WO2024235145A1 - Preparation and use of n-substituted phenyl-2-pyridone and endoperoxide - Google Patents

Abstract

The preparation and use of N-substituted phenyl-2-pyridone and an endoperoxide, wherein the endoperoxide can be converted into N-substituted phenyl-2-pyridone and simultaneously release singlet oxygen and triplet oxygen. The N-substituted phenyl-2-pyridone and the endoperoxide have a great effect of resisting several types of fibrosis and can significantly inhibit inflammatory factors, and can not only inhibit the proliferation and migration of cancer cells at the cellular level, but can also inhibit the growth of various tumors at the living-body level.

Description

Preparation and application of a class of N-substituted phenyl-2-pyridones and endoperoxides Technical Field

The invention belongs to the technical field of organic compound synthesis and medicine, and specifically relates to the preparation and application of a class of N-substituted phenyl-2-pyridones and endoperoxides.

Background Art

Pulmonary fibrosis refers to a type of lung disease in which fibroblasts proliferate and extracellular matrix accumulate in large quantities, accompanied by inflammatory damage and tissue structure destruction. It is also regarded as a scar formed by abnormal structure after normal alveolar tissue is damaged and repaired abnormally. Up to now, the cause of most patients with pulmonary fibrosis is still unclear, which is also called idiopathic pulmonary fibrosis. The average survival time after diagnosis of idiopathic pulmonary fibrosis is short, and the mortality rate is higher than that of most cancers, which seriously affects people's respiratory function and daily life. Lung cancer is one of the most common malignant tumors, and the global incidence of lung cancer remains high. Among them, non-small cell lung cancer accounts for more than 80% of the total number, and most patients are diagnosed in the middle and late stages, which increases the difficulty of clinical treatment for patients. Lung cancer is also one of the important complications of patients with idiopathic pulmonary fibrosis. Among patients with idiopathic pulmonary fibrosis, the incidence of lung cancer is as high as 48%, which is significantly higher than that of the general population. Up to now, there is no specific drug for idiopathic pulmonary fibrosis, and the only approved drugs are pirfenidone and nintedanib. Among them, pirfenidone is a cytokine inhibitor that can inhibit transforming growth factor TGF-β, fibroblast growth factor bFGF, etc., thereby inhibiting the activity of fibroblasts, reducing cell proliferation and the synthesis of matrix collagen. Pirfenidone can delay lung function failure and alleviate the deterioration of the disease, but it still cannot reverse the course of pulmonary fibrosis. Chemotherapy is the main means of treating non-small cell lung cancer, but drug resistance often leads to treatment failure. The treatment of pulmonary fibrosis combined with lung cancer is more difficult, and there is currently no effective means. Since this type of disease often occurs in the elderly, patients often have problems such as poor lung function, low tolerance to surgery and chemotherapy. Conventional chemotherapy often leads to problems such as lung infection, neutropenia and respiratory insufficiency. In addition, the mortality rate of patients with pulmonary fibrosis combined with lung cancer is several times higher than that of patients with simple pulmonary fibrosis, and there is currently no specific drug. The development and treatment of other cancers will also be accompanied by the occurrence of fibrosis, such as liver cancer accompanied by liver fibrosis. Therefore, there is an urgent need for a class of drugs that can treat fibrosis and cancer.

Pirfenidone, 5-methyl-1-phenyl-2-pyridone, is an oral anti-pulmonary fibrosis drug. It has anti-inflammatory, antioxidant and anti-fibrotic effects. Since its launch, pirfenidone can delay lung failure and alleviate the deterioration of the disease. In addition, studies have shown that pirfenidone has certain anti-cancer effects and can slow down liver fibrosis, kidney fibrosis and other effects. However, pirfenidone has low efficacy, often requires higher doses, and is prone to adverse reactions in the gastrointestinal tract and skin, and has certain phototoxicity. By modifying and improving the structure of pirfenidone, the development of new anti-cancer and fibrosis-alleviating drugs has important practical application value.

Singlet oxygen ( ¹ O ₂ ) is an excited oxygen molecule that participates in many physiological processes as a signal and stimulus molecule. In addition, singlet oxygen is also a highly reactive substance that can destroy tumor cell membranes, proteins and other biological molecules under a certain dosage, causing tumor cell apoptosis, vascular damage or inducing immune response to eliminate tumor cells. Studies have shown that singlet oxygen can react with naphthalene, anthracene and pyridone through a [4+2] cycloaddition reaction to generate endoperoxides. Endoperoxides can undergo a reverse Diels-Alder reaction at a certain temperature to convert into starting materials and release singlet oxygen. When endoperoxides release singlet oxygen, they also release a portion of molecular oxygen (triplet oxygen). Singlet oxygen can also be converted into molecular oxygen after being quenched under physiological conditions. Molecular oxygen can alleviate tumor hypoxia, and oxygen therapy is a frequently used solution for patients with fibrosis. Singlet oxygen delivery systems designed based on endoperoxides can provide a new solution for the treatment of various diseases such as fibrosis, cancer, bacterial infection, Alzheimer's disease, coagulation disorders, and ischemic injury.

Summary of the invention

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

Wherein, R ¹ and R ³ are each independently selected from hydrogen, deuterated methyl, C1-C10 alkyl, carboxyl, C1-C5 fluoroalkyl, nitro, and C1-C10 ester.

R ⁴ -R ⁸ 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.

R is independently a C1-C5 alkyl group.

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.

The pharmaceutical composition further comprises a carrier and/or a pharmaceutical excipient.

The preparation method of the compound comprises the following steps:

(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 light irradiation to obtain compound E.

wherein R ¹ , R ³ , R ⁴ -R ⁸ have the same definitions as in formula I;

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 temperature of the reflux reaction is between 150-200°C. The amount of iodobenzene 2 and potassium carbonate is 1-3 times (molar amount) of 2-pyridone. The amount of cuprous iodide is 2%-20% (molar amount) of 2-pyridone.

