A class of N-substituted phenyl-2-pyridone endoperoxides and their applications

Abstract

A class of N-substituted phenyl-2-pyridone endoperoxides and their applications belong to the field of organic compound synthesis and pharmaceutical technology. The present invention discloses that this class of endoperoxide compounds can be converted into N-substituted phenyl-2-pyridone at a certain temperature, while releasing singlet oxygen and triplet oxygen. The series of endoperoxides have excellent anti-pulmonary fibrosis effects and can significantly inhibit inflammatory factors, and the effect is significantly better than the marketed drug pirfenidone. The results of anti-lung cancer experiments show that the series of compounds can not only inhibit the proliferation and migration of cancer cells at the cellular level, but also inhibit the growth of tumors at the living level. The safety assessment results show that the series of endoperoxides have good safety, and can treat pulmonary fibrosis, lung cancer, and pulmonary fibrosis combined with lung cancer without obvious damage to other organs. This class of endoperoxides combines multiple therapeutic factors in one, and is expected to be developed into a first-line drug for the treatment of pulmonary fibrosis, lung cancer, and pulmonary fibrosis combined with lung cancer.

Description

Technical Field

[0001] The invention belongs to the technical field of organic compound synthesis and medicine, and particularly relates to a class of N-substituted phenyl-2-pyridone endoperoxides and applications thereof. Background Art

[0002] The lungs are essential respiratory organs in the human body, exchanging gases between the body and the external environment through respiration, thereby sustaining life. Lung disease can severely impact human health. Pulmonary fibrosis is a lung disease characterized by fibroblast proliferation and massive accumulation of extracellular matrix, accompanied by inflammatory damage and tissue structural destruction. It is also considered to be scarring caused by abnormal repair of damaged alveolar tissue. Currently, the cause of pulmonary fibrosis remains unknown in most patients, and this condition is also known as idiopathic pulmonary fibrosis. The average survival after diagnosis of idiopathic pulmonary fibrosis is short, with a mortality rate higher than that of most cancers, severely impacting respiratory function and daily life. Lung cancer is one of the most common malignant tumors, with a high global incidence. Non-small cell lung cancer accounts for over 80% of cases, and most patients are diagnosed in the advanced stage, complicating clinical treatment. Lung cancer is also a significant comorbidity in patients with idiopathic pulmonary fibrosis. The incidence of lung cancer in patients with idiopathic pulmonary fibrosis is as high as 48%, significantly higher than in the general population. To date, there is no specific treatment for idiopathic pulmonary fibrosis. The only approved drugs are pirfenidone and nintedanib. Pirfenidone is a cytokine inhibitor that inhibits transforming growth factor (TGF-β) and fibroblast growth factor (bFGF), thereby suppressing fibroblast activity, reducing cell proliferation, and matrix collagen synthesis. Pirfenidone can delay lung function failure and mitigate disease progression, but it still cannot reverse the progression of pulmonary fibrosis. Chemotherapy is the mainstay of treatment for non-small cell lung cancer, but drug resistance often leads to treatment failure. Treatment of pulmonary fibrosis combined with lung cancer is challenging, and currently no effective treatment exists. Because these diseases often occur in the elderly, patients often suffer from poor lung function and poor tolerance to surgery and chemotherapy. Conventional chemotherapy often leads to lung infections, neutropenia, and respiratory insufficiency. Furthermore, the mortality rate in patients with pulmonary fibrosis and lung cancer is several times higher than in patients with pulmonary fibrosis alone, and no specific treatment is currently available. Therefore, there is an urgent need to develop drugs that can treat pulmonary fibrosis, lung cancer, and pulmonary fibrosis combined with lung cancer.

[0003] Singlet oxygen 1O2), also known as excited oxygen molecules, act as signaling and stimulatory molecules in many physiological processes. Furthermore, singlet oxygen is a highly reactive species that, at certain doses, can damage tumor cell membranes, proteins, and other biomolecules, causing apoptosis, vascular damage, or inducing immune responses to eliminate tumor cells. Studies have shown that singlet oxygen reacts with naphthalene, anthracene, and pyridone via a [4+2] cycloaddition reaction to form endoperoxides. Under certain temperatures, endoperoxides undergo a reverse Diels-Alder reaction to convert to the starting material and release singlet oxygen. While releasing singlet oxygen, endoperoxides also release some molecular oxygen (triplet oxygen). Singlet oxygen, after being quenched under physiological conditions, can also be converted to molecular oxygen. Molecular oxygen can alleviate tumor hypoxia, and oxygen therapy is a common treatment option for patients with pulmonary fibrosis. Singlet oxygen delivery systems designed based on endoperoxides may provide novel treatment options for diseases such as cancer, bacterial infections, Alzheimer's disease, coagulation disorders, and ischemic injury. Summary of the Invention

[0004] One object of the present invention is to provide a compound of formula I or a pharmaceutically acceptable salt thereof:

[0005]

[0006] Among them, R 1 、R 2 、R 3 Each independently selected from hydrogen, deuterated methyl, C1-C10 alkyl, carboxyl, C1-C5 fluoroalkyl, nitro, and C1-C10 ester;

[0007] R 4 -R 8 Each independently selected from hydrogen, C1-C5 deuterated alkyl, C1-C5 fluoroalkyl, halogen, carboxyl, sulfonic acid, aminosulfonic acid, hydroxyl, amino, -COOR, -CONR, C1-C10 alkylamino, nitro, cyano, C1-C10 alkyl, C1-C10 alkoxy, C1-C10 thioether;

[0008] R is independently a C1-C5 alkyl group.

[0009] Another object of the present invention is to provide a pharmaceutical composition comprising one or more of the above-mentioned compounds or pharmaceutically acceptable salts thereof.

[0010] The pharmaceutical composition further comprises a carrier and / or pharmaceutical excipients.

[0011] The preparation method of the compound comprises the following steps:

[0012]

[0013] (1) Compound 1 and Compound 2 react in the presence of potassium carbonate and cuprous iodide to obtain Compound P;

[0014] (2) Compound P reacts in the presence of a photosensitizer, an oxygen environment, and light irradiation to obtain compound E.

