A lung fibrosis-resistant prodrug compound and a preparation method and application thereof

Abstract

The present application relates to the technical field of medicine, and particularly relates to an anti-pulmonary fibrosis prodrug compound and a preparation method and application thereof. The prodrug compound is a novel prodrug, and the structure comprises: a) an active drug unit selected from pirfenidone and nintedanib; b) a lung targeting unit selected from diphenyl chloroiodonium salt and a mimic thereof 521; and c) a linker unit which is a responsive linkage sensitive to a biomarker specifically overexpressed in a pulmonary fibrosis microenvironment. The prodrug is stable in the systemic circulation and has no activity or low activity, and can be actively targeted to a pulmonary fibrosis lesion; under the specific stimulation of the PFM, the linker is broken, and the original drug molecule is accurately released, so that the concentration of the drug in the lesion site is significantly increased, the toxic side effects caused by systemic exposure are reduced, and the anti-pulmonary fibrosis effect is enhanced.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to an anti-pulmonary fibrosis prodrug compound, its preparation method, and its application. Background Technology

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

[0003] Pulmonary fibrosis (PF) is a serious respiratory disease with a high mortality rate. Infections by pathogenic microorganisms such as bacteria and viruses, drug-induced PF, and aging are all high-risk factors. Idiopathic pulmonary fibrosis (IPF), characterized by diffuse alveolitis and alveolar structural disorder ultimately leading to interstitial fibrosis, has a median survival of only 2–5 years and is also known as a neoplastic disease, with no specific treatment. The etiology of IPF is complex and not fully understood, involving multiple factors such as genetic susceptibility, abnormal repair, repeated epithelial cell damage, cellular senescence, and activation of abnormal signaling pathways (e.g., TGF-β, PDGF, and FGF signaling pathways).

[0004] Currently, pirfenidone and nintedanib are globally approved for the treatment of idiopathic pulmonary fibrosis. Their anti-inflammatory and anti-fibrotic effects are widely recognized, and multiple clinical trials have shown that both drugs can effectively improve lung function and prognosis in various interstitial lung diseases. Pirfenidone is a pleiotropic pyridone compound with anti-fibrotic, anti-inflammatory, and antioxidant effects. Nintedanib is an orally administered small-molecule tyrosine kinase inhibitor that inhibits the production of growth factors such as PDGF, FGF, and VEGF, thereby inhibiting the proliferation of fibroblasts and their transdifferentiation into myofibroblasts mediated by these factors.

[0005] However, existing pirfenidone and nintedanib drugs have obvious limitations, which seriously affect clinical efficacy and patient compliance. The main limitations are as follows: (1) Severe systemic toxic side effects. Pirfenidone: Common adverse reactions include photosensitive rash, gastrointestinal reactions (nausea, vomiting, anorexia, indigestion), abnormal liver function, and dizziness. Nintedanib: The most common and serious adverse reactions are gastrointestinal reactions, such as diarrhea (incidence >60%), nausea, vomiting, as well as increased liver enzymes and increased risk of cardiovascular events (such as myocardial infarction). These side effects also limit their long-term and adequate use. (2) Lack of targeting and low bioavailability. Pirfenidone and nintedanib are administered orally. After being absorbed by the gastrointestinal tract, very little of the drug reaches the lesion after oral administration. High doses are usually required, which further aggravates the toxic side effects. (3) Obstruction of the pulmonary fibrosis microenvironment. Pulmonary fibrosis lesions have a unique pathological microenvironment (PFM), such as a dense and rigid ECM barrier, abnormally high interstitial fluid pressure, and avascular areas. This severely hinders the penetration and diffusion of drugs from the blood into the depths of the lesions, making it difficult for even small amounts of drugs to reach the lungs and effectively distribute to the core fibrotic areas.

[0006] Existing technologies such as nanoparticle / liposome delivery systems, inhalation drug delivery, and traditional prodrug strategies all have significant shortcomings. Nanoparticle / liposome delivery systems suffer from problems such as carrier material toxicity, poor stability, and difficulties in large-scale production; inhalation drug delivery struggles to reach the lesion area and can cause airway irritation and inflammatory responses; and traditional prodrug strategies lack targeting specificity. Summary of the Invention

[0007] In view of this, the present invention provides an anti-pulmonary fibrosis prodrug compound, its preparation method and application.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides an anti-pulmonary fibrosis prodrug compound comprising an active unit D, a lung-targeting unit T, and a linker unit L, wherein the anti-pulmonary fibrosis prodrug compound has a structure shown in general formula (I): TLD (I).

[0009] Furthermore, D is selected from pirfenidone and nintedanib. D is the active unit, which is the effector molecule that exerts the final anti-fibrotic effect.

[0010] Further, T is a lung-targeting ligand; the lung-targeting ligand is selected from diphenyl iodide chloride (DPI) and its analogue 521; the analogue 521 is a dinitro-substituted DPI. Preferably, the structural formulas of DPI and analogue 521 are respectively... and .

[0011] The T unit is a lung-targeting ligand with high affinity for integrins (especially αvβ3, αvβ5, and αvβ6) and intracellular reactive oxygen species (ROS) overexpressed in pulmonary fibrosis lesions. These integrins and ROS are specifically highly expressed on the surface of activated fibroblasts, myofibroblasts, and pathological vascular endothelial cells, but their expression levels are very low in normal tissues. The T unit endows the entire prodrug molecule with the ability to actively seek targets, enabling it to efficiently accumulate in fibrotic lesion cells through receptor-ligand-mediated endocytosis. Simultaneously, this invention modifies the structure of DPI by adding two nitro groups to the diphenyl group of DPI, which reduces the toxicity of the prodrug molecule.

[0012] Further, L (Linker) is the linker subunit, L is PEG4, or L1-L2-L3, where L1 is PEG4, L2 is 5-hexynoic acid; L3 is compound 2-6; the structural formula of compound 2-6 is: .

[0013] The connecting subunit is a breakable linker that responds to specific stimuli in the pulmonary fibrosis microenvironment (PFM). The specific stimuli are selected from: Chemical substances: Reactive oxygen species (ROS), such as H2O2. Persistent oxidative stress exists in PFM, leading to abnormally elevated ROS levels.

[0014] Physical conditions: Low pH value. Due to ischemia, hypoxia and inflammatory cell metabolism, PFM is usually acidic (pH 6.5-7.0).

