Application of 2-amino-6-methylphenol derivatives as ferroptosis inhibitors in the preparation of drugs for the prevention and treatment of osteoarthritis

Abstract

This application relates to the field of biomedical technology, specifically to the application of a ferroptosis inhibitor in the preparation of drugs for the prevention and treatment of osteoarthritis. The present invention applies a 2-amino-6-methylphenol (formula (I)) derivative to human C28 chondrocytes and live mice, and finds that the above compound can act as a ferroptosis inhibitor, significantly inhibiting Erastin and RSL3-induced ferroptosis, and exhibiting a long-term stable inhibitory effect. Therefore, the 2-amino-6-methylphenol derivative can serve as a targeted inhibitor of ferroptosis, and provides the use of the above compound in the prevention and treatment of osteoarthritis.

Description

Technical Field

[0001] This application relates to the field of biomedical technology, specifically to the application of 2-amino-6-methylphenol derivatives as ferroptosis inhibitors in the preparation of drugs for the prevention and treatment of osteoarthritis. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Ferroptosis is a recently discovered type of programmed cell death that differs from apoptosis, necrosis, and autophagy in morphology, biochemistry, and genetics. It is named ferroptosis because it depends on the presence of iron ions. Its mechanism involves an imbalance between the generation and degradation of reactive oxygen species (ROS) in intracellular membrane lipids, leading to iron-dependent, oxidative, non-apoptotic programmed cell death. Typical characteristics include smaller mitochondria, increased bilayer membrane density, and an increase in ROS free radicals in cell membrane lipids.

[0004] Osteoarthritis (OA) is a common joint disease primarily affecting the articular cartilage, eventually leading to degeneration, fibrosis, fracture, and defects of the articular cartilage, as well as damage to the entire articular surface. Symptoms include joint pain, stiffness, enlargement, and limited range of motion. It commonly affects weight-bearing joints such as the knee, hip, cervical spine, and lumbar spine, as well as the distal interphalangeal joints, proximal interphalangeal joints, first carpometacarpal joint, and first metatarsophalangeal joint. Osteoarthritis is prevalent in middle-aged and elderly individuals and is a leading cause of disability in the elderly.

[0005] Currently, the factors that induce osteoarthritis and their underlying mechanisms remain unclear. However, existing studies have shown that ferroptosis promotes the progression of osteoarthritis by participating in chondrocyte loss, extracellular matrix dissolution, and synovitis.

[0006] Ferrostatin-1 and Liproxstatin-1 are first-generation small-molecule ferroptosis inhibitors that function by scavenging lipid free radical damage to cell membranes and blocking ferroptosis. These inhibitors have well-defined mechanisms of action and structure-activity relationships. However, both compounds suffer from drawbacks, namely short half-lives and relatively high toxicity.

[0007] Summary of the Invention

[0008] To overcome the above problems, this invention provides the application of a 2-amino-6-methylphenol derivative as a ferroptosis inhibitor in the preparation of drugs for the prevention and treatment of osteoarthritis. Based on research on the mechanism of ferroptosis, this invention, through virtual screening and cell activity testing, discovered that a 2-amino-6-methylphenol derivative can act as an inhibitor targeting ferroptosis, and provides the use of the above compound in the prevention and treatment of osteoarthritis.

[0009] To achieve the above technical objectives, the present invention adopts the following technical solution:

[0010] In a first aspect, the invention provides the use of a 2-amino-6-methylphenol derivative as a ferroptosis inhibitor, said 2-amino-6-methylphenol derivative having the structural formula shown in Formula (I):

[0011]

[0012] Wherein, R is selected from hydrogen, ethyl, n-butyl, benzyl, 3-cyano-benzyl, 3-nitro-benzyl, 4-chloro-benzyl, 4-methyl-benzyl, 4-methoxy-benzyl, or methyl 4-benzoate. 4-Methyl-benzyl is further preferred.

[0013] 2-Amino-6-methylphenol derivatives can block the chain reaction of lipid free radicals, reduce the damaging effect of free radicals on cell membranes, prevent ferroptosis, and protect cells, thereby achieving a therapeutic effect on diseases related to ferroptosis mechanisms.

[0014] It should be noted that, in addition to the small molecule entities having the above-described structure, the hydrates, solvates, pharmaceutical salts, and pharmaceutical esters of the 2-amino-6-methylphenol derivatives described in this invention also fall under the same concept as the first aspect of this invention and are part of the technical content protected by this invention.