[0015] In some specific preparation methods, the reaction solvent in step (1) is one or more high-boiling-point solvents such as N,N-dimethylformamide and dimethyl sulfoxide. The reflux reaction temperature is between 150-200°C. The amount of iodobenzene 2 and potassium carbonate used is 1-3 times the amount of 2-pyridone (by mole). The amount of cuprous iodide used is 2%-20% (by mole) of 2-pyridone.

[0016] In some specific preparation methods, the reaction solvent in step (2) can be one or more solvents such as chloroform, dichloromethane, ether, methanol, ethanol, toluene, benzene, DMF, ethyl acetate, water, deuterated reagents, etc. The reaction temperature is -20°C to room temperature, preferably an ice-water bath, 0°C, an ice bath, a room temperature water bath or below 0°C. The reaction can be carried out at room temperature, but the high temperature evaporation of the solvent due to red light irradiation should be avoided. The irradiation light source can also use a light source of other wavelengths (425-750nm). The gas environment can be oxygen, a gas containing oxygen, or a direct open reaction. Methylene blue / red light can be replaced with other common photosensitizers and light sources of corresponding wavelengths, such as BODIPY / red light.

[0017] The deuterated reagent is selected from one or more of deuterated chloroform, deuterated methanol, deuterated benzene, and deuterated water;

[0018] The photosensitizer is selected from methylene blue, fluoroborane, hematoporphyrin, dihydrochlorin e6, pyropheophorbide-a, pyropheophorbide-a hexyl ether, phenothiazine derivatives, phenoxazine derivatives, porphyrin photosensitizers or phthalocyanine photosensitizers.

[0019] The present invention also provides the use of a compound of formula I or a pharmaceutically acceptable salt thereof in preparing a medicament:

[0020] Use of a compound of formula I or a pharmaceutically acceptable salt thereof in the preparation of a drug that is converted into N-substituted phenyl-2-pyridone in vivo and releases singlet oxygen and triplet oxygen:

[0021]

[0022] Use of a compound of formula I or a pharmaceutically acceptable salt thereof in the preparation of the following medicaments:

[0023] Drugs that inhibit the expression of transforming growth factor-β (TGF-β), monocyte chemoattractant protein-1 (MCP-1), and serum inflammatory factor interleukin-1β (IL-1β), while also upregulating heme oxygenase-1 (HO-1); or

[0024] Drugs for treating or improving diseases or inflammation involving one or more factors including TGF-β, MCP-1, IL-1β, HO-1, TNF-α, IL-6, IFN-γ, bFGF, PDGF, NLRP3, CAT, and SOD.

[0025] Use of a compound of formula I or a pharmaceutically acceptable salt thereof in the preparation of a drug for improving or treating fibrosis.

[0026] The fibrosis is pulmonary fibrosis, renal interstitial fibrosis or liver fibrosis, in particular idiopathic pulmonary fibrosis.

[0027] Use of the compound of formula I or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating cancer or inflammation.

[0028] The cancer is lung cancer or non-small cell lung cancer.

[0029] Use of a compound of formula I or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating pulmonary fibrosis combined with lung cancer, especially idiopathic pulmonary fibrosis combined with lung cancer.

[0030] The medicine in the application can be prepared into tablets, capsules, granules, powders, oral preparations, injections, microcapsule preparations, suppositories, pills, aerosols, sprays, powder inhalers, syrups, wine preparations, tinctures, lotions, and films, and one or more of the above dosage forms can be selected for treatment.

[0031] The drugs used in the application include:

[0032] At least one compound of formula I; and / or

[0033] carrier; and / or

[0034] pharmaceutical excipients;

[0035] The carrier is selected from one or more of metal nanocarriers, non-metal nanocarriers, liposomes, lactose, sucrose, gelatin, hard magnesium sulfate, and stearic acid;

[0036] The pharmaceutical excipients are selected from one or more of diluents, binders, disintegrants, lubricants, glidants, flavoring agents, coating agents, gelatin capsule shells, latent solvents, propellants, surfactants, preservatives, and freeze-drying protective agents.

[0037] Advantages and beneficial effects of the present invention:

[0038] The present invention relates to the synthesis and application of N-substituted phenyl-2-pyridone endoperoxides. A series of compounds can be prepared in batches by a simple chemical synthesis method, and at the same time have good stability and can be stored for a long time. This type of endoperoxide integrates multiple therapeutic factors such as endoperoxide, N-substituted phenyl-2-pyridone, singlet oxygen and triplet oxygen, that is, it can exert the therapeutic effect of a single factor and simultaneously achieve the purpose of synergistic treatment of several factors. The endoperoxide can inhibit the expression of factors such as transforming growth factor-β (TGF-β), monocyte chemoattractant protein-1 (MCP-1), serum inflammatory factor interleukin-1β (IL-1β) and upregulate heme oxygenase-1 (HO-1), and its anti-pulmonary fibrosis effect is better than that of the marketed drug pirfenidone. The endoperoxide of the present invention achieves the treatment of pulmonary fibrosis, lung cancer, and lung cancer combined with lung cancer through multiple therapeutic factors independently or in collaboration, is suitable for multiple dosage forms and carriers, and is expected to become a first-line drug for the treatment of pulmonary fibrosis, lung cancer, and lung cancer combined with lung cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 is the structural formula of the compound of the present invention.

[0040] Figure 2 This is a half-life test graph of the release of P5, singlet oxygen and triplet oxygen from the representative compound E5.

[0041] Figure 3 It is a test graph of the yield of P5, singlet oxygen and triplet oxygen released by the representative compound E5.

[0042] Figure 4 This is a study chart of singlet oxygen release of representative compounds E5 and P5.

[0043] Figure 5 This is a graph showing the hydroxyproline content in the serum of mice undergoing an anti-pulmonary fibrosis experiment.

[0044] Figure 6 This is a HE staining image of lung tissue sections from mice in the anti-pulmonary fibrosis experiment.

[0045] Figure 7 This is a MASSON staining image of lung tissue sections from mice in the anti-pulmonary fibrosis experiment.

[0046] Figure 8 This is an immunofluorescence image of lung tissue sections from mice undergoing an anti-pulmonary fibrosis experiment.

