CN112426422A

CN112426422A - Application of pyridine carboxamide derivative AMSP-30m in preparation of drugs for preventing and treating rheumatoid arthritis

Info

Publication number: CN112426422A
Application number: CN202011401663.6A
Authority: CN
Inventors: 李�荣; 刘明明; 蔡莉; 沐玉荣; 周梦媛; 孟博; 刘方园
Original assignee: Anhui Medical University
Current assignee: Anhui Medical University
Priority date: 2020-12-02
Filing date: 2020-12-02
Publication date: 2021-03-02

Abstract

The invention discloses an application of a pyridine carboxamide derivative AMSP-30m in preparing a medicament for preventing and treating rheumatoid arthritis, wherein the chemical structural formula of the AMSP-30m is as follows:

the invention provides the application of AMSP-30m in preventing and treating rheumatoid arthritis, explores the new medicinal prospect of AMSP-30m and develops a new medicinal field; in-vivo pharmacodynamic experiments and in-vitro cell experiments prove that the AMSP-30m has the function of efficiently preventing and treating the rheumatoid arthritis, the AMSP-30m shows better effect of treating the rheumatoid arthritis, no obvious side effect is seen, and a powerful basis is provided for preparing the AMSP-30m into an innovative medicine for preventing and treating the rheumatoid arthritis.

Description

Application of pyridine carboxamide derivative AMSP-30m in preparation of drugs for preventing and treating rheumatoid arthritis

Technical Field

The invention relates to a pyridine carboxamide derivative, in particular to an application of a pyridine carboxamide derivative AMSP-30m in preparing a medicament for preventing and treating rheumatoid arthritis.

Background

Rheumatoid Arthritis (RA) is a common chronic, systemic, progressive autoimmune disease, characterized mainly by extensive and persistent synovial inflammation and irreversible cartilage/bone destruction, ultimately leading to joint deformity and loss of function in patients with high disability rates. The global incidence of RA is about 1%, which seriously harms human health and reduces the quality of life. Currently, RA treatment is mainly for relieving pain, controlling inflammation, improving joint function and the like, and clinical RA treatment measures are mainly for drug therapy, including glucocorticoids, non-steroidal anti-inflammatory drugs, disease condition-improving antirheumatic drugs, biological agents and the like, but over 30% of patients have no obvious curative effect on common clinical treatment methods, and long-term administration often produces toxic and side effects such as gastrointestinal discomfort, ulcer, osteoporosis, opportunistic infection and the like, so that development of a novel RA treatment drug aiming at a specific target spot and having high safety is urgently needed.

The excessive proliferation of RA fibroblast-like synoviocytes (FLS) leads to the increase of oxygen consumption of synovium, pannus formation, abnormal blood supply, inflammatory cell infiltration, inflammatory factor release and the like exist in synovial tissue, and various factors lead to the local hypoxia of joints, and the chronic hypoxia microenvironment of the synovial tissue is one of important pathological features of RA. Hypoxia inducible factor-1 alpha (HIF-1 alpha) is a core factor for regulating cell hypoxia response, the stable and accumulated HIF-1 alpha under hypoxia causes the change of the transcription level of target genes, and the high and low expression of HIF-1 alpha is a mark of the activation degree of HIF-1 alpha. RA synovial tissue hypoxia microenvironment continuously activates HIF-1 alpha channel, promotes transcription of downstream target genes such as vascular endothelial growth factor, matrix metalloproteinase and the like, induces angiogenesis, promotes proliferation of RAFLS, apoptosis inhibition and invasion, promotes secretion of inflammatory factors and the like, and is closely involved in pathological processes such as synovial inflammation, synovial hyperplasia, cartilage/bone destruction and the like. Therefore, HIF-1 alpha is a novel target with great potential for preventing and treating RA, and the intervention of HIF-1 alpha signal pathway has potential therapeutic significance for RA. At present, relevant research reports that HIF-1 alpha targeted inhibitors are not available in the treatment of RA show that the development of novel HIF-1 alpha inhibitors has important clinical value for preventing and treating RA.