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 by red light irradiation should be avoided. The irradiation light source can also use light sources of other wavelengths (425-750nm). The gas environment can be oxygen, a gas containing oxygen, or a direct open reaction into the reaction vessel. Methylene blue/red light can be replaced by other common photosensitizers and light sources of corresponding wavelengths, such as BODIPY/red light, etc.

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

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

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

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:

wherein R ¹ , R ³ , R ⁴ -R ⁸ have the same definitions as in formula I;

Use of a compound of formula I or a pharmaceutically acceptable salt thereof in the preparation of the following drugs:

Drugs that inhibit the expression of 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); or

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.

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

The fibrosis is pulmonary fibrosis, liver fibrosis, renal fibrosis, myocardial fibrosis, pulmonary fibrosis especially refers to idiopathic pulmonary fibrosis, renal fibrosis especially refers to renal interstitial fibrosis. In addition, it also has therapeutic effects on kidney damage diseases including acute kidney injury, chronic kidney disease and end-stage renal disease.

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

The cancers include lung cancer, non-small cell lung cancer, breast cancer, liver cancer, prostate cancer, and pancreatic cancer.

The medicine can alleviate non-alcoholic fatty hepatitis, non-alcoholic fatty liver disease, and diseases such as inflammation, cavitation and necrosis of the liver.

Use of the 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.

The drug 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, dews, and films, and one or more of the above dosage forms can be selected for treatment. In addition, it can also be prepared into liposomes or micelles for administration.

The drug is administered orally, intravenously, by inhalation through the respiratory tract, topically, or sublingually.

The drugs used in the application include:

At least one of the compounds of formula I; and/or

carrier; and/or

Pharmaceutical excipients;

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

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

The drug used in the application is prepared into a liposome or micelle form.

Another object of the present invention is to provide a class of N-substituted phenyl-2-pyridone compounds for use in treating cancer and alleviating fibrosis.

Use of the compound of formula II in the preparation of drugs for treating liver fibrosis, kidney fibrosis and myocardial fibrosis.

The compound of formula II is used in the preparation of drugs for treating lung cancer, breast cancer, liver cancer, prostate cancer and pancreatic cancer.

Here, R ⁹ , R ¹⁰ , R ¹¹ , R ¹² , and R ¹³ are each independently a hydrogen atom, a deuterated methyl group, a halogen group, a hydroxyl group, a cyano group, an amino group, a nitro group, a trifluoromethyl group, a carboxyl group, an amide group having 1 to 6 carbon atoms, an alkyl group having 1 to 6 carbon atoms, an alkoxy group having 1 to 6 carbon atoms, a thioether group having 1 to 6 carbon atoms, or the like.

In some specific compounds, R ⁹ , R ¹⁰ , R ¹¹ , R ¹² and R ¹³ are each independently a hydrogen atom, a methyl group, an ethyl group, a deuterated methyl group, a trifluoromethyl group, a butyl group, a halogen group, a hydroxyl group, a cyano group, an amino group, a carboxyl group, a methoxy group, an ethoxy group or a methylthio group.

In some specific compounds, R ⁹ , R ¹⁰ , R ¹¹ , R ¹² and R ¹³ are each independently a hydrogen atom, a methyl group, an ethyl group, a deuterated methyl group or a trifluoromethyl group.

Specifically, the compound is selected from the following structures:

A drug for treating liver fibrosis, kidney fibrosis, myocardial fibrosis, lung cancer, breast cancer, liver cancer, prostate cancer or pancreatic cancer, comprising at least one of the compounds of formula II or pharmaceutically acceptable salts thereof.

The drug dosage form is tablets, capsules, granules, powders, oral preparations, injections, microcapsule preparations, suppositories, and can also be prepared into liposomes or micelles for administration. The drug can be used alone or in combination with other drugs.

The drug is administered orally, intravenously, by inhalation through the respiratory tract, topically, or sublingually.

The cancers treated by the drug include one or more of lung cancer, breast cancer, liver cancer, prostate cancer, pancreatic cancer and the like.

The drug can alleviate one or more diseases such as pulmonary fibrosis, liver fibrosis, renal fibrosis, myocardial fibrosis, or diseases induced by fibrosis. In addition, it also has a therapeutic effect on kidney damage diseases including acute kidney injury, chronic kidney disease, and end-stage renal disease.

The medicine can alleviate non-alcoholic fatty hepatitis, non-alcoholic fatty liver disease, and diseases such as inflammation, cavitation and necrosis of the liver.

The present invention studies the effects of N-substituted phenyl-2-pyridone compounds with different substituents on the benzene ring, especially compounds without substituents on the pyridone structure but with substituents on the benzene ring, in treating cancer and alleviating fibrotic diseases. The anti-pulmonary fibrosis effect of such compounds has been reported in the patents previously applied for by the inventor. This patent discloses the relevant results of such compounds in treating cancer, especially lung cancer, breast cancer, liver cancer, prostate cancer, pancreatic cancer, and alleviating fibrotic diseases such as liver fibrosis, kidney fibrosis, and myocardial fibrosis.

The present invention provides a class of N-substituted phenyl-2-pyridone compounds for use in treating cancer and alleviating fibrosis, including a class of N-substituted phenyl-2-pyridone compounds and medically acceptable salts thereof, which can also be added with conventional adjuvants in the field of preparations to form conventional dosage forms such as tablets, capsules, granules, powders, oral liquids, injections, etc. It can also include pharmaceutically acceptable adjuvants, auxiliary ingredients or other carriers such as solvents, diluents, adhesives, disintegrants, lubricants, glidants, flavoring agents, coating agents, gelatin capsule shells, latent solvents, propellants, surfactants, preservatives, freeze-drying protective agents, etc. In addition, it can also be prepared into liposomes or micelles for administration.