[0047] Figure 9 This is a graph showing the expression of TGF-β mRNA in mice undergoing anti-pulmonary fibrosis experiments.

[0048] Figure 10 This is a graph showing the expression of MCP-1 mRNA in mice undergoing an anti-pulmonary fibrosis experiment.

[0049] Figure 11 This is a graph showing the expression of IL-1β mRNA in mice undergoing an anti-pulmonary fibrosis experiment.

[0050] Figure 12 This is a graph showing the expression of HO-1 mRNA in mice undergoing an anti-pulmonary fibrosis experiment.

[0051] Figure 13 This is a graph of AST, ALT, AKP, BUN, and SCR levels in the serum of mice undergoing an anti-pulmonary fibrosis experiment.

[0052] Figure 14 This is a graph showing the lung coefficient (lung weight / body weight) of mice in an anti-pulmonary fibrosis experiment.

[0053] Figure 15 This is the weight change curve of mice in the anti-pulmonary fibrosis experiment.

[0054] Figure 16 This is a HE staining image of mouse liver tissue section.

[0055] Figure 17 This is a HE staining image of mouse spleen tissue section.

[0056] Figure 18 This is a HE staining image of mouse kidney tissue section.

[0057] Figure 19 This is a HE staining image of mouse heart tissue section.

[0058] Figure 20 Representative MTT assay of endoperoxides on A549 lung cancer cells.

[0059] Figure 21 Representative endoperoxide singlet oxygen release assay in A549 lung cancer cells.

[0060] Figure 22 Representative endoperoxide-induced apoptosis in A549 lung cancer cells.

[0061] Figure 23 Representative endoperoxide inhibition of A549 lung cancer cell migration. DETAILED DESCRIPTION

[0062] Pharmaceutically acceptable salts of compounds of formula (I) are formed by adding pharmaceutically acceptable acids. Examples of salts include, but are not limited to, nitrates, hydrochlorides, hydrobromides, sulfates, bisulfates, phosphates, hydrogen phosphates, acetates, benzoates, succinates, fumarates, maleates, lactates, citrates, tartrates, gluconates, pyrosulfinates, benzenesulfonates, and p-toluenesulfonates.

[0063] The term "halogen" represents a fluorine, chlorine, bromine or iodine atom.

[0064] The term "C1-C10 alkyl" refers to a straight-chain or branched, saturated, aliphatic hydrogen-carbon group having 1 to 10 carbon atoms. Examples of C1-C10 alkyl groups are methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl and tert-butyl, n-pentyl, n-hexyl, n-octyl, and n-octyl.

[0065] The term "C1-C5 fluoroalkyl" refers to a linear or branched, saturated, aliphatic hydrogen carbon group having 1 to 5 carbon atoms and containing one or more fluorine atom substituents. Examples of C1-C5 fluoroalkyl are trifluoromethyl, 1,1,1,2-tetrafluoroethyl, perfluoroethyl, perfluoropropyl, and perfluorohexyl.

[0066] The term "C1-C10 ester group" refers to an ester group having 1 to 10 carbon atoms obtained by dehydration of a linear or branched, saturated, fatty alcohol or a linear or branched, fatty acid. Examples of C1-C10 ester groups are wait.

[0067] The term "C1-C10 alkoxy" refers to a straight or branched, saturated, aliphatic alkoxy group having 1 to 10 carbon atoms. Examples of C1-C10 alkoxy are methoxy, ethoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, and the like.

[0068] The term "C1-C10 thioether group" refers to a linear or branched, saturated, aliphatic thioether group having 1 to 10 carbon atoms. Examples of C1-C10 thioether groups are methylthio, ethylthio, isopropylthio, n-butylthio, isobutylthio, sec-butylthio, and the like.

[0069] The term "C1-C5 deuterated alkyl" refers to a straight-chain or branched, saturated, aliphatic hydrocarbon group having 1 to 5 carbon atoms and containing at least one deuterium atom. Examples of C1-C5 deuterated alkyl groups are deuterated methyl, deuterated ethyl, deuterated propyl, deuterated n-butyl, deuterated isobutyl, deuterated n-pentyl, and the like.

[0070] -COOR and -CONR refer to

[0071] The term "C1-C10 alkylamino" refers to a straight-chain or branched, saturated, aliphatic alkylamino group having 1 to 10 carbon atoms. Examples of C1-C10 alkylamino groups are methylamino, ethylamino, isopropylamino, n-butylamino, isobutylamino, sec-butylamino, tert-butylamino, and the like.

[0072] The following examples are provided to help understand the present invention, but they are not intended to limit the present invention.

[0073] A class of drugs capable of treating pulmonary fibrosis, lung cancer, and pulmonary fibrosis combined with lung cancer is disclosed, along with a method for synthesizing such compounds. The present invention creatively develops N-substituted phenyl-2-pyridone endoperoxides, which release N-substituted phenyl-2-pyridone (pirfenidone analogs or pirfenidone), singlet oxygen, and triplet oxygen in vivo to treat pulmonary fibrosis, lung cancer, and pulmonary fibrosis combined with lung cancer.

[0074] Specifically, N-substituted phenyl-2-pyridone endoperoxide or a pharmaceutically acceptable salt thereof has the following structure:

[0075]

[0076] wherein R1, R2, and R3 are each independently selected from hydrogen, deuterated methyl, C1-C10 alkyl, carboxyl, trifluoromethyl, nitro, and C1-C10 ester;

[0077] R4-R8 are each independently selected from hydrogen, C1-C5 deuterated alkyl, C1-C5 fluoroalkyl, halogen, carboxyl, sulfonic acid, aminosulfonic acid, hydroxyl, amino, -COOR, -CONR, C1-C10 alkylamino, nitro, cyano, C1-C10 alkyl, C1-C10 alkoxy, C1-C10 thioether;

[0078] R is independently a C1-C5 alkyl group.