The pyridine carboxamide derivative AMSP-30m is a novel HIF-1 alpha inhibitor, and the chemical structure of the derivative is shown as follows:

there are studies showing that: AMSP-30m inhibits the expression of tumor cell HIF-1 alpha protein and HIF-1 alpha signal channel under anaerobic culture, inhibits the invasion, migration and angiogenesis of tumor cell under anaerobic culture in vitro and inhibits the formation of metastasis of breast cancer in the lung of a nude mouse in vivo, and shows better antitumor effect. The abnormal proliferation and high invasiveness of the RAFLS are similar to tumor cells, and the AMSP-30m is reasonably supposed to possibly exert potential antirheumatic effect by inhibiting the expression of HIF-1 alpha protein in RA hypoxic synovial tissue, but the pharmacological effect of the AMSP-30m on preventing and treating rheumatoid arthritis is not reported, and the invention is based on the fact that the invention provides the AMSP-30m with the effect of inhibiting the expression of the HIF-1 alpha protein in RA hypoxic synovial tissue.

Disclosure of Invention

The invention aims to solve the technical problem that the relation between AMSP-30m and rheumatoid arthritis is lack in the existing research, and provides an application of AMSP-30m in preparing a medicament for preventing and treating rheumatoid arthritis, and a new way for preventing and treating rheumatoid arthritis.

In order to achieve the technical purpose, the technical scheme of the invention is realized as follows:

application of pyridine carboxamide derivative AMSP-30m in preparing medicine for preventing and treating rheumatoid arthritis, wherein the chemical name of AMSP-30m is N- (2-fluorophenethyl) -5- (2-methoxyphenyl) -pyridine carboxamide, and the molecular formula is C₂₁H₁₉FN₂O₂Molecular weight is 350.39, and its chemical structure is shown as follows:

further, the medicine for preventing and treating rheumatoid arthritis comprises AMSP-30m with effective dose and a pharmaceutically acceptable carrier.

Further, the AMSP-30m is prepared into a pharmaceutical preparation suitable for gastrointestinal tract and parenteral administration.

Further, the AMSP-30m is prepared into a pharmaceutical preparation suitable for gastrointestinal tract administration, and the pharmaceutical preparation is in the form of conventional tablets, capsules, controlled-release preparations and sustained-release preparations.

Further, in the pharmaceutical preparation prepared from AMSP-30m and suitable for gastrointestinal and parenteral administration, the content of AMSP-30m in the pharmaceutical preparation is 1 wt% -99 wt%.

Furthermore, in the pharmaceutical preparation prepared from AMSP-30m and suitable for gastrointestinal administration, the content of AMSP-30m in the pharmaceutical preparation is 10 wt% -90 wt%.

Further, the preparation method of the pharmaceutical preparation comprises the steps of directly preparing the AMSP-30m into the preparation, or preparing the AMSP-30m into the pharmaceutical preparation respectively or/and mixing the AMSP-30m with auxiliary materials, and then packaging the pharmaceutical preparation to obtain the pharmaceutical preparation.

Further, the administration dosage of the pharmaceutical composition varies according to the administration subject, the administration route or the form of the pharmaceutical preparation, but is based on the premise that the pharmaceutical composition can achieve an effective blood concentration in the body of the subject.

The technical scheme of the invention researches the treatment effect of the intragastric administration of AMSP-30m on rats with adjuvant arthritis by an in-vivo animal experiment method. The AMSP-30m can reduce secondary foot swelling and arthritis indexes of rats with adjuvant arthritis, relieve joint lesion and cartilage destruction and reduce the level of serum inflammatory cytokines, the effects are related to the inhibition of HIF-1 alpha expression in synovial tissues by the AMSP-30m, and pharmacodynamic experiments provide sufficient experimental basis for discussing the prevention and treatment of rheumatoid arthritis by the AMSP-30 m.

The technical scheme of the invention also researches the influence of AMSP-30m on the proliferation, migration and invasion capacity of human fibroblast synovial cells (MH7A) cultured in an anoxic way in vitro and the expression of HIF-1 alpha protein by using an in vitro cell experiment method. The AMSP-30m externally administered inhibits the proliferation, migration and invasion capacity of MH7A cells cultured in an anoxic culture mode, and inhibits the expression of HIF-1 alpha protein in MH7A cells cultured in the anoxic culture mode, which indicates that the AMSP-30m is taken as an HIF-1 alpha inhibitor to be externally administered to obviously inhibit the abnormal activation of MH7A cells cultured in the anoxic culture mode, and the AMSP-30m can be one of action mechanisms of preventing and treating rheumatoid arthritis.

