Use of compounds having a 4-quinolinecarboxamide nucleus for the preparation of a medicament for the treatment of rheumatoid arthritis

Abstract

The application provides application of a compound with a 4-quinoline carboxamide as a mother nucleus in preparation of a medicine for resisting rheumatoid arthritis, and belongs to the field of medicinal chemistry. The application finds, for the first time, that the compound AK968-1,2 with a 4-quinoline carboxamide as a mother nucleus is aimed at nicotinamide nucleotide transhydrogenase as a drug target, and the compound AK968-1,2 has a novel action mechanism of inhibiting differentiation of inflammatory immune cells (Th1 / Th17) by inhibiting nicotinamide nucleotide transhydrogenase, and shows significant anti-rheumatoid arthritis activity. The finding provides a novel intervention node for RA treatment, and has important scientific value and clinical conversion potential. The application provides a new candidate medicine for early intervention of rheumatoid arthritis, and is expected to play an important role in the field of RA prevention and treatment.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical chemistry, and particularly relates to the application of compounds with a 4-quinoline carboxamide core in the preparation of drugs for treating rheumatoid arthritis. Background Technology

[0002] Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic, erosive synovitis. The pathogenesis of RA is complex, involving multiple factors including genetics, environment, and abnormal immune regulation. The global prevalence of RA is approximately 0.5%-1%. RA can affect multiple joints throughout the body, and without timely and effective intervention, it can lead to irreversible joint destruction, loss of function, and involvement of multiple systems throughout the body, severely impacting patients' quality of life and work capacity. Currently, clinical treatment for RA mainly includes nonsteroidal anti-inflammatory drugs (NSAIDs), glucocorticoids, disease-modifying antirheumatic drugs (DMARDs, such as methotrexate), and biologics (such as TNF-alpha inhibitors, IL-6 receptor antagonists, and IL-17 inhibitors). However, current treatments still have many limitations: some patients do not respond well to existing treatments (refractory RA); biologics are expensive and require injection; long-term use of immunosuppressants can increase the risk of infection and tumors; current treatments mainly target the inflammatory symptoms of end-stage RA, with limited intervention options for early immune imbalances. Therefore, developing novel small-molecule anti-RA drugs with novel structures, clear mechanisms of action, and oral efficacy remains an important research direction in this field.

[0003] In recent years, immunological research has revealed the core role of T lymphocytes in the pathogenesis of rheumatoid arthritis (RA). Abnormal differentiation of CD4+ naive T cells is a key driver of RA immunopathological damage. In RA patients and animal models, the differentiation of naive CD4+ T cells into pro-inflammatory Th17 cells is abnormally enhanced, leading to a Th17 / Treg immune imbalance. Th17 cells, through the secretion of large amounts of the pro-inflammatory cytokine IL-17A, activate synovial fibroblasts, macrophages, and osteoclasts, driving synovial proliferation, inflammatory cell infiltration, cartilage erosion, and bone destruction. Therefore, restoring the Th17 / Treg balance has become an important strategy for RA treatment. Nicotinamide nucleotide transhydrogenase is a core regulator for maintaining the homeostasis of reduced nicotinamide adenine dinucleotide phosphate (NADPH) in the mitochondrial matrix. It utilizes the mitochondrial membrane potential (Δψ) to drive the conversion of hydrogen ions from NADH to NADP. +Translocation and NADPH generation. Maintaining NADPH homeostasis within the mitochondrial matrix is ​​crucial for clearing leaked hydrogen peroxide from the respiratory chain, maintaining the glutathione and thioredoxin reduction systems, and supporting mitochondrial biosynthesis. Redox signaling is closely related to multiple aspects of T cell biology, including balancing pro-inflammatory and anti-inflammatory immune responses. Short-term and highly active ROS act as second messengers, controlling cell proliferation and differentiation.