[0079] In some specific N-substituted phenyl-2-pyridone endoperoxides, R1, R2, and R3 are each independently selected from hydrogen, deuterated methyl, C1-C5 alkyl, and trifluoromethyl;

[0080] R4-R8 are each independently selected from hydrogen, C1-C5 deuterated alkyl, C1-C5 fluoroalkyl, halogen, carboxyl, sulfonic acid, aminosulfonic acid, hydroxyl, amino, -COOR, -CONR, C1-C10 alkylamino, nitro, cyano, C1-C10 alkyl, C1-C10 alkoxy, C1-C10 thioether;

[0081] R is independently a C1-C5 alkyl group.

[0082] In some specific N-substituted phenyl-2-pyridone endoperoxides, R1, R2, and R3 are each independently selected from hydrogen, deuterated methyl, trifluoromethyl, and methyl;

[0083] R4-R8 are each independently selected from hydrogen, C1-C5 deuterated alkyl, C1-C5 fluoroalkyl, halogen, carboxyl, sulfonic acid, aminosulfonic acid, hydroxyl, amino, -COOR, -CONR, C1-C10 alkylamino, nitro, cyano, C1-C10 alkyl, C1-C10 alkoxy, C1-C10 thioether;

[0084] R is independently a C1-C5 alkyl group.

[0085] In some specific N-substituted phenyl-2-pyridone endoperoxides, R1, R2, and R3 are each independently selected from hydrogen and methyl;

[0086] R4-R8 are each independently selected from hydrogen, C1-C5 deuterated alkyl, C1-C5 fluoroalkyl, halogen, carboxyl, sulfonic acid, aminosulfonic acid, hydroxyl, amino, -COOR, -CONR, C1-C10 alkylamino, nitro, cyano, C1-C10 alkyl, C1-C10 alkoxy, C1-C10 thioether;

[0087] R is independently a C1-C5 alkyl group.

[0088] In some specific N-substituted phenyl-2-pyridone endoperoxides, R1, R2, and R3 are each independently selected from hydrogen, deuterated methyl, trifluoromethyl, and methyl;

[0089] R4-R8 are each independently selected from hydrogen, deuterated methyl, trifluoromethyl, halogen, carboxyl, sulfonic acid, aminosulfonic acid, hydroxyl, amino, -COOR, -CONR, C1-C5 alkylamino, nitro, cyano, C1-C5 alkyl, C1-C5 alkoxy, C1-C5 thioether;

[0090] R is independently a C1-C5 alkyl group.

[0091] Some specific N-substituted phenyl-2-pyridone endoperoxides are selected from the following structures:

[0092]

[0093]

[0094]

[0095] Preparation method of N-substituted phenyl-2-pyridone endoperoxide

[0096] The synthetic route is as follows:

[0097]

[0098] (1) 2-pyridone 1, iodobenzene 2, potassium carbonate, cuprous iodide and DMF were placed in a reaction flask and refluxed under argon protection. After the reaction was completed, the mixture was cooled to room temperature, filtered, and the filtrate was decolorized by adding activated carbon. The decolorized solution was evaporated to dryness under reduced pressure, 10% acetic acid was added, and the mixture was stirred at 70°C. After standing for stratification, the upper aqueous phase was taken and sodium hydroxide solution was added to adjust the pH to 13, and the mixture was placed in a refrigerator overnight for crystallization. The crude product was heated to reflux with ethyl acetate, filtered while hot, and the filtrate was cooled for crystallization. Filtered, the filter cake was dried under reduced pressure to obtain pure product P.

[0099] (2) Compound P, a catalytic amount of methylene blue, and chloroform were placed in a reaction flask, irradiated with red light (620-625 nm) under oxygen protection, stirred in an ice-water bath, and the reaction was monitored by TLC. After the reaction, the methylene blue was removed by filtration using silica gel (or activated carbon). The filtrate was evaporated to dryness under reduced pressure to obtain pure compound E.

[0100] Application of N-substituted phenyl-2-pyridone endoperoxides

[0101] The endoperoxide developed by the present invention can release N-substituted phenyl-2-pyridone, singlet oxygen, and triplet oxygen at a certain temperature (such as 37°C in the human body). The N-substituted phenyl-2-pyridone endoperoxide is mainly used to treat pulmonary fibrosis, lung cancer, and pulmonary fibrosis combined with lung cancer.

[0102] Pulmonary fibrosis in mice was induced using bleomycin and treated with the endoperoxides developed in this invention. Following treatment, lung tissue was stained with HE, Masson's, and immunofluorescence staining, and serum hydroxyproline levels were measured. The results demonstrated that the compounds exhibited excellent anti-fibrotic effects, with some, such as E5, demonstrating superior anti-fibrotic effects compared to the marketed drug pirfenidone.

[0103] The marketed drug pirfenidone reduces cell proliferation and matrix collagen synthesis by inhibiting factors such as TGF-β, while exerting anti-inflammatory and antioxidant effects by inhibiting the secretion of IL-1β and other inflammatory mediators and reducing lipid peroxidation. Compared with pirfenidone, the endoperoxides involved in the present invention can significantly inhibit the expression of factors that promote pulmonary fibrosis, such as TGF-β and MCP-1. It is worth noting that, as a singlet oxygen carrier, the endoperoxides involved in the present invention not only do not upregulate inflammatory factors, but are more excellent in anti-inflammatory effect than pirfenidone, further verifying the clinical application potential of the series of compounds in anti-pulmonary fibrosis. In addition, heme oxygenase-1 (HO-1) in the pulmonary fibrosis model group was significantly downregulated compared with the blank group, while representative endoperoxides can significantly upregulate heme oxygenase-1, close to normal physiological levels, and the upregulation effect is far superior to pirfenidone.

[0104] The anti-lung cancer properties of endoperoxides were evaluated at both the cellular and animal levels. The experimental results demonstrated that the endoperoxides described in this invention can efficiently release singlet and triplet oxygen within cancer cells. Singlet oxygen capture probes were used to successfully verify the release of singlet oxygen within cells. Apoptosis experiments demonstrated singlet oxygen-induced apoptosis. MTT experiments confirmed the potent anti-cancer properties of the compounds. Cell migration experiments demonstrated that endoperoxides can successfully inhibit the migration of lung cancer cells. Animal anti-lung cancer experimental results demonstrated that N-substituted phenyl-2-pyridone endoperoxides can effectively inhibit tumor growth in living animals.