The application of AMSP-30m in preparing the medicine for preventing and treating rheumatoid arthritis provided by the invention has the following advantages: the invention explores the new medicinal prospect of AMSP-30m and develops a new medicinal field; through in vivo pharmacodynamic experiments and in vitro cell experiments, the AMSP-30m is proved to have the function of efficiently preventing and treating the rheumatoid arthritis, and provides a powerful basis for developing the AMSP-30m into a new medicament for preventing and treating the rheumatoid arthritis.

Drawings

FIG. 1 is a graph showing the effect of AMSP-30m on secondary foot swelling in adjuvant arthritis rats in example 1.

FIG. 2 is a graph showing the effect of AMSP-30m on the arthritis index of adjuvant arthritis rats in example 1.

FIG. 3 is a graph showing the effect of AMSP-30m on the pathological changes of the ankle joint tissues of rats with adjuvant arthritis in example 1.

FIG. 4 is a graph showing the effect of AMSP-30m on the injury of ankle cartilage in rats with adjuvant arthritis in example 1.

FIG. 5 is a graph showing the effect of AMSP-30m on serum inflammatory factor levels in rats with adjuvant arthritis according to example 1.

FIG. 6 is a graph showing the effect of AMSP-30m on HIF-1 α expression in synovium of adjuvant arthritic rats in example 1.

FIG. 7 is a graph showing the effect of AMSP-30m on proliferation of human fibroblast-like synoviocytes MH7A cultured in the absence of oxygen in example 2.

FIG. 8 is a graph showing the effect of AMSP-30m on the migration of human fibroblast-like synoviocytes MH7A in hypoxia culture in example 2.

FIG. 9 is a graph showing the effect of AMSP-30m on the invasion of human fibroblast-like synoviocytes MH7A cultured in the absence of oxygen in example 2.

FIG. 10 is a graph of the effect of AMSP-30m on HIF-1 α expression in human fibroblast-like synoviocytes MH7A in hypoxic culture in example 2.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1

Pharmacodynamic study of pyridine carboxamide derivative AMSP-30m on rats with adjuvant arthritis

1. Experimental methods

1.1 adjuvant-induced arthritis (AIA) establishment and experimental grouping of rats:

BCG vaccine is inactivated at 80 ℃ for 1h, and then is fully milled and uniformly mixed with autoclaved liquid paraffin to prepare 10g/L emulsion (Freund's complete adjuvant), SD rat left hind foot is injected with 0.1mL of Freund's complete adjuvant in intradermal mode to cause inflammation, and an AIA rat model is prepared. Rats in the normal control group were injected with an equal volume of physiological saline intradermally into the left hind paw. On day 12 of molding (d12), rats without secondary symptoms of swelling of the lateral joints were discarded, and rats exhibiting AIA symptoms were randomly divided into a model group, AMSP-30m (30,60mg/kg) group and a positive drug methotrexate (MTX,0.5mg/kg/3 day) control group. AMSP-30m and MTX were formulated with 0.5% sodium carboxymethyl cellulose (CMC-Na) as suspensions of the appropriate concentrations just prior to use. D13-26 after the drug combination is used for model building is continuously administered by intragastric administration (10mL/kg) according to the corresponding dose once a day for 14 days continuously, and the normal control group is administered by intragastric administration with 0.5% CMC-Na with equal volume.

1.2 secondary foot swelling and arthritis index determination:

the hind paw volumes on the secondary side (non-injected side, right side) of the rats were measured before (d0) and after (d12,15,18,21,24,27) respectively, and secondary paw swelling (Δ mL ═ paw volume at some time point after inflammation-pre-inflammatory paw volume) was calculated. And performing an arthritis index score at the above time points: 0-no red swelling; 1-mild redness and swelling of the limited foot or ankle joint; 2-mild swelling of joints and toes; total swelling of the toes and ankle, 4-ankle to total leg redness or joint deformity, with a maximum of 4 points per paw, a total of 12 points for the secondary hind and two forepaws.

1.3 hematoxylin-eosin (HE) staining and histological scoring:

a model d28 is made to kill rats, the right hind ankle joints are taken down, 4% paraformaldehyde is used for fixation, 10% nitric acid is used for decalcification, the ankle joints are longitudinally split open, paraffin is embedded and sliced, HE is used for staining, pathological changes and radiographing are observed under a microscope, and grading (0-no change, 1-mild degree, 2-moderate degree, 3-severe degree and 4-very severe degree) is carried out according to pathological index changes such as synovium hyperplasia, inflammatory cell infiltration, pannus formation and the like.