[0004] Quinoline carboxamide compounds have been disclosed in numerous patent documents for the treatment of autoimmune diseases. For example, international application PCT / SE99 / 01270 (WO00 / 03991) discloses quinoline-3-carboxamide derivatives for the treatment of multiple sclerosis, insulin-dependent diabetes mellitus, systemic lupus erythematosus, and rheumatoid arthritis. International application PCT / SE00 / 02055 (WO01 / 30758) further discloses quinoline carboxamides for the treatment of solid tumors. Bayer Pharma AG's authorized patent US 10,189,788 B2 discloses substituted N,2-diarylquinoline-4-carboxamide derivatives and their use as anti-inflammatory agents, wherein the N-terminal substituent of the amide is aryl or substituted aryl, primarily for the treatment of fibrosis and / or inflammatory diseases.

[0005] However, existing quinoline carboxamide anti-inflammatory compounds have the following shortcomings: First, their targets and mechanisms of action are limited. The anti-inflammatory activities of existing quinoline carboxamide compounds mainly involve S100A9 binding and FP receptor antagonism, and their association with nicotinamide nucleotide transhydrogenase (NNT) has not yet been revealed. As a core enzyme in mitochondrial redox regulation, NNT plays a crucial role in regulating immune cell function and chronic inflammation, but its potential as a therapeutic target for rheumatoid arthritis (RA) has not yet been explored.

[0006] Second, the limitations of the N-terminal substituent of amides. In the prior art, the N-terminus of quinoline-4-carboxamide compounds is mostly aryl or substituted aryl (such as US 10,189,788 B2), while the effects of introducing five-membered nitrogen-containing heterocycles such as isoxazolyl or thiadiazolyl on NNT inhibitory activity and anti-RA efficacy have never been disclosed or revealed.

[0007] Third, there is a lack of precise regulation targeting the differentiation of inflammatory immune cells. Most existing RA treatments directly inhibit the production of inflammatory factors or signal transduction, lacking precise intervention strategies that selectively affect the differentiation of pathogenic immune cells (Th1 / Th17) by regulating mitochondrial redox metabolism.

[0008] Therefore, there is an urgent need to develop novel anti-RA drugs with novel structures, clear mechanisms of action, and the ability to precisely regulate the differentiation of inflammatory immune cells. Summary of the Invention

[0009] In view of this, the object of the present invention is to provide the use of compounds with a parent nucleus of 4-quinoline carboxamide in the preparation of medicaments for treating rheumatoid arthritis.

[0010] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides the use of a compound with a 4-quinoline carboxamide core in the preparation of a drug for treating rheumatoid arthritis, the general formula of which is shown in Formula I:

[0011] Formula I; Preferably, the compound further includes a pharmaceutically acceptable salt thereof.

[0012] Preferably, R¹ in Formula I includes hydrogen, halogen, C1-C6 alkyl, C1-C6 alkoxy, halogenated C1-C6 alkyl, halogenated C1-C6 alkoxy, hydroxyl, cyano, nitro, amino, C1-C6 alkylamino, diC1-C6 alkylamino, C3-C7 cycloalkyl, aryl or heteroaryl. R² includes hydrogen, halogen, C1-C6 alkyl, C1-C6 alkoxy, halo-C1-C6 alkyl or halo-C1-C6 alkoxy; R³ includes hydrogen, halogen, C1-C6 alkyl, C1-C6 alkoxy, halo-C1-C6 alkyl, halo-C1-C6 alkoxy, hydroxyl, cyano, nitro or amino; R 4 Including hydrogen, C1-C6 alkyl groups, or pharmaceutically acceptable cations; Het includes 5- or 6-membered heteroaryl groups.

[0013] Preferably, the heteroaryl group is substituted with one or more substituents; The substituents include halogens, C1-C4 alkyl groups, C1-C4 alkoxy groups, halogenated C1-C4 alkyl groups, hydroxyl groups, cyano groups, or amino groups.

[0014] Preferably, the aryl group is substituted with one or more substituents; The substituents include halogens, C1-C4 alkyl groups, C1-C4 alkoxy groups, halogenated C1-C4 alkyl groups, hydroxyl groups, cyano groups, or amino groups.

[0015] Preferably, the compound whose parent nucleus is 4-quinoline carboxamide includes 2-(3-ethoxyphenyl)-N-(5-methyl-3-isoxazolyl)-4-quinoline carboxamide or 2-(3,4-dichlorophenyl)-N-(1,3,4-thiadiazol-2-yl)-4-quinoline carboxamide.