Advantages and beneficial effects of the present invention:

The N-substituted phenyl-2-pyridone endoperoxide provided by the present invention can be prepared in batches by a simple chemical synthesis method, and has good stability and can be stored for a long time. This type of endoperoxide integrates multiple therapeutic factors of 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 chemotactic protein-1 (MCP-1), serum inflammatory factor interleukin-1β (IL-1β) and upregulate heme oxygenase-1 (HO-1), and the anti-pulmonary fibrosis effect is better than the listed drug pirfenidone. The endoperoxide of the present invention independently or collaboratively realizes the treatment of pulmonary fibrosis, liver fibrosis, renal fibrosis, myocardial fiber, lung cancer, lung cancer combined with lung cancer, breast cancer, liver cancer, prostate cancer, and pancreatic cancer through multiple therapeutic factors, and is suitable for multiple dosage forms and carriers, and is expected to become a first-line drug for the treatment of multiple fibrosis and cancer.

The new therapeutic application of a class of N-substituted phenyl-2-pyridone compounds provided by the present invention proves that compounds without substituents on the pyridone and with methyl substituents on the benzene ring can effectively treat cancer, especially lung cancer, breast cancer, liver cancer, prostate cancer, pancreatic cancer, and alleviate fibrosis, especially liver fibrosis, kidney fibrosis, and myocardial fibrosis. In addition, the safety assessment proves that the developed drugs also have good safety, and have no obvious damage to other organs while exerting therapeutic effects.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is the general structural formula of the compound of the present invention.

FIG2 is a graph showing the yield of P5, singlet oxygen and triplet oxygen released by a representative compound E5.

FIG3 is a graph showing singlet oxygen release studies of representative compound E5;

Among them, (3a) is the singlet oxygen release graph using SOSG as a probe; (3b) is the singlet oxygen release graph using DPBF as a probe.

FIG4 is a graph showing the results of the hydroxyproline content in serum of mice in an anti-pulmonary fibrosis experiment, lung coefficient (lung weight/body weight), and TGF-β1 mRNA expression;

Among them, (4a) is a graph showing the hydroxyproline content; (4b) is a graph showing the lung coefficient (lung weight/body weight) results; and (4c) is a graph showing the TGF-β1 mRNA expression.

FIG. 5 is an image of α-SMA immunofluorescence, HE staining and MASSON staining of lung tissue sections of mice in the anti-pulmonary fibrosis experiment.

Figure 6 is a graph showing the levels of AST, ALT, AKP, BUN and SCR in the serum of mice in the anti-pulmonary fibrosis experiment.

FIG7 is a graph showing the MTT test, HIF-1α expression test, singlet oxygen release test, and apoptosis test of A549 lung cancer cells by endoperoxide E5;

Among them, (7a) is the MTT test graph of E5 on A549 cancer cells; (7b) is the HIF-1α expression graph of E5 on A549 cancer cells; (7c) is the singlet oxygen release test graph of E5 on A549 cancer cells; (7d) is the apoptosis double staining experiment graph of E5 on A549 cancer cells.

FIG8 is a graph showing the tumor volume, tumor weight, and HE and immunofluorescence sections of tumor tissues in mice during the anticancer experimental treatment process;

Among them, (8a) is a graph showing the changes in mouse tumor volume; (8b) is a graph showing the changes in mouse tumor weight; (8c) is a HE staining graph of mouse tumor tissue; (8d) is a graph showing immunofluorescence sections of mouse tumors

FIG9 is a diagram of serum biochemical analysis of mice in the anticancer experiment and HE staining of mouse organs;

Among them, (9a) is a biochemical analysis diagram of mouse serum; (9b) is a HE staining diagram of mouse lung tissue, liver tissue, spleen tissue, kidney tissue and heart tissue sections.

Figure 10 is a curve of mouse tumor volume changes in the anticancer experiment of compound P10, a picture of mouse tumor after treatment, and a picture of tumor volume

FIG11 is HE staining images of other organs of mice after treatment (11a), the ALT, AST, BUN and SCr levels in mouse serum (11b) and the weight change curve of mice during treatment (11c).

DETAILED DESCRIPTION

The pharmaceutically acceptable salts of the 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.

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

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

The term "C1-C5 fluoroalkyl" refers to a straight 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, perfluorohexyl.

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 and a linear or branched, fatty acid. Examples of C1-C10 ester groups are wait.

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.

The term "C1-C10 thioether group" refers to a straight chain 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.

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

-COOR and -CONR refer to

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

The following examples are provided to help understand the present invention, but they cannot limit the content of the present invention.

A class of drugs capable of treating pulmonary fibrosis, lung cancer, and pulmonary fibrosis combined with lung cancer and a method for synthesizing such compounds is provided. 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.

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

Wherein, R ¹ and R ³ are each independently selected from hydrogen, deuterated methyl, C1-C10 alkyl, carboxyl, trifluoromethyl, nitro, and C1-C10 ester.

R ⁴ -R ⁸ 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.

R is independently a C1-C5 alkyl group.

In some specific N-substituted phenyl-2-pyridone endoperoxides, R ¹ and R ³ are each independently selected from hydrogen, deuterated methyl, C1-C5 alkyl, and trifluoromethyl.

R ⁴ -R ⁸ 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.

R is independently a C1-C5 alkyl group.

In some specific N-substituted phenyl-2-pyridone endoperoxides, R ¹ and R ³ are each independently selected from hydrogen, deuterated methyl, trifluoromethyl, and methyl.

R ⁴ -R ⁸ 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.

R is independently a C1-C5 alkyl group.

In some specific N-substituted phenyl-2-pyridone endoperoxides, R ¹ and R ³ are each independently selected from hydrogen and methyl;

R ⁴ -R ⁸ 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.

R is independently a C1-C5 alkyl group.

In some specific N-substituted phenyl-2-pyridone endoperoxides, R ¹ and R ³ are each independently selected from hydrogen, deuterated methyl, trifluoromethyl, and methyl.

R ⁴ -R ⁸ 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.

R is independently a C1-C5 alkyl group.

In some specific N-substituted phenyl-2-pyridone endoperoxides, R ¹ and R ³ are each independently selected from hydrogen, deuterated methyl, and methyl.