[0105] Safety assessment results showed that after endoperoxide treatment, multiple serum indicators (AST, ALT, AKP, BUN, and SCR) in mice remained normal. Other organs, such as the heart, liver, spleen, and kidneys, showed no significant damage. The lung coefficient and overall body weight changes were also normal. These results demonstrate that the drug developed by the present invention has excellent safety and bioavailability, and has potential for clinical application.

[0106] Example 1: Preparation of representative compounds P5, E5

[0107]

[0108] Compound 1a (20 g), iodobenzene 2a (68.8 g, 1.5 equiv.), potassium carbonate (32.0 g, 1.1 equiv.), cuprous iodide (4 g, 10 mol%), and DMF (300 mL) were placed in a 500 mL reaction flask and refluxed at 150°C for 10 hours under argon protection. After the reaction, the mixture was cooled to room temperature and filtered. The filter cake was washed three times with DMF. The filtrates were combined and decolorized with activated carbon. The decolorized solution was evaporated to dryness under reduced pressure, 100 mL of 10% acetic acid was added, and the mixture was stirred at 70°C for 30 minutes. After standing for separation, the upper aqueous phase was collected, and the lower oily substance was extracted twice with 10% acetic acid. The aqueous phases were combined. Sodium hydroxide solution was added to the aqueous phase to adjust the pH to 13 and the mixture was placed in a refrigerator overnight for crystallization. 100 mL of ethyl acetate was added to the crude product obtained by vacuum drying, and the mixture was heated to reflux. The mixture was filtered while hot and the filtrate was cooled for crystallization. The filter cake was filtered and dried under reduced pressure to obtain pure product P5: 1 H NMR (400MHz, CDCl3) δ7.49–7.46(m,2H),7.42–7.37(m,3H),7.26(d,J=6.9Hz,1H),7.23(d,J=6.9Hz,1H),6.16(t,J=6.8Hz,1H),2.19(s,3H); 13 CNMR (100MHz, CDCl3) δ162.8,141.4,136.9,135.4,130.8,129.2,128.2,126.6,105.6,17.4.

[0109] Compound P5 (15 g), methylene blue (5 mg), and chloroform (100 mL) were placed in a reaction flask. Under oxygen protection, the mixture was irradiated with red light (620-625 nm). The reaction was stirred in an ice-water bath and monitored by TLC. After completion of the reaction, the methylene blue was removed by filtration using silica gel (or activated carbon), and the filtrate was evaporated to dryness under reduced pressure to obtain compound E5. Yield: 100%. 1 H NMR (400MHz, CDCl3) δ7.43–7.39(m,2H),7.32–7.27(m,3H),6.97–6.93(m,1H),6.59–6.57(m,1H),6.08–6.06(m,1H),1.69(s,3H); 13 C NMR (100MHz, CDCl3) δ168.3,138.2,134.0,129.4,126.9,123.7,86.1,82.1,14.7.

[0110] Example 2: Half-life and yield analysis of P5, singlet oxygen, and triplet oxygen release from compound E5 and singlet oxygen capture experiments

[0111]

[0112] Half-life of release of P5, singlet oxygen and triplet oxygen: Endoperoxide E5 releases P5, singlet oxygen and triplet oxygen through reverse cycloaddition reaction. The whole process follows the first-order kinetic rate equation, which is expressed by t 1 / 2 =0.693 / k formula can be used to calculate the half-life. The specific experimental procedure is as follows: weigh about 2 mg of endoperoxide E5, dissolve it in deuterated water and place it in a 37°C water bath. The nuclear magnetic resonance hydrogen spectrum of the solution is tested at 6 hours, 10 hours, 23 hours and 35 hours respectively. The half-life of the reverse cycloaddition reaction is calculated based on the characteristic peaks of E5 and P5 between 6.25-6.75 ppm: t 1 / 2 = 11.9 hours (37 ° C, deuterated water, such as Figure 2 shown).

[0113]

[0114] The yields of P5, singlet oxygen, and triplet oxygen released: Tetramethylethylene was used as a singlet oxygen trapping agent, and the yields of P5, singlet oxygen, and triplet oxygen released by the reverse cycloaddition reaction of E5 were calculated by H NMR spectroscopy according to the above method. The results showed that after 1 mol of E5 underwent complete reverse reaction, 1 mol of P5, 0.5 mol of singlet oxygen, and 0.5 mol of triplet oxygen (such as Figure 3 shown).

[0115] Furthermore, these results demonstrate that compounds such as the endoperoxide E5 exhibit moderate water solubility and are fully converted to P5 in aqueous solution, releasing both singlet and triplet oxygen. The reverse cycloaddition of the endoperoxides produces no other impurities, demonstrating the high potential for drug development and clinical application of these compounds.

[0116] Singlet oxygen capture experiment: DPBF was used as a singlet oxygen capture agent to monitor the release of singlet oxygen from the compound. The experimental process is as follows: the representative compound E5 (750 μM) and DPBF (singlet oxygen capture agent, 37.5 μM) were dissolved in DMF. At 37 ° C, in the dark, the absorption at 417 nm was monitored using a UV-visible spectrophotometer at different time periods to analyze the release of singlet oxygen. The results showed that endoperoxides can efficiently release singlet oxygen (such as Figure 4 shown).

[0117] Example 3: Preparation of Compounds E1-E4, E6-E160

[0118] The synthesis methods of compounds E1–E4, E6–E160 refer to the synthesis of compound E5, where the structures of 2-pyridone and iodobenzene used are as follows:

[0119] 2. Pyridone

[0120]

[0121] Iodobenzene

[0122]

[0123] The structures of compounds E1-E4 and E6-E160 were all identified.