[0016] Preferably, the antirheumatoid arthritis includes inhibition of nicotinamide nucleotide transhydrogenase activity.

[0017] Preferably, the anti-rheumatoid arthritis includes relieving symptoms of rheumatoid arthritis such as joint swelling, synovial hyperplasia, and bone destruction, or reducing serum inflammatory factor levels.

[0018] Preferably, the antirheumatoid arthritis includes rheumatoid arthritis that does not respond adequately to methotrexate treatment.

[0019] Preferably, the drug includes disease-modifying antirheumatic drugs, nonsteroidal anti-inflammatory drugs, corticosteroids, or biological agents.

[0020] Compared with the prior art, the present invention has the following beneficial effects: This invention is the first to discover two novel small molecule compounds, AK968-1 and AK968-2, targeting nicotinamide nucleotide transhydrogenase (NNT) with 4-quinoline carboxamide as the parent nucleus. Through two rounds of virtual screening from a spec library, these compounds exhibit a novel mechanism of action: inhibiting NNT function and thus suppressing the differentiation of inflammatory immune cells (Th1 / Th17). They demonstrate significant anti-rheumatoid arthritis (RA) activity, providing a novel intervention point for RA treatment and possessing significant scientific value and clinical translational potential. Both compounds, AK968-1 and AK968-2, introduce a five-membered nitrogen-containing heterocycle (isoxazole group and thiadiazole group) at the N-terminus of the amide, significantly differentiating them from existing quinoline-4-carboxamide derivatives with an aryl group at the N-terminus, thus endowing them with unique NNT inhibitory activity and anti-RA efficacy. The AK968-1 and AK968-2 provided by this invention are small molecule compounds, offering advantages over biologics such as lower production costs, higher oral bioavailability, and better patient compliance. This invention validated the in vivo efficacy of AK968-1 and 2 using the CIA animal model system, providing reliable preclinical data to support its further clinical development. This invention offers a novel drug candidate for early intervention in rheumatoid arthritis (RA) and is expected to play an important role in the prevention and treatment of RA. Attached Figure Description

[0021] Figure 1 The MTS assay results and cell morphology images show the effect of AK968-1 on inhibiting the activation and proliferation of JKT cells. In the figures, A is the chemical structure of AK968-1, B is the IC50 value of AK968-1, C is the percentage of cell survival after treatment with different concentrations (0µM, 5µM, 10µM, 15µM), D is cell morphology images after treatment with different concentrations (0µM, 5µM, 10µM, 15µM) for DAY1-DAY3, and E shows the NADPH / NADP production of cells treated with different concentrations (0µM, 5µM, 10µM). + The ratio of .

[0022] Figure 2 The MTS assay results and cell morphology images show the effect of AK968-2 on inhibiting the activation and proliferation of JKT cells. In the figures, A is the chemical structure of AK968-2, B is the IC50 value of AK968-2, C is the percentage of cell survival after treatment with different concentrations (0µM, 20µM, 30µM), D is cell morphology images after treatment with different concentrations (0µM, 20µM, 30µM) for DAY1-DAY3, and E shows the NADPH / NADP ratio generated by cells treated with different concentrations (0µM, 20µM, 30µM). + The ratio of .

[0023] Figure 3 Figure 1 shows the experimental results of AK968-1 and AK968-2 inhibiting in vitro induced Th17 differentiation; Figure 2 shows the RORC mRNA expression level detected by qPCR after using AK968-1; Figure 3 shows the IL-17A secretion level detected by ELISA after using AK968-1; Figure 4 shows the RORC mRNA expression level detected by qPCR after using AK968-2; Figure 5 shows the IL-17A secretion level detected by ELISA after using AK968-2.

[0024] Figure 4 The experimental results of AK968-1 in alleviating the progression of arthritis in CIA mice are shown. A is a flowchart of CIA modeling and drug administration. B is a curve of CIA incidence and joint clinical score. C is a photograph of the joint appearance and HE pathological section (arrows indicate areas of synovial hyperplasia and inflammatory cell infiltration).

[0025] Figure 5 The results of AK968-1 inhibiting pro-inflammatory Th17 differentiation in CIA mice are shown in Figure 1; A shows the flow cytometry results of the Th17 / Treg ratio in the joints of CIA mice (top row: Foxp3+Treg; bottom row: RORgammat+Th17); B shows the quantitative results of RORgammat expression level and IL-17A secretion level in the joints of CIA mice.