R ⁴ -R ⁸ are each independently selected from hydrogen, deuterated methyl, methyl, ethyl, tert-butyl, trifluoromethyl, F, Cl, carboxyl, sulfonic acid, aminosulfonic acid, hydroxyl, -COOCH ₃ , -COOtBu, -CONHCH ₃ , methoxy, ethoxy, nitro, and cyano.

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

The preparation method of N-substituted phenyl-2-pyridone endoperoxide, the synthetic route is as follows:

(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 activated carbon was added to the filtrate for decolorization. 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.

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

Application of N-substituted phenyl-2-pyridone endoperoxides

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 of human body). The application of N-substituted phenyl-2-pyridone endoperoxide is mainly to treat pulmonary fibrosis, lung cancer, and pulmonary fibrosis combined with lung cancer.

Bleomycin was used to induce pulmonary fibrosis in mice, and the endoperoxide developed by the present invention was used for treatment. After the treatment, HE staining, Masson staining, and immunofluorescence staining were performed on the lung tissues of the mice, and the hydroxyproline content in the serum of the mice was measured. The results showed that the series of compounds had excellent anti-fibrosis effects, and some compounds (such as E5) had better anti-fibrosis effects than the marketed drug pirfenidone.

The marketed drug pirfenidone inhibits factors such as TGF-β, reduces cell proliferation and matrix collagen synthesis, and exerts anti-inflammatory and antioxidant effects by inhibiting the secretion of IL-1β and other inflammatory mediators, reducing lipid peroxidation, etc. 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 the representative endoperoxides can significantly upregulate heme oxygenase-1, close to normal physiological levels, and the upregulation effect is far superior to pirfenidone.

The anti-lung cancer performance of endoperoxides was evaluated at the cellular level and the animal level, and the experimental results showed that the endoperoxides of the present invention can efficiently release singlet oxygen and triplet oxygen in cancer cells. The release of singlet oxygen in cells was successfully verified using a singlet oxygen capture probe, and the cell apoptosis experiment proved that the cell apoptosis caused by singlet oxygen, the MTT experiment proved that the series of compounds had good anti-cancer ability, and the cell migration experiment proved that endoperoxides could successfully inhibit the migration of lung cancer cells. The anti-lung cancer experimental results at the animal level showed that N-substituted phenyl-2-pyridone endoperoxides can effectively inhibit the growth of tumors in living animals.

The safety evaluation results showed that after endoperoxide treatment, multiple indicators (AST, ALT, AKP, BUN, SCR) in the serum of mice were normal, and other organs of mice such as heart, liver, spleen, and kidney had no obvious damage, and the lung coefficient and total body changes of mice were normal. The above results show that the drug developed by the present invention has excellent safety and bioavailability, and has clinical application potential.

Example 1: Preparation of representative compounds P5, E5

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 bottle and refluxed at 150 ° C for 10 hours under argon protection. After the reaction was completed, it was cooled to room temperature, filtered, and the filter cake was washed three times with DMF. The filtrate was combined and activated carbon was added for decolorization. The decolorized solution was evaporated to dryness under reduced pressure, 100 mL of 10% acetic acid was added, and stirred at 70 ° C for 30 minutes. After standing and stratification, the upper aqueous phase was taken, and the lower oily matter was extracted twice with 10% acetic acid, and the aqueous phases were combined. Sodium hydroxide solution was added to the aqueous phase to adjust the pH to 13, and it was placed in a refrigerator overnight for crystallization. 100 mL of ethyl acetate was added to the crude product obtained by vacuum drying, heated to reflux, 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 (400 MHz, CDCl ₃ ) δ7.49–7.46 (m, 2H), 7.42–7.37 (m, 3H), 7.26 (d, J=6.9 Hz, 1H), 7.23 (d, J=6.9 Hz, 1H), 6.16 (t, J=6.8 Hz, 1H), 2.19 (s, 3H); ¹³ C NMR (100 MHz, CDCl ₃ ) δ162.8, 141.4, 136.9, 135.4, 130.8, 129.2, 128.2, 126.6, 105.6, 17.4.

Compound P5 (15 g), methylene blue (5 mg) and chloroform (100 mL) were placed in a reaction bottle, 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 was completed, the methylene blue was removed by suction filtration using silica gel (or activated carbon), and the filtrate was evaporated to dryness under reduced pressure to obtain compound E5. Yield: 100%. ^C _​ ^​ NMR (100MHz, CDCl ₃ ) δ168.3,138.2,134.0,129.4,126.9,123.7,86.1,82.1,14.7.

Example 2: Half-life and yield analysis of P5, singlet oxygen and triplet oxygen released by compound E5 and singlet oxygen capture experiment

Half-life of releasing P5, singlet oxygen and triplet oxygen: Endoperoxide E5 releases P5, singlet oxygen and triplet oxygen by reverse cycloaddition reaction. The whole process follows the first-order kinetic rate equation. The half-life can be calculated by the formula t _1/2 =0.693/k. The specific experimental process is as follows: About 2 mg of endoperoxide E5 is weighed and dissolved in deuterated water and placed 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 reverse cycloaddition reaction half-life is calculated by the characteristic peaks of E5 and P5 between 6.25-6.75 ppm: t _1/2 =11.9 hours (37°C, in deuterated water).

Yields of released P5, singlet oxygen and triplet oxygen: Tetramethylethylene was used as a singlet oxygen capture agent, and the yields of released P5, singlet oxygen and triplet oxygen from the reverse cycloaddition reaction of E5 were calculated using hydrogen nuclear magnetic resonance spectroscopy according to the above method. The results showed that after a complete reverse reaction of 1 mol of E5, 1 mol of P5, 0.5 mol of singlet oxygen and 0.5 mol of triplet oxygen were released (as shown in FIG2 ).

In addition, the above results prove that the series of compounds, such as endoperoxide E5, have a certain water solubility and are completely converted into P5 in the aqueous phase, while releasing singlet oxygen and triplet oxygen. No other impurities are produced during the endoperoxide reverse cycloaddition process, proving that the series of compounds have extremely high drug development potential and clinical application value.

