Use of a small molecule compound for the preparation of an IL-6R inhibitor

Abstract

The application discloses an application of a small molecule compound in preparation of an IL-6R inhibitor and belongs to the technical field of medicines. Through various calculation methods and in-vitro experiments, two novel small molecule compounds targeting IL-6R are successfully identified, and are named as Z1268663363 and Z1268663684 respectively. The small molecule compound can inhibit IAV and SARS-CoV-2 induced IL-6 signaling pathway, and phosphorylation of STAT3, JAK2 and gp130 induced by IL-6. In addition, the small molecule compound can inhibit IL-6 stimulated TF-1 cell proliferation and block the binding of IL-6 and IL-6R. In addition, MD simulation confirms the spontaneous and sustained binding between the compound and IL-6R. The research reveals the flexible binding site of IL-6R, and determines a promising small molecule binder, and promotes the development of new drugs for IL-6 related diseases.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medicine, and in particular to a small molecule compound for use in the preparation of an IL-6R inhibitor. BACKGROUND

[0002] IL-6 is an important pleiotropic cytokine with multiple physiological functions, and plays an important role in inflammatory response, immune regulation and metabolic balance. In the human body, IL-6 needs to first bind to the specific receptor IL-6R, and then form a hexameric signal complex with glycoprotein gp130 to exert its wide range of biological functions. The biological effects of IL-6 are mainly mediated by its interaction with its receptor interleukin-6 receptor (IL-6R, also known as IL-6Rα). This receptor 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 called trans-signaling and is widely present in human serum, and plays a central role in the transition from acute to chronic states of some diseases, including autoimmune diseases, inflammatory diseases, cardiovascular diseases, central nervous system diseases and tumors, etc. The interaction between IL-6 and IL-6R promotes the formation of a hexamer through the recruitment of gp130 and the subsequent homodimerization process. Subsequently, Janus kinases (JAKs), particularly JAK1, JAK2, JAK3 or Tyk2, phosphorylate the cytoplasmic domain of gp130, ultimately leading to the activation of signal transducers and activators of transcription 3 (STAT3).

[0003] Inhibitors targeting IL-6 and IL-6R provide an important therapeutic approach for IL-6-related diseases. Monoclonal antibodies targeting IL-6 such as siltuximab have been approved for the treatment of Castleman's disease patients, while clazakizumab and olokizumab are in phase III trials for antibody-mediated rejection (ABMR) and RA, respectively. Tocilizumab, sarilumab and satralizumab target IL-6R and 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] Biological agents 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, it is still urgent to develop small molecule inhibitors that can block the formation of IL-6 / IL-6R complex, improve their safety, convenience and cost-effectiveness. SUMMARY

[0005] In order to solve the above-mentioned deficiencies existing in the prior art, the purpose of the present application is to provide the 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 scheme of the present application for solving the above technical problems is as follows: the application of a small molecule compound in the preparation of an IL-6R inhibitor, the molecular formula of the small molecule compound Z1268663363 is C 16 H 19 N3O2, the structural formula is shown as formula I or the molecular formula of the small molecule compound Z1268663684 is C 17 H 21 N3O2, the structural formula is shown as formula II.

[0007] .

[0008] The present application provides an IL-6R inhibitor, the IL-6R inhibitor comprising at least one of the above-mentioned small molecule compounds.

[0009] The present application also provides the application of the above-mentioned IL-6R inhibitor in the preparation of a drug for treating autoimmune diseases, inflammatory diseases, cardiovascular diseases and tumors.

[0010] Further, the IL-6R inhibitor can inhibit the binding of IL-6 and IL-6R.

[0011] Further, the IL-6R inhibitor can inhibit the phosphorylation of STAT3, JAK2 and gp130 induced by IL-6.

[0012] Further, the IL-6R inhibitor can inhibit the proliferation of TF-1 cells stimulated by IL-6.

[0013] Further, the autoimmune disease includes rheumatoid arthritis and systemic lupus erythematosus.

[0014] Further, the inflammatory disease includes Crohn's disease and Castleman's disease.

[0015] Further, the cardiovascular disease includes atherosclerosis and myocardial infarction.

[0016] Further, the central nervous system disease includes depression.

[0017] Further, the tumor includes multiple myeloma and prostate cancer.

[0018] The present application has the following beneficial effects: The present application successfully identifies two novel small molecule compounds targeting IL-6R through various computational methods and in vitro experiments, which 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 the activation of IL-6 induced by influenza virus (IAV) and SARS-CoV-2. Western Blot experiments show that these compounds inhibit the phosphorylation of STAT3, JAK2 and gp130. Cell proliferation experiments show that Z1268663363 and Z1268663684 inhibit the proliferation of IL-6-stimulated TF-1 cells. Surface plasmon resonance (SPR) experiments confirm the affinity of Z1268663363 and Z1268663684 for rIL-6Rα, and the obtained dissociation constants (Kd) are 14.83 μM and 8.27 μM, respectively. MD simulation verifies that Z1268663363 and Z1268663684 can spontaneously re-enter the previously predicted binding site, and spontaneously and continuously bind to IL-6R, which provides a verified site of IL-6R for future drug development work. The two small molecule compounds of the present application provide promising candidate drugs for the treatment of IL-6-related diseases. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 RMSD values of the complex of the small molecule compound evolved over time binding to IL-6R in the second round of virtual screening relative to the protein backbone atoms (black), ligand heavy atoms (red) and active site residues (blue) of the initial coordinates;

[0020] Figure 2 Binding mode and residue analysis diagram of Z1268663363 / IL-6R and Z1268663684 / IL-6R complex;

[0021] Figure 3 Spontaneous binding of Z1268663363 and Z1268663684 to IL-6R;

[0022] Figure 4 Inhibition of IL-6 on STAT3 activation in RAW264.7 cells;

[0023] Figure 5To evaluate the expression level of p-STAT3 in RAW264.7 cells with different concentrations of Z1268663363 and Z1268663684;

[0024] Figure 6 To evaluate the inhibitory effect of Z1268663363 and Z1268663684 on IL-6-induced TF-1 cell proliferation;

[0025] Figure 7 To evaluate the inhibitory effect of Z1268663363 and Z1268663684 on the interaction of IL-6 / IL-6Ra complex by directly binding to IL-6Ra. DETAILED DESCRIPTION

[0026] The following examples are only used to explain the present application, and are not intended to limit the scope of the present application. If the specific conditions are not specified in the examples, the conventional conditions or the conditions recommended by the manufacturer are used. If the reagents or instruments used are not specified by the manufacturer, they are all conventional products that can be purchased on the market.

[0027] The present application discovers two small molecule inhibitors targeting IL-6R, Z1268663363 and Z1268663684, by a method combining computer simulation screening and in vitro verification. The two small molecule compounds Z1268663363 and Z1268663684 of the present application are purchased from Enamine Company (https: / / www.enamine-genez.com / ).

[0028] Example 1: Molecular dynamics simulation (MD simulation)

[0029] (1) System preparation: The docking pose of the small molecule compound / IL-6R complex was prepared using the GPU-accelerated PMEMD module in AMBER18 software, and MD simulation was performed. The force field parameters of the small molecule compound and the 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. This process includes assigning force field parameters, adding counterions, and placing the structure in a rectangular periodic box of TIP3P water with an edge length of 10.0 Å.

[0030] (2) Simulation settings: MD simulation was simulated using GPU acceleration in AMBER18 software. Each simulation started with two stages of energy minimization. Initially, all atoms were subjected to a 10.0 kcal·mol -1·Å -2 harmonic restraints, followed by unconstrained minimization. The minimization procedure included 5000 steps of steepest descent followed by 5000 steps of conjugate gradient 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, an equilibration simulation was performed at 310.0 K for 500 ps by releasing all atomic restraints. Finally, all production MD simulations were performed for all selected systems in the docking results in the NPT ensemble at a temperature of 310.0 K and a pressure of 1.0 atm. A total of 78.35 μs of MD simulation was performed, with 100 simulations in the first round and 753 simulations in the second round, each lasting 50 ns. The simulation time of small molecule compounds with calculated binding energy lower than -20 kcal / mol (50 compounds in the first round) and lower than -30 kcal / mol (188 compounds in the second round) was extended to 200 ns.

[0031] (3) Analysis of MD simulation trajectories: In the AMBER18 software, the structure characteristics (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. To elucidate the binding interactions between the inhibitor and IL-6R, a representative structure was extracted from the equilibrium trajectory of the 200 ns simulation. Using the mmpbsa.pl tool, the binding free energy between the IL-6R protein and the small molecule compound was determined by the generalized Born surface area (MM / GBSA) method, and then the free energy decomposition calculation was performed to determine the key residues involved in the interaction. These structure and energy analyses were performed automatically using scripts. The formula for calculating the binding energy is as follows:

[0032] Δ𝐺 calc =Δ𝐸 vdW +Δ𝐸 ele +Δ𝐺 sol_pol +Δ𝐺 sol_nonpol

[0033] Where 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.

[0034] Example 2: In vitro experiments

[0035] (1) Luciferase assay: RAW264.7 cells were transfected with p-STAT3 luciferase reporter vector for 24 hours. Then, cells were stimulated / infect 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 candidate compounds. Subsequently, cells were lysed for luciferase assay.

[0036] (2) Western blot assay (WB): RAW264.7 cells were seeded at 1 x 105cells per well in 6-well plates. After seeding, cells were incubated with Z1268663363 or Z1268663684 for 1 hour, then stimulated with IL-6 (10 ng / mL) for 10 minutes. Then, cells were harvested and lysed to obtain cell lysates. Cell lysates were separated by SDS-PAGE and transferred to PVDF membranes. Blots were detected with the following 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). 6 Cells were seeded in 6-well plates at a density. After seeding, cells were incubated with Z1268663363 or Z1268663684 for 1 hour, then stimulated with IL-6 (10 ng / mL) for 10 minutes. Then, cells were harvested and lysed to obtain cell lysates. Cell lysates were separated by SDS-PAGE and transferred to PVDF membranes. Blots were detected with the following 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).

[0037] (3) Cell proliferation assay: TF-1 cells were seeded in 96-well microplates at a density of 50,000 cells per well in the presence of tocilizumab (10 nM) or different concentrations of compounds, and stimulated with IL-6 (1 ng / mL) for 72 hours. Cell proliferation was detected using CyQUANT™ MTT Cell Proliferation Assay Kit (V13154, Invitrogen).

[0038] (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. 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, with a dissociation time of 240 s / 120 s, and a dissociation time of 900 s / 600 s. Recombinant IL-6Rα protein (rIL-6Rα) was immobilized on a CM5 chip, and different concentrations of Z1268663363 or Z1268663684 were injected. The dissociation rate constant (Kd) at each concentration was calculated using steady-state affinity data, with a flow rate of 20 mL / min, a dissociation time of 240 s, and a dissociation time of 900 s.

[0039] Results analysis

[0040] (1) Molecular mechanism of inhibitors Z1268663363 and Z1268663684 binding to IL-6R

[0041] Z1268663363 and Z1268663684 were identified as inhibitors of the IL-6 signaling pathway through in vitro experiments, in order to further understand the interaction between the two compounds and IL-6R, the MD simulation of Z126866336 / IL-6R and Z1268663684 / IL-6R for 200 ns was analyzed.

[0042] ① Overall structural properties of compound / IL-6R complexes

[0043] The RMSD of protein main chain atoms, ligand heavy atoms, and residues located within 5 Å of the binding pocket was calculated throughout the 200 ns simulation (see Figure 1 ). The results of the analysis showed that the structure fluctuated very little, indicating that both complexes reached a state of convergence through simulation, with RMSD values of 2.54 Å and 2.72 Å for Z1268663363 and Z1268663684, 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 can form stable and sustained interactions at specific binding sites of IL-6R.

[0044] ② Energy basis for small molecule compounds binding to IL-6R

[0045] The binding energy (Δ𝐺 calc) of -38.34 kcal / mol and -40.95 kcal / mol, respectively, as shown in Table 1. Detailed analysis of these binding energies revealed that they can be decomposed into several contributing terms. The van der Waals (AG vdW ) and electrostatic (AE ele ) interactions are the main driving forces for the binding of these compounds to IL-6R, in contrast, the polar solvation free energy (AG sol_pol ) plays a hindering role in the binding process. Several key residues that contribute to the binding were identified by the decomposition of free energy calculations (see Figure 2 C). The energy contributions of residues F248, Y249, E296, and F298 are all less than -1.0 kcal / mol. In the Z1268663363 / IL-6R and Z1268663684 / IL-6R complexes, the energy contribution of residue F248 is significant, at -5.28 kcal / mol and 1.13 kcal / mol, respectively. Similarly, residue E296 also contributes significantly to both complexes, with an energy contribution value of -2.49 kcal / mol for Z1268663363 / IL-6R and -7.69 kcal / mol for 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 the structural and energetic levels.

[0046] Table 1. Binding energies (AG calc , kcal / mol) of small molecule compounds with IL-6R

[0047]

[0048] wherein 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 spontaneous binding process by 2 μs MD simulation.

[0049] iii. Binding modes of small molecule compounds with IL-6R

[0050] Z1268663363 and Z1268663684 share structural similarities, particularly the 5,6,7,8- tetrahydroimidazol[l,2-a]pyridinium moiety occupying almost identical positions in the binding pocket. The nitrogen atom within the five-membered ring is able to form a hydrogen bonding interaction with E296 from the S4 pocket (see Figure 2 B and 2D), and establishes a hydrophobic interaction with F249 from the S3 pocket. However, the presence of a chiral center in these compounds leads to different orientations. The 2,3-dihydro-l,4-benzodioxin moiety of Z1268663684 is uniquely positioned at the C211 site of the SI subpocket, with the nitrogen atom at the ring-ring junction forming a hydrogen interaction with E296. In contrast, the 2h-l,3-benzenediol-4-ylmethyl group of Z1268663363 forms 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 small molecule binding to IL6R. This study provides important insights into the structural features and binding mechanisms of small molecules targeting IL-6R, thus supporting future drug development programs.

[0051] (2) Spontaneous binding of small molecule compounds to IL-6R

[0052] Since SPR experiments indicated that compounds Z1268663363 and Z1268663684 have the ability to bind to IL-6R, the binding modes of these two compounds to IL-6R were predicted by analyzing the 200 ns MD simulations. To further investigate the binding pathway between the compounds and IL-6R, long-time MD simulations were performed. As shown in Figure 3 A, at the beginning of the simulation, the small molecules were placed at a distance of approximately 20 Å from IL-6R. The RMSD values of the 2 μs simulations were calculated with the equilibrated representative conformation of the 200 ns simulation trajectories (Z1268663363 / IL-6R and Z1268663684 / IL-6R) as reference.

[0053] ① Spontaneous binding of Z1268663363 to IL-6R

[0054] Figure 3B indicates that Z1268663363 successfully reached the previously predicted binding site around 200 ns and made a conformational adjustment to optimize its binding mode. Throughout the simulation trajectory, the RMSD value of Z1268663363 was observed to be consistently below 5 A for a duration of approximately 560 ns with the predicted Z1268663363 / IL-6R complex as the reference. This observation suggests that the small molecule maintained a certain degree of structural stability in relation to IL6R during the MD simulation. At around 580 ns, we captured a binding mode very similar to the predicted conformation (see Figure 3 B) with an RMSD value of only 0.48 A. This finding further confirms the accuracy represented by the 200 ns MD simulation. For this equilibrated trajectory, the calculated binding energy was -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.

[0055] 2) Spontaneous binding of Z1268663684 to IL-6R

[0056] During the MD simulation, Z1268663684 required approximately 500 ns to explore and locate the appropriate binding site (see Figure 3 C). Subsequently, the compound gradually approached a small molecule binding orientation consistent with the predicted conformation of the Z1268663684 / IL-6R complex and eventually reached a stable state at the binding site. Throughout the simulation, the RMSD value of Z1268663684 remained below 5 A for a duration of approximately 1,480 ns with the 200 ns MD simulated Z1268663684 / IL-6R as the reference. The results indicate that the small molecule compound stably bound to IL-6R for most of the simulation time. Furthermore, within the 2 μs simulation time, a binding site very matching the expected conformation (see Figure 3 C) was effectively observed at around 1275 ns with an RMSD value of only 0.47 A, showing a high degree of structural similarity. The calculated binding energy for this equilibrated trajectory was -39.62 kcal / mol (see Table 1), which is almost identical to the previously predicted -40.95 kcal / mol. This result further validates the accuracy of the Z1268663684 binding mode prediction to IL-6R.

[0057] (3) Luciferase reporter gene assay to identify small molecule compounds as IL-6 signaling inhibitors

[0058] To identify compounds that inhibit the IL-6 signaling pathway, we screened 19 commercially available compounds by assessing their effects on IL-6-induced luciferase expression in p-STAT3-Luc-transfected mouse leukemia RAW264.7 cells. Specifically, p-STAT3-Luc-transfected RAW264.7 cells were stimulated with IL-6 in the presence of these compounds, and the subsequent luciferase activity was measured. From Figure 4 As can be seen, Z1268663363 and Z1268663684 had a significant inhibitory effect on the IL-6 signaling pathway; at a concentration of 100 μΜ, Z1268663363 ( Figure 4 B) and Z1268663684 ( Figure 4 C) could reduce IL-6-induced luciferase activity by about 100%.

[0059] (4) Inhibition of virus-induced IL-6 signaling by small molecule compounds

[0060] It is known that infection by SARS-CoV-2 and influenza A / PR8 / 34 (IAV) can enhance the inflammatory response, leading to increased levels of IL-6 and subsequent activation of the IL-6 signaling pathway. To assess the potential of the compounds Z1268663363 and Z1268663684 to modulate the IL-6 signaling pathway during viral infection, the effects of these small molecule compounds on luciferase expression at different concentrations were investigated, which is an indicator of p-STAT3 activation in response to IL-6 stimulation induced by SARS-CoV-2 and IAV infection. From Figure 4 As can be seen, Z1268663363 and Z1268663684 were both able to effectively inhibit the activation of the IL-6 signaling pathway under viral infection, and the inhibitory effect was more pronounced as the concentration of the compounds increased.

[0061] (5) Inhibition of IL-6-induced STAT3, JAK2, and gp130 phosphorylation by small molecule compounds

[0062] 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 the level of IL-6-induced p-STAT3 was dose-dependently decreased after pre-incubation with Z1268663363 and Z1268663684 at 1, 3, 10, 30 and 100 μΜ. In addition, the present application also explored whether the inhibitory effect of Z1268663363 and Z1268663684 on STAT3 phosphorylation occurred upstream of the IL-6 signaling cascade. Since IL-6-induced pSTAT3 activation depends on p-JAK2, the effect of Z1268663363 and Z1268663684 on JAK2 phosphorylation was evaluated. The results showed that Z1268663363 and Z1268663684 inhibited the phosphorylation of JAK2 and gp130 at an IL-6 concentration of 30 μΜ. These findings indicated that Z1268663363 and Z1268663684 inhibited JAK2 phosphorylation, which in turn led to reduced gp130 phosphorylation and JAK2 / gp130-mediated STAT3 phosphorylation (see Figure 5 ).

[0063] (6) Small molecule compounds inhibit IL-6-induced proliferation of human erythroleukemia cell line TF-1

[0064] Z1268663363 and Z1268663684 were serially diluted in the presence of IL-6 (1 ng / ml) to TF-1 cells, and proliferation experiments were performed after 72 hours of incubation. Tocilizumab, an anti-IL-6Ra monoclonal antibody with immunosuppressive properties, was used as a positive control. It was found that Figure 6 both Z1268663363 and Z1268663684 significantly inhibited cell proliferation, thereby confirming their ability to attenuate the IL-6 signaling pathway.

[0065] (7) Small molecule compounds inhibit the formation of IL-6 / IL-6R complex by binding to IL-6R

[0066] To test whether Z1268663363 and Z1268663684 could 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 μΜ (see Figure 7A-7C). After we confirmed that Z1268663363 and Z1268663684 blocked the formation of IL-6 / IL-6R complex, further, we monitored the binding affinity between these compounds and IL-6R by SPR experiments. Recombinant IL-6Rα (rIL-6Rα) was covalently immobilized on a dextran matrix of a 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 constant Kd values of Z1268663363, Z1268663684 and rIL6Rα were 14.83 μM and 8.27 μM, respectively.

[0067] In summary, the above data showed that the two small molecule compounds Z1268663363 and Z1268663684 of the present application could inhibit the activation of IL-6 signaling pathway by targeting IL-6R, and were promising candidate drugs for IL-6 dependent inflammation.

[0068] The above description is merely preferred embodiments of the present application, but not to limit the present application, any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. The use of an IL-6R inhibitor in the preparation of drugs for treating leukemia and inflammatory diseases, characterized in that, The molecular formula of the IL-6R inhibitor is C 16 H 19 N3O2, with the structural formula shown in Formula I, or the molecular formula of the IL-6R inhibitor is C 17 H 21 N3O2, the structural formula is shown in Formula II; 。

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