[0026] Figure 6 The experimental results of AK968-2 in alleviating the progression of arthritis in CIA mice are shown. A is a flowchart of CIA modeling and drug administration. B is a curve showing the incidence of CIA and the clinical score of the joint. C is a photograph of the joint appearance and an HE pathological section (arrows indicate areas of synovial hyperplasia and inflammatory cell infiltration).

[0027] Figure 7The results of AK968-2 inhibiting pro-inflammatory Th17 differentiation in CIA mice are shown in Figure 1. A shows the flow cytometry results of the Th17 / Treg ratio in the joints of CIA mice (top row: Foxp3+Treg; bottom row: RORgammat+Th17). B shows the quantitative results of RORgammat expression level and IL-17A secretion level in the joints of CIA mice. Detailed Implementation

[0028] This invention provides the use of a compound with a 4-quinoline carboxamide core in the preparation of a drug for treating rheumatoid arthritis, the general formula of which is shown in Formula I:

[0029] Formula I; In this invention, the compound also includes a pharmaceutically acceptable salt thereof.

[0030] In this invention, R¹ in Formula I preferably includes hydrogen, halogen, C1-C6 alkyl, C1-C6 alkoxy, halogenated C1-C6 alkyl, halogenated C1-C6 alkoxy, hydroxyl, cyano, nitro, amino, C1-C6 alkylamino, diC1-C6 alkylamino, C3-C7 cycloalkyl, aryl or heteroaryl, more preferably includes C1-C4 alkoxy or halogen, and even more preferably includes ethoxy or chlorine; R² is located at the 4 position of the benzene ring, preferably including hydrogen, halogen, C1-C6 alkyl, C1-C6 alkoxy, halo-C1-C6 alkyl or halo-C1-C6 alkoxy, and more preferably including halogen; R³ preferably includes hydrogen, halogen, C1-C6 alkyl, C1-C6 alkoxy, halo-C1-C6 alkyl, halo-C1-C6 alkoxy, hydroxyl, cyano, nitro or amino, and more preferably includes hydrogen. R 4 Preferably, it includes hydrogen, C1-C6 alkyl, or pharmaceutically acceptable cations; Het preferably comprises a 5- or 6-membered heteroaryl group, more preferably comprising isoxazolyl, thiadiazolyl, oxadiazolyl, triazolyl, tetrazolyl, pyrazolyl, or imidazolyl, and even more preferably comprising isoxazolyl or thiadiazolyl; the heteroaryl group is preferably substituted with one or more substituents, the substituents preferably comprising halogen, C1-C4 alkyl, C1-C4 alkoxy, halogenated C1-C4 alkyl, hydroxyl, cyano, or amino, more preferably comprising one or more C1-C4 alkyl groups, and even more preferably methyl.

[0031] In this invention, the compound whose parent nucleus is 4-quinoline carboxamide preferably includes 2-(3-ethoxyphenyl)-N-(5-methyl-3-isoxazolyl)-4-quinoline carboxamide or 2-(3,4-dichlorophenyl)-N-(1,3,4-thiadiazol-2-yl)-4-quinoline carboxamide.

[0032] In this invention, the anti-rheumatoid arthritis preferably includes inhibiting nicotinamide nucleotide transhydrogenase activity, regulating the redox balance of mitochondria in immune cells, and / or inhibiting the differentiation of inflammatory immune cells, wherein the inflammatory immune cells preferably include Th1 cells and / or Th17 cells.

[0033] In this invention, the anti-rheumatoid arthritis includes relieving symptoms of rheumatoid arthritis such as joint swelling, synovial hyperplasia, and bone tissue destruction, or reducing serum inflammatory factor levels.

[0034] In this invention, the antirheumatoid arthritis preferably includes rheumatoid arthritis that does not respond adequately to methotrexate treatment.

[0035] In this invention, the drug preferably includes a disease-modifying antirheumatic drug, a nonsteroidal anti-inflammatory drug, a corticosteroid, or a biological agent.