[0015] The L-unit is the core of this invention for achieving "intelligent" response. It remains stable in systemic circulation and normal tissues, preventing premature drug release; once the prodrug is guided to the pulmonary fibrosis site by the T-unit, the L-unit is rapidly cleaved by the aforementioned specific stimulus, thereby releasing the original drug D, achieving precise, explosive drug delivery to the lesion site.

[0016] Furthermore, the anti-pulmonary fibrosis prodrug compound is selected from:

[0017] .

[0018] In a second aspect, the present invention provides a method for preparing the anti-pulmonary fibrosis prodrug compound described in the first aspect, comprising the following steps: (1) Synthesize carboxylated lung-targeting unit T; (2) The carboxylated lung-targeting unit T was modified and a breakable linker L was introduced into its molecule to obtain the intermediate TL; (3) In the presence of catalyst SmI2 / isopropanethiol, the TL from step (2) is coupled with the active drug D through a reaction to obtain the target prodrug compound TLD; (4) Purify the crude product; or, (a) Synthesizing carboxylated lung-targeting unit T; (b) The carboxylated lung-targeting unit T was modified by introducing a breakable linker L1 onto its molecule to obtain intermediate T-L1; (c) Modify the active drug D by introducing breakable linkers L2 and L3 onto its molecule to obtain intermediate L2-L3-D; (d) In the presence of copper sulfate and sodium ascorbate, T-L1 from step (b) and L2-L3-D from step (c) are coupled by reaction to obtain the target prodrug compound T-L1-L2-L3-D; (e) Purify the crude product.

[0019] Furthermore, the carboxylated lung-targeting unit T is (DPI-COOH) or (521-COOH).

[0020] Furthermore, the synthesis method of DPI-COOH is as follows: Compound a was prepared by reacting 1,2-diiodobenzene with ethyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxoboronyl-2-yl)benzoate in the presence of a palladium catalyst and a base. Compound a is hydrolyzed under alkaline conditions to give compound b; Compound b was reacted in the presence of mCPBA and TfOH to prepare DPI-COOH.

[0021] Preferably, the molar ratio of 1,2-diiodobenzene to ethyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxoboronyl-2-yl)benzoate is 1-3:1; the reaction temperature is 65-75 °C; the reaction time is 10-15 h; and the solvent is a mixed solution of THF and water.

[0022] Preferably, the hydrolysis reaction temperature is 45-55 ℃.

[0023] Preferably, the molar ratio of compound b to mCPBA is 1:1.3-1.8; the molar ratio of compound b to TfOH is 1:25-35; the reaction temperature is 20-30 °C; and the reaction time is 1-3 h. The synthetic route is as follows: .

[0024] Furthermore, the synthesis method of 521-COOH is as follows: Compound 1-1 was prepared by reacting 1,2-diiodo-3-nitrobenzene with pinene 4-methoxycarbonyl-2-nitrobenzeneboronic acid in the presence of a palladium catalyst and a base. Compound 1-1 was hydrolyzed under alkaline conditions to prepare compound 1-2; Compounds 1-2 were reacted in the presence of mCPBA and TfOH to prepare 521-COOH. The synthetic route is as follows:

[0025] Preferably, the molar ratio of 1,2-diiodo-3-nitrobenzene to pinene 4-methoxycarbonyl-2-nitrobenzeneboronic acid pinene ester is 1-3:1; the reaction temperature is 65-75 °C; the reaction time is 10-15 h; and the solvent is a mixed solution of THF and H2O.

[0026] Preferably, the hydrolysis reaction temperature is 45-55 ℃.

[0027] Preferably, the molar ratio of compound 1-2 to mCPBA is 1:1.3-1.8; the molar ratio of compound 1-2 to TfOH is 1:25-35; the reaction temperature is 20-30 °C; and the reaction time is 1-3 h.

[0028] Furthermore, the active drug D is pirfenidone or nintedanib.

[0029] Furthermore, in step (2), L is PEG4.

[0030] Furthermore, in step (b), L1 is PEG4; in step (c), L2 is 5-hexyneic acid; and L3 is compounds 2-6.

[0031] Furthermore, the synthetic methods for compounds 2-6 are as follows: Compound 2-1 was prepared by oxidation of 2,6-dimethyl-1-nitrobenzene under alkaline conditions. Compound 2-1 was dissolved in THF and reacted with borane-THF solution to prepare compound 2-2; Compound 2-2 was reacted with hydrazine hydrate in methanol in the presence of a palladium on carbon catalyst to prepare compound 2-3; Compound 2-3 and imidazole were dissolved in a solvent and reacted with TBSCl to prepare compound 2-4; Compound 2-5 was prepared by reacting compound 2-4 and compound 2-4-1 under alkaline conditions; Compound 2-5 was reacted with p-toluenesulfonic acid to prepare compound 2-6. The synthetic route is as follows:

[0032] Preferably, the oxidant used in the oxidation reaction is potassium permanganate; the molar ratio of potassium permanganate to 2,6-dimethyl-1-nitrobenzene is 3.5-4.5:1.

[0033] Preferably, the molar ratio of compound 2-2 to hydrazine hydrate is 1:2.5-3.5; the reaction temperature is 65-75 °C; and the reaction time is 6-10 h.

[0034] Preferably, the molar ratio of compound 2-3 to imidazole is 1:3.5-4.5; and the molar ratio of compound 2-3 to TBSCl is 1:2.5-3.5.

[0035] Preferably, the molar ratio of compound 2-4 to compound 2-4-1 is 1:0.9-1.1; the reaction solvent for compounds 2-4 and 2-4-1 is toluene; the reaction temperature is 100-120 °C; and the reaction time is 10-15 h.

[0036] Preferably, the molar ratio of compounds 2-5 to p-toluenesulfonic acid is 1:1.5-2.5; the reaction temperature is 20-30 °C; and the reaction time is 1-3 h.

[0037] Furthermore, the synthesis method of the prodrug PR-DPI is as follows: Pirfenidone reacts with compounds 2-6, and after the reaction is complete, HCl solution is added to give compound 3-1; compound 3-1 reacts with 5-hexyneic acid to synthesize compound 3-2. Compound 3-3 was prepared by reacting DPI-COOH with 1-amino-11-azido-3,6,9-trioxaundecan. Compounds 3-3 and 3-2 were reacted in the presence of copper sulfate and sodium ascorbate to prepare the crude prodrug PR-DPI; the crude product was then purified. The synthetic route is as follows:

[0038] Preferably, the molar ratio of pirfenidone to compounds 2-6 is 1:10-20; NaBH4 is also added during the reaction of pirfenidone with compounds 2-6; the molar ratio of NaBH4 to pirfenidone is 1:1-3.