[0124] Example 4: Anti-pulmonary fibrosis experiment

[0125] 4.1 Experimental process of endoperoxide anti-pulmonary fibrosis

[0126] Mice were randomly divided into a blank control group, a model group, a pirfenidone-treated group, an N-substituted phenyl-2-pyridone-treated group, and an N-substituted phenyl-2-pyridone endoperoxide-treated group. After acclimation for 3 days (weighing approximately 20 g), surgical modeling was performed on the 4th day. The procedure was as follows: After weighing, the mice were anesthetized with an intraperitoneal injection of sodium pentobarbital. The anesthetized mice were placed in the supine position and secured with a mouse board, with the limbs and head elevated, to fully expose the neck skin. The anterior neck of the mice was routinely depilated and disinfected. A midline longitudinal incision was performed, with blunt dissection of muscle and tissue until the trachea was exposed. A bleomycin solution (2 mg / ml) was prepared in sterile saline. The syringe was inserted into the trachea through the interstitial tracheal cartilage rings toward the heart. After air was withdrawn and no resistance was detected, 50 μL of the solution was slowly injected into the trachea. The blank control group received the same volume of saline. After injection, the mouse board was upright and rapidly rotated to evenly distribute the solution throughout the lungs. The incision was sutured, and the mice were placed in the right lateral position and allowed to recover naturally. Three days after modeling, oral administration began. The drug was diluted with 0.5% sodium carboxymethylcellulose (CMC-Na) and administered to each mouse at a dose of 400 mg / kg. The blank control group and model group received the same dose of CMC-Na solution. During oral administration, mouse body weight and survival were measured. Sixteen days after administration, mice were sacrificed by eye removal, blood collection, and cervical dislocation. Lungs and other organs were collected. A portion of lung tissue and other tissues were fixed in 4% neutral formalin, and a portion was treated with liquid nitrogen and stored at -80°C for histological observation and subsequent testing of relevant indicators.

[0127] 4.2 Analysis of the experimental results of endoperoxides against pulmonary fibrosis

[0128] 4.2.1 Analysis of Hydroxyproline Content in Mouse Serum

[0129] Hydroxyproline (HYP) is a non-essential amino acid unique to collagen and one of the main components of collagen tissue. It can be used to assess the degree of fibroblast activation. The hydroxyproline content was determined using a hydroxyproline standard curve and the absorbance of mouse serum samples at 560 nm. The results of the serum HYP content test in each group of mice are shown in Figure 2. Figure 5 As shown. Compared with the HYP content in the serum of mice in the blank group (206±4ng / ml), the HYP content of mice in the model group (360±7ng / ml) increased significantly, proving that the fibrosis of the mice was more serious. The HYP content of mice in the pirfenidone treatment group was 286±4ng / ml, showing a better effect in alleviating pulmonary fibrosis. The HYP content of mice in the P5 treatment group was 317±5ng / ml, proving that P5 has a certain anti-pulmonary fibrosis effect. The HYP content of mice in the E5 treatment group was 218±4ng / ml, close to that of the blank group (206±4ng / ml), indicating that the anti-pulmonary fibrosis effect of E5 is better than that of the marketed drug pirfenidone, and the hydroxyproline content after medication is close to the normal value.

[0130] The anti-pulmonary fibrosis efficacy of compounds E1–E4 and E6–E160 was higher than that of pirfenidone. The test data of some compounds are given in the following table, where the test process and method refer to Examples 4.1 and 4.2.

[0131] Table 1 Anti-pulmonary fibrosis experimental results of a series of compounds

[0132]

[0133]

[0134]

[0135] Note: A represents the serum HYP content of mice after treatment is 200-250 ng / ml; B represents the serum HYP content of mice after treatment is 250-300 ng / ml; C represents the serum HYP content of mice after treatment is 300-360 ng / ml; D represents the serum HYP content of mice after treatment is greater than 360 ng / ml.

[0136] 4.2.2 Analysis of lung tissue pathological changes

[0137] HE staining, MASSON staining and α-SMA immunofluorescence staining were performed on lung tissue sections to compare the pathological changes of lung tissue in the blank group, model group, pirfenidone treatment group, P5 treatment group and E5 treatment group. Figure 6-8 As shown in the results, the lung tissue structure of the blank group was clear, with no thickening of the alveolar septa, no water-filled edema, no obvious myofibroblasts, no inflammation and pulmonary fibrosis, and no obvious exudation in the alveolar cavity. The alveolar structure of the model group was severely damaged, with severe atrophy and collapse, a significant increase in collagen lung fibers, a large number of inflammatory cell infiltration, and a large number of myofibroblasts. The degree of alveolar structure damage in the pirfenidone treatment group was alleviated, collagen lung fibers were slightly reduced, fibroblasts were reduced, and the degree of pulmonary fibrosis was alleviated. The degree of alveolar structure damage in the P5 treatment group was alleviated, collagen lung fibers were slightly reduced, fibroblasts were reduced, and the degree of pulmonary fibrosis was also alleviated. The lung tissue structure of the E5 treatment group was clearer, collagen lung fibers were greatly reduced, fibroblasts were greatly reduced, and the overall state of the lungs was close to that of the blank group. The above data show that the pirfenidone analog P5 developed by the present invention has a certain effect in alleviating pulmonary fibrosis. Although the chemical structure of P5 has been reported, its anti-pulmonary fibrosis research has not yet been carried out. The endoperoxide E5 developed based on P5 has a more excellent anti-pulmonary fibrosis effect, which is more significant than the marketed drug pirfenidone. After treatment, the degree of fibrosis in the lung tissue is greatly alleviated.

[0138] 4.2.3 mRNA expression analysis

[0139] After treatment, mRNA levels were analyzed by fluorescence quantitative PCR. The results showed that TGF-β, MCP-1, IL-1β and other factors were significantly upregulated in the bleomycin-induced pulmonary fibrosis model ( Figure 9-11 ), indicating the formation of pulmonary fibrosis and the occurrence of inflammation. Heme oxygenase-1 (HO-1) in the pulmonary fibrosis model was significantly downregulated compared with the blank group (e.g. Figure 12 This conclusion is consistent with the results of previous reports, indicating that the oxidative stress response is imbalanced after the occurrence of pulmonary fibrosis. The anti-pulmonary fibrosis results showed that the endoperoxide treatment group was able to significantly downregulate factors such as TGF-β, MCP-1, and IL-1β, and the inhibitory effect was better than that of the listed drug pirfenidone. In addition, endoperoxides can significantly upregulate HO-1, close to normal physiological levels, and by regulating the oxidative stress response and protecting the body, the effect is far better than pirfenidone. The above results show that the endoperoxides of the present invention can effectively resist pulmonary fibrosis and inhibit inflammatory-related factors.

