Application of small molecule compound in preparation of IL-6R inhibitor

Abstract

The invention discloses application of a small molecule compound in preparation of an IL-6R inhibitor, and belongs to the technical field of medicines. According to the research, two novel small molecule compounds targeting IL-6R are successfully identified through multiple calculation methods and in-vitro experiments, the two novel small molecule compounds are respectively named as Z1268663363 and Z1268663684, and the small molecule compounds can inhibit IAV and SARS-CoV-2 induced IL-6 signal channels and IL-6 induced phosphorylation of STAT3, JAK2 and gp130, and can be used for inhibiting IL-6R signal channels induced by IAV and SARS-CoV-2 induced IL-6 signal channels induced by IAV and SARS-CoV-2 induced IL-6 signal channels induced by IAV and SARS-CoV-2. The TF-1 cell proliferation stimulated by IL-6 can also be inhibited, and the combination of IL-6 and IL-6R is blocked. In addition, the MD simulation demonstrates spontaneous and sustained binding between the compound and IL-6R. The research reveals a flexible binding site of IL-6R, determines a promising small molecule conjugate, and promotes the development of new drugs for IL-6 related diseases.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to an application of a small molecule compound in the preparation of an IL-6R inhibitor. Background Art

[0002] IL-6 is an important pleiotropic cytokine with multiple physiological functions, and is of great significance in inflammatory response, immune regulation and metabolic balance. In the human body, IL-6 must first bind to the specific receptor IL-6R and then form a hexameric signaling complex with the glycoprotein gp130 to exert its extensive biological functions. The biological effects of IL-6 are mainly mediated by the interaction with its receptor interleukin-6 receptor (IL-6R, also known as IL-6Rα), which has two forms: transmembrane (mIL-6R) and soluble (sIL-6R). IL-6R lacks intrinsic signaling ability, and the IL-6 / IL-6R complex needs to bind to glycoprotein 130 (gp130, also known as IL-6Rβ) to initiate intracellular signaling pathways. The interaction between IL-6 and mIL-6R exists in specific cell types, such as hepatocytes, leukocytes and epithelial cells, and is called classical signaling. In contrast, the interaction between IL-6 and sIL-6R is known as trans-signaling, which is widely present in human serum and plays a central role in the transition from acute to chronic states in several diseases, including autoimmune diseases, inflammatory diseases, cardiovascular diseases, central nervous system diseases, and tumors. The interaction between IL-6 and IL-6R promotes the formation of hexamer by recruiting gp130 and subsequent homodimerization. Subsequently, Janus kinases (JAKs), especially JAK1, JAK2, JAK3, or Tyk2, phosphorylate the cytoplasmic domain of gp130, ultimately leading to the activation of signal transducer and activator of transcription 3 (STAT3).

[0003] Inhibitors targeting IL-6 and IL-6R offer an important therapeutic avenue for IL-6-related diseases. Monoclonal antibodies such as siltuximab targeting IL-6 have been approved for the treatment of patients with Castleman disease, while clazakizumab and olokizumab are in Phase III trials for antibody-mediated rejection (ABMR) and RA, respectively. Tocilizumab, sarilumab, and satralizumab, which target IL-6R, are FDA-approved drugs for the treatment of RA and neuromyelitis optica spectrum disorder (NMOSD), and tocilizumab has been approved in China for the treatment of severe COVID-19 with elevated IL-6 levels.

[0004] Biologics that interfere with the IL-6 / IL-6R complex are effective, but they are often accompanied by significant adverse reactions and face challenges related to management, cost, and production. Therefore, the development of small molecule inhibitors that can block the formation of the IL-6 / IL-6R complex and improve their safety, convenience, and cost-effectiveness remains an urgent need. Summary of the invention

[0005] In order to solve the above-mentioned deficiencies in the prior art, the purpose of the present invention is to provide an application of a small molecule compound in the preparation of an IL-6R inhibitor, so as to provide a new small molecule inhibitor targeting IL-6R.

[0006] The technical solution of the present invention to solve the above technical problems is as follows: Provided is a use of a small molecule compound in the preparation of an IL-6R inhibitor, wherein the molecular formula of the small molecule compound Z1268663363 is C 16 H 19 N3O2, the structural formula is shown in Formula I or the molecular formula of the small molecule compound Z1268663684 is C 17 H 21 N3O2, the structural formula is shown in Formula II; .

[0007] The present invention provides an IL-6R inhibitor, which includes at least one of the above-mentioned small molecule compounds.

[0008] The present invention also provides a use of the above IL-6R inhibitor in the preparation of drugs for treating autoimmune diseases, inflammatory diseases, cardiovascular diseases and tumors.

[0009] Furthermore, IL-6R inhibitors can inhibit the binding between IL-6 and IL-6R.

[0010] Furthermore, IL-6R inhibitors can inhibit IL-6-induced phosphorylation of STAT3, JAK2, and gp130.

[0011] Furthermore, IL-6R inhibitors can inhibit IL-6-stimulated TF-1 cell proliferation.

[0012] Further, autoimmune diseases include rheumatoid arthritis and systemic lupus erythematosus.

[0013] Further, inflammatory diseases include Crohn's disease and Castleman's disease.

[0014] Further, cardiovascular diseases include atherosclerosis and myocardial infarction.

[0015] Further, central nervous system diseases include depression.

[0016] Further, tumors include multiple myeloma and prostate cancer.

[0017] The present invention has the following beneficial effects: This study successfully identified two new small molecule compounds targeting IL-6R through multiple computational methods and in vitro experiments. The small molecule compounds can block the binding of IL-6 to IL-6R and are named Z1268663363 and Z1268663684, respectively. Z1268663363 and Z1268663684 have inhibitory effects on the IL-6 signaling pathway and can inhibit IL-6 activation induced by influenza virus (IAV) and SARS-CoV-2. Protein imprinting (Western Blot) experiments showed that these compounds inhibited the phosphorylation of STAT3, JAK2 and gp130. Cell proliferation experiments showed that Z1268663363 and Z1268663684 inhibited the proliferation of TF-1 cells stimulated by IL-6. Further surface plasmon resonance (SPR) experiments confirmed the affinity of Z1268663363 and Z1268663684 for rIL-6Rα, and the obtained dissociation constants (Kd) were 14.83 μM and 8.27 μM, respectively. MD simulations verified that Z1268663363 and Z1268663684 were able to spontaneously re-enter the previously predicted binding site and spontaneously and continuously bind to IL-6R, which provides a validation site for IL-6R for future drug development. The two small molecule compounds of the present invention provide promising candidate drugs for the treatment of IL-6-related diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The RMSD values ​​of the protein backbone atoms (black), ligand heavy atoms (red), and active site residues (blue) of the complex of the small molecule compound bound to IL-6R relative to the initial coordinates during the second round of virtual screening; Figure 2 The binding mode and residue analysis diagram of Z1268663363 / IL-6R and Z1268663684 / IL-6R complexes; Figure 3 for the spontaneous binding of Z1268663363 and Z1268663684 to IL-6R; Figure 4 for the inhibitory effect of IL-6 on STAT3 activation in RAW264.7 cells; Figure 5 To evaluate the expression level of p-STAT3 in RAW264.7 cells at different concentrations of Z1268663363 and Z1268663684; Figure 6 The inhibitory effects of Z1268663684 and Z1268663363 on IL-6-induced TF-1 cell proliferation; Figure 7 Z1268663363 and Z1268663684 inhibit the interaction of IL-6 / IL-6Ra complex by directly binding to IL-6Ra. DETAILED DESCRIPTION

[0019] The following examples are only used to explain the present invention and are not intended to limit the scope of the present invention. If no specific conditions are specified in the examples, the conditions are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0020] The present invention discovered two small molecule inhibitors Z1268663363 and Z1268663684 targeting IL-6R by combining computer simulation screening and in vitro verification. The two small molecule compounds Z1268663363 and Z1268663684 of the present invention were purchased from Enamine (https: / / www.enamine-genez.com / ).

[0021] Example 1: Molecular dynamics simulation (MD simulation) (1) System preparation: The GPU-accelerated PMEMD module in AMBER18 software was used to prepare the docking poses of the small molecule compound / IL-6R complex and perform MD simulations. The force field parameters of the small molecule compound and protein were generated by the following method: the charge of the small molecule was calculated using the Antechamber suite and the AM1-BCC charge model, and the missing parameters were obtained using the parmchk2 program. The protein was characterized using the AMBER ff14SB force field. Subsequently, the promtop and inpcrd files of the simulation system were generated for each complex using the LEaP module. The process included assigning force field parameters, adding counterions, and placing the structure in a rectangular periodic box in the TIP3P water area with an edge length of 10.0 Å.

[0022] (2) Simulation settings: MD simulations were performed in AMBER18 software using GPU acceleration. Each simulation started with a two-stage energy minimization. Initially, all atoms were minimized using a force constant of 10.0 kcal·mol -1 ·Å -2The minimization process consisted of 5000 steps using the steepest descent method followed by 5000 steps using the conjugate gradient method with a nonbonded cutoff of 10.0 Å. After minimization, the complex was heated in two phases: 2500 steps from 0 K to 100.0 K and 50000 steps from 100.0 K to 310.0 K with a force constant of 10.0 kcal·mol -1 ·Å -2 . Subsequently, equilibrium simulations were performed at 310.0 K for 500 ps by releasing all atomic constraints. Finally, all production MD simulations were performed for all systems selected in the docking results at a temperature of 310.0 K and a pressure of 1.0 atm in the NPT ensemble. A total of 78.35 μs MD simulations were performed, with 100 simulations in the first round and 753 in the second round, each lasting 50 ns. The simulation time was extended to 200 ns for small molecules with calculated binding energies below -20 kcal / mol (50 compounds in the first round) and below -30 kcal / mol (188 compounds in the second round).

[0023] (3) MD simulation trajectory analysis: In AMBER18 software, the structural properties (such as stability) in each trajectory were initially analyzed using CPPTRAJ. The RMSD of each snapshot relative to the initial structure was calculated to evaluate the convergence of the simulation. In order to elucidate the binding interaction between the inhibitor and IL-6R, a representative structure was extracted from the equilibrium trajectory of the 200 ns simulation. The mmpbsa.pl tool was used to determine the binding free energy between the IL-6R protein and the small molecule compound using the generalized Bonn surface area (MM / GBSA) method, and then the decomposition free energy calculation was performed to determine the key residues involved in the interaction. These structural and energy analyses were performed automatically using scripts. The binding energy calculation formula is as follows: Δ𝐺 calc =Δ𝐸 vdW +Δ𝐸 ele +Δ𝐺 sol_pol +Δ𝐺 sol_nonpol Among them, Δ𝐺 calc is the binding free energy of the complex; Δ𝐸 vdW is the van der Waals force; Δ𝐸 ele is the electrostatic energy; Δ𝐺 sol_pol is the polar solvation free energy; Δ𝐺 sol_nonpol is the nonpolar solvation free energy.

[0024] Example 2: In vitro experiments (1) Luciferase assay: RAW264.7 cells were transfected with the p-STAT3 luciferase reporter vector for 24 hours. Then, the cells were stimulated / infected with IL-6 (10 ng / mL), LIF (10 ng / mL), Influenza A / PR8 / 34 (MOI = 0.1), or SARS-CoV-2 (MOI = 0.05) for 3 hours in the presence or absence of the candidate compound. The cells were then lysed for luciferase assay.

[0025] (2) Western blot (WB): RAW264.7 cells were cultured at 1×10 6 Cells were seeded at the density of 10 μg / mL in 6-well plates. After seeding, cells were incubated with Z1268663363 or Z1268663684 for 1 hour and then stimulated with IL-6 (10 ng / mL) for 10 minutes. Cells were then harvested and lysed to obtain cell lysates. Cell lysates were separated by SDS-PAGE and transferred to PVDF membranes. Blots were probed with the indicated antibodies: 1 / 500 anti-p-stat3 (9131, Cell Signaling Technology), 1 / 500 anti-stat3 (MA1-13042, Invitrogen), 1 / 500 anti-p-jak2 (AF3024, Affinity), 1 / 500 anti-jak2 (AHO1352, Invitrogen), 1 / 500 anti-p-gp130 (sc-377572, Santa Cruz Biotechnology), 1 / 500 anti-gp130 (3732, Cell Signaling Technology), and 1 / 1000 anti-gapdh (AF1186, Beyotime).

[0026] (3) Cell proliferation assay: TF-1 cells were seeded in 96-well microplates at a density of 50,000 cells per well and stimulated with IL-6 (1 ng / mL) for 72 hours in the presence of tocilizumab (10 nM) or different concentrations of compounds. Cell proliferation was detected using the CyQUANT™ MTT Cell Viability Assay Kit (V13154, Invitrogen).

[0027] (4) Surface plasmon resonance: Surface plasmon resonance (SPR) was used to detect the effect of compounds on the binding affinity of IL-6 to IL-6R. The purified IL-6R protein was immobilized on a CM5 chip, and different concentrations of IL-6 were injected into the flow cell with or without 200 μM Z1268663363 or Z1268663684 at a flow rate of 20 mL / min, a separation time of 240 s / 120 s, and a separation time of 900 s / 600 s. Recombinant IL-6Rα protein (rIL-6Rα) was immobilized on a CM5 chip and injected with different concentrations of Z1268663363 or Z1268663684. The dissociation rate constant (Kd) of each concentration was calculated using the steady-state affinity data, with a flow rate of 20 mL / min, a separation time of 240 s, and a separation time of 900 s.

[0028] Results Analysis (1) Molecular mechanism of the binding of inhibitors Z1268663363 and Z1268663684 to IL-6R Two compounds, Z1268663363 and Z1268663684, were identified as inhibitors of IL-6 signaling pathway by in vitro experiments. To gain a deeper understanding of the interaction between these two compounds and IL-6R, MD simulations of Z126866336 / IL-6R and Z1268663684 / IL-6R for 200 ns were analyzed.

[0029] ① Overall structural properties of the compound / IL-6R complex During the entire 200 ns simulation, the RMSD of protein backbone atoms, ligand heavy atoms, and residues within 5 Å of the binding pocket were calculated (see Figure 1 ). The analysis results showed that the structural fluctuations were minimal, indicating that both complexes reached a convergence state through simulation. Among them, the RMSD values ​​of Z1268663363 and Z1268663684 were 2.54 Å and 2.72 Å, respectively. For residues adjacent to the binding site, the RMSD values ​​were 2.26 Å and 2.83 Å, respectively. These results demonstrate that both compounds are able to form stable and sustained interactions at the specific binding site of IL-6R.

[0030] ②Energy basis for small molecule compounds to bind to IL-6R The complex binding energy of Z1268663363 and Z1268663684 with IL-6R (∆𝐺 calc ) are -38.34 kcal / mol and -40.95 kcal / mol, respectively, as shown in Table 1. A detailed analysis of these binding energies shows that they can be decomposed into several contributing terms.vdW ) and electrostatics (∆𝐸 ele ) interaction is the main driving force for the binding of these compounds to IL-6R, whereas the polar solvation free energy (Δ𝐺 sol_pol ) hinders the binding process. By calculating the decomposition free energy (see Figure 2 C) Several key residues that contribute to binding were identified. The energetic contributions of residues F248, Y249, E296, and F298 are all less than -1.0 kcal / mol. Residue F248 has a significant energetic contribution of -5.28 kcal / mol and 1.13 kcal / mol in the Z1268663363 / IL-6R and Z1268663684 / IL-6R complexes, respectively. Similarly, residue E296 contributes significantly to both complexes, with an energetic contribution of -2.49 kcal / mol in Z1268663363 / IL-6R and -7.69 kcal / mol in Z1268663684 / IL-6R. In addition, residues C211 (-0.60 kcal / mol for Z1268663684 / IL-6R), R250 (0.70 kcal / mol for Z1268663363 / IL-6R), E297 (-4.23 kcal / mol for Z1268663363 / IL-6R), and Q300 (-5.25 kcal / mol for Z1268663684 / IL-6R) were also identified as key residues. The observed differences in the energy contributions of these key residues can be attributed to changes in their binding modes. These findings provide valuable insights into optimizing the interaction of small molecules with IL-6R at both the structural and energetic levels.

[0031] Table 1 Binding energy of small molecule compounds to IL-6R (∆𝐺 calc , kcal / mol)

[0032] Among them, a is the complex of small molecule compound / IL-6R in 200 ns MD simulation; b is the complex of small molecule compound / IL-6R in the spontaneous binding process through 2 μs MD simulation.

[0033] ③ Binding mode of small molecule compounds to IL-6R Z1268663363 and Z1268663684 are structurally similar, especially the 5,6,7,8-tetrahydroimidazo[1,2-a]pyridin-1-ium moiety that occupies almost the same position in the binding pocket. The nitrogen atom within the five-membered ring is able to form hydrogen bonding interactions with E296 from the S4 pocket (see Figure 2B and 2D), and establish hydrophobic interactions with F249 from the S3 pocket. However, the presence of chiral centers in these compounds leads to different orientations. The 2,3-dihydro-1,4-benzenedioxopyrimidine moiety of Z1268663684 is uniquely located at the C211 position of the S1 subpocket, and the nitrogen atom at the ring-ring junction forms a hydrogen interaction with E296. In contrast, the 2h-1,3-benzenediol-4-ylmethyl group of Z1268663363 undergoes a π-cation interaction with R250 of the S2 pocket, while the nitrogen atom at the ring-ring junction forms a hydrogen interaction with F248 of S2. Overall, these findings highlight the critical role of the S3 and S4 subpockets in the binding of small molecules to IL6R. This study provides important insights into the structural features and binding mechanisms of small molecules targeting IL-6R, thereby supporting future drug development plans.

[0034] (2) Spontaneous binding of small molecule compounds to IL-6R Since the SPR experiment showed that compounds Z1268663363 and Z1268663684 have the ability to bind to IL-6R, the binding modes of these two compounds with IL-6R were predicted by analyzing the 200 ns MD simulation. In order to further study the binding pathway between the compounds and IL-6R, a long-term MD simulation was performed. Figure 3 As shown in A, at the beginning of the simulation, the small molecule was placed approximately 20 Å away from IL-6R, and the RMSD value of the 2 μs simulation was calculated using the equilibrium representative conformations (Z1268663363 / IL-6R and Z1268663684 / IL-6R) of the 200 ns simulation trajectory as a reference.

[0035] ① Spontaneous binding of Z1268663363 to IL-6R Figure 3 B shows that Z1268663363 successfully reached the previously predicted binding site around ~200 ns and made conformational adjustments to optimize its binding mode. Throughout the simulation trajectory, taking the predicted Z1268663363 / IL-6R complex as a reference, it was observed that the RMSD value of Z1268663363 was always less than 5 Å for about 560 ns. This observation suggests that the small molecule maintains a certain degree of structural stability associated with IL6R during the MD simulation. During the simulation process of up to 2 μs, we captured a binding mode very similar to the predicted conformation around 580 ns ( Figure 3B), with an RMSD value of only 0.48 Å. This finding further confirms the accuracy of the 200 ns MD simulation representation. For this equilibrium trajectory, the calculated binding energy is -40.19 kcal / mol (see Table 1), which is very close to the initially predicted -38.34 kcal / mol. This consistency supports the validity of the predicted binding site and binding mode.

[0036] ② Spontaneous binding of Z1268663684 to IL-6R During the MD simulation, Z1268663684 required approximately 500 ns to explore and position itself to find a suitable binding site (see Figure 3 C). Subsequently, the compound gradually approaches a small molecule binding orientation consistent with the expected conformation of the Z1268663684 / IL-6R complex, and eventually reaches a stable state at the binding site. Throughout the simulation, the RMSD value of Z1268663684 / IL-6R representation of the 200 ns MD simulation was used as a reference, and the duration of Z1268663684 was maintained below 5 Å for approximately 1,480 ns. The results indicate that the small molecule compound stably binds to IL-6R for most of the simulation time. In addition, within the 2 μs simulation time, a conformation consistent with the expected conformation (see Figure 3 C) A very well-matched binding site with an RMSD value of only 0.47 Å, showing a high degree of structural similarity. The calculated binding energy of this equilibrium trajectory is -39.62 kcal / mol (see Table 1), which is almost the same as the previously predicted -40.95 kcal / mol. This result further verifies the accuracy of the prediction of the binding mode of Z1268663684 to IL-6R.

[0037] (3) Luciferase reporter gene assay to identify small molecule compounds as IL-6 signaling inhibitors To identify compounds that inhibit the IL-6 signaling pathway, we screened 19 commercially available compounds by evaluating their effects on IL-6-induced luciferase expression in mouse leukemia RAW264.7 cells transfected with p-STAT3-Luc. Specifically, RAW264.7 cells transfected with p-STAT3-Luc were stimulated with IL-6 in the presence of these compounds, and the subsequent luciferase activity was measured. Figure 4 A shows that Z1268663363 and Z1268663684 have significant inhibitory effects on the IL-6 signaling pathway; at a concentration of 100 μM, Z1268663363 ( Figure 4 B) and Z1268663684 ( Figure 4 C) It can reduce IL-6-induced luciferase activity by approximately 100%.

[0038] (4) Inhibitory effect of small molecule compounds on virus-induced IL-6 signaling Infections caused by SARS-CoV-2 and influenza A / PR8 / 34 (IAV) are known to enhance inflammatory responses, leading to increased IL-6 levels and subsequent activation of the IL-6 signaling pathway. To evaluate the potential of compounds Z1268663363 and Z1268663684 to modulate the IL-6 signaling pathway during viral infection, the effects of these small molecule compounds at different concentrations on luciferase expression, an indicator of p-STAT3 activation in response to IL-6 stimulation induced by SARS-CoV-2 and IAV infection, were investigated. Figure 4 D-4G shows that both Z1268663363 and Z1268663684 can effectively inhibit the activation of the IL-6 signaling pathway under viral infection, and the inhibitory effect becomes more obvious with the increase of compound concentration.

[0039] (5) Small molecule compounds inhibit IL-6-induced STAT3, JAK2 and gp130 phosphorylation RAW264.7 cells were treated with Z1268663363 and Z1268663684 for 1 hour and then treated with IL-6 (10 ng / mL) for 10 minutes. WB analysis showed that IL-6-induced p-STAT3 levels decreased in a dose-dependent manner after pre-incubation with Z1268663363 and Z1268663684 at concentrations of 1, 3, 10, 30, and 100 μM. In addition, the present invention also explored whether the inhibitory effects of Z1268663363 and Z1268663684 on STAT3 phosphorylation occurred upstream of the IL-6 signaling cascade. Given that IL-6-induced pSTAT3 activation depends on p-JAK2, the effects of Z1268663363 and Z1268663684 on JAK2 phosphorylation were evaluated. The results showed that Z1268663363 and Z1268663684 inhibited the phosphorylation of JAK2 and gp130 at an IL-6 concentration of 30 μM. These findings suggest that Z1268663363 and Z1268663684 inhibit JAK2 phosphorylation, which in turn leads to decreased gp130 phosphorylation and JAK2 / gp130-mediated STAT3 phosphorylation (see Figure 5 ).

[0040] (6) Small molecule compounds inhibit IL-6-induced proliferation of human erythroleukemia cell line TF-1 Z1268663363 and Z1268663684 were serially diluted into TF-1 cells in the presence of IL-6 (1 ng / ml) and cultured for 72 hours before proliferation assay. Tocilizumab, an anti-IL-6Rα monoclonal antibody with immunosuppressive properties, was used as a positive control. Figure 6 It can be seen that both Z1268663363 and Z1268663684 can significantly inhibit cell proliferation, thereby confirming that they have the ability to weaken the IL-6 signaling pathway.

[0041] (7) Small molecule compounds inhibit the formation of IL-6 / IL-6R complex by binding to IL-6R To test whether Z1268663363 and Z1268663684 can disrupt the interaction between IL-6 and IL-6R, we performed SPR experiments. The results showed that Z1268663363 and Z1268663684 inhibited the binding of IL-6 to IL-6R at a concentration of 200 μM (see Figure 7 A-7C). After we confirmed that Z1268663363 and Z1268663684 blocked the formation of IL-6 / IL-6R complex, we further performed SPR experiments to monitor the binding affinity between these compounds and IL-6R. Recombinant IL-6Rα (rIL-6Rα) was covalently immobilized on the dextran matrix of the CM5 sensor chip. Subsequently, different concentrations of Z1268663363 and Z1268663684 were introduced to the functionalized surface and the reference surface, respectively. The results showed that Z1268663363 and Z1268663684 could effectively bind to rIL-6Rα (see Figure 7 D-7G), the dissociation rate constants Kd values ​​of Z1268663363 and Z1268663684 from rIL6Rα were 14.83 μM and 8.27 μM, respectively.

[0042] In summary, the above data indicate that the two small molecule compounds Z1268663363 and Z1268663684 of the present invention can inhibit the activation of the IL-6 signaling pathway by targeting IL-6R, and are promising candidate drugs for IL-6-dependent inflammation.

[0043] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. Use of a small molecule compound in the preparation of an IL-6R inhibitor, characterized in that: The molecular formula of the small molecule compound is C 16 H 19 N3O2, the structural formula is shown in Formula I or the molecular formula of the small molecule compound is C 17 H 21 N3O2, the structural formula is shown in Formula II; 。 2. An IL-6R inhibitor, characterized in that The IL-6R inhibitor comprises at least one of the small molecule compounds according to claim 1.

3. Use of the IL-6R inhibitor according to claim 2 in the preparation of drugs for treating autoimmune diseases, inflammatory diseases, cardiovascular diseases, central nervous system diseases and tumors.

4. The use according to claim 3, characterized in that: The IL-6R inhibitor can inhibit the binding between IL-6 and IL-6R.

5. The use according to claim 3, characterized in that: The IL-6R inhibitor can inhibit the phosphorylation of STAT3, JAK2 and gp130 induced by IL-6.

6. The use according to claim 3, characterized in that: The IL-6R inhibitor can inhibit the proliferation of TF-1 cells stimulated by IL-6.

7. The use according to claim 3, characterized in that: The autoimmune diseases include rheumatoid arthritis and systemic lupus erythematosus.

8. The use according to claim 3, characterized in that: Such inflammatory diseases include Crohn's disease and Castleman's disease.

9. The use according to claim 3, characterized in that: The central nervous system disease includes depression.

10. The use according to claim 3, characterized in that: Such tumors include multiple myeloma and prostate cancer.

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