[0039] Preferably, the HCl solution has a mass fraction of 10-20%; the reaction time is 3-5 h after adding the HCl solution; and the reaction temperature is 50-70 °C.

[0040] Preferably, the molar ratio of compound 3-1 to 5-hexyneic acid in the reaction of compound 3-1 is 1:1-1.2; the condensing agents are DMAP and DCC; and the molar ratio of DMAP and DCC is 1:9-11.

[0041] Preferably, the molar ratio of amino-11-azido-3,6,9-trioxaundecan to DPI-COOH is 1:1.2-1.8; the condensing agent is HATU and DIPEA; the molar ratio of HATU and DIPEA is 1:2-4.

[0042] Preferably, the molar ratio of compound 3-3 to compound 3-2 is 1:0.9-1.1; the molar ratio of copper sulfate to compound 3-2 is 0.3-0.4:1; the molar ratio of sodium ascorbate to compound 3-2 is 1.3-1.7:1; the reaction temperature is 65-75 °C; and the reaction time is 15-20 h.

[0043] Furthermore, the synthesis method of the prodrug molecule PE-DPI is as follows: Compounds 3-4 were prepared by condensation reaction of 15-amino-4,7,10,13-tetraoxapentadecanal with DPI-COOH. The prodrug molecule PE-DPI was prepared by reacting pirfenidone with compound 3-4. The synthetic route is as follows:

[0044] Preferably, the molar ratio of 15-amino-4,7,10,13-tetraoxapentadecanal to DPI-COOH is 1:1.3-1.7; the condensation reagents are HATU and DIPEA; the molar ratio of 15-amino-4,7,10,13-tetraoxapentadecanal to HATU is 1:0.9-1.1; the molar ratio of 15-amino-4,7,10,13-tetraoxapentadecanal to DIPEA is 1:2.5-3.5; the condensation reaction temperature is 20-30℃, and the reaction time is 20-25 h.

[0045] Preferably, during the reaction of pirfenidone with compound 3-4, the molar ratio of pirfenidone to compound 3-4 is 1:1.3-1.5; and during the reaction of pirfenidone with compound 3-4, the catalyst SmI2 / isopropanethiol is also added.

[0046] Furthermore, the synthesis method of the prodrug molecule NR-DPI is as follows: Nydanib reacted with compounds 2-6, and after the reaction was complete, HCl solution was added to react again to prepare compound 4-1. Compound 4-1 was condensed with 5-hexyneic acid to prepare compound 4-2; Compounds 3-3 and 4-2 were reacted in the presence of copper sulfate and sodium ascorbate to prepare the prodrug molecule NR-DPI. The synthetic route is as follows:

[0047] Preferably, the molar ratio of nintedanib to compound 2-6 is 1:1-2; the reaction temperature of nintedanib and compound 2-6 is 15-25 °C, and the reaction time is 2-3 h; NaBH4 is added during the reaction of nintedanib and compound 2-6; the molar ratio of NaBH4 to compound 2-6 is 0.01:8-10.

[0048] Preferably, the HCl solution has a mass fraction of 10-20%; the reaction time is 3-5 h after adding the HCl solution; and the reaction temperature is 50-70 °C.

[0049] Preferably, the molar ratio of compound 4-1 to 5-hexyneic acid is 1:1-1.2; the condensation reagents in the condensation reaction are DMAP and DCC; and the molar ratio of DMAP to DCC is 1-1.5:9.

[0050] Preferably, the molar ratio of compound 3-3 to compound 4-2 is 1:0.9-1.1; the molar ratio of compound 4-2 to copper sulfate is 4-6:1; and the molar ratio of compound 4-2 to sodium ascorbate is 1:1-2.

[0051] Furthermore, the synthesis method of the prodrug molecule NE-DPI is as follows: Nintedanib was reacted with compound 3-4 to prepare the prodrug molecule NE-DPI. The synthetic route is as follows:

[0052] Preferably, the molar ratio of nintedanib to compound 3-4 is 1:1.3-1.5; the catalyst SmI2 / isopropanethiol is added during the reaction of nintedanib with compound 3-4.

[0053] Furthermore, the synthesis method of the prodrug molecule PR-521 is as follows: Compound 5-1 was prepared by condensation reaction of 1-amino-11-azido-3,6,9-trioxaundecane with 521-COOH. Compound 5-1 and compound 3-2 were reacted in the presence of copper sulfate and sodium ascorbate to prepare the prodrug molecule PR-521. The synthetic route is as follows:

[0054] Preferably, the molar ratio of 1-amino-11-azido-3,6,9-trioxaundecane to 521-COOH is 1:1.3-1.7; the condensing agents in the condensation reaction are HATU and DIPEA; the molar ratio of HATU to 521-COOH is 1:1.3-1.7; and the molar ratio of 521-COOH to DIPEA is 1:1-3.

[0055] Preferably, the molar ratio of compound 5-1 to compound 3-2 is 1:0.9-1.1; the molar ratio of compound 3-2 to copper sulfate is 4-6:1; and the molar ratio of compound 3-2 to sodium ascorbate is 1:1.3-1.7.

[0056] Preferably, the reaction temperature of compound 5-1 with compound 3-2 is 65-75 °C, and the reaction time is 15-20 h.

[0057] Furthermore, the synthesis method of the prodrug molecule PE-521 is as follows: Compound 5-2 was prepared by the condensation reaction of 15-amino-4,7,10,13-tetraoxapentadecanal with 521-COOH. The prodrug molecule PE-521 was prepared by reacting pirfenidone with compound 5-2. The synthetic route is as follows:

[0058] Preferably, the molar ratio of 15-amino-4,7,10,13-tetraoxapentadecanal to 521-COOH is 1:1.3-1.7; the condensing agents in the condensation reaction are HATU and DIPEA; the molar ratio of 15-amino-4,7,10,13-tetraoxapentadecanal to HATU is 1:0.9-1.1; and the molar ratio of 15-amino-4,7,10,13-tetraoxapentadecanal to DIPEA is 1:2.6-3.2.