1.4 safranin O/fast Green staining and Mankins scoring:

taking ankle joint sections for safranin O/fast green staining, carrying out Mankins scoring on articular cartilage damage, and scoring from cartilage structures (0-6 points), chondrocyte morphologies (0-3 points), staining evaluation (0-4 points), tide line integrity (0-1 points) and the like, wherein the highest score is 14 points, and the lowest score is 0 point.

1.5 ELISA for inflammatory factor levels:

blood was collected after sacrifice, centrifuged and the supernatant stored at-80 ℃. ELISA kit detects the levels of proinflammatory cytokines such as TNF-alpha, IL-1 beta and the like in the serum of rats of each group.

1.6 Western blot (western blot) to detect the expression of HIF-1. alpha. protein in synovial tissue:

taking the synovial tissue of the knee joint of the rat, adding RIPA lysate containing PMSF for full lysis, centrifuging, taking the supernatant and packaging. Adding equal volume of sample buffer solution (2 x) into the protein supernatant, boiling in water bath for 5min, adding 30 μ g protein per well, performing SDS-PAGE electrophoresis, and transferring membrane by semi-dry transfer method. 5% skimmed milk powder was blocked for 2h, the corresponding HIF-1. alpha. primary antibody was added, incubated overnight at 4 ℃ and washed with TBST. Adding a horseradish peroxidase-labeled secondary antibody, reacting for 2h at room temperature, and washing with TBST. Immersing in the aqueous solution containing chemiluminescent reagent A, B to excite fluorescence, pressing X-ray film in dark room, developing, and fixing. The negative was scanned and semi-quantitative analysis was performed by ImageJ software.

1.7 statistical analysis:

the data analysis adopts SPSS 16.0 software, the data result is expressed by mean +/-standard deviation, the multi-group comparison adopts One-factor analysis of variance (One-Way ANOVA test), the pairwise comparison adopts Tukey HSD for analysis, and P <0.05 shows that the difference has statistical significance.

2. Results of the experiment

2.1 general conditions of experimental animals:

no rats died during the experiment. The rats in the normal group had flexible movement, good mental status, normal diet and free joint movement. The AIA model rat is listened, diet is reduced, joint movement is obviously obstructed, joints of limbs are swollen, and joint mobility is small. The mental state of the rats in the AMSP-30m (30mg/kg) group is general, the eating condition is better than that of the rats in the AIA model group, the joint movement is limited, part of joints are red and swollen, and the mobility is partially recovered. The rats in the AMSP-30m (60mg/kg) group and the positive drug MTX group have the advantages of obviously better diet condition than that of the AIA model group, better mental state, slightly red and swollen joints and no obvious movement disorder.

2.2 effect of AMSP-30m on adjuvant arthritis secondary foot swelling in rats:

secondary hind paw volumes were measured at various time points for 14 days post-molding following d13-26 gavage, and the results are shown in fig. 1, with a significant increase in secondary paw swelling in the right hind paw of rats in the AIA model group at d12, d 15, d 18, d 21, d24 and d 27 (P <0.01), peaking at d24 and progressively regressing thereafter, compared to the normal group. Compared with the AIA model group, AMSP-30m (30mg/kg) obviously inhibits the secondary foot swelling of AIA rats at d 21, d24 and d 27; AMSP-30m (60mg/kg) has obvious inhibition effect on d 18, d 21, d24 and d 27, and AMSP-30m (60mg/kg) and positive drug MTX (0.5mg/kg/3d) have similar action strength.

2.3 effect of AMSP-30m on the arthritis index of adjuvant arthritis rats:

as shown in FIG. 2, the right hind paw and the front paw of the AIA rat were red, swollen and deformed, some tail nodules were visible, and the arthritis index peaked at d24 days. The arthritis indexes of the rats in the AIA model group are obviously increased in d12, d 15, d 18, d 21, d24 and d 27 compared with the normal group (P < 0.01); the AMSP-30m (30,60mg/kg) can obviously reduce the arthritis indexes of AIA rats at d 21, d24 and d 27, the arthritis indexes of MTX rats at d 18, d 21, d24 and d 27 are obviously reduced compared with those of a model group, and the AMSP-30m (60mg/kg) and a positive drug MTX (0.5mg/kg/3d) have similar action intensity.