Singlet oxygen capture experiment: SOSG and DPBF were used as singlet oxygen capture agents to monitor the release of singlet oxygen from the compounds. The experimental process is as follows: Representative compound E5 (375 μM) and SOSG (singlet oxygen capture agent, 12.5 μM) were dissolved in PBS. At 37°C and in the dark, a fluorimeter was used to detect fluorescence emission at different time periods. The increase in the 530nm peak intensity indicates that endoperoxides can efficiently release singlet oxygen (as shown in Figure 3a). Representative compound E5 (750 μM) and DPBF (singlet oxygen capture agent, 37.5 μM) were dissolved in DMF. At 37°C and in the dark, a UV-visible spectrophotometer was used to monitor the absorption at 417nm at different time periods to analyze the release of singlet oxygen. The change in the 417nm peak indicates that endoperoxides can efficiently release singlet oxygen (as shown in Figure 3b).

Example 3: Preparation of Compounds E1-E4, E6-E18, E37-E72, E73a-E124a

The synthesis methods of compounds E1-E4, E6-18, E37-E72, E73a-E124a refer to the synthesis of compound E5, wherein the structures of 2-pyridone and iodobenzene used are as follows:

Compounds E1–E4, E6–E18, E37-E72, and E73a-E124a were all structurally identified.

Example 4: Anti-pulmonary fibrosis experiment

4.1 Experimental process of endoperoxides against pulmonary fibrosis

Mice were randomly divided into a blank control group, a model group, a pirfenidone treatment group, an N-substituted phenyl-2-pyridone treatment group, and an N-substituted phenyl-2-pyridone endoperoxide treatment group. After the mice were adapted for 3 days (weight about 20g), bleomycin solution (2mg/ml) was used for surgical modeling on the 4th day. Oral administration began 3 days after modeling. The drug was prepared into a solution with 0.5% sodium carboxymethyl cellulose (CMC-Na). Each mouse was administered at 400mg/KG. The blank control group and the model group were orally administered with the same dose of CMC-Na solution. The weight and survival of the mice were detected during the oral administration. The mice were killed after 16 days of medication. The mice were killed by dislocating the neck after removing the eyeballs for blood collection, and the lungs and other organs were collected. Part of the lung tissue and other tissues were fixed in 4% neutral formalin, and part of them were placed in a -80℃ refrigerator after liquid nitrogen treatment for tissue morphology observation and subsequent detection of related indicators.

4.2 Analysis of the results of endoperoxide anti-pulmonary fibrosis experiment

4.2.1 Analysis of Hydroxyproline Content in Mouse Serum

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 evaluate the degree of activation of fibroblasts. The results of the test results of serum HYP content in each group of mice are shown in Figure 4a. 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 mice was more serious. The HYP content of mice in the pirfenidone treatment group was 286±4ng/ml, showing a good effect of 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 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.

The anti-pulmonary fibrosis efficacy of compounds E1–E18, E37-E41, E44-E49, E51, E55-E59, E62-E63, E66, E70, and E73a-E124a are all higher than that of pirfenidone. The test data of some compounds are given in the form of the following table, where the test process and method refer to Examples 4.1 and 4.2.

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


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

4.2.2 Lung coefficient

Lung coefficient (lung weight/body weight) is a key animal experimental measurement indicator. As shown in Figure 4b, the lung coefficient of the model group was improved to a certain extent compared with the blank group. Compared with the blank group, the three treatment groups (pirfenidone treatment group, P5 treatment group, and E5 treatment group) had no obvious changes in lung coefficient, further indicating the excellent safety and therapeutic effect of E5.

4.2.3 Analysis of lung coefficient mRNA expression

After the treatment, the mRNA content was analyzed by fluorescence quantitative PCR, and the 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 marketed drug pirfenidone (Figure 4c). In addition, endoperoxides can significantly upregulate HO-1, close to normal physiological levels, and by regulating oxidative stress reactions 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 inflammation-related factors.

4.2.4 Analysis of pathological changes in lung tissue

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. As shown in Figure 5, the lung tissue structure of the blank group was clear, the alveolar septum was not thickened, there was no water 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, the atrophy and collapse were severe, the collagen lung fibers increased significantly, a large number of inflammatory cells infiltrated, and a large number of myofibroblasts appeared. In the pirfenidone treatment group, the degree of alveolar structure damage was reduced, the collagen lung fibers were slightly reduced, the fibroblasts were reduced, and the degree of pulmonary fibrosis was alleviated. In the P5 treatment group, the degree of alveolar structure damage was reduced, the collagen lung fibers were slightly reduced, the fibroblasts were reduced, and the degree of pulmonary fibrosis was also alleviated. The lung tissue structure of the E5 treatment group was clearer, the collagen lung fibers were greatly reduced, the fibroblasts were greatly reduced, and the overall state of the lungs was close to the blank group. The above data show that the pirfenidone analogue P5 developed by the present invention has a certain effect of 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, and its anti-pulmonary fibrosis effect is more significant than the listed drug pirfenidone, and the degree of fibrosis of the lung tissue after treatment has been greatly alleviated.

4.2.5 Safety Assessment

The serum indexes were analyzed by the standard comparison method to evaluate the drug safety. Compared with the model group, 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 all decreased significantly, and were the same as the blank group, proving that the drug E5 has good safety (Figure 6).

The changes in the weight of mice during the experiment can evaluate the effects of drug use on mice. Compared with the blank group, the three treatment groups (pirfenidone treatment group, P5 treatment group, and E5 treatment group) did not have significant weight loss, further demonstrating the excellent safety of the new drug E5.

Example 5: Anti-lung cancer experiment

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 completely adhere to the wall. Gradient concentrations of endoperoxide E were then added to the cells and incubated for 24 hours. Finally, the cytotoxicity of the compounds was tested using the MTT method. As can be seen from the results shown in Figure 7a, the representative endoperoxide E5 exhibited good anticancer properties with an IC ₅₀ of 119 μM. The series of compounds all showed good anticancer properties, and the anticancer ability tests of some compounds are shown in Table 2.