[0059] Preferably, the molar ratio of pirfenidone to compound 5-2 is 1:1.2-1.5; and the catalyst SmI2 / isopropanethiol is added during the reaction of pirfenidone and compound 5-2.

[0060] Furthermore, the synthesis method of the prodrug molecule NR-521 is as follows: Compound 5-3 was prepared by the condensation reaction of 1-amino-11-azido-3,6,9-trioxaundecane with 521-COOH. Compounds 4-2 and 5-3 were hardened in the presence of copper sulfate and sodium ascorbate to prepare the prodrug molecule NR-521. The synthetic route is as follows:

[0061] Preferably, the molar ratio of 1-amino-11-azido-3,6,9-trioxaundecan to 521-COOH is 1:1.3-1.7; the condensing agents in the condensation reaction are HATU and DIPEA; the molar ratio of 1-amino-11-azido-3,6,9-trioxaundecan to HATU is 1:0.8-1.2; and the molar ratio of 1-amino-11-azido-3,6,9-trioxaundecan to DIPEA is 1:2.5-3.2.

[0062] Preferably, the molar ratio of compound 4-2 to compound 5-3 is 1:0.9-1.1; the molar ratio of compound 4-2 to copper sulfate is 3-6:1; and the molar ratio of compound 4-2 to sodium ascorbate is 1:1-2.

[0063] Furthermore, the synthesis method of the prodrug molecule NE-521 is as follows: NE-521 was prepared by reacting nintedanib with compounds 3-4. The synthetic route is as follows:

[0064] Preferably, the molar ratio of nintedanib to compound 3-4 is 1:1.3-1.5; the catalyst SmI2 / isopropanethiol is also added during the reaction of nintedanib with compound 3-4.

[0065] Thirdly, the present invention provides a pharmaceutical composition comprising the anti-pulmonary fibrosis prodrug compound described in the first aspect or a pharmaceutically acceptable salt thereof.

[0066] Furthermore, the composition further includes one or more pharmaceutically acceptable carriers, diluents, or excipients.

[0067] Furthermore, the dosage form of the pharmaceutical composition can be any form suitable for systemic or local administration, preferably an oral formulation (such as tablets or capsules), an inhaled formulation (such as dry powder inhalers, aerosols, or nebulized solutions), or an injectable formulation (such as liposomes or nanoparticle injections). Inhalation administration can further synergistically enhance the lung targeting of the drug.

[0068] Fourthly, the present invention provides the use of the anti-pulmonary fibrosis prodrug compound described in the first aspect or the pharmaceutical composition described in the third aspect in the preparation of a medicament for the prevention and / or treatment of pulmonary fibrosis and related diseases.

[0069] Furthermore, the pulmonary fibrosis includes idiopathic pulmonary fibrosis (IPF), silicosis, radiation-induced pulmonary fibrosis, and drug-induced pulmonary fibrosis.

[0070] Compared with the prior art, the present invention has achieved the following beneficial effects: (1) Precise targeting and improved efficacy: The prodrug compound provided by this invention can be efficiently enriched in pulmonary fibrosis lesions through active targeting mediated by T unit, thereby increasing the local drug concentration in the lesions and significantly enhancing the anti-fibrotic efficacy.

[0071] (2) Intelligent response and reduced toxicity: The design of the L unit in the prodrug compound provided by this invention ensures that the prodrug specifically releases the original drug only in the PFM, while remaining inert in normal tissues and systemic circulation. This reduces the drug exposure to non-target organs (such as the gastrointestinal tract, liver, and skin), fundamentally avoiding the systemic toxicity of pirfenidone and nintedanib, and broadening the therapeutic window.

[0072] (3) Synergistic effect: This invention combines advanced targeted delivery technology with prodrug design concept. It is not a simple dosage form improvement, but a brand-new chemical entity with synergistic effect. Attached Figure Description

[0073] The accompanying drawings, which form part of this specification, are used to provide a further understanding of the invention. The illustrative embodiments and descriptions of the invention are used to explain the invention and do not constitute an undue limitation thereof. Obviously, those skilled in the art can obtain other drawings based on these drawings without any inventive effort.

[0074] Figure 1 This is a graph showing the lung-targeted delivery efficiency of the single drug and prodrug of this invention; Figure 2 This is a diagram showing the effects of the single drug and prodrug of this invention on pulmonary fibrosis in mice; where (A) is an H&E staining diagram; (B) is a quantitative result diagram of the Ashcroft score; (C) is a Sirius red staining diagram; and (D) is a quantitative analysis diagram of collagen. Figure 3 These are the expression diagrams of the single drug and prodrug of the present invention inhibiting fibrosis-related proteins; (A) is the expression diagram of the single drug and prodrug of the present invention inhibiting Fibronectin, Vimentin, Collagen I, and α-SMA; (B) is the expression diagram of Fibronectin; (C) is the expression diagram of Vimentin; (D) is the expression diagram of Collagen I; (E) is the expression diagram of α-SMA. Figure 4 The diagram shows the effects of the single drug and prodrug of this invention on physiological functions and inflammation; (A) is the effect on body weight; (B) is the effect on forced vital capacity; (C) is the effect on lung compliance; (D) is the expression of TNF-α in lung tissue; and (E) is the expression of TGF-β in lung tissue. Detailed Implementation

[0075] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0076] The technical solution of the present invention will be further described below with reference to specific embodiments. The present invention does not impose any special restrictions on the source of reagents used in the following embodiments; commercially available products well known to those skilled in the art can be used.

[0077] Example 1 (1) Synthesis of DPI-COOH

[0078] Synthesis of compound a: Under Ar protection, a THF / H2O mixture (4 mL + 4 mL) containing 1,2-diiodobenzene (660 mg, 2 mmol), ethyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxoboronyl-2-yl)benzoate (276 mg, 1 mmol), PdCl2[PPh3] (35 mg, 0.05 mmol), and Na2CO3 (318 mg, 3 mmol) was stirred overnight at 70 °C. The reaction mixture was then extracted with ethyl acetate and washed three times with saturated sodium chloride solution. The resulting organic layer was dried over anhydrous sodium sulfate and concentrated under vacuum. Finally, the residue was purified by column chromatography to give compound a (300 mg, 0.85 mmol, 85% yield).

[0079] Synthesis of compound b: Compound a (300 mg, 0.85 mmol) was completely dissolved in THF (3 mL), followed by dropwise addition of 3 M sodium hydroxide solution. The mixture was then stirred at 50 °C until the reaction was complete. After the reaction was complete, the reaction system was neutralized with 3 M HCl solution to adjust the pH to neutral. The reaction mixture was then extracted with ethyl acetate to separate the organic layer. The organic layer was washed three times with saturated sodium chloride solution and then dried with anhydrous sodium sulfate. Finally, the organic layer was concentrated under vacuum to obtain the desired residue. This residue did not require further purification and could be used directly in subsequent experimental steps.

[0080] Synthesis of compound DPI-COOH: mCPBA (25.8 mg, 0.15 mM) and TfOH (45 mg, 3 mM) were added to a suspension of dichloromethane (1 mL) containing compound b (32.3 mg, 0.1 mM). The reaction system was stirred at room temperature for 2 h, and then the solvent was removed under vacuum. The resulting residue was resuspended in dry diethyl ether, and the solid was collected by filtration, washed with diethyl ether, and dried under vacuum to give a pale white solid DPI-COOH (41 mg, 0.087 mM, 87%), without further purification.

[0081] (2) Synthesis of 521-COOH

[0082] Synthesis of Compound 1-1: Under Ar protection, a THF / H2O mixture (4 mL + 4 mL) containing 1,2-diiodo-3-nitrobenzene (750 mg, 2 mmol), pinene 4-methoxycarbonyl-2-nitrobenzeneboronic acid (321 mg, 1 mmol), PdCl2[PPh3] (35 mg, 0.05 mmol), and Na2CO3 (318 mg, 3 mmol) was stirred overnight at 70 °C. The reaction mixture was then extracted with ethyl acetate and washed three times with saturated sodium chloride solution. The resulting organic layer was dried over anhydrous sodium sulfate and concentrated under vacuum. Finally, the residue was purified by column chromatography to give compound 1-1 (376 mg, 0.85 mmol, 85% yield).

[0083] Synthesis of Compounds 1-2: Compound 1-1 (376 mg, 0.85 mmol) was completely dissolved in THF (3 mL), followed by dropwise addition of 3 M sodium hydroxide solution. The mixture was then stirred at 50 °C until the reaction was complete. After the reaction was complete, the reaction system was neutralized with 3 M HCl solution to adjust the pH to neutral. The reaction mixture was then extracted with ethyl acetate to separate the organic layer. The organic layer was washed three times with saturated sodium chloride solution and then dried with anhydrous sodium sulfate. Finally, the organic layer was concentrated under vacuum to obtain the desired residue. This residue did not require further purification and could be used directly in subsequent experimental steps.

[0084] Synthesis of compound 521-COOH: mCPBA (25.8 mg, 0.15 mM) and TfOH (45 mg, 3 mM) were added to a suspension of dichloromethane (1 mL) containing compounds 1-2 (32.3 mg, 0.1 mM). The reaction system was stirred at room temperature for 2 h, and then the solvent was removed under vacuum. The resulting residue was resuspended in dry diethyl ether, and the solid was collected by filtration, washed with diethyl ether, and dried under vacuum to give a pale white solid 521-COOH (47 mg, 0.085 mM, 85%).

[0085] Example 2 Linker Synthesis

[0086] Synthesis of compound 2-1: 2,6-Dimethyl-1-nitrobenzene (4 g, 27 mmol) was dissolved in 300 mL of water, and sodium hydroxide (1.6 g, 40 mmol) was added at room temperature. The mixture was heated to 95 °C, and potassium permanganate (17 g, 106 mmol) was added in portions over 3 h. After reacting overnight, TLC monitoring showed that the reaction was complete. The reaction was stopped and cooled to room temperature. Solid impurities were removed by filtration, and hydrochloric acid was slowly added to the filtrate, precipitating a large amount of solid. Filtration yielded a white solid product 2-1 (5.2 g, 86% yield).

[0087] Synthesis of compound 2-2: Compound 2-1 (5 g, 23.7 mmol) was dissolved in 40 mL of THF and cooled to 0 °C in an ice bath. Under nitrogen protection, 1 M borane-THF solution (120 mL, 120 mmol) was slowly added through a dropping funnel. After naturally warming to room temperature, the mixture was stirred for 48 h. The reaction was monitored by TLC until it was complete. The mixture was then cooled to 0 °C in an ice bath, and the reaction was quenched by slowly adding 30 mL of methanol.

[0088] Synthesis of compound 2-3: Compound 2-2 (3 g, 16.3 mmol) and 5% Pd / C catalyst (0.1 g) were added to 10 mL of methanol. Under nitrogen protection, hydrazine hydrate (N2H4·H2O, 2.5 mL, 50 mmol) was slowly added. The mixture was heated to 70 °C and stirred for 8 h. TLC monitoring showed that the reaction was complete. After cooling to room temperature, the catalyst was removed by filtration. During vacuum filtration, the catalyst cake was kept away from drying (dry Pd / C is flammable). The filtrate was collected and concentrated under reduced pressure. Purification by silica gel column chromatography yielded a pale yellow solid 2-3 (2 g, yield 78%).

[0089] Synthesis of compounds 2-4: Compound 2-3 (10 g, 65.3 mmol) and imidazole (17.8 g, 261 mmol) were dissolved in 50 mL DMF and cooled to 0 °C in an ice bath. A solution of TBSCl (30 g, 196 mmol) in 50 mL DMF was slowly added dropwise over 1 h. After the addition was complete, the mixture was allowed to rise naturally to room temperature and stirred for another 5 h. The reaction was monitored by TLC (thin-layer chromatography) to indicate completion. 400 mL H2O was added to the reaction mixture, and the mixture was extracted with ethyl acetate (80 mL × 3). The organic phases were combined and washed with saturated brine (400 mL × 3). After drying with anhydrous Na2SO4, the mixture was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography to give compound 2-4 (24 g, 96% yield) as a pale yellow oil.

[0090] Synthesis of compound 2-5: Compound 2-4 (10 g, 26.2 mmol) and compound 2-4-1 (9.2 g, 26.2 mmol) were dissolved in 50 mL of toluene, and K2CO3 (10.6 g, 67.4 mmol) was added. The mixture was heated to 110 °C under nitrogen protection and stirred for 12 h. After TLC monitoring showed complete consumption of compound 2-4, the mixture was cooled to room temperature. Solid impurities (mainly excess K2CO3 and possible byproducts) were removed by filtration. The filtrate was concentrated under reduced pressure and purified by silica gel column chromatography to give a brown solid 2-5 (1.5 g, yield 9.6%).

[0091] Synthesis of compounds 2-6: Compound 2-5 (600 mg, 0.87 mmol) was dissolved in 5 mL of methanol, and p-toluenesulfonic acid (p-TsOH) (300 mg, 1.73 mmol) was added. The mixture was stirred at room temperature for 1 h, and after most of the methanol had evaporated, 30 mL of saturated NaHCO3 solution was added. The solution was purified by silica gel column chromatography to give a pale blue solid 2-6 (400 mg, 99.5%).

[0092] Example 3: (1) Synthesis of prodrug molecule PR-DPI

[0093] Synthesis of compound 3-1: 0.02 mol pirfenidone was added in portions to a suspension of 0.3 mol of compound 2-6 containing 0.01 mol NaBH4. The reaction mixture was stirred at 20 °C for 2.5 h. 3.7 mL of 10% HCl aqueous solution was added to the reaction mixture, and the mixture was stirred at 60 °C for 4 h. The reaction mixture was cooled and poured into 200 mL of ice water. The product was extracted with diethyl ether (2 × 40 mL). The organic phases were combined and washed with water. The organic layer was dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation. Benzyl ether compounds were separated by column chromatography. The water-insoluble alcohol co-reactants were removed from the diethyl ether extract by vacuum distillation to obtain compound 3-1 (6.33 g, 50%).

[0094] Synthesis of Compound 3-2: Dichloromethane (30 mL), anhydrous ethanol (1.33 mL, 22.48 mmol), and 4-dimethylaminopyridine (DMAP, 151 mg, 1.23 mmol) were added to the starting materials of Compound 3-1 (12.33 mmol) and 5-hexyneic acid (12.33 mmol), and the mixture was cooled to 0 °C in an ice bath. N,N'-dicyclohexylcarbodiimide (DCC, 2.55 g, 12.36 mmol) was added, and the mixture was stirred at 0 °C for 5 min, then heated to room temperature and stirred for 3 h. The precipitated dicyclohexylurea (DCU) was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was redissolved in dichloromethane (250 mL), and the residue solid was removed by a second filtration. The organic phase was washed successively with 0.5 M HCl solution (80 mL) and saturated NaHCO3 solution (80 mL), the organic layer was dried over anhydrous magnesium sulfate, the solvent was removed by rotary evaporation under reduced pressure, and the compound 3-2 (5.39 g, 60%) was purified by silica gel column chromatography (elution gradient: dichloromethane / methanol 100:0 → 100:1).

[0095] Synthesis of Compound 3-3: Under nitrogen protection, 1-amino-11-azido-3,6,9-trioxaundecan (2.52 g, 11.58 mmol, 1.0 equivalent) and DPI-COOH (8.16 g, 17.34 mmol, 1.5 equivalent) were dissolved in anhydrous DMF (96 mL) and stirred at room temperature. After activation with a condensing agent, HATU (4.4 g, 11.58 mmol, 1.0 equivalent) and DIPEA (6 mL, 34.74 mmol, 3 equivalent) were added sequentially to the above solution, and the reaction mixture was continuously stirred at room temperature. The reaction was monitored by TLC (evolving solvent: dichloromethane / methanol = 9:1, phosphomolybdic acid ethanol solution for color development), with a reaction time of approximately 24 h. The reaction was quenched with deionized water (120 mL), the aqueous phase was extracted with ethyl acetate (180 mL), the organic phases were combined, dried over anhydrous sodium sulfate, and the crude product was concentrated under reduced pressure. The target product 3-3 (6 g, 75%) was obtained by rapid column chromatography purification (elution gradient: dichloromethane / methanol 92:8 → 95:5).

[0096] Synthesis of compound PR-DPI: Compound 3-2 (1.0 equivalent, 1.5 mmol) and copper sulfate pentahydrate (CuSO4·5H2O, 20 mol%, 0.3 mmol) were added sequentially to a stirred solution of the corresponding azide compound 3-3 (1.0 equivalent, 1.5 mmol). Sodium ascorbate (1.5 equivalent, 2.25 mmol) was then added in portions. The reaction mixture was heated in an oil bath at 70 °C for 18 h. After cooling to room temperature, the reaction was quenched with concentrated ammonia solution (~25%) to complex residual copper ions. The aqueous phase was extracted with dichloromethane (3 × 100 mL), and the organic phases were combined. The mixture was washed with 0.02 M EDTA solution until colorless (at least 3 times) to completely remove copper, and finally washed with saturated brine. The organic phase was dried over anhydrous magnesium sulfate. Filter, concentrate under reduced pressure onto diatomaceous earth support, and purify by silica gel rapid column chromatography with an eluent gradient of dichloromethane (100%) → dichloromethane / methanol (95:5). The solvent was evaporated under reduced pressure to obtain the product PR-DPI (1.89 g, 90%).

[0097] (2) Synthesis of the prodrug molecule PE-DPI

[0098] Synthesis of compounds 3-4: Under nitrogen protection, 15-amino-4,7,10,13-tetraoxapentadecanal (962 mg, 3.86 mmol, 1.0 equivalent) and DPI-COOH (2.72 g, 5.78 mmol, 1.5 equivalent) were dissolved in anhydrous DMF (16 mL) and stirred at room temperature. After activation with a condensing agent, HATU (1.468 g, 3.86 mmol, 1.0 equivalent) and DIPEA (2 mL, 11.58 mmol, 3 equivalent) were added sequentially to the above solution, and the reaction mixture was continuously stirred at room temperature. The reaction was monitored by TLC (evolving solvent: dichloromethane / methanol = 9:1, phosphomolybdic acid ethanol solution for color development), with a reaction time of approximately 24 h. The reaction was quenched with deionized water (40 mL), the aqueous phase was extracted with ethyl acetate (60 mL), the organic phases were combined, dried over anhydrous sodium sulfate, and the crude product was concentrated under reduced pressure. Rapid column chromatography was used to purify the target product 3-4 (2.1 g, 77%) (elution gradient: dichloromethane / methanol 92:8 → 95:5).

[0099] Synthesis of compound PE-DPI: Pirfenidone (1.0 mmol), SmI2 / isopropanethiol (approximately 3 mol%) dissolved in 10 mL THF, and 3–4 (1.0 g, 1.4 mmol) of THF solution (50 mL) were premixed in an oven-dried syringe to form a yellow solution. This yellow solution was then slowly added dropwise to a THF master reaction solution (140 mL) containing SmI2 / isopropanethiol (50 / 40 mol%) to obtain the target product PE-DPI (1.2 g, 95%).

[0100] (3) Synthesis of the prodrug molecule NR-DPI:

[0101] Synthesis of compound 4-1: 6 mmol of nintedanib was added fractionally to a suspension of 9 mmol of compound 2-6 containing 0.01 mol NaBH4. The reaction mixture was stirred at 20 °C for 2.5 h. 3.7 mL of 10% HCl aqueous solution was added to the reaction mixture, and the mixture was stirred at 60 °C for 4 h. The reaction mixture was cooled and poured into 60 mL of ice water. The product was extracted with diethyl ether (2 × 12 mL). The organic phases were combined and washed with water. The organic layer was dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation. Benzyl ether compounds were separated by column chromatography. The water-insoluble alcohol co-reactants were removed from the ether extract by vacuum distillation to obtain compound 4-1 (5.8 g, 65%).

[0102] Synthesis of Compound 4-2: Dichloromethane (30 mL), anhydrous ethanol (1.33 mL, 22.48 mmol), and 4-dimethylaminopyridine (DMAP, 151 mg, 1.23 mmol) were added to the starting materials of Compound 4-1 (7.39 mmol) and 5-hexyneic acid (7.39 mmol), and the mixture was cooled to 0 °C in an ice bath. N,N'-dicyclohexylcarbodiimide (DCC, 1.85 g, 9 mmol) was added, and the mixture was stirred at 0 °C for 5 min, then heated to room temperature and stirred for another 3 h. The precipitated dicyclohexylurea (DCU) was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was redissolved in dichloromethane (250 mL), and the residue solid was removed by a second filtration. The organic phase was washed successively with 0.5 M HCl solution (80 mL) and saturated NaHCO3 solution (80 mL), the organic layer was dried over anhydrous magnesium sulfate, the solvent was removed by rotary evaporation under reduced pressure, and the compound was purified by silica gel column chromatography (elution gradient: dichloromethane / methanol 100:0 → 100:1 → 100:2) to obtain compound 4-2 (4.8 g, 60%).

[0103] Synthesis of compound NR-DPI: Compound 4-2 (1.0 equivalent, 2 mmol) and copper sulfate pentahydrate (CuSO4·5H2O, 20 mol%, 0.4 mmol) were added sequentially to a stirred solution of the corresponding azide compound 3-3 (1.0 equivalent, 2 mmol). Sodium ascorbate (1.5 equivalent, 3 mmol) was then added in portions, and the reaction mixture was heated in a 70 °C oil bath for 18 h. After cooling to room temperature, the reaction was quenched with concentrated ammonia solution (~25%) to complex residual copper ions. The aqueous phase was extracted with dichloromethane (3 × 100 mL), and the organic phases were combined. The organic phases were washed with 0.02 M EDTA solution until colorless (at least 3 times) to completely remove copper, and finally washed with saturated brine. The organic phase was dried over anhydrous magnesium sulfate. The solution was filtered, concentrated under reduced pressure onto a diatomaceous earth support, and purified by rapid silica gel column chromatography with an eluent gradient of dichloromethane (100%) → dichloromethane / methanol (95:6). The solvent was evaporated under reduced pressure to obtain the product NR-DPI (3.4 g, 92%).

[0104] (4) Synthesis of the prodrug molecule NE-DPI:

[0105] Synthesis of compound NE-DPI: Nintedanib (1.0 mmol), SmI2 / isopropanethiol (approximately 3 mol%, dissolved in 1 mL THF), and a 3-4 (1 g, 1.4 mmol) THF solution (5 mL) were premixed in an oven-dried syringe to form a yellow solution. This yellow solution was then slowly added dropwise to a THF master reaction solution (14 mL) containing SmI2 / isopropanethiol (50 / 40 mol%) to obtain the target product NE-DPI (1.65 g, 95%).

[0106] Example 4 (1) Synthesis of the prodrug molecule PR-521:

[0107] Synthesis of Compound 5-1: Under nitrogen protection, 1-amino-11-azido-3,6,9-trioxaundecan (2.52 g, 1.93 mmol, 1.0 equivalent) and 521-COOH (9.75 g, 2.89 mmol, 1.5 equivalent) were dissolved in anhydrous DMF (96 mL) and stirred at room temperature. After activation with a condensing agent, HATU (4.4 g, 1.93 mmol, 1.0 equivalent) and DIPEA (6 mL, 5.79 mmol, 3 equivalent) were added sequentially to the above solution, and the reaction mixture was stirred continuously at room temperature. The reaction was monitored by TLC (evolving solvent: dichloromethane / methanol = 9:1, phosphomolybdic acid ethanol solution for color development), and the reaction time was approximately 24 h. The reaction was quenched with deionized water (120 mL), the aqueous phase was extracted with ethyl acetate (180 mL), the organic phases were combined, dried over anhydrous sodium sulfate, and the crude product was concentrated under reduced pressure. The target product 3-3 (6.61 g, 75%) was obtained by rapid column chromatography purification (elution gradient: dichloromethane / methanol 92:8 → 95:5).

[0108] Synthesis of compound PR-521: Compound 3-2 (1.0 equivalent, 1.5 mmol) and copper sulfate pentahydrate (CuSO4·5H2O, 20 mol%, 0.3 mmol) were added sequentially to a stirred solution of the corresponding azide compound 5-1 (1.0 equivalent, 1.5 mmol). Sodium ascorbate (1.5 equivalent, 2.25 mmol) was then added in portions. The reaction mixture was heated in a 70 °C oil bath for 18 h. After cooling to room temperature, the reaction was quenched with concentrated ammonia (~25%) to complex residual copper ions. The aqueous phase was extracted with dichloromethane (3 × 100 mL). The organic phases were combined and washed with 0.02 M EDTA solution until colorless (at least 3 times) to completely remove copper. Finally, the organic phase was washed with saturated brine. The organic phase was dried over anhydrous magnesium sulfate. Filter, concentrate under reduced pressure onto diatomaceous earth support, and purify by silica gel rapid column chromatography with an eluent gradient of dichloromethane (100%) → dichloromethane / methanol (95:5). The solvent was evaporated under reduced pressure to obtain product PR-521 (2.01 g, 90%).

[0109] (2) Synthesis of the prodrug molecule PE-521:

[0110] Synthesis of Compound 5-2: Under nitrogen protection, 15-amino-4,7,10,13-tetraoxapentadecanal (962 mg, 3.86 mmol, 1.0 equivalent) and 521-COOH (3.25 g, 5.78 mmol, 1.5 equivalent) were dissolved in anhydrous DMF (16 mL) and stirred at room temperature. After activation with a condensing agent, HATU (1.468 g, 3.86 mmol, 1.0 equivalent) and DIPEA (2 mL, 11.58 mmol, 3 equivalent) were added sequentially to the above solution, and the reaction mixture was continuously stirred at room temperature. The reaction was monitored by TLC (evolving solvent: dichloromethane / methanol = 9:1, phosphomolybdic acid ethanol solution for color development), with a reaction time of approximately 24 h. The reaction was quenched with deionized water (40 mL), the aqueous phase was extracted with ethyl acetate (60 mL), the organic phases were combined, dried over anhydrous sodium sulfate, and the crude product was concentrated under reduced pressure. The target product 5-2 (3.3 g, 75%) was obtained by rapid column chromatography purification (elution gradient: dichloromethane / methanol 92:8 → 95:5).

[0111] Synthesis of compound PE-521: Pirfenidone (1.0 mmol), SmI2 / isopropanethiol (approximately 3 mol%) dissolved in 10 mL THF, and a THF solution of 5-2 (1.1 g, 1.4 mmol) (50 mL) were premixed in an oven-dried syringe to form a yellow solution (characteristic color development of trivalent samarium ions). This yellow solution was slowly added dropwise to a THF master reaction solution (140 mL) containing SmI2 / isopropanethiol (50 / 40 mol%) to obtain the target product PE-521 (1.3 g, 95%).

[0112] (3) Synthesis of the prodrug molecule NR-521:

[0113] Synthesis of Compound 5-3: Under nitrogen protection, 1-amino-11-azido-3,6,9-trioxaundecan (2.52 g, 11.58 mmol, 1.0 equivalent) and 521-COOH (9.74 g, 17.34 mmol, 1.5 equivalent) were dissolved in anhydrous DMF (96 mL) and stirred at room temperature. After activation with a condensing agent, HATU (4.4 g, 11.58 mmol, 1.0 equivalent) and DIPEA (6 mL, 34.74 mmol, 3 equivalent) were added sequentially to the above solution, and the reaction mixture was continuously stirred at room temperature. The reaction was monitored by TLC (evolving solvent: dichloromethane / methanol = 9:1, phosphomolybdic acid ethanol solution for color development), with a reaction time of approximately 24 h. The reaction was quenched with deionized water (120 mL), the aqueous phase was extracted with ethyl acetate (180 mL), the organic phases were combined, dried over anhydrous sodium sulfate, and the crude product was concentrated under reduced pressure. Rapid column chromatography was used to purify the target product 5-3 (9.25 g, 70%) (elution gradient: dichloromethane / methanol 92:8 → 95:5).

[0114] Synthesis of compound NR-521: Compound 4-2 (1.0 equivalent, 2 mmol) copper sulfate pentahydrate (CuSO4·5H2O, 20 mol%, 0.4 mmol) was added sequentially to a stirred solution of the corresponding azide compound 5-3 (1.0 equivalent, 2 mmol). Sodium ascorbate (1.5 equivalent, 3 mmol) was then added in portions. The reaction mixture was heated in a 70 °C oil bath for 18 h. After cooling to room temperature, the reaction was quenched with concentrated ammonia solution (~25%) to complex residual copper ions. The aqueous phase was extracted with dichloromethane (3 × 100 mL), and the organic phases were combined. The organic phases were washed with 0.02 M EDTA solution until colorless (at least 3 times) to completely remove copper, and finally washed with saturated brine. The organic phase was dried over anhydrous magnesium sulfate. Filter, concentrate under reduced pressure onto diatomaceous earth support, and purify by silica gel rapid column chromatography with an eluent gradient of dichloromethane (100%) → dichloromethane / methanol (95:6). The solvent was evaporated under reduced pressure to obtain product NR-521 (3.5 g, 95%).

[0115] (4) Synthesis of the prodrug molecule NE-521:

[0116] Synthesis of compound NE-521: Nintedanib (1.0 mmol), SmI2 / isopropyl mercaptan (approximately 3 mol%, dissolved in 1 mL THF), and a THF solution of 5-2 (1.11 g, 1.4 mmol) (5 mL) were premixed in an oven-dried syringe to form a yellow solution. This yellow solution was then slowly added dropwise to a THF master reaction solution (14 mL) containing SmI2 / isopropyl mercaptan (50 / 40 mol%) to obtain the target product NE-521 (1.57 g, 95%).

[0117] Example 5 1. Drug Targeting Evaluation CY3 fluorescent dye was covalently linked to each of the single drugs (DPI, 521, pirfenidone, nintedanib) and the eight prodrugs prepared in Examples 3 and 4, respectively. Equimolar doses (based on CY3) of each labeled drug were injected into mice via tail vein injection. After injection, the drugs were allowed to freely distribute in vivo for 12 h to ensure sufficient tissue penetration and accumulation. After 12 h, the mice were euthanized, and tissues from five major organs—heart, liver, spleen, lung, and kidney—were immediately collected. Each tissue was weighed and added to pre-chilled phosphate-buffered saline (PBS, pH 7.4) at a specific ratio (1 g tissue added to 9 mL pre-chilled PBS). The homogenate was thoroughly homogenized using a high-speed tissue homogenizer under ice bath conditions to prepare a homogenous tissue homogenate. After centrifugation at 4 °C, the supernatant was collected, and the CY3 fluorescence channel was scanned using an IVIS Spectrum system with uniform scanning parameters to compare the distribution differences of different drugs in various organs.

[0118] 2. In vivo pharmacodynamic evaluation: Bleomycin-induced mouse pulmonary fibrosis model was used.

[0119] Groups: Solvent control group, model group, nintedanib group, pirfenidone group, DPI group, compound 521 group, compound PR-DPI group, compound PE-DPI group, compound PR-521 group, compound PE-521 group, compound NR-DPI group, compound NE-DPI