2.4 Effect of AMSP-30m on pathological changes of ankle joint tissues of rats with adjuvant arthritis:

FIG. 3 shows the results of HE staining, in which the synovial lining layer of the ankle joint of normal rats consists of 1-2 layers of synovial cells, the lower layer of the synovial lining is composed of adipocytes and a small amount of blood vessels, the joint surface is smooth, no exudation occurs in the joint cavity, and inflammatory cell infiltration is not observed. The joint lesions of rats in the AIA model group are characterized in that synovial tissues obviously proliferate, synovial lining layer cells increase to 3-4 layers or more, a large amount of inflammatory cells infiltrate into synovial capsules, the synovial tissues have obvious pannus formation, articular cartilage is damaged, and the basic pathological characteristics of human RA are realized. AMSP-30m (30,60mg/kg) and MTX (0.5mg/kg/3d) have various degrees of improvement effects on the above pathological changes of ankle joint tissues of AIA rats. Further, histopathological scoring is carried out on the aspects of synovial tissue hyperplasia, inflammatory cell infiltration, pannus generation and the like, the result shows that the histopathological scoring of various pathological indexes of the rats in the AIA model group is obviously higher than that of the rats in the normal group, and AMSP-30m (30 and 60mg/kg) can reduce the synovial tissue hyperplasia, inflammatory cell infiltration and pannus formation in ankle joints of the AIA rats in a dose-dependent manner, so that the AMSP-30m obviously improves the joint injury of the AIA rats. AMSP-30m (60mg/kg) has similar action intensity with positive drug MTX (0.5mg/kg/3 d).

2.5 Effect of AMSP-30m on adjuvant arthritis rat ankle cartilage injury:

FIG. 4 shows the results of safranin O-fast green staining, from which, AIA rat articular cartilage damage is severe, which is manifested by decreased staining intensity of cartilage matrix, cartilage surface fracture and incomplete tide line, and AMSP-30m (30,60mg/kg) gastric lavage can reduce the severity of cartilage damage to different degrees. The Mankin scoring system is used to assess cartilage pathology to quantify the severity of cartilage damage. The results show that both AMSP-30m (30,60mg/kg) and MTX can significantly reduce cartilage damage in AIA rats compared to the AIA group. AMSP-30m (60mg/kg) has similar action intensity with positive drug MTX (0.5mg/kg/3 d).

2.6 Effect of AMSP-30m on serum inflammatory factor levels in adjuvanted arthritic rats:

FIG. 5 shows ELISA test results, in which the levels of TNF- α and IL-1 β in the serum of AIA rats are significantly higher than those in the normal group (P <0.01), and compared with the AIA group, AMSP-30m (30,60mg/kg) can reduce the levels of TNF- α and IL-1 β in the serum of AIA rats dose-dependently, showing better anti-inflammatory effect. AMSP-30m (60mg/kg) has similar action intensity with positive drug MTX (0.5mg/kg/3 d).

2.7 Effect of AMSP-30m on HIF-1 α expression in synovial tissue of adjuvanted arthritic rats:

FIG. 6 shows the result of Western blot, HIF-1 alpha protein expression in synovial tissue of rats in AIA model group is significantly higher than that in normal group, and compared with the AIA model group, AMSP-30m (30,60mg/kg) and MTX significantly reduce HIF-1 alpha protein expression in synovial tissue of AIA rats, indicating that AMSP-30m can inhibit HIF-1 alpha signal channel in synovial of AIA rats. AMSP-30m (60mg/kg) has similar action intensity with positive drug MTX (0.5mg/kg/3 d).

3. Conclusion

The administration of the pyridine carboxamide derivative AMSP-30m through gastric lavage can obviously reduce the joint inflammation of an AIA rat, obviously reduce the joint injury and cartilage destruction of the AIA rat, obviously reduce the serum inflammatory cytokine level of the AIA rat, and obviously inhibit the HIF-1 alpha protein expression in the synovial tissue of the AIA rat, which indicates that the compound AMSP-30m as an HIF-1 alpha inhibitor is a medicament for preventing and treating rheumatoid arthritis.

