CD19 antibody mixture in the kit, add 100 μL of the antibody mixture to the sample, gently mix with the sample and incubate at room temperature for 3 min. Vortex the RapidSpheres TM Magnetic beads for 30 seconds until in a uniformly dispersed state, aspirate 100 μL and add it to the sample, mix well and incubate at room temperature for 3 min. Add dilution buffer to make the volume of the sample up to 2.5 mL, gently pipette and mix well. Place the flow tube in the magnetic pole and incubate at room temperature for 3 min. Pick up the magnetic pole, continuously rotate the magnetic pole and the test tube, pour out the supernatant, and then take out the test from the magnetic pole. Repeat the above steps twice, then add Releasebuffer (1×) to the flow tube to make the volume of the cell sample up to 2.5 mL and incubate at room temperature for 3 min. Place the flow tube in the magnetic pole and incubate at room temperature for 5 min. Pick up the magnetic pole and transfer the supernatant to a new test tube to obtain CD19 + B cells for standby.

[0062] 2. B cell culture and induced differentiation

[0063] B cells were taken from the peripheral blood of SLE diagnosed patients. The specific extraction and separation steps were the same as before. The subjects signed the informed consent form, which met the ethical requirements. Prepare 1640 medium containing IL-2 with a liquid volume of 1 / 100 of the medium volume and R848 with a volume of 1 / 200 of the medium volume, add 10% FBS and penicillin with a final concentration of 100 U / mL and streptomycin with a concentration of 100 μg / mL. Count the B cells and dilute the cells to 2×10 6 cells / mL with the above-mentioned 1640 medium containing stimulatory factors. Transfer the cells to a culture dish and place it in an incubator at 37 °C and culture at a position close to the water tank for 5 days.

[0064] 3. Pretreatment and coating of the PVDF membrane in a 96-well plate

[0065] In a biosafety cabinet, pre-wash the PVDF membrane (MSIPS4510, Millipore) in each well of the 96-well plate with 25 μL of 70% ethanol and incubate at room temperature for 1 min. Use a pipette to remove the ethanol, rinse each well with sterile PBS twice, empty the PBS in the well plate, and pat it on absorbent paper to ensure complete removal. Add 50 μL of diluted coating antibody solution (anti-IgG) or 50 μL of specific antigen (dsDNA) at 100 μg / μL to each well. Paste the covering film, cover the plate cover, and incubate overnight at 4 °C under sterile conditions.

[0066] 4. Blocking of the PVDF membrane in the 96-well plate

[0067] First, remove the antibody solution, and rinse each well with 200 μL of sterile PBS three times, then forcefully flick out the residual sterile PBS on the well plate. Add 200 μL of blocking buffer (1×) to each well. Cover the plate lid and incubate at room temperature for 1 h. During the incubation step, start preparing the cell sample suspension. Once the cell suspension is ready, forcefully flick out the blocking buffer (1×) without washing the well plate.

[0068] 5. Cell seeding and drug administration

[0069] Transfer the cells to a sterile centrifuge tube, centrifuge at 500 g for 8 min, discard the supernatant, and wash twice with 1640 medium without FBS and induction substances. Resuspend the B cells in 1 mL of 1640 complete medium without stimulatory factors, count the cells, and dilute to 5×10 6 cells / mL (for anti-dsDNA IgG detection), or 5×10 5 cells / mL (for total IgG detection). Add 100 μL of cell suspension per well, i.e., 5×10 5 cells per well, or 5×10 4 cells per well.

[0070] Drug administration protocol: Protocol 1, when inducing and stimulating the differentiation of cells, simultaneously add compound L3011 pre-diluted with DMSO to the 48-well plate according to different groups, with the final concentration of the compound being 10 μmol / L and 2 replicates. Protocol 2, 5 days after induction, add compound L3011 pre-diluted with DMSO to each well of the cells, with the final concentration of the compound being 10 μmol / L and 2 replicates. Cover the lid and incubate at 37 °C, 5% carbon dioxide, and 90% humidity for 24 h.

[0071] 6. Detection antibody incubation

[0072] Remove from the biosafety cabinet and forcefully flick out a large amount of suspended memory B cells in the well plate at once. Rinse each well with 200 μL of sterile PBS twice to thoroughly remove the residual suspended cells on the PVDF membrane. Dilute the wash buffer to 1× with dH2O according to the kit instructions, and wash the PVDF membrane 5 times with 250 μL of wash buffer per well. Add 100 μL of the diluted detection antibody to each well. Seal the plate with an adhesive coverslip and incubate overnight at 4 °C.