[0036] In this invention, the drug is preferably an injection, oral tablet, oral capsule, or intra-articular administration formulation; the drug also preferably includes one or more excipients selected from pharmaceutically acceptable fillers, disintegrants, binders, lubricants, and solubilizers; the drug is preferably administered orally, intravenously, subcutaneously, or intra-articularly, in a single or multiple doses, and the clinical dosage is preferably 0.1~100 mg / kg / day, more preferably 1~10 mg / kg / day, and even more preferably 5 mg / kg / day.

[0037] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0038] Example 1

[0039] AK968-1 and AK968-2 inhibited JKT cell activation and proliferation in an MTS assay and inhibited NNT function, respectively.

[0040] (1.1) Experimental materials: human JKT cell line (T lymphocyte line); compounds AK968-1 and 2, purity >98%, prepared into stock solution with DMSO; MTS cell proliferation assay kit; RPMI 1640 medium; fetal bovine serum (FBS).

[0041] (1.2) Experimental grouping: JKT cells were randomly divided into 6 groups: 1) Control group (0 microM, containing an equal amount of DMSO solvent); 2) AK968-1 5µM group; 3) AK968-1 10µM group; 4) AK968-1 15µM group; 5) AK968-2 20µM group; 6) AK968-2 30µM group. Each group had 3 replicates.

[0042] (1.3) Experimental methods: (1.3.1) MTS assay: JKT cells were seeded at an appropriate density in 96-well plates and RPMI 1640 complete medium containing 10% FBS was added. A control group and a treatment group were set up. The treatment group was treated with two concentrations of AK968. The plates were incubated at 37°C in a 5% CO2 incubator for 3 days. At fixed daily detection times, MTS reagent was added, and after incubation at 37°C for 2 hours, the absorbance (OD490) at 490 nm was measured using a microplate reader. Cell morphology photographs were taken under an inverted microscope on DAY1, DAY2, and DAY3.

[0043] (1.3.2) NADPH / NADP + Ratio detection experiment: Similar to the MTS experiment, cells with different treatments were placed in a 37°C, 5% CO2 incubator and cultured for 2 days. Two days later, cells were collected by centrifugation. Cells from different treatment groups were seeded at appropriate densities in 96-well plates with white walls and clear bottoms, with three replicates per well. The following steps were then performed: ① Add an alkaline solution containing 1% DTAB to each well containing PBS, and briefly shake the plate to ensure homogenization and cell lysis; ② Add 50 μL of each sample to a blank well, followed by 25 μL of 0.4 N HCl to each well; ③ Cover the 96-well plate and incubate all samples at 60°C for 15 minutes; ④ Remove the 96-well plate and equilibrate at room temperature for 10 minutes; ⑤ Add 25 μL of 0.5 M Trizma® base to each well containing acid-treated cells for neutralization, and add 50 μL of HCl / Trizma® solution to each well containing alkaline-treated samples; ⑥ Add an equal volume of NADP / NADPH-Glo™ assay reagent to each well, and gently shake the plate to mix thoroughly. Incubate at room temperature for 30 to 60 minutes; ⑦ Record the luminescence value using a luminescence detector.

[0044] (1.4) Experimental results: such as Figure 1As shown in Figure 2, MTS assay results revealed that, compared to the Control group, the 10µM and 15µM AK968-1 treatment groups exhibited significant cell proliferation inhibition from Day 1, in a concentration-dependent manner. The 15µM group showed stronger inhibitory effects than the 10µM group. By Day 3, cell survival was approximately 70% in the 5µM group, 50% in the 10µM group, and 35% in the 15µM group; while AK968-2 survival was 70% and 52% at 20µM and 30µM, respectively. Cell morphology photographs also clearly showed that the cell density in the treatment groups was significantly lower than that in the control group, suggesting that AK968-1 and AK968-2 can effectively inhibit the activation and proliferation of JKT cells, with AK968-1 showing better efficacy. NADPH / NADP + Ratio analysis showed that AK968-1,2 treatment significantly reduced this ratio, indicating a shift from NADH to NADP. + The NADPH production pathway is inhibited.