Example 2

Effect of the Pyridinecarboxamide derivative AMSP-30m on the biological Properties of human fibroblast-like synoviocytes MH7A cultured in vitro in anoxic culture

1. Experimental methods

1.1 MTT method for detecting cell proliferation:

the cells were grouped as follows: hypoxia (1% O)₂) Group, hypoxia + DMSO solvent control group, hypoxia + AMSP-30m (1)0 μ M) group. A suspension of human fibroblast-like synoviocytes (MH7A) was seeded in 96-well plates (100. mu.L/well) at a cell density of about 4000 cells/well at 37 ℃ with 5% CO₂Adherent culture was performed, and the original culture was discarded, and 100. mu.L of DMEM containing AMSP-30M (final concentration: 10. mu.M) was added thereto, and the untreated group and the DMSO solvent control group were separately prepared. Culturing in an anoxic incubator for 48h, adding 10 μ L MTT (5g/L) into each well, culturing for 4h, removing supernatant, adding 100 μ L dimethyl sulfoxide into each well, shaking, detecting absorbance A (490nm) with an enzyme labeling instrument, and detecting relative proliferation capacity of cells with the control of the non-added medicines.

1.2 scratch assay to detect cell migration:

the cells were grouped as follows: hypoxia (1% O)₂) Group, hypoxia + DMSO solvent control group, hypoxia + AMSP-30M (10 μ M) group. Collecting cell suspension, counting by counting plate, adjusting cell density to 1 × 10⁸Per L, pipette 2mL into 6-well plates. When the growth reaches 90%, use

The tip was drawn straight perpendicular to the cell layer surface. PBS is used for washing, DMEM culture solution containing AMSP-30m is added into the PBS, the DMEM culture solution is placed into an anoxic incubator for culture, and samples are taken at the time points of 0 hour and 24 hours for photographing. Based on the scratch distance of 0h (L)_0h) The variation (L) of the scratch distance in 24h of culture was calculated_0h-L_24h) And calculating a migration index: (L)_0h-L_24h)/L_0h。

1.3 Transwell assay to detect cell invasion:

the cells were grouped as follows: hypoxia (1% O)₂) Group, hypoxia + DMSO solvent control group, hypoxia + AMSP-30M (10 μ M) group. The cell suspension was prepared by placing a Transwell chamber in a 24-well plate and spreading Matrigel gel, counting the plate and adding 1X 10 to the upper chamber⁸100 mu L of serum-free cell suspension, 500 mu L of 10% FBS culture medium containing different concentrations of AMSP-30m is added into the lower chamber, and the mixture is placed in an anoxic culture box for 24 hours. The upper chamber was removed, washed with PBS, fixed with 4% paraformaldehyde at 200. mu.L for 30min, and stained with 0.1% crystal violet for 20 min. Sucking out crystal violet, lightly wiping the inner surface of the membrane with a cotton swab, washing with PBS, drying, and observing under a microscopeAnd images were collected and 5 fields were randomly counted for cell count, and the results were expressed as the average of the number of invading cells per field.

1.4 Western blot detection of HIF-1. alpha. protein expression in cells:

the cells were grouped as follows: hypoxia (1% O)₂) Group, hypoxia + DMSO solvent control group, hypoxia + AMSP-30M (10 μ M) group. Taking MH7A cells of each group, adding RIPA lysate containing PMSF for full lysis, centrifuging, taking supernatant and packaging. Adding equal volume of sample buffer solution (2 x) into the protein supernatant, boiling in water bath for 5min, adding 30 μ g protein per well, performing SDS-PAGE electrophoresis, and transferring membrane by semi-dry transfer method. 5% skimmed milk powder was blocked for 2h, the corresponding HIF-1. alpha. primary antibody was added, incubated overnight at 4 ℃ and washed with TBST. Adding a horseradish peroxidase-labeled secondary antibody, reacting for 2h at room temperature, and washing with TBST. Immersing in the aqueous solution containing chemiluminescent reagent A, B to excite fluorescence, pressing X-ray film in dark room, developing, and fixing. The negative was scanned and semi-quantitative analysis was performed by ImageJ software.

1.5 statistical analysis:

2. Results of the experiment

2.1 Effect of AMSP-30m on proliferation of human fibroblast-like synoviocytes MH7A in hypoxic culture:

fig. 7 shows MTT experimental results, in which DMSO (0.1%) solvent control had no effect on the proliferation potency of hypoxia-cultured MH7A cells compared to non-drug-treated MH7A cells under hypoxic culture conditions, whereas AMSP-30M (10 μ M) in vitro administration significantly inhibited the proliferation potency of hypoxia-cultured MH7A cells for 48h (P < 0.01).