[0015] Preferably, the application of the above-mentioned 2-amino-6-methylphenol derivative as a ferroptosis inhibitor includes, but is not limited to, any of the following methods:

[0016] (1) It is used to prevent, improve or treat diseases related to the ferroptosis pathway;

[0017] (2) Used in the preparation of drugs for the prevention, improvement or treatment of diseases related to the ferroptosis pathway;

[0018] (3) Applied to the preparation of ferroptosis pathway inhibition model.

[0019] In the applications of (1) and (2) above, the iron death-related diseases include, but are not limited to, cancer, cerebral hemorrhage, tumors, ischemia-reperfusion injury, traumatic brain injury, Parkinson's disease, plant heat stress, liver and kidney injury, biliary tract disease, osteoarthritis, etc.; in one embodiment verified by the present invention, the above-mentioned 2-amino-6-methylphenol derivative is used to prevent, improve or treat osteoarthritis.

[0020] In the above (3) applications, the above compounds are used as model drugs in the preparation of basic research models, such as the in vitro C28 human chondrocyte ferroptosis pathway inhibition model, or to inhibit Erastin and RSL3-induced cell ferroptosis.

[0021] A third aspect of the present invention provides a pharmaceutical composition comprising an active dose of the above-described 2-amino-6-methylphenol derivative.

[0022] In the above-described pharmaceutical composition, the dosage of the compound can be routinely adjusted according to factors such as the purpose of drug administration and the condition of the subject. The dosage of the compound in the pharmaceutical composition should be an effective dose, and the pharmaceutical composition should be in a dosage form that facilitates precise administration.

[0023] When the pharmaceutical composition is used to prepare an in vivo pharmaceutical formulation, the pharmaceutical formulation shall be sterile. Methods for achieving sterility of the pharmaceutical formulation should be known to those skilled in the art, such as filtration through a sterile filter membrane. Those skilled in the art can also select a suitable pharmaceutically acceptable carrier according to the required dosage form of the pharmaceutical composition to prepare it into different dosage forms, such as gastrointestinal dosage forms or non-gastrointestinal dosage forms.

[0024] Furthermore, the aforementioned gastrointestinal dosage forms include powders, tablets, granules, capsules, sustained-release preparations, solutions, dry suspensions, effervescent tablets, emulsions, and suspensions.

[0025] Furthermore, non-gastrointestinal drug delivery formulations include injectable drug delivery formulations (e.g., injections, including various injections such as intravenous injections, intramuscular injections, subcutaneous injections, intradermal injections, and intracavitary injections); respiratory drug delivery formulations (e.g., sprays, aerosols, powder inhalers, etc.); skin drug delivery formulations (e.g., topical solutions, lotions, liniments, ointments, plasters, pastes, patches, etc.); mucosal drug delivery formulations (e.g., eye drops, nasal drops, ophthalmic ointments, mouthwashes, sublingual tablets, adhesive tablets, films, etc.); and cavity drug delivery formulations (e.g., suppositories, aerosols, effervescent tablets, drops, pills, etc., used in the rectum, vagina, urethra, nasal cavity, ear canal, etc.).

[0026] Furthermore, the aforementioned drug is an injectable form.

[0027] A fourth aspect of the invention provides the use of the above-described 2-amino-6-methylphenol derivative and / or the pharmaceutical composition described in the third aspect in the preparation of a medicament for the prevention, improvement or treatment of osteoarthritis or related diseases.

[0028] In summary, this invention provides the application of 2-amino-6-methylphenol derivatives in the treatment of osteoarthritis. Specifically, the ferroptosis inhibitor 2-amino-6-methylphenol derivative can be used to prepare drugs for treating / relieving osteoarthritis, or as a supplement for the prevention of osteoarthritis when used in combination with ferroptosis inhibitors.

[0029] The beneficial effects of this invention are mainly reflected in the following aspects: This invention provides evidence of the inhibitory effect of 2-amino-6-methylphenol derivatives on chondrocyte ferroptosis, providing a theoretical basis for the treatment of osteoarthritis targeting ferroptosis, and in particular, providing a basis for its combined use with ferroptosis inhibitors in clinical practice. Attached Figure Description

[0030] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0031] Figure 1 The 2-amino-6-methylphenol derivative has a significant inhibitory effect on Erastin-induced ferroptosis.