[0045] Example 2

[0046] Experiments using AK968-1 and AK968-2 to inhibit in vitro induced Th17 differentiation

[0047] (2.1) Experimental materials: C57BL / 6 mouse spleen naive CD4+ T cells; compound AK968; anti-CD3 antibody, anti-CD28 antibody; recombinant mouse IL-6, TGF-beta, IL-23, IL-1beta; RORC qPCR primers; mouse IL-17A ELISA kit.

[0048] (2.2) Experimental groups: 1) Th0 control group (without Th17 differentiation inducing factor); 2) Control group (with Th17 differentiation inducing factor, without AK968-1, 2); 3) AK968-1 group (with Th17 differentiation inducing factor and 10µM AK968-1); 4) AK968-2 group (with Th17 differentiation inducing factor and 20µM AK968-2).

[0049] (2.3) Experimental methods: Naive CD4+ T cells were sorted from mouse spleens using immunomagnetic bead sorting. Naive CD4+ T cells were added to culture plates pre-coated with anti-CD3 (5 microg / ml). The Th0 group received only anti-CD28 (2 microg / ml); the Th17 differentiation group received anti-CD28, IL-6 (20 ng / ml), TGF-beta (5 ng / ml), IL-23 (20 ng / ml), and IL-1beta (10 ng / ml) to induce Th17 differentiation, and was also treated with AK968-1,2. Cells and culture supernatant were collected after 72 hours of culture.

[0050] (2.4) qPCR detection of RORC expression: Total RNA was extracted using the TRIzol method, reverse transcribed into cDNA, and real-time quantitative PCR was performed using the SYBR Green method to detect the expression level of RORC mRNA, with beta-actin as an internal control.

[0051] (2.5) ELISA detection of IL-17A: Collect cell culture supernatant and perform the IL-17A secretion level according to the instructions of the mouse IL-17A ELISA kit.

[0052] (2.6) Experimental results: such as Figure 3 As shown, compared with the Th0 control group, RORC expression and IL-17A secretion were significantly increased under Th17 differentiation conditions (p<0.001), indicating successful Th17 differentiation induction. Compared with the control group, the AK968-1 and AK968-2 treatment groups showed significantly decreased RORC mRNA expression levels (approximately 60%-70% decrease, p<0.001) and significantly decreased IL-17A secretion levels (approximately 40%-50% decrease, p<0.001). These results suggest that AK968-1 and AK968-2 can effectively inhibit Th17 cell differentiation in vitro.

[0053] Example 3

[0054] Establishment of a CIA mouse model of arthritis and AK968-1 and AK968-2 dosing regimens

[0055] (3.1) Laboratory animals: C57 / 6N male mice, 8-10 weeks old, weighing 18-22g, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. All animal experiments were approved by the institution's animal ethics committee.

[0056] (3.2) CIA modeling method: On day 0, mice were initially immunized by intradermal injection of a complete Freund's adjuvant emulsion containing bovine type II collagen (Collagen + CFA) at the base of the tail; on day 14, a booster immunization was performed with an incomplete Freund's adjuvant containing collagen. The modeling procedure is as follows: Figure 4 As shown in A in 6.

[0057] (3.3) Administration regimen: Starting from day 1 after booster immunization (approximately day 15), mice with successfully established models were randomly divided into three groups: CIA control group, CIA AK968-1 group, and CIA AK968-2 group. The CIA AK968-1 group was treated with intraperitoneal injection of 30 mg / kg AK968-1, and the CIA AK968-2 group was treated with intraperitoneal injection of 30 mg / kg AK968-2, for 30 consecutive days. The CIA control group received an equal volume of solvent as a control. A normal control group without modeling was also included.

[0058] (3.4) Arthritis assessment: From the date of administration, clinical arthritis scores were assessed 2-3 times per week in a blinded manner using a 0-4 point scale: 0 points = no redness or swelling; 1 point = mild redness and swelling; 2 points = moderate redness and swelling; 3 points = severe redness and swelling involving the entire joint; 4 points = joint stiffness or deformity. The incidence rate (percentage of mice exhibiting arthritis symptoms) was also recorded for each group. Mice were sacrificed after administration (days 60-70), and ankle joints and footpads were collected for further analysis.

[0059] Example 4

[0060] Effects of AK968-1 and 2 on the incidence and clinical score of arthritis in CIA mice

[0061] (4.1) Experimental methods: The CIA model was established according to the method in Example 3 and the mice were divided into groups and administered drugs. The incidence of arthritis and changes in clinical scores of mice in each group were recorded.