5.2 Endoperoxides inhibit HIF-1α expression and relieve tumor hypoxia: Under hypoxic conditions, A549 cells were cultured in 96-well plates overnight. Endoperoxides were then added to the cells and incubated for 24 hours, and HIF-1α expression was analyzed using Western blotting. The results showed that E5 could effectively inhibit HIF-1α (Figure 7b). Experiments using NaN ₃ as a singlet oxygen quencher proved that triplet oxygen released by E5 was the main component that inhibited HIF-1α.

5.3 Singlet oxygen release of endoperoxides in A549 human lung cancer cells: A549 cells were cultured in a confocal culture dish overnight. After adding endoperoxide E and raw material control P (40μM) and continuing to incubate for 5 hours, the cells were washed with PBS. After staining the cells with 10μM DCFH-DA solution for 45 minutes, the cells were then incubated with 10μg/mL Hoechst solution at room temperature for 20 minutes and photographed under a fluorescence microscope. As can be seen from the results shown in Figure 7c, there is significant singlet oxygen release in the cells treated with compound E5, while no detailed singlet oxygen release was observed in the control compound.

5.4 Experiment on endoperoxide-promoted apoptosis of A549 human lung cancer cells: The cells were cultured overnight in a confocal culture dish. Then, endoperoxide E and raw material control P (160 μM, respectively) were added to the cells and incubated for 9 hours, then the drugs were removed and the cells were washed with PBS. Annexin V-FITC/PI staining solution was then added, and the cells were incubated at room temperature in the dark for 20 minutes, and then photographed under a fluorescence microscope. As shown in the results in Figure 7d, compound E5 significantly promoted apoptosis of A549 cells.

5.5 Establishment of A549 nude mouse model and anticancer effect of endoperoxides in vivo

Balb/c nude mice were selected, and A549 (6×10 ⁵ ) cell suspension was inoculated subcutaneously near the axilla of the right dorsal side of the nude mice to make models. 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 began. The drug was prepared into a solution with 0.5% sodium carboxymethyl cellulose (CMC-Na), and the model control group was gavaged with the same volume of sodium carboxymethyl cellulose solution. The drug was administered once every two days and the tumor volume and mouse body weight were monitored. After the treatment, the mice were killed and the tumors were removed and weighed. As shown in Figure 8, the tumor volume of mice in the N-substituted phenyl-2-pyridone P5 treatment group showed a decreasing trend (compared with the model group, the tumor volume decreased by about 10%), while the tumor volume of mice in the N-substituted phenyl-2-pyridone endoperoxide E5 treatment group was significantly reduced (compared with the model group, the tumor volume decreased by about 80%) (Figures 8a-8b). The results of tumor section staining all proved that E5 has good anti-cancer and tumor hypoxia relief capabilities (Figures 8c-8d). In addition, the weight of mice in the two treatment groups, serum biochemical studies, and HE staining of other organs did not change significantly compared with the model control group (Figures 9a-9b). The above results verify that the N-substituted phenyl-2-pyridone endoperoxide developed by the present invention has excellent in vivo treatment of lung cancer and excellent safety. The anti-lung cancer experimental results of other compounds are shown in Table 2.

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


Note: A represents the compound with IC ₅₀ <100μM; B represents the compound with IC ₅₀ of 100-150μM; C represents the compound with IC 50 of
IC ₅₀ is 150-200 μM; D represents IC ₅₀ >200 μM of the compound; 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.

Example 6

The synthesis of the series of compounds refers to patent document CN114716365A:

Example 7

Anti-cancer experiments:

A mouse lung cancer model was established by subcutaneously inoculating A549 cell suspension (1×10 ⁷ cell/mL) in the right forelimb axilla of 6-8-week-old Balb/c male nude mice. The A549 tumor-bearing mice were randomly divided into two groups, a 0.5% sodium carboxymethylcellulose group (control group) and a drug treatment group, with a dose of 300 mg/kg. After 14 days of administration, the tumor-bearing mice were killed by bleeding from their eyes, and blood, tumors and other organs were collected to analyze the anti-cancer ability of the compound.

Analysis of the anti-lung cancer results of the representative compound P10

The results showed that the volume of both groups of mice showed an increasing trend, among which the growth of the P10-treated group was slow. As shown in Figures 10a-10c, the growth of tumors in the treated mice was significantly inhibited, proving that the drug P10 can alleviate tumor growth and has anti-cancer efficacy.

Safety evaluation of representative compound P10

HE staining was performed on the heart, liver, lung, spleen and kidney tissues of the blank group and P10 treatment group mice to evaluate the effects of the drug on other organs and prove the safety of the drug (Figure 11a). Compared with the blank group, continuous use of P10 did not cause obvious damage to other organs of the mice, proving that P10 has good safety. There was no significant difference in the levels of AST, ALT, BUN and SCR in the plasma of the treated mice and the blank group, proving that the drug P10 has good safety (Figure 11b). Compared with the blank group, there was no obvious weight loss in the P10 treatment group, further indicating the excellent safety of the new drug P10 (Figure 11c).

Example 8

The anti-lung cancer test data of other compounds are given in the form of the following table, wherein the test process and method refer to Example 7, and corresponding cancer models are constructed using breast cancer cells MCF7, liver cancer cells HepG2, prostate cancer cells LNCap and pancreatic cancer cells PANC-1 to evaluate the anti-cancer universality of the series of compounds.

Table 3 Anticancer data of series of compounds


In anti-cancer experiments, "yes" means that the drug can inhibit the growth of tumors, and "no" means that it cannot inhibit the growth of tumors.