[0032] Figure 2 The 2-amino-6-methylphenol derivative increased Erastin-induced GPX4 levels in cells;

[0033] Figure 3 The 2-amino-6-methylphenol derivative has a significant inhibitory effect on Erastin-induced cellular lipid peroxidation;

[0034] Figure 4 The 2-amino-6-methylphenol derivative showed a sustained inhibitory effect on RSL3-induced ferroptosis (A: cell viability; B: lipid peroxidation);

[0035] Figure 5 Joint damage in mice treated with 2-amino-6-methylphenol derivatives (HE staining 10 / 20X). Detailed Implementation

[0036] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0037] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0038] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0039] Compound 2-amino-6-methylphenol was purchased from Specs, and Ferrostatin-1, Erastin, and RSL3 were purchased from Sigma. All compounds were dissolved in sterile dimethyl sulfoxide (DMSO) to prepare the desired concentrations. The preparation methods for the derivatives of 2-amino-6-methylphenol are shown in Examples 1-9 of this invention.

[0040] C28 human chondrocyte cell line culture conditions: DMEM high-glucose medium (GIBCO) containing 10% FBS (GIBCO), 37℃, 5% CO2 saturated humidity incubator.

[0041] The statistical analysis used in this invention employed R software, and experimental data are expressed as Mean ± SEM. Tukey's test (ANOVA) was used for inter-group comparisons in cell and animal experiments, while Student's test was used for comparisons between two groups. A p-value < 0.05 was considered statistically significant; different letters indicate p < 0.05.

[0042] Example 1: Synthesis of compound 2: 2-(ethylamino)-6-methylphenol

[0043]

[0044] 2-Amino-6-methylphenol (1 mmol, 123 mg), acetaldehyde (1 mmol, 44 mg, 1 eq.), and dichloroethane (5 mL) were added to a flask under N2 atmosphere. After stirring at room temperature for 2 hours, the reaction solution was cooled to 0 °C in an ice bath. Sodium triacetoxyborohydride (1 mmol, 212 mg, 1 eq.) was added to the reaction solution in four portions over 30 minutes. The reaction mixture was stirred at room temperature for 12 hours. When the reaction was complete as monitored by TLC, the reaction was quenched with water and extracted with ethyl acetate in three fractions of 10 mL each. The extracted organic phases were combined. The combined organic phases were washed with water, dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to give the crude product. The crude product was purified by silica gel column chromatography. 2-(ethylamino)-6-methylphenol (compound 2) (95 mg) was synthesized in 62.9% yield as a yellow solid. 1 H NMR (400MHz, DMSO) δ7.94(s,1H),6.59(t,J=7.7Hz,1H),6.38–6.30(m,2H),4.52(s,1H),3.04(q,J=7.1Hz,2H),2.11(s,3H),1.17(t,J=7.1Hz,3H).

[0045] Example 2: Synthesis of compound 3: 2-(butylamino)-6-methylphenol

[0046]

[0047] 2-Amino-6-methylphenol (1 mmol, 123 mg), n-butyraldehyde (1 mmol, 72 mg, 1 eq.), and dichloroethane (5 mL) were added to a flask under N2 atmosphere. After stirring at room temperature for 2 hours, the reaction solution was cooled to 0°C in an ice bath. Sodium triacetoxyborohydride (1 mmol, 212 mg, 1 eq.) was added to the reaction solution in four portions over 30 minutes. The reaction mixture was stirred at room temperature for 12 hours. When the reaction was complete as monitored by TLC, the reaction was quenched with water and extracted with ethyl acetate in three fractions of 10 mL each. The extracted organic phases were combined. The combined organic phases were washed with water, dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to give the crude product. The crude product was purified by silica gel column chromatography. 2-(butylamino)-6-methylphenol (compound 3) (107 mg) was synthesized in 59.8% yield as a yellow liquid. 1H NMR (400MHz, DMSO) δ7.97(s,1H),6.59(t,J=7.7Hz,1H),6.34(dd,J=13.0,7.7Hz,2H),4.56(s,1H),3.02(t,J = 6.9Hz, 2H), 2.12 (s, 3H), 1.55 (dt, J = 14.7, 7.2Hz, 2H), 1.38 (dq, J = 14.4, 7.3Hz, 2H), 0.92 (t, J = 7.3Hz, 3H).