[0062] (4.2) Experimental results: such as Figure 4 As shown in B of Figure 6, CIA control mice began to develop arthritis symptoms approximately 20-30 days after modeling, with the incidence rate reaching 60%-70% by day 40-50, and the clinical score continuously increasing to approximately 3 points. In contrast, the incidence rates in the CIA AK968-1 and CIA AK968-2 groups were significantly lower, at 38% in the CIA AK968-1 group and 42% in the CIA AK968-2 group, with clinical scores remaining at a lower level (approximately 1 point), and significantly lower than the CIA control group throughout the observation period. These results indicate that both AK968-1 and AK968-2 can significantly inhibit the occurrence and development of arthritis in CIA mice, with AK968-1 showing better efficacy.

[0063] Example 5

[0064] (5.1) Experimental methods: After drug administration, ankle and footpad tissues of mice in each group were taken, fixed in 4% paraformaldehyde, decalcified, embedded in paraffin, and prepared into 5µM thick tissue sections for HE staining. The tissues were observed and photographed under an optical microscope to evaluate the degree of synovial hyperplasia, inflammatory cell infiltration, cartilage erosion, and bone destruction. At the same time, photographs of the joint appearance were also taken.

[0065] (5.2) Experimental results: such as Figure 4As shown in C in Figure 6 and C in Figure 6, the joints of mice in the CIA control group were significantly swollen and red, with limited joint movement. HE pathological sections revealed significant synovial hyperplasia, extensive inflammatory cell infiltration, cartilage erosion, and bone destruction, with pannus formation observed in some areas. In contrast, the joints of mice in the CIA AK968-1 and 2 groups appeared nearly normal, with significantly reduced swelling. HE staining showed a significant reduction in synovial hyperplasia, a marked decrease in inflammatory cell infiltration, better cartilage surface integrity, and a significant improvement in bone erosion. These results confirm that AK968-1 and 2 can effectively alleviate joint pathological damage in CIA mice.

[0066] Example 6

[0067] Flow cytometry analysis of the Th17 / Treg ratio in the joints of CIA mice

[0068] (6.1) Experimental Methods: Under aseptic conditions, tissue from the ankle joint and surrounding draining lymph nodes of CIA mice was collected and ground to prepare a single-cell suspension. After removing erythrocytes using erythrocyte lysis buffer, flow cytometry staining was performed. Surface staining: anti-CD3-APC, anti-CD4-FITC; Intracellular staining: after fixation and membrane rupture, anti-Foxp3-PE (Treg marker) and anti-RORgammat-PE (Th17 marker) antibodies were added. The proportions of Foxp3+Treg and RORgammat+Th17 cells in CD4+ T cells were detected and analyzed using flow cytometry.

[0069] (6.2) Experimental results: such as Figure 5 As shown in A in Figure 7, compared with the CIA control group, the proportion of Foxp3+Treg cells in CD4+ T cells in the joint region of mice in the CIA AK968-1 and 2 groups was significantly increased (approximately from 2% to 9% in AK968-1, and from approximately 1.5% to 7% in AK968-2, p<0.001), while the proportion of RORgammat+Th17 cells was significantly decreased (approximately from 4% to 2% in AK968-1, and from approximately 5% to 2% in AK968-2, p<0.01). These results suggest that both AK968-1 and 2 can restore the Th17 / Treg balance in the joint region by upregulating Treg cells and downregulating Th17 cells.

[0070] Example 7

[0071] Detection of RORC expression and IL-17A secretion in the joints of CIA mice

[0072] (7.1) Experimental methods: Synovial tissue of CIA mice was collected, total RNA was extracted by TRIzol method, and RORC mRNA expression level was detected by qPCR after reverse transcription (with beta-actin as internal control). At the same time, the supernatant of joint tissue homogenate was collected and IL-17A protein concentration was detected by ELISA.