[0140] 4.2.4 Safety Assessment

[0141] a. Liver and kidney function evaluation

[0142] By comparing with the standard sample, serum indicators are analyzed to evaluate drug safety. Figure 13 As shown in the figure, the AST, ALT, AKP, BUN and SCR levels in the serum of mice in the pirfenidone treatment group, P5 treatment group and E5 treatment group were significantly decreased compared with those in the model group, and were the same as those in the blank group, proving that the drug E5 has good safety.

[0143] b. Lung coefficient

[0144] Lung coefficient (lung weight / body weight) is a key measurement indicator in animal experiments. Figure 14 As shown in the figure, the lung coefficient of the model group was improved to a certain extent compared with the blank group. However, compared with the blank group, the three treatment groups (pirfenidone treatment group, P5 treatment group, and E5 treatment group) did not show significant changes in the lung coefficient, further demonstrating the excellent safety of E5.

[0145] c. Mouse weight

[0146] The changes in the body weight of mice during the experiment can be used to evaluate the effects of drug use on mice. Figure 15 As shown, compared with the blank group, the three treatment groups (pirfenidone treatment group, P5 treatment group, and E5 treatment group) had no significant weight loss, further demonstrating the excellent safety of the new drug E5.

[0147] d. HE staining of heart, liver, spleen and kidney tissues

[0148] HE staining was performed on the heart, liver, spleen and kidney tissues of the blank group, model group, pirfenidone treatment group, P5 treatment group and E5 treatment group to evaluate the effects of the drug on other organs and prove the safety of the drug. Figure 16-19 It can be seen that compared with the blank group, continuous use of pirfenidone, P5, and E5 had no obvious damage to other organs of mice, which once again proved that E5 has good safety.

[0149] Example 5: Anti-lung cancer experiment

[0150] 5.1 Test of the killing ability of endoperoxides on A549 human lung cancer cells: A549 cells were cultured in 96-well plates overnight to allow the cells to fully adhere. Gradient concentrations of endoperoxide E and raw material control P were then added to the cells and incubated for another 24 hours. Finally, the cytotoxicity of the compounds was tested using the MTT assay. Figure 20 The results show that the representative endoperoxide E5 exhibits good anticancer properties, IC 50 The series of compounds all showed good anticancer properties, and the anticancer ability tests of some compounds are shown in Table 2.

[0151] 5.2 Singlet oxygen release of endoperoxides in A549 human lung cancer cells: A549 cells were cultured in confocal culture dishes overnight to allow the cells to completely adhere to the wall. Endoperoxide E and raw material control P (40 μM each) were then added to the cells and incubated for 5 hours, and then the cells were washed with PBS. The cells were then stained with 10 μM DCFH-DA solution for 45 minutes, the culture medium was removed, and the cells were washed with PBS. The cells were then incubated with 10 μg / mL Hoechst solution at room temperature for 20 minutes, and the excess dye was washed with PBS. Fresh culture medium was added to each culture dish, and finally, the cells were photographed under a fluorescence microscope. Figure 21 The results show that there is significant singlet oxygen release in the cells treated with compound E5, while no significant singlet oxygen release is observed in the control compound.

[0152] 5.3 Experiment on endoperoxide-promoted apoptosis of A549 human lung cancer cells: The cells were cultured in a confocal culture dish overnight to allow the cells to completely adhere to the wall. Endoperoxide E and raw material control P (160 μM each) were then added to the cells and incubated for 9 hours before drug removal and cell washing with PBS. Annexin V-FITC / PI staining solution was then added and the cells were incubated in the dark at room temperature for 20 minutes, then placed in an ice bath and finally photographed under a fluorescence microscope. Figure 22 The results show that compound E5 significantly promotes apoptosis of A549 cells.

[0153] 5.4 Experiment on the inhibition of A549 human lung cancer cell migration by endoperoxides: A549 cells were seeded in 6-well plates and cultured into a monolayer of cells. A 10 μL pipette tip was used to scratch the monolayer of cells and washed three times with PBS. Three parallel experiments were conducted, namely the control group, the endoperoxide E-administered group, and the raw material control P-administered group, with the dosage of 80 μM respectively. After incubation for a period of time, the culture medium was aspirated and the photos were taken. The results showed that ( Figure 23 As shown in the Figure 3, cell migration in the P5-administered group was inhibited, while the degree of inhibition of cell migration in the E5-administered group was more significant, indicating that the endoperoxides developed by the present invention have the ability to inhibit the metastasis and proliferation of lung cancer cells.

[0154] 5.5 Establishment of the A549 nude mouse model and in vivo anticancer effects of endoperoxides

[0155] Balb / c nude mice were selected and A549 (6×10 5 ) cell suspension was inoculated subcutaneously on the right dorsal side of nude mice near the axilla to create a model. The tumor-bearing mice were randomly divided into three groups (model control group, N-substituted phenyl-2-pyridone treatment group, and N-substituted phenyl-2-pyridone endoperoxide treatment group), with five mice in each group. When the tumor grew to 100 cm 3 Treatment was started at the beginning of the experiment, and the drug was prepared into a solution with 0.5% sodium carboxymethylcellulose (CMC-Na). The model control group was gavaged with the same volume of sodium carboxymethylcellulose solution. The drug was administered once every two days and the tumor volume and mouse weight were monitored. After the treatment, the mice were killed and the tumors were removed and weighed. The results showed that the tumor volume of mice in the N-substituted phenyl-2-pyridone P5 treatment group showed a decreasing trend (the tumor volume decreased by about 10% compared with the model group), while the tumor volume of mice in the N-substituted phenyl-2-pyridone endoperoxide E5 treatment group was significantly reduced (the tumor volume decreased by about 80% compared with the model group). In addition, the weight of mice and HE staining of other organs in the two treatment groups did not change significantly compared with the model control group. The above results verify that the N-substituted phenyl-2-pyridone endoperoxide developed by the present invention has excellent in vivo treatment ability for lung cancer and excellent safety.

[0156] Table 2 Anti-lung cancer experimental results of a series of compounds

[0157]

[0158]

[0159]

[0160] Note: A represents the IC of the compound 50 <100 μM; B represents the IC of the compound 50 100-150 μM; C represents the IC of the compound50 150-200 μM; D represents the IC of the compound 50 >200μM; Whether it has in vivo anti-lung cancer ability is evaluated by inhibiting tumor growth in tumor-bearing mice, and "yes" indicates that it can inhibit the growth of nude mouse A549 tumor.

Claims

1. A compound of formula I or a pharmaceutically acceptable salt thereof: in, R 1 、R 2 、R 3 Each independently selected from hydrogen, deuterated methyl, and C1-C10 alkyl; R 4 -R 8 Each independently selected from hydrogen, C1-C5 deuterated alkyl, C1-C5 fluoroalkyl, halogen, carboxyl, sulfonic acid, aminosulfonic acid, hydroxyl, -COOR, -CONR, nitro, cyano, C1-C10 alkyl, C1-C10 alkoxy; R is independently a C1-C5 alkyl group.

2. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that R 1 、R 2 、R 3 Each independently selected from hydrogen, deuterated methyl, and C1-C5 alkyl; R 4 -R 8 Each independently selected from hydrogen, C1-C5 deuterated alkyl, C1-C5 fluoroalkyl, halogen, carboxyl, sulfonic acid, aminosulfonic acid, hydroxyl, -COOR, -CONR, nitro, cyano, C1-C10 alkyl, C1-C10 alkoxy; R is independently a C1-C5 alkyl group.

3. The compound according to claim 2 or a pharmaceutically acceptable salt thereof, characterized in that R 1 、R 2 、R 3 Each is independently selected from hydrogen, deuterated methyl, and methyl.

4. The compound according to claim 2 or a pharmaceutically acceptable salt thereof, characterized in that R 1 、R 2 、R 3 Each is independently selected from hydrogen and methyl.

5. The compound according to claim 3 or a pharmaceutically acceptable salt thereof, characterized in that R 4 -R 8 Each is independently selected from hydrogen, deuterated methyl, trifluoromethyl, halogen, carboxyl, sulfonic acid, aminosulfonic acid, hydroxyl, -COOR, -CONR, nitro, cyano, C1-C5 alkyl, C1-C5 alkoxy; R is independently a C1-C5 alkyl group.

6. The compound according to claim 5 or a pharmaceutically acceptable salt thereof, characterized in that The compound is selected from the following structures:

7. The compound according to claim 5 or a pharmaceutically acceptable salt thereof, characterized in that The compound is selected from the following structures:

8. The method for preparing the compound according to any one of claims 1 to 7, characterized in that: The following steps are involved: (1) Compound 1 and Compound 2 react in the presence of potassium carbonate and cuprous iodide to obtain Compound P; (2) Compound P reacts in the presence of a photosensitizer, an oxygen environment, and irradiation to produce compound E; In the step (2), the reaction solvent is selected from one or more of chloroform, dichloromethane, ether, methanol, ethanol, toluene, benzene, DMF, ethyl acetate, water, and deuterated reagents; The deuterated reagent is selected from one or more of deuterated chloroform, deuterated methanol, deuterated benzene, and deuterated water; The reaction temperature is -20°C to room temperature; The wavelength of the red light source is 425-750nm, and the oxygen environment is oxygen, oxygen-containing gas or direct open reaction; The photosensitizer is selected from methylene blue, fluoroboron pyrrole, hematoporphyrin, dihydrochlorin e6, pyropheophorbide-a, and pyropheophorbide-a hexyl ether.

9. The method according to claim 8, characterized in that The reaction was carried out in an ice-water bath, an ice bath, a room temperature water bath or at 0°C; The photosensitizer is methylene blue, and the wavelength of the red light source is 620-625 nm.

10. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the compound according to any one of claims 1 to 7 or a pharmaceutically acceptable salt thereof.

11. Use of the compound according to claim 1 or a pharmaceutically acceptable salt thereof in the preparation of a drug for improving or treating fibrosis.

12. The use according to claim 11, characterized in that The fibrosis is pulmonary fibrosis, renal interstitial fibrosis or liver fibrosis.

13. The use according to claim 11, characterized in that The pulmonary fibrosis is idiopathic pulmonary fibrosis.

14. The use according to claim 11, characterized in that: The compound or a pharmaceutically acceptable salt thereof is converted into N-substituted phenyl-2-pyridone in vivo and releases singlet oxygen and triplet oxygen:

15. The use according to claim 11, characterized in that: The compound or a pharmaceutically acceptable salt thereof inhibits transforming growth factor-β (TGF-β), monocyte chemoattractant protein-1 (MCP-1), and serum inflammatory factor interleukin-1β (IL-1β), while upregulating heme oxygenase-1 (HO-1); or The compound or a pharmaceutically acceptable salt thereof regulates one or more factors selected from the group consisting of TGF-β, MCP-1, IL-1β, HO-1, TNF-α, IL-6, IFN-γ, bFGF, PDGF, NLRP3, CAT, and SOD.

16. Use of the compound according to claim 1 or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating cancer.

17. The use according to claim 16, characterized in that The cancer is non-small cell lung cancer.

18. Use of the compound according to claim 1 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating idiopathic pulmonary fibrosis combined with lung cancer.

19. The use according to any one of claims 13 to 18, characterized in that: The medicine is one or more of tablets, capsules, granules, powders, oral preparations, injections, microcapsule preparations, suppositories, pills, aerosols, sprays, powder inhalers, syrups, alcoholic preparations, tinctures, lotions, and membrane preparations.

20. The use according to any one of claims 13 to 18, characterized in that: The drug comprises: at least one compound of formula I; and a carrier; The carrier is selected from one or more of metal nanocarriers, non-metal nanocarriers, liposomes, lactose, sucrose, gelatin, hard magnesium sulfate, and stearic acid.

21. The use according to any one of claims 13 to 18, characterized in that: The drug comprises: at least one compound of formula I; and pharmaceutical excipients; The pharmaceutical excipients are selected from one or more of diluents, binders, disintegrants, lubricants, glidants, flavoring agents, coating agents, gelatin capsule shells, latent solvents, propellants, surfactants, preservatives, and freeze-drying protective agents.

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