Example 9

Liver fibrosis treatment trial

Carbon tetrachloride (CCl ₄ ) was used to induce liver fibrosis in mice, and the therapeutic effect of compound P10 on liver fibrosis was evaluated. The specific method was as follows: male mice were randomly divided into 3 groups, 10 mice in each group, namely: blank group, CCl ₄ model group, and P10 treatment group. The CCl ₄ model group and the P10 treatment group used CCl ₄ (3mL/kg, twice a week, for 4 consecutive weeks) for 4 weeks to establish a liver fibrosis model. During the treatment period, the P10 treatment group was administered with a 5% sodium carboxymethyl cellulose (CMC-Na) solution of P10 (400mg/kg), and the blank group and the CCl ₄ model group were gavaged with the same dose of CMC-Na solution. The weight and survival of mice were tested during oral administration. The mice were killed 14 days after medication. The eyeballs of the mice were removed to collect blood and the serum was separated. The alanine aminotransferase ALT and aspartate aminotransferase AST indicators were tested to determine the degree of liver damage. The LN (laminin) and total bilirubin (TBIL) indicators were tested to evaluate the degree of liver fibrosis. The mouse livers were removed and the hydroxyproline HYP content was tested.

Analysis of the results of treatment of liver fibrosis

As shown in Table 4, the ALT and AST levels in the serum of mice in the P10 treatment group were significantly lower than those in the CCl ₄ model group. P10 can significantly inhibit the increase of ALT and AST in mice induced by CCl ₄ and reduce liver function damage. The LN (laminin) and total bilirubin (TBIL) of mice in the treatment group were significantly reduced. Compared with the blank control group mice, the hydroxyproline content in the liver of the CCl ₄ model group mice was significantly increased. After the administration of P10, the hydroxyproline content was significantly reduced, indicating that P10 can reduce the content of hydroxyproline in liver tissue, inhibit the formation of collagen fibers in the liver of mice, and thus inhibit liver fibrosis.

Table 4 Data of representative compound P10 on alleviating liver fibrosis

Example 10

The test data of other compounds for treating liver fibrosis are given in the form of the following table, wherein the test process and method refer to Example 8.

Table 5 Data of other compounds for the treatment of liver fibrosis


In the liver fibrosis experiment, "yes" means that the drug can inhibit liver fibrosis, and "no" means that it cannot inhibit liver fibrosis.

Embodiment 11

Experimental study on the treatment of renal fibrosis and analysis of the results

Rats were randomly divided into 3 groups, 10 in each group, namely: blank group, model group, and P10 treatment group. The model group and P10 treatment group used unilateral ureteral ligation method to establish the model: the rats were anesthetized, the abdomen was disinfected, the hair was shaved, the skin was exposed, and the left abdominal renal area incision was made in the lateral position to enter the abdominal cavity, the lower edge of the kidney was exposed, the ureter was found, and the ureter was ligated at the proximal and distal ends of the kidney and the wound was sutured. After three weeks, a renal fibrosis model was established. During the treatment period, the P10 treatment group was administered with 5% sodium carboxymethylcellulose (CMC-Na) solution of P10 (400 mg/kg), and the blank group and model group were gavaged with the same dose of CMC-Na solution. The weight and survival of the mice were detected during gavage. The mice were killed 14 days after medication, the eyeballs of the mice were removed to collect blood and separate the serum, and the content of urea nitrogen (BUN) and creatinine (Scr) was detected to analyze the degree of renal fibrosis. As shown in Table 4, the levels of BUN and Scr in the serum of mice in the P10 treatment group were significantly lower than those in the model group, which proved that P10 could improve the damage of glomeruli and renal tubules and reduce renal fibrosis and infiltration of inflammatory cells.

Table 6 Data of representative compound P10 on alleviating renal fibrosis

Example 12

The test data of other compounds for alleviating renal fibrosis are given in the form of the following table, wherein the test process and method refer to Example 11.

Table 7 Data of other compounds on alleviating renal fibrosis

In the renal fibrosis experiment, "yes" means that the drug can reduce renal fibrosis and the infiltration of inflammatory cells, and "no" means that it cannot reduce renal fibrosis and the infiltration of inflammatory cells.

Example 13

Experimental study on the treatment of myocardial fibrosis and analysis of the results

TGF-β1 was used to induce mouse cardiac fibroblasts to construct an in vitro myocardial fibrosis model. The experiment was divided into a control group, a model group, and a P10 treatment group. The cells in the model group were incubated with TGF-β1 (10 ng/ml), and the cells in the P10 treatment group were incubated with TGF-β1 (10 ng/ml) and compound P10 at the same time. After the experiment, RNA was extracted for reverse transcription and amplification, and the expression levels of α-SMA, collagen I, and collagen III mRNA were determined.

As shown in Table 8, the levels of α-SMA, collagen I and collagen III in cardiac fibroblasts treated with TGF-β1 were significantly increased, proving the establishment of a cardiac fibrosis model. The above factors were significantly inhibited in the treatment group, close to normal levels, proving that compound P10 can inhibit the expression of myocardial fibrosis biomarkers and has an anti-myocardial fibrosis effect.

Table 8 Data of representative compound P10 on alleviating myocardial fibrosis

Embodiment 14

The test data of other compounds for treating myocardial fibrosis are given in the form of the following table, wherein the test process and method refer to Example 13.

Table 9 Data of other compounds for the treatment of myocardial fibrosis

In the myocardial fibrosis experiment, "yes" means that the drug can inhibit the expression of myocardial fibrosis biomarkers and has an anti-myocardial fibrosis effect, and "no" means that it has an anti-myocardial fibrosis effect.

Claims (32)