[0048] Example 3: Synthesis of compound 4: 2-(benzylamino)-6-methylphenol

[0049]

[0050] 2-Amino-6-methylphenol (1 mmol, 123 mg), benzaldehyde (1 mmol, 106 mg, 1 eq.), and dichloroethane (5 mL) were added to a flask under N2 atmosphere. After stirring at room temperature for 2 hours, the reaction solution was cooled to 0 °C in an ice bath. Sodium triacetoxyborohydride (1 mmol, 212 mg, 1 eq.) was added to the reaction solution in four portions over 30 minutes. The reaction mixture was stirred at room temperature for 12 hours. When the reaction was complete as monitored by TLC, the reaction was quenched with water and extracted with ethyl acetate in three fractions of 10 mL each. The extracted organic phases were combined. The combined organic phases were washed with water, dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to give the crude product. The crude product was purified by silica gel column chromatography. 2-(benzylamino)-6-methylphenol (compound 4) (121 mg) was synthesized in 56.8% yield as a yellowish-brown solid. 1 H NMR (400MHz, DMSO) δ8.10 (d, J=15.8Hz, 1H), 7.37–7.33 (m, 2H), 7.33–7.28 (m, 2H), 7.24–7.18 (m, 1H), 6.51 (t, J= 7.7Hz, 1H), 6.33–6.29 (m, 1H), 6.27 (d, J = 7.9Hz, 1H), 5.35 (t, J = 5.6Hz, 1H), 4.29 (d, J = 5.6Hz, 2H), 2.13 (s, 3H).

[0051] Example 4: Synthesis of compound 5: 2-methyl-6-((3-cyanobenzyl)amino)phenol

[0052]

[0053] 2-Amino-6-methylphenol (1 mmol, 123 mg), 3-cyanobenzaldehyde (1 mmol, 131 mg, 1 eq.), and dichloroethane (5 mL) were added to a flask under N2 atmosphere. After stirring at room temperature for 2 hours, the reaction solution was cooled to 0 °C in an ice bath. Sodium triacetoxyborohydride (1 mmol, 212 mg, 1 eq.) was added to the reaction solution in four portions over 30 minutes. The reaction mixture was stirred at room temperature for 12 hours. When the reaction was complete as monitored by TLC, the reaction was quenched with water and extracted with ethyl acetate in three fractions of 10 mL each. The extracted organic phases were combined. The combined organic phases were washed with water, dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to give the crude product. The crude product was purified by silica gel column chromatography. 2-Methyl-6-((3-cyanobenzyl)amino)phenol (compound 5) (154 mg) was synthesized in 64.7% yield as a yellow solid. 1 H NMR (400MHz, DMSO) δ8.12(s,1H),7.76(d,,J=11.6Hz,1H),7.73–7.63(m,2H),7.52(t,J=7.7Hz,1H),6.50(t,J=7. 7Hz, 1H), 6.32 (d, J = 7.4Hz, 1H), 6.21 (d, J = 7.7Hz, 1H), 5.63 (t, J = 6.2Hz, 1H), 4.36 (d, J = 6.2Hz, 2H), 2.13 (s, 3H).

[0054] Example 5: Synthesis of compound 6: 2-methyl-6-((3-nitrobenzyl)amino)phenol

[0055]

[0056] 2-Amino-6-methylphenol (1 mmol, 123 mg), 3-nitrobenzaldehyde (1 mmol, 151 mg, 1 eq.), and dichloroethane (5 mL) were added to a flask under N2 atmosphere. After stirring at room temperature for 2 hours, the reaction solution was cooled to 0 °C in an ice bath. Sodium triacetoxyborohydride (1 mmol, 212 mg, 1 eq.) was added to the reaction solution in four portions over 30 minutes. The reaction mixture was stirred at room temperature for 12 hours. When the reaction was complete as monitored by TLC, the reaction was quenched with water and extracted with ethyl acetate in three fractions of 10 mL each. The extracted organic phases were combined. The combined organic phases were washed with water, dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to give the crude product. The crude product was purified by silica gel column chromatography. 2-Methyl-6-((3-nitrobenzyl)amino)phenol (compound 6) (157 mg) was synthesized in 60.9% yield as a brown solid. 1H NMR (400MHz, DMSO) δ8.17(s,1H),8.05(dd,J=8.1,1.0Hz,1H),7.71–7.63(m,1H),7.58(d,J=7.0Hz,1H),7.55–7.45(m,1H),6 .48(t,J=7.7Hz,1H), 6.33(d,J=7.1Hz,1H), 6.14(d,J=7.1Hz,1H), 5.58(t,J=6.3Hz,1H), 4.62(d,J=6.3Hz,2H), 2.14(s,3H).