2.2 effects of AMSP-30m on migration of human fibroblast-like synoviocytes MH7A in hypoxic culture:

fig. 8 shows the results of the scratch test, in which the DMSO (0.1%) solvent control had no effect on the migration ability of the MH7A cells cultured under hypoxia culture compared to MH7A cells not treated with drug, whereas AMSP-30M (10 μ M) administered in vitro significantly reduced the migration index and migration ability of MH7A cells cultured under hypoxia for 24h (P < 0.01).

2.3 Effect of AMSP-30m on invasion of human fibroblast-like synoviocytes MH7A in hypoxic culture:

fig. 9 shows the results of Transwell experiments, in which DMSO (0.1%) solvent control had no effect on the invasive potential of the hypoxic MH7A cells compared to non-drug-treated MH7A cells under hypoxic culture conditions, whereas AMSP-30M (10 μ M) in vitro administration significantly reduced the number of cells that were passed through Matrigel by the hypoxic 24h MH7A cells and decreased the invasive potential thereof (P < 0.01).

2.4 Effect of AMSP-30m on HIF-1 α expression in human fibroid synoviocytes MH7A in hypoxic culture:

FIG. 10 shows the results of Western blot assay, in the hypoxic culture condition, compared with MH7A cells without drug treatment, DMSO (0.1%) solvent control has no effect on HIF-1 α protein expression in MH7A cells in hypoxic culture, while AMSP-30M (10 μ M) in vitro administration can significantly inhibit HIF-1 α protein expression (P <0.01) in MH7A cells in hypoxic culture for 24h, indicating that AMSP-30M can inhibit HIF-1 α signal pathway in MH7A cells in hypoxic condition.

3. Conclusion

The pyridine carboxamide derivative AMSP-30m serving as a HIF-1 alpha inhibitor remarkably inhibits the proliferation, transfer and invasion capabilities of human fibroblast synovial cells MH7A cultured in an anoxic culture in vitro, and obviously inhibits the abnormal activation of MH7A cells cultured in the anoxic culture, which is probably one of action mechanisms of AMSP-30m for preventing and treating rheumatoid arthritis.

The above-described examples 1 and 2 are to be understood as merely illustrative of the present invention and not as limiting the scope of the present invention. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The application of the pyridine carboxamide derivative AMSP-30m in preparing a medicament for preventing and treating rheumatoid arthritis is characterized in that the medicament for preventing and treating rheumatoid arthritis comprises an effective dose of AMSP-30m and a pharmaceutically acceptable carrier, and the chemical structural formula of the compound AMSP-30m is as follows:

2. the use of the pyridine carboxamide derivative AMSP-30m as claimed in claim 1 for the preparation of a medicament for the prophylaxis or treatment of rheumatoid arthritis, characterized in that the compound AMSP-30m is used as active compound in the preparation of a pharmaceutical preparation suitable for gastrointestinal and parenteral administration.

3. The use of the pyridine carboxamide derivative AMSP-30m as claimed in claim 1 for the preparation of a medicament for the prophylaxis and treatment of rheumatoid arthritis, characterized in that the compound AMSP-30m is used as the active compound to prepare pharmaceutical preparations suitable for gastrointestinal administration in the form of conventional tablets, capsules, controlled-release preparations and sustained-release preparations.

4. The use of the pyridine carboxamide derivative AMSP-30m as claimed in claim 2 for the preparation of a medicament for the prophylaxis and treatment of rheumatoid arthritis, characterized in that the content of AMSP-30m in the pharmaceutical preparation prepared from AMSP-30m is 1% to 99% by weight, the pharmaceutical preparation being suitable for gastrointestinal and parenteral administration.

5. The use of the pyridine carboxamide derivative AMSP-30m as claimed in claim 3 for the preparation of a medicament for the prophylaxis and treatment of rheumatoid arthritis, characterized in that the content of AMSP-30m in the pharmaceutical preparation prepared from AMSP-30m and suitable for gastrointestinal administration is 10% to 90% by weight.

6. The use of the pyridine carboxamide derivative AMSP-30m as claimed in claim 4 or 5 for the preparation of a medicament for the prevention or treatment of rheumatoid arthritis, characterized in that the pharmaceutical preparation is prepared by directly formulating AMSP-30m or by separately or/and mixing with auxiliary substances to form a pharmaceutical preparation and then packaging.