A compound of formula I or a pharmaceutically acceptable salt thereof: Wherein, R ¹ and R ³ are each independently selected from hydrogen, deuterated methyl, C1-C10 alkyl, carboxyl, C1-C5 fluoroalkyl, nitro, and C1-C10 ester; R ⁴ -R ⁸ 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; R is independently a C1-C5 alkyl group. The compound or pharmaceutically acceptable salt thereof according to claim 1, characterized in that R ¹ and R ³ are each independently selected from hydrogen, deuterated methyl, C1-C5 alkyl, trifluoromethyl, and carboxyl; R ⁴ -R ⁸ 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; R is independently a C1-C5 alkyl group. [Corrected 30.07.2024 in accordance with Article 91]
The compound or pharmaceutically acceptable salt thereof according to claim 2, characterized in that R ¹ and R ³ are each independently selected from hydrogen, deuterated methyl, trifluoromethyl, methyl, and carboxyl. [Corrected 30.07.2024 in accordance with Article 91]
The compound or pharmaceutically acceptable salt thereof according to claim 3, characterized in that R ⁴ -R ⁸ 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; R is independently a C1-C5 alkyl group. The compound or pharmaceutically acceptable salt thereof according to claim 2, characterized in that R ¹ and R ³ are each independently selected from hydrogen and methyl. The compound or pharmaceutically acceptable salt thereof according to claim 5, characterized in that R ⁴ -R ⁸ are each independently selected from hydrogen, deuterated methyl, methyl, ethyl, tert-butyl, trifluoromethyl, F, Cl, carboxyl, sulfonic acid, aminosulfonic acid, hydroxyl, -COOCH ₃ , -COOtBu, -CONHCH ₃ , methoxy, ethoxy, nitro, and cyano. The compound or pharmaceutically acceptable salt thereof according to claim 6, characterized in that the compound is selected from the following structures: The compound or pharmaceutically acceptable salt thereof according to claim 7, characterized in that the compound is selected from the following structures: The method for preparing the compound according to any one of claims 1 to 8, characterized in that it comprises the following steps: (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 conditions to obtain compound E; wherein R ¹ , R ³ , R ⁴ -R ⁸ have the same definitions as in formula I; The preparation method according to claim 9, characterized in that 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 a deuterated reagent; 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, fluoroborax, hematoporphyrin, dihydrochlorin e6, pyropheophorbide-a, pyropheophorbide a hexyl ether, phenothiazine derivatives, phenoxazine derivatives, porphyrin photosensitizers or phthalocyanine photosensitizers. The method according to claim 10, characterized in that the reaction is carried out in an ice water bath, an ice bath, a room temperature water bath or 0°C; The photosensitizer is methylene blue, and the wavelength of the red light source is 620-625nm. A pharmaceutical composition, characterized in that the pharmaceutical composition comprises the compound according to any one of claims 1 to 8 or a pharmaceutically acceptable salt thereof. Use of the compound according to claim 1 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: wherein R ¹ , R ³ , R ⁴ -R ⁸ have the same definitions as in formula I; Use of the compound according to claim 1 or a pharmaceutically acceptable salt thereof in the preparation of the following medicaments: Drugs that inhibit the expression of 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); or 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. 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. The use according to claim 15 is characterized in that the fibrosis is pulmonary fibrosis, renal interstitial fibrosis, liver fibrosis, renal fibrosis, and myocardial fibrosis. The use according to claim 16, characterized in that the pulmonary fibrosis is idiopathic pulmonary fibrosis. Use of the compound according to claim 1 or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating cancer. The use according to claim 18 is characterized in that the cancer is non-small cell lung cancer, breast cancer, liver cancer, prostate cancer, and pancreatic cancer. Use of the compound according to claim 1 or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating idiopathic pulmonary fibrosis combined with lung cancer. The use according to any one of claims 14 to 20 is characterized in that the drug is in the form of tablets, capsules, granules, powders, oral preparations, injections, microcapsule preparations, suppositories, pills, aerosols, sprays, powder inhalers, syrups, wine preparations, tinctures, dews, and films, and one or more of the above dosage forms are selected for treatment. The use according to any one of claims 14 to 20, characterized in that the drug comprises: At least one of the compounds of formula I; and/or carrier; and/or Pharmaceutical excipients; The carrier is selected from one or more of metal nanocarriers, non-metal nanocarriers, liposomes, lactose, sucrose, gelatin, hard magnesium sulfate, and stearic acid; The pharmaceutical excipients are selected from one or more of diluents, adhesives, disintegrants, lubricants, glidants, flavoring agents, coating agents, gelatin capsule shells, latent solvents, propellants, surfactants, preservatives, and freeze-drying protective agents. The use according to any one of claims 14 to 20 is characterized in that the drug is prepared in the form of liposomes or micelles. Use of the compound of formula II in the preparation of drugs for treating liver fibrosis, kidney fibrosis, myocardial fibrosis, lung cancer, breast cancer, liver cancer, prostate cancer, and pancreatic cancer: Here, R ⁹ , R ¹⁰ , R ¹¹ , R ¹² and R ¹³ are each independently a hydrogen atom, a methyl group, an ethyl group, a deuterated methyl group, a trifluoromethyl group, a butyl group, a halogen group, a hydroxyl group, a cyano group, an amino group, a carboxyl group, a methoxy group, an ethoxy group or a methylthio group. The use according to claim 24, characterized in that the compound of formula II is selected from the following structures: The use according to claim 24, characterized in that the compound of formula II is selected from the following structures: The use according to claim 24, characterized in that the compound of formula II is selected from the following structures: A drug for treating liver fibrosis, renal fibrosis, myocardial fibrosis, lung cancer, breast cancer, liver cancer, prostate cancer or pancreatic cancer, characterized in that it contains at least one compound of formula II or a pharmaceutically acceptable salt thereof. The use according to any one of claims 24 to 28 is characterized in that the dosage form of the drug is tablets, capsules, granules, powders, oral preparations, injections, microcapsule preparations or suppositories. The use according to any one of claims 24-28 is characterized in that the drug is administered orally, intravenously, by respiratory inhalation, topically or sublingually. [Corrected 30.07.2024 in accordance with Article 91]
The use according to any one of claims 24-28, characterized in that the drug is prepared in the form of a liposome or micelle; the liposome or micelle drug comprises at least one of the compounds of formula II; and/or carrier; and/or Pharmaceutical excipients; The carrier is selected from one or more of metal nanocarriers, non-metal nanocarriers, micelles, liposomes, lactose, sucrose, gelatin, hard magnesium sulfate, and stearic acid; The pharmaceutical excipients are selected from one or more of diluents, adhesives, disintegrants, lubricants, glidants, flavoring agents, coating agents, gelatin capsule shells, latent solvents, propellants, surfactants, preservatives, and freeze-drying protective agents. [Corrected 30.07.2024 in accordance with Article 91]
The use according to any one of claims 1, characterized in that the drug is prepared in the form of liposomes or micelles.