[0057] Example 6: Synthesis of compound 7: 2-((4-chlorobenzyl)amino)-6-methylphenol

[0058]

[0059] 2-Amino-6-methylphenol (1 mmol, 123 mg), 4-chlorobenzaldehyde (1 mmol, 140 mg, 1 eq.), and dichloroethane (5 mL) were added to a flask under N2 atmosphere. After stirring at room temperature for 2 hours, the reaction solution was cooled to 0°C in an ice bath. Sodium triacetoxyborohydride (1 mmol, 212 mg, 1 eq.) was added to the reaction solution in four portions over 30 minutes. The reaction mixture was stirred at room temperature for 12 hours. When the reaction was complete as monitored by TLC, the reaction was quenched with water and extracted with ethyl acetate in three fractions of 10 mL each. The extracted organic phases were combined. The combined organic phases were washed with water, dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to give the crude product. The crude product was purified by silica gel column chromatography. 2-((4-chlorobenzyl)amino)-6-methylphenol (compound 7) (142 mg) was synthesized in 57.2% yield as a yellowish-brown solid. 1 H NMR (400MHz, DMSO) δ8.08 (s, 1H), 7.36 (s, 4H), 6.49 (t, J = 7.7Hz, 1H), 6.30 (d, J = 7 .1Hz, 1H), 6.21 (d, J = 7.7Hz, 1H), 5.48 (s, 1H), 4.29 (d, J = 6.0Hz, 2H), 2.12 (s, 3H).

[0060] Example 7: Synthesis of compound 8: 2-methyl-6-((4-methylbenzyl)amino)phenol

[0061]

[0062] 2-Amino-6-methylphenol (1 mmol, 123 mg), p-methylbenzaldehyde (1 mmol, 120 mg, 1 eq.), and dichloroethane (5 mL) were added to a flask under N2 atmosphere. After stirring at room temperature for 2 hours, the reaction solution was cooled to 0 °C in an ice bath. Sodium triacetoxyborohydride (1 mmol, 212 mg, 1 eq.) was added to the reaction solution in four portions over 30 minutes. The reaction mixture was stirred at room temperature for 12 hours. When the reaction was complete as monitored by TLC, the reaction was quenched with water and extracted with ethyl acetate in three fractions of 10 mL each. The extracted organic phases were combined. The combined organic phases were washed with water, dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to give the crude product. The crude product was purified by silica gel column chromatography. 2-Methyl-6-((4-methylbenzyl)amino)phenol (compound 8) (161 mg) was synthesized in 70.9% yield as a yellow solid. 1 H NMR (400MHz, DMSO) δ8.05(s,1H),7.22(d,J=7.9Hz,2H),7.11(d,J=7.9Hz,2H),6.50(t,J=7.7Hz,1H),6. 30(d,J=7.2Hz,1H),6.26(d,J=7.8Hz,1H),5.29(s,1H),4.23(d,J=4.6Hz,2H),2.26(s,3H),2.11(s,3H).

[0063] Example 8: Synthesis of compound 9: 2-((4-methoxybenzyl)amino)-6-methylphenol

[0064]

[0065] 2-Amino-6-methylphenol (1 mmol, 123 mg), 4-methoxybenzaldehyde (1 mmol, 136 mg, 1 eq.), and dichloroethane (5 mL) were added to a flask under N2 atmosphere. After stirring at room temperature for 2 hours, the reaction solution was cooled to 0 °C in an ice bath. Sodium triacetoxyborohydride (1 mmol, 212 mg, 1 eq.) was added to the reaction solution in four portions over 30 minutes. The reaction mixture was stirred at room temperature for 12 hours. When the reaction was complete as monitored by TLC, the reaction was quenched with water and extracted with ethyl acetate in three fractions of 10 mL each. The extracted organic phases were combined. The combined organic phases were washed with water, dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to give the crude product. The crude product was purified by silica gel column chromatography. 2-((4-methoxybenzyl)amino)-6-methylphenol (compound 9) (137 mg) was synthesized in 56.4% yield as a brown solid. 1HNMR(400MHz,DMSO)δ8.03(s,1H),7.26(d,J=8.0Hz,2H),6.87(d,J=8.0Hz,2H),6.51(t,J =7.6Hz,1H),6.29(s,,2H),5.23(s,1H),4.20(d,J=4.9Hz,2H),3.72(s,3H),2.11(s,3H).

[0066] Example 9: Synthesis of compound 10: methyl 4-(((2-hydroxy-3-methylphenyl)amino)methyl)benzoate

[0067]

[0068] 2-Amino-6-methylphenol (1 mmol, 123 mg), methyl 4-formylbenzoate (1 mmol, 164 mg, 1 eq.), and dichloroethane (5 mL) were added to a flask under N2 atmosphere. After stirring at room temperature for 2 hours, the reaction solution was cooled to 0 °C in an ice bath. Sodium triacetoxyborohydride (1 mmol, 212 mg, 1 eq.) was added to the reaction solution in four portions over 30 minutes. The reaction mixture was stirred at room temperature for 12 hours. When the reaction was complete as monitored by TLC, the reaction was quenched with water and extracted with ethyl acetate in three fractions of 10 mL each. The extracted organic phases were combined. The combined organic phases were washed with water, dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to give the crude product. The crude product was purified by silica gel column chromatography. Methyl 4-(((2-hydroxy-3-methylphenyl)amino)methyl)benzoate (compound 10) (193 mg) was synthesized in 71.2% yield as an orange solid. 1 H NMR(400MHz,,DMSO)δ8.10(s,1H),7.90(d,J=8.3Hz,2H),7.47(d,J=8.3Hz,2H),6.47(t,J=7.7Hz,1H),6.30(d ,J=6.8Hz,1H),6.17(d,J=7.8Hz,1H),5.57(t,J=6.3Hz,1H),4.39(d,J=6.2Hz,2H),3.83(s,3H),2.12(s,3H).

[0069] Experimental Example 1: 2-Amino-6-methylphenol derivatives inhibit Erastin-induced ferroptosis

[0070] After the C28 human chondrocytes were cultured and adhered to the culture medium, DMSO, Erastin (10 μM, final concentration), Erastin (10 μM) + 2-amino-6-methylphenol derivative (1 μM), and Erastin (10 μM) + Ferrostatin-1 (0.1 μM) were added to the medium, respectively. Cell viability was assessed by MTT assay after 24 hours of treatment. The results are shown in Table 1 (data obtained after three repetitions of the experiment). The final cell survival rate results are as follows. Figure 1 As shown (data obtained after repeating the experiment 3 times). Simultaneously, the levels of GPX4 and cell membrane lipid peroxidation were measured before and after cell treatment, and the final results are shown below. Figure 2 and Figure 3 As shown.

[0071] Cell viability assay (MTT): C28 cells treated with the drug for 24 hours were viable using the MTT assay. The MTT kit was purchased from Beijing Solarbio Science & Technology Co., Ltd. Method for measuring cell membrane lipid peroxidation level: C28 cells treated with the drug for 12 hours were digested into single-cell suspensions with trypsin. The suspensions were incubated with C11-BODIPY (10 μM) at room temperature in the dark for 60 minutes, washed three times with PBS, and analyzed by flow cytometry.

[0072] Table 1. Cell viability results of 2-amino-6-methylphenol derivatives

[0073]

[0074] The experimental results showed that: Figure 1 As shown, compared with the control group, the ferroptosis inducer Erastin significantly killed cells; while the 2-amino-6-methylphenol derivative significantly inhibited Erastin-induced cell death (p<0.01), and its activity was comparable to that of the positive control Ferrostatin-1, indicating that the 2-amino-6-methylphenol derivative has a significant inhibitory effect on Erastin-induced cell ferroptosis.

[0075] like Figure 2 As shown, compared with Erastin, the GPX4 content in cells treated with the 2-amino-6-methylphenol derivative was significantly increased; Figure 3 As shown, compared with Erastin, the degree of lipid peroxidation in cells treated with 2-amino-6-methylphenol derivative was significantly reduced, thus demonstrating the therapeutic effect of 2-amino-6-methylphenol derivative on chondrocyte ferroptosis.

[0076] Experiment 2: Inhibition of RSL3-induced ferroptosis in C28 cells by 2-amino-6-methylphenol derivative. After C28 human chondrocytes adhered to the culture medium, DMSO, RSL3 (3 μM, final concentration), RSL3 (3 μM) + Ferrostatin-1 (0.1 μM), and RSL3 (3 μM) + 2-amino-6-methylphenol derivative (1 μM) were added to the medium, respectively. Cell viability was assessed by MTT assay after 24 hours of treatment. The results are shown below. Figure 4 As shown in Figure A, the level of cell membrane lipid peroxidation was simultaneously detected, and the final inhibitory effect was determined. Figure 4 As shown in B (data obtained after 3 repetitions of the experiment). Cell viability assay (MTT): C28 cells treated with the drug for 24 hours were viable using the MTT assay. The MTT kit was purchased from Beijing Solarbio Science & Technology Co., Ltd. Cell membrane lipid peroxidation level assay: C28 cells treated with the drug for 12 hours were digested into single-cell suspensions with trypsin. The cells were incubated with C11-BODIPY (10 μM) at room temperature in the dark for 60 minutes, washed 3 times with PBS, and analyzed by flow cytometry.

[0077] The experimental results showed that: Figure 4 As shown, compared with the control group, stimulation by the ferroptosis inducer RSL3 significantly killed cells; while the 2-amino-6-methylphenol derivative significantly inhibited RSL3-induced cell death (p<0.01), and its activity was comparable to that of the positive control Ferrostatin-1. Simultaneously, the 2-amino-6-methylphenol derivative significantly scavenged RSL3-induced lipid free radicals (p<0.01). This indicates that the 2-amino-6-methylphenol derivative significantly inhibits RSL-3-induced ferroptosis.

[0078] Experimental Example 3: Alleviating effect of 2-amino-6-methylphenol derivatives on osteoarthritis in mice.

[0079] Animal grouping: Thirty C57BL / 6 mice aged 8-10 weeks were selected and randomly divided into three groups, half male and half female. Ten C57 mice were used as the control group; ten C57 mice were used as the DMM model group; and ten C57 mice were used as the DMM model + 2-amino-6-methylphenol derivative (20 mg / kg) group.

[0080] Animal handling: The 2-amino-6-methylphenol derivative was dissolved in DMSO and administered intramuscularly or intravenously. One week after drug injection, the mice were dissected, and the knee joint was immediately removed for subsequent histological section staining. The final results are as follows: Figure 5 As shown.

[0081] Osteogenic function of chondrocytes: C28 cells treated with the drug for 12 hours were digested into single-cell suspensions with trypsin, and the cells were directly separated by low-speed centrifugation. Cell lysis buffer was added, and after lysis, the protein supernatant was collected by centrifugation and Western blot analysis was performed to detect indicators such as glutathione peroxidase (GPX4), alkaline phosphatase (ALP), type I collagen (Collenge-1), and osteogenic transcription factor (Runx2).

[0082] The experimental results showed that: Figure 5 As shown, compared with the normal DMM model group, the knee joint damage of mice treated with 2-amino-6-methylphenol derivatives was significantly reduced, indicating that 2-amino-6-methylphenol derivatives have a therapeutic and alleviating effect on arthritis.

[0083] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

The application of 1,2-amino-6-methylphenol derivatives in the preparation of drugs for the prevention, improvement, or treatment of osteoarthritis, characterized in that, The structural formula of the 2-amino-6-methylphenol derivative is shown below: 。 2. The application as described in claim 1, characterized in that, It also includes pharmaceutical salts of the 2-amino-6-methylphenol derivative.

3. The use of a pharmaceutical composition in the preparation of a medicament for the prevention, improvement, or treatment of osteoarthritis, characterized in that, The composition comprises an active dose of a 2-amino-6-methylphenol derivative; the structural formula of the 2-amino-6-methylphenol derivative is shown below: 。 4. The application as described in claim 3, characterized in that, The dosage of the pharmaceutical composition should be an effective dosage, and the pharmaceutical composition should be in a dosage form that facilitates precise administration; Alternatively, when the pharmaceutical composition is used to prepare a pharmaceutical formulation for in vivo administration, the pharmaceutical formulation shall be sterile; the pharmaceutical composition further includes a pharmaceutically acceptable carrier.

5. The application as described in claim 4, characterized in that, The dosage form includes a gastrointestinal dosage form or a non-gastrointestinal dosage form; The gastrointestinal dosage forms include powders, tablets, granules, capsules, dry suspensions, effervescent tablets, emulsions, and suspensions; The non-gastrointestinal dosage forms include injectable dosage forms, respiratory dosage forms, skin dosage forms, and mucosal dosage forms.

6. The application as described in claim 5, characterized in that, The above-mentioned drugs are in the form of injectable dosage forms.

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