[0073] 7. HRP binding

[0074] Empty the plate, remove the plastic trough at the bottom of the plate, wash both sides of the PVDF membrane with wash buffer 5 times. Add 100 μL of diluted HRP-conjugate to each well. Install the bottom plastic trough, seal the plate with an adhesive coverslip, and incubate at room temperature in the dark for 1 h.

[0075] 8. Coloring

[0076] Empty the plate, remove the plastic trough at the bottom of the plate, rinse both sides of the PVDF membrane with wash buffer 5 times. Take out and discard the plastic leak at the bottom of the plate, rinse the back of the membrane with wash buffer, and perform additional washing on the back of the PVDF membrane to further reduce background staining. Add 100 μL of freshly prepared AEC solution to each well. Cover the lid and incubate at room temperature in the dark for 30 min. Empty the plate, stop the reaction, and thoroughly rinse both sides of the PVDF membrane with deionized water. Dry naturally at room temperature in the dark.

[0077] 9. Detection by machine

[0078] In this experiment, a CTL enzyme-linked immunosorbent spot analyzer was used to count the spots, and the SmartSpot function of the software was used to automatically identify the spots at single-cell resolution. The Quality Control function was used to approve the identified area and spot area and manually calibrate the clusters for accurate ELISPOT counting.

[0079] III. Experimental Results

[0080] Anti-dsDNA IgG is an immunoglobulin G type autoantibody targeting double-stranded DNA (dsDNA) and is a specific marker for systemic lupus erythematosus (SLE).

[0081] The experimental results showed that when compound L3011 was administered according to Protocol 1 and intervened simultaneously with the induction and differentiation conditions, that is, when compound L3011 targeted the B cell differentiation stage, it could significantly inhibit the change in the number of ELISpot spots of specific anti-dsDNA IgG ( Figure 2 ) and total IgG ( Figure 3 A in). However, after the differentiation of primary B cells from SLE patients was completed, the addition of compound L3011 had no significant effect on the ability of single cells to produce antibodies (producing specific anti-dsDNA IgG and total IgG) ( Figure 3 B and C in). In summary, compound L3011 inhibits antibody secretion by inhibiting B cell differentiation.

[0082] Example 4: Compound L3011 Inhibits the Differentiation of B Cells from SLE Patients into Plasmablasts in Vitro

[0083] I. Experimental Materials

[0084] Biological safety cabinet, Ficoll (Cytiva), pipette, 10 mL pipette, RIPM-1640 medium, fetal bovine serum, PBS, R848, IL-2, IL-10, flow antibodies: flow anti-human antibodies CD19 (1:200), CD27 (1:100), CD38 (1:100); flow tubes, flow cytometer (BD FACSCalibur), tabletop centrifuge, cell counting plate, cell counter, CO2 incubator, 10 μL, 100 - 200 μL multi-channel pipette, etc.

[0085] Compound L3011 was prepared in Example 1.

[0086] II. Experimental methods

[0087] 1. Inducing differentiation of cells into plasmablasts

[0088] Total B cells were taken from the peripheral blood of SLE patients. The specific extraction and separation steps were the same as before. The subjects signed the informed consent form, which met the ethical requirements. Cell counting was performed, and the total B cells were diluted to 2×10 5 cells / mL with 1640 medium (containing 10% FBS). In a sterile 48-well plate, 500 μL of the B cell suspension was added to each well. The plate was placed in a 5% CO2, 37 °C constant temperature and humidity cell incubator for 2 h. After drug administration, it was incubated for 30 min, the pre-diluted inducer R848 was added, and it was continued to be incubated in the incubator for 30 min. Then the human-derived IL-2 and IL-10 inducers were added. The induction protocol is shown in Table 1.

[0089] Table 1 Protocol for in vitro induction of B cells into plasmablasts

[0090]

[0091] 2. Drug administration protocol

[0092] A blank control group (mock), a model control group (mimic), a vehicle control group, a positive drug control group, and an experimental group were set up. Since cepharanthine (CEP) was considered to be able to intervene in the mitotic stage of the B cell cycle in previous studies, the drug in the positive drug control group of this experiment was CEP. Each group had 3 replicate wells, and the drug administration protocol is shown in Table 2. After adding the drug, the 48-well plate was placed in the cell incubator and incubated for 30 min. Then the differentiation inducers were added according to Table 1.

[0093] Table 2 Drug administration protocol

[0094]

[0095]

[0096] 3. In vitro induction of plasmablast detection

[0097] Take out the cell suspension from the cell incubator, centrifuge at 1500 rpm for 5 min, then add 500 μL of sterile PBS solution containing 2% FBS, centrifuge again at the same speed for 5 min to completely remove the culture medium. Vitality dye: Add FVS (1:2000) to 1×PBS, 50 μL for each flow cytometry tube, incubate in the dark at room temperature for 10 min, add 300 μL of 1×PBS solution (containing 2% FBS) to stop the staining, centrifuge at 1500 rpm for 5 min, and pour off to remove the remaining antibody. Flow cytometry antibodies: Add flow cytometry anti-human antibodies CD19 (1:200), CD27 (1:100), CD38 (1:100) to 1×PBS solution (containing 2% FBS) and mix well, 50 μL for each tube. At the same time, prepare single-stained and blank tubes, place them in the dark at 4°C for 30 min of staining, add 300 μL / tube of 1×PBS buffer (containing 2% FBS), centrifuge at 1500 rpm for 5 min to remove the remaining antibody. 300 μL / tube of buffer is used to elute the excess antibody, centrifuge at 1500 rpm for 5 min, add 500 μL / tube of buffer to resuspend the cells after removing the antibody, and perform flow cytometry detection on the machine. The cell population markers are shown in Table 3.

[0098] Table 3 Flow cytometry staining protocol for plasmablasts

[0099]

[0100] III. Experimental results

[0101] Overactivation of plasmablasts can produce autoantibodies (such as anti-dsDNA antibodies), which participate in organ damage in diseases such as systemic lupus erythematosus (SLE). As Figure 4 shown, different concentrations of compound L3011 can significantly inhibit the differentiation of B cells into PB, and when the concentration of compound L3011 is 0.06 μmol / L, it can still block the differentiation of B cells into PB to a certain extent. In summary, compound L3011 has good safety and effectiveness in vitro, and can further inhibit the secretion of autoantibodies mediated by the anti-dsDNA IgG+ B cell subtype by inhibiting the process of in vitro differentiation of B cells into plasmablasts.

[0102] Example 5: In vivo inhibition of SLE phenotype in lupus mice by compound L3011

[0103] I. Experimental materials

[0104] SPF-grade MRL / MpJ-Fas lpr / J and MRL / MpJ strain female mice, SPF-level breeding barrier, DMSO, tween-20, PBS, ultrasonic water bath, laminar flow hood, pipette, isoflurane, inhalation anesthetic machine, electronic balance, ophthalmic forceps, ophthalmic scissors, 1.5 mL ep tube, 1.0 mL syringe, centrifuge, mouse immunoglobulin G (IgG) enzyme-linked immunosorbent assay kit (Wuhan Huamei Bio), mouse anti-double-stranded DNA antibody IgG enzyme-linked immunosorbent assay kit (Wuhan Huamei Bio), multi-spectrum microplate reader, 37 °C incubator, tin foil, BSA, 50 mL centrifuge tube, 70 μm cell filter, ice box, steel ruler, red blood cell lysis buffer (Beyotime), vortex mixer, cryopreservation tube, dry ice, pipette, urinary protein quantification test kit (Nanjing Jian Gong), -80 °C chromatography cabinet, 96-well microplate, formalin fixative (4% paraformaldehyde), bright-field optical microscope, high-temperature oven, xylene, 100%, 95%, 85%, 75% alcohol, citrate antigen retrieval solution, microwave oven, TBST buffer, Triton x-100, 3% H2O2, IFN-γ rabbit anti-mouse primary antibody (thermo), wet box, 4 °C chromatography cabinet, secondary antibody, chromogenic solution, hematoxylin, neutral resin mounting, cover glass, etc.

[0105] Compound L3011 was prepared in Example 1.

[0106] II. Experimental methods

[0107] 1. Animal breeding

[0108] SPF-level MRL / MpJ-Fas lpr / J strain mice (hereinafter referred to as MRL / lpr) were used as the lupus erythematosus disease model animals in this experiment, and the mice in the blank control group were of the SPF-level MRL / MpJ strain. The mice were provided by Cyagen Biosciences (Suzhou) Inc., and the age was 4 to 5 weeks old. This project has been approved by the Animal Ethics Committee of West China Hospital in accordance with the relevant regulations of the "Guidelines for the Ethical Review of Laboratory Animal Welfare" of the country (filing number: 20231101008). The mice were bred in an SPF-level breeding barrier, and were randomly assigned to different experimental groups and control groups before the experiment, and were housed in cages with 4 mice per cage, and the free diet and drinking water scheme was adopted. The animals were bred at a constant temperature (21 - 24 °C) and a constant humidity (40 - 55%), and the light simulated the day and night alternation every 12 h.

[0109] 2. Drug dissolution

[0110] 5% DMSO + 10% tween-20 + 85% sterile 1×PBS buffer solution, water bath at 25°C, ultrasonic treatment for 10 min. The blank control mice were female SPF-grade MRL / MpJ, and the mice in the model control group, positive control group and experimental group were MRL / lpr female mice. The mice were weighed every week, the drugs for that week were dissolved and prepared, and stored at -80°C. The specific grouping and dosing regimen are shown in Table 4. The drugs were intervened in the mice starting from 8 weeks of age, continuously intervened for 6 weeks, and administered daily.

[0111] Table 4 Grouping and dosing regimen of animal experiments

[0112]

[0113] 3. Detection

[0114] In this experiment, the experimental endpoint was that 80% of the mice in the model control group had facial hair loss and peripheral lymph node enlargement. The body weight of the mice was weighed before euthanasia, with the unit of g, and the data was retained to two decimal places. The mice were anesthetized by inhaling 5% (v / v) isoflurane for anesthesia induction. The hind limb toes of the animals were squeezed with fingers for a deep pain test. When the mice had completely lost consciousness and had no pain reflex, blood collection was quickly performed and the mice were decapitated by cervical dislocation for subsequent sampling and detection.

[0115] 3.1. Determination of the contents of total IgG and anti-dsDNA-IgG in the peripheral blood of mice

[0116] In this experiment, the terminal blood collection protocol was used for the experimental mice. The deeply anesthetized mice were fixed on the operating table in the supine position, the fur on the chest area was cut off, the chest cavity was dissected after skin disinfection, and then the injection needle was inserted into the right ventricle and blood was drawn immediately, and then the mice were decapitated by cervical dislocation. The blood collection volume for each mouse was 1.0 mL each time, collected in a 1.5 mL centrifuge tube without anticoagulant, and placed at 25°C for 4 h to ensure that the blood cells coagulated under the action of the prothrombin cascade. Centrifuge at 6000 rpm for 10 min at 4°C, collect the serum and store it at -80°C.

[0117] The collected mouse serum samples were thawed on ice and gently pipetted to ensure the uniformity of the sample contents. According to the requirements of the ELISA instruction manual, the total IgG and anti-ds-DNA specific antibodies in the serum samples were detected. First, take out the enzyme-linked immunosorbent assay plate pre-coated with the detection antigen, dilute the standard sample curve and the serum samples, add them to the reaction plate and incubate at room temperature in the dark for 2 h. Subsequently, washing and incubation with solutions A and B were carried out. Finally, horseradish peroxidase was conjugated, incubated at 37°C in the dark for 30 min, the stop solution was added, and the OD value of the samples was detected at 450 nm using a BioTek microplate reader, and the sample standard curve results were calculated using the Huamei Bio special CurveExpert software.

[0118] 3.2 Isolation of Mouse Spleen and Determination of Spleen Index

[0119] Isolation and Pretreatment of Peripheral Lymph Nodes: Peripheral lymph nodes are one of the main sites for the colonization of mature B cells. In this experiment, the cervical, axillary, and inguinal lymph nodes of experimental mice were mainly isolated for subsequent detection. Superficial cervical lymph nodes: The mice were fixed in the supine position on the operating table. The skin was cut along the midline from the thoracic cavity opening towards the neck to expose the submandibular gland. The submandibular gland was lifted to find the superficial cervical lymph nodes, which were light yellow, clear, and had clear edges, and were wrapped by a small amount of connective tissue on both sides. Axillary lymph nodes: The skin was cut along the midline towards the tail, and the chest muscles were cut along the sternum to expose the axillary adipose tissue. The encapsulated oval axillary lymph nodes were found. Inguinal lymph nodes: The skin was cut along the midline of the ilium of the mouse. The epithelium and peritoneum were carefully bluntly separated with scissors and ophthalmic forceps. There was a clear oval structure, one on each side, wrapped in the submucosa near the inguinal area, which was the inguinal lymph node. The lymph nodes at each location were bluntly separated with forceps, measured and photographed using an experimental steel ruler, and then placed in PBS buffer containing 2% BSA and stored on ice. The lymph was ground on ice until there were no obvious lumps. The ground sample was resuspended in PBS buffer containing 2% BSA, and the single-cell suspension was collected by filtering through a cell strainer with a pore size of 70 μm for subsequent detection.

[0120] Isolation of Spleen and Determination of Spleen Index: The peritoneum and muscle tissues of the mouse were bluntly separated to expose the abdominal cavity. A smooth, long oval, dark red parenchymal organ behind the stomach in the left abdominal cavity was found, which was the mouse spleen. The connective tissue at the proximal end of the spleen was separated with forceps and the spleen was removed. The spleen was weighed, measured, and photographed, and then placed in PBS buffer containing 2% BSA and stored on ice. Calculate the spleen index: the ratio of spleen weight to body weight. The spleen was evenly divided into two parts along the transverse axis, and the top 1 / 2 was placed in a cryotube and quickly frozen in liquid nitrogen. The bottom 1 / 2 of the spleen was ground on ice until there were no obvious lump tissues. The ground sample was resuspended in PBS buffer containing 2% BSA, and the single-cell suspension was collected by filtering through a cell strainer with a pore size of 70 μm. At 4°C, it was centrifuged at 1500 rpm for 5 min, and the supernatant was completely discarded. Five times the volume of red blood cell lysate was added, and it was lysed at room temperature for 8 min, during which it was vortexed twice with a vortex mixer. The bottom stem cells were retained by centrifugation, the supernatant was discarded, and it was resuspended in PBS (2% BSA) for subsequent detection.

[0121] 3.3 Determination of Mouse Urinary Protein

[0122] Use 2.0 mL cryogenic tubes to continuously collect urine samples from experimental mice. Taking the first drug administration time as day 1, collect morning urine from mice on days 0, 7, 14, 21, 28, 35, 36, 37, ……, 42 respectively. Gently press the bladder of the mouse with the thumb to help the mouse urinate, and avoid causing anxiety in the experimental animals during the sampling process. The collected urine samples are immediately frozen with liquid nitrogen and stored in liquid nitrogen, and then transported to a BSL-1 laboratory for urine protein detection.

[0123] The urine samples to be tested are thawed on ice. The detection is based on a urine protein quantification test kit (CBB method). The CBB reagent can bind to the amino groups of proteins and cause a color change of the reagent from brown to blue in an acidic medium. Preparation of the CBB working solution: Dilute the CBB reagent 5-fold with double-distilled water to obtain the detection working solution. The preparation of the detection reagents is shown in Table 5. Set 3 detection replicates for each sample. After the color change stabilizes, pipette 100 μL of the sample to be tested from each well into a 96-well microplate, and use a BioTek microplate reader to measure the absorbance of the samples (OD595) at 595 nm. Calculate the average urine protein concentration per day for each sample. Taking the urine protein concentration of the urine sample on day 0 as the baseline, calculate the relative change trend of urine protein concentration in each mouse after 6 weeks of intervention. The formula for calculating urine protein concentration is as follows:

[0124]

[0125] Table 5 Reagent preparation table for CBB detection method

[0126]

[0127] 3.4. Glomerular and tubular lesions in mice

[0128] Use ophthalmic forceps and a hook needle to bluntly separate the peritoneal and abdominal muscle layer tissues of the mouse, expose the abdominal cavity, gently push the intestines to one side, expose the posterior part of the abdominal cavity, and find the dark red broad bean-shaped kidneys on both sides of the spine in the posterior part of the abdominal cavity, close to the dorsal wall. Lift the kidney gently with forceps, carefully separate the covering fat and connective tissue with scissors, expose the renal hilum, cut short the renal artery, vein and ureter, and gently take out the complete kidney. Rinse the bloodstains with pre-cooled PBS buffer containing 2% BSA, put the left kidney into a cryogenic tube and freeze it quickly with liquid nitrogen, and put the right kidney into 5 times the volume of formalin fixative, and fix it at 4 °C in the dark for 12 h.

[0129] This experiment was commissioned by Hubei Bioscience Co., Ltd. to perform sample embedding, sectioning, and staining of paraffin sections (4 mm) with periodic acid-Schiff reagent (PAS). We blindly evaluated glomerular pathology by assessing 20 glomerular cross-sections of each kidney and scored each glomerulus according to a semi-quantitative scale. The PAS scoring criteria refer to previous literature reports (Chen K, Deng Y, Shang S, et al. Complement factor B inhibitor LNP023 improves lupus nephritis in MRL / lpr mice [J]. Biomed Pharmacother, 2022, 153: 113433.): The scores for glomerular mesangial cell hyperplasia and basement membrane thickening were 0 - 4 points (0, none; 1, mild; 2, mild to moderate; 3, moderate; 4, severe); the score for glomerular PAS+ deposition was 0 - 4 (same criteria as above); the score for tubulointerstitial lesions was 0 - 4 (0, none; 1, <25%; 2, 25 - 50%; 3, 50 - 75%; 4, >75%); the score for crescent lesions was 0 - 4 (0, none; 1, <25%; 2, 25 - 50%; 3, 50 - 75%; 4, >75%).

[0130] 3.5 IFN-γ production level in mouse kidneys

[0131] First, bake the slides in an oven at 65 °C for 6 h to ensure firm adhesion of the kidney tissue sections to the slides. Then, dewax with xylene (three times, 15 min each), rehydrate with alcohol (soak twice in 100% and 95% alcohol, once in 85% and 75% alcohol, 5 min each), and hydrate (rinse gently with tap water for 30 min). Then, immerse the sections in citrate antigen retrieval solution (pH = 6.0) and perform antigen heat retrieval with high-power microwave for a total of 15 min (avoiding over-boiling and overflow of the liquid). Take out the sections in the box and cool them in water to room temperature, then wash with TBST buffer. Cover the tissue with Triton x-100 for 5 min to make holes, wash with TBST, 3 times; cover the tissue with 3% H2O2 and incubate for 7 min to block and inactivate endogenous peroxidase, wash with TBST, 3 times; block with 5% PBS buffer containing BSA at room temperature for 30 min.

[0132] Dilute the IFN-γ rabbit anti-mouse primary antibody with 2% BSA-containing PBS buffer at a ratio of 1:500. For each tissue, add 100 μL to completely cover the tissue, and place it in a wet box at 4°C overnight. Take out the sections from the chromatography cabinet, warm them to room temperature for 30 min, dilute the secondary antibody and incubate at room temperature for 1.5 h, and wash with TBST 3 times for a total of 15 min. Drop the chromogenic solution and observe under the microscope until the tissue in the field of view turns brown. After staining for 2 min, thoroughly wash off the chromogenic solution. Stain with hematoxylin for 20 s, wash several times, and heat in a microwave oven for 30 s to blue. Wait for the sections to dry, seal them with neutral resin, and observe under bright field of the microscope.

[0133] III. Experimental Results

[0134] 1. Compound L3011 reduces the levels of total IgG and anti-dsDNA-IgG in the peripheral blood of mice

[0135] In the SLE mouse model, the elevation of autoantibody levels such as anti-double-stranded DNA (anti-dsDNA) is closely related to pathological manifestations such as kidney damage and neutropenia. These antibodies activate the complement system by forming immune complexes, leading to multi-organ inflammatory damage. ELISA was used to detect the levels of total IgG antibody and autoantibody in the sera of mice in different treatment groups, and the results are as Figure 5 shown. After intervention with the positive drug and compound L3011, the level of total IgG in the peripheral blood of mice decreased to no significant difference compared with the blank control group, indicating that the abnormal activation of B cells in the mouse body was alleviated. At the same time, the compound L3011 intervention group could significantly reduce the level of anti-dsDNA specific antibody in the peripheral blood circulation of MRL / lpr mice, while no significant decrease in the level of autoantibody was observed in the positive drug group, indicating that the phenomenon of autoimmunity attack in the experimental group of mice was improved, and compound L3011 could inhibit the disease activity of SLE mice by reducing the level of autoantibody.

[0136] 2. Compound L3011 inhibits splenomegaly and peripheral lymph node hyperplasia in mice

[0137] Compound L3011 was administered to MRL / lpr mice at doses of 2 mg / kg and 10 mg / kg respectively. After continuous administration for 6 weeks, it was observed that the degree of swelling of the spleen appearance in the L3011 intervention group of mice was lower than that in the MRL / lpr model control group ( Figure 6 A in). Weighed the body weight and wet weight of the spleen of mice to obtain the spleen index of each mouse ( Figure 6 B in). The results showed that there was a significant difference in the spleen index between the blank control group of mice and the model group of mice, indicating that the spleen of MRL / lpr was abnormally enlarged compared with normal mice. The spleen index of the prednisolone intervention group was significantly lower than that of the model group, and there was also a difference in the spleen index between the L3011 (2 mg / kg) intervention group and the model group, indicating that compound L3011 could inhibit the degree of splenomegaly (Table 6).

[0138] Table 6 Record Table of Mouse Spleen and Body Weight

[0139]

[0140] Note: The body weight of the mice was weighed and recorded immediately after induced anesthesia. The spleen index was equal to the ratio of spleen weight to body weight (w / w).

[0141] Both the spleen and peripheral lymph nodes are important secondary immune organs of the body and are the main sites for lymphocyte differentiation and maturation. It is Figure 7 known that compound L3011 can alleviate the hyperplasia of peripheral lymph nodes during the progression of MRL / lpr lupus disease.

[0142] 3. Compound L3011 Slows Down the Increase in Urinary Protein in Mice

[0143] The urinary protein content of mice in different treatment groups was detected by the CBB method (see Table 7). The urinary protein level on the day before drug intervention (day 0) was used as the baseline for the increase in urinary protein in mice, and a trend graph of the increase in urinary protein in mice was drawn (see Figure 8 ). The results showed that compared with the blank control group of mice (MRL / MpJ), the trend of the increase in urinary protein in the urine of MRL / lpr mice was significantly increased without drug intervention after six weeks; the positive drug prednisolone could inhibit the increase in urinary protein in lupus mice; after intervention with compound L3011, both the high-dose group and the low-dose group could make the increase in urinary protein return to the baseline level (MRL / MpJ), and there was no significant difference in the increase in urinary protein in lupus mice in the non-intervention group (P > 0.05).

[0144] Table 7 Average Urinary Protein in Mice

[0145]

[0146] 4. Compound L3011 Inhibits Glomerular and Tubular Lesions in Mice

[0147] Periodic Acid-Schiff stain (PAS) was used to analyze the pathological changes of the kidneys, and the morphological structure of the mouse kidney tissue and the situation of glycogen deposition (PAS+) were observed. The pathological analysis results showed that in the model group, there were mesangial cell hyperplasia, basement membrane thickening (black "→"), glycogen deposition (blue "→"), crescent formation (red "→") and detachment of tubular epithelial cells (brown "→"). No significant glomerular and tubular lesions were found in the positive drug control group (prednisolone) and the L3011 intervention group ( Figure 9 A in). The sections were blindly scored and statistically analyzed, and the analysis results are shown in Figure 9In group B, compared with the blank control group, MRL / lpr mice showed significant mesangial hyperplasia and tubular lesions in the glomeruli, a significant increase in glycogen deposition, and fibrotic changes and crescent formation in some glomeruli, indicating that the disease progression of systemic lupus erythematosus mice can involve the kidney organs. At the same time, the renal lesion condition of the compound L3011 intervention group mice was lower than that of the model group. At the administration dose of 2 mg / kg, compound L3011 could significantly relieve the renal pathological changes caused by the development of lupus disease.

[0148] 5. Compound L3011 inhibits the production of IFN-gamma in the kidneys of mice

[0149] The increased expression of IFN-γ in SLE patients can be considered an indicator of SLE disease activity. In this experiment, anti-IFN-γ antibody was used to examine the expression level of IFN-γ in mouse kidney sections by immunohistochemical staining. The results are as Figure 10 shown. Compared with the kidneys of the model group mice, the compound L3011 (2 mg / kg and 10 mg / kg) intervention group had a significant effect of reducing IFN-γ.

[0150] Example 6: Compound L3011 reduces the proportion of plasmablasts in the spleens and peripheral lymph nodes of lupus mice in vivo

[0151] I. Experimental materials

[0152] Flow cytometry antibodies: APC-Cy7 Rat Anti-Mouse CD19 Antibody, BV786 Rat Anti-Mouse IgM Antibody, BV510 Rat Anti-Mouse IgD Antibody, BV605 Rat Anti-Mouse CD38 Antibody, PE-Cy7 Rat Anti-Mouse CD23 Antibody, BB515 Rat Anti-Mouse CD138 Antibody, APC Rat anti-Mouse CD21 / CD35 Antibody, PE Hamster Anti-Mouse CD80 Antibody, PerCP-Cy5.5 Rat Anti-Mouse CD45R / B220 Antibody, BV421 Rat Anti-Mouse T- and B-Cell Activation Antigen (GL7); PBS, FVS (BD), flow cytometry tubes, centrifuges, 70 μm pore size cell sieves.

[0153] II. Experimental methods

[0154] Detection of B cell subsets in the spleen and lymph nodes:

[0155] Cell count, each flow cytometry tube contains 1×10 6 Total cells, centrifuged at 1500rpm for 5min, removed the supernatant, resuspended the cells in 500μL PBS solution containing 2% FBS, centrifuged at 1500rpm for 5min, and removed the buffer. Dead and alive dye: FVS (1:2000) was added to 1×PBS, 50μL per flow tube, incubated at room temperature in the dark for 10min, added 300μL 1×PBS solution (containing 2% FBS) to stop staining, centrifuged at 1500rpm for 5min, and removed the remaining antibodies. Live and dead fluorescent dye: FVS (1:2000), add to 1×PBS solution and mix well, 50 μL per tube, cover with tin foil and stain at room temperature for 10 minutes; flow cytometry antibodies: flow cytometry anti-human antibodies CD19 (1:200), CD45R / B220 (1:200), CD80 (1:50), CD23 (1:100), CD21 (1:100), CD38 (1:50), CD138 (1:50), IgM (1:200), IgD (1:200), GL7 (1:50), add to 1×PBS solution (containing 2% FBS) and mix well, 50 μL per tube, prepare single staining and blank tubes at the same time, put them in a 4°C chromatography cabinet, cover with tin foil and stain for 30 minutes. Add 300 μL / tube 1×PBS buffer (containing 2% FBS) to dilute the dye, centrifuge at 1500rpm for 5 min, discard the supernatant antibody, add 500 μL / tube buffer (containing 2% FBS) to resuspend the cells, filter the cell clumps with a 70 μm pore size cell sieve, and perform flow cytometry detection. The markers of each cell are shown in the table below.

[0156]

[0157] 3. Experimental Results

[0158] The results of flow cytometry showed that in the spleen, there was a difference between the proportion of PB in the blank control group (MRL / MpJ) and the model group (MRL / lpr). Since PB is the main contributor to the disease activity of SLE patients, the increased proportion of PB proves that the spleen of MRL / lpr lupus spontaneous mice is hyperfunctional. After six consecutive weeks of drug intervention, the positive drug prednisolone, one of the commonly used drugs in the clinical treatment of systemic lupus erythematosus, not only did not inhibit PB differentiation in this experiment, but instead caused the final proportion of PB to be higher than that of the model group, indicating that prednisolone's inhibition of splenic parenchymal enlargement may be due to inflammation suppression, and it does not directly act on the key lymphocytes of SLE disease. However, the proportion of PB to Pan-B in the spleen of the L3011 experimental group was significantly different from that of the model group ( Figure 11), it is proved that L3011 can effectively inhibit the differentiation and maturation of B cells into PB in the spleen in vivo, that is, L3011 may play a pharmacological role in improving the SLE phenotype by inhibiting the abnormal differentiation of B cells in MRL / lpr mice in vivo.

[0159] In the peripheral lymph nodes, compared with the blank control group, there was a very significant abnormal development of immune function in the peripheral lymph nodes of the model group. After continuous administration for six weeks, the L3011 intervention group could significantly block the differentiation of PB in the lymph nodes ( Figure 12 ). The above experiments indicate that L3011 can directly act on the process of B cell differentiation and functional development (antibody secretion).

[0160] In summary, in vitro experiments show that the compound L3011 has good safety and effectiveness, and can further inhibit the secretion of autoantibodies mediated by the anti-dsDNA IgG+B cell subtype by inhibiting the process of in vitro differentiation of B cells into plasmablasts. The mechanism of the compound L3011 in inhibiting the activation of primary B cells in SLE patients in vitro may be related to its inhibitory effect on the variable domain gene of the immunoglobulin heavy locus. Animal experiments show that the compound L3011 can effectively reduce the contents of total IgG and anti-dsDNA-IgG in the peripheral blood of mice, inhibit splenomegaly and peripheral lymph node hyperplasia in mice, slow down the increase of urinary protein in mice, inhibit glomerular and tubular lesions in mice, inhibit the production of IFN-gamma in the kidneys of mice, and inhibit the differentiation of plasmablasts in the spleen and peripheral lymph nodes of mice, that is, directly act on the process of B cell differentiation and functional development (antibody secretion), and has the prospect of being developed into a drug for the treatment of systemic lupus erythematosus.

[0161] The role of the above embodiments is to specifically introduce the substantial content of the present invention, but those skilled in the art should know that the protection scope of the present invention should not be limited to this specific embodiment.

Claims

1. A compound of the following structure or a pharmaceutically acceptable salt or solvate thereof:

2. Use of the compound according to claim 1 or a pharmaceutically acceptable salt or solvate thereof in the preparation of a medicament for treating systemic lupus erythematosus.

3. According to the use of claim 2, the medicament uses the compound according to claim 1 or a pharmaceutically acceptable salt or solvate thereof as an active ingredient and is made into a pharmaceutically acceptable dosage form through pharmaceutically acceptable excipients.

4. The application according to claim 3, characterized in that: The excipients are solids, liquids or semi - solids.

5. The application according to claim 3, characterized in that: The dosage forms include tablets, capsules, injections and pills.

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