[0073] (7.2) Experimental results: such as Figure 5 As shown in B in Figure 7, compared with the CIA control group, the expression levels of RORC transcription factors in the joints of the CIA AK968-1 and AK968-2 groups were significantly downregulated (p<0.01), and the secretion levels of IL-17A were also significantly reduced (AK968-1 decreased from approximately 33 pg / ml to approximately 16 pg / ml, and AK968-2 decreased from approximately 34 pg / ml to approximately 20 pg / ml, p<0.05). These results are consistent with the results of flow cytometry, further confirming that both AK968-1 and AK968-2 exert their anti-rheumatoid arthritis effects by inhibiting Th17 differentiation and IL-17A production.

[0074] In summary, this invention systematically demonstrated, through MTS cell proliferation experiments, in vitro Th17 differentiation induction experiments, a CIA mouse arthritis model, and various experimental techniques including flow cytometry, qPCR, and ELISA, that compounds AK968-1 and AK968-2, with 4-quinoline carboxamide as the parent nucleus, possess significant inhibitory activity against inflammatory immune cell differentiation and anti-rheumatoid arthritis activity. Both AK968-1 and AK968-2 effectively alleviate joint inflammation and pathological damage in CIA mice through multiple mechanisms, including inhibiting T cell activation and proliferation, downregulating the key Th17 differentiation transcription factor RORC, reducing IL-17A secretion, and restoring the Th17 / Treg balance. This invention provides important candidate compounds and scientific evidence for the development of novel anti-rheumatoid arthritis drugs.

[0075] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The use of compounds with a 4-quinoline carboxamide core in the preparation of drugs for treating rheumatoid arthritis, characterized in that, The general formula structure of the compound is shown in Formula I: Formula I.

2. The application according to claim 1, characterized in that, The compound also includes pharmaceutically acceptable salts of the compound.

3. The application according to claim 2, characterized in that, R¹ in Formula I includes hydrogen, halogen, C1-C6 alkyl, C1-C6 alkoxy, halo-C1-C6 alkyl, halo-C1-C6 alkoxy, hydroxyl, cyano, nitro, amino, C1-C6 alkylamino, diC1-C6 alkylamino, C3-C7 cycloalkyl, aryl or heteroaryl. R² includes hydrogen, halogen, C1-C6 alkyl, C1-C6 alkoxy, halo-C1-C6 alkyl or halo-C1-C6 alkoxy; R³ includes hydrogen, halogen, C1-C6 alkyl, C1-C6 alkoxy, halo-C1-C6 alkyl, halo-C1-C6 alkoxy, hydroxyl, cyano, nitro or amino; R 4 Including hydrogen, C1-C6 alkyl groups, or pharmaceutically acceptable cations; Het includes 5- or 6-membered heteroaryl groups.

4. The application according to claim 3, characterized in that, The heteroaryl group is substituted by one or more substituents; The substituents include halogens, C1-C4 alkyl groups, C1-C4 alkoxy groups, halogenated C1-C4 alkyl groups, hydroxyl groups, cyano groups, or amino groups.

5. The application according to claim 4, characterized in that, The aryl group is substituted by one or more substituents; The substituents include halogens, C1-C4 alkyl groups, C1-C4 alkoxy groups, halogenated C1-C4 alkyl groups, hydroxyl groups, cyano groups, or amino groups.

6. The application according to claim 5, characterized in that, The compounds whose parent core is 4-quinoline carboxamide include 2-(3-ethoxyphenyl)-N-(5-methyl-3-isoxazolyl)-4-quinoline carboxamide or 2-(3,4-dichlorophenyl)-N-(1,3,4-thiadiazol-2-yl)-4-quinoline carboxamide.

7. The application according to claim 6, characterized in that, The anti-rheumatoid arthritis treatment includes inhibiting nicotinamide nucleotide transhydrogenase activity.

8. The application according to claim 7, characterized in that, The anti-rheumatoid arthritis treatment includes relieving symptoms of rheumatoid arthritis such as joint swelling, synovial hyperplasia, and bone destruction, or reducing serum inflammatory factor levels.

9. The application according to claim 8, characterized in that, The antirheumatoid arthritis treatment includes rheumatoid arthritis that does not respond adequately to methotrexate therapy.

10. The application according to claim 9, characterized in that, The drugs include disease-modifying antirheumatic drugs, nonsteroidal anti-inflammatory drugs, corticosteroids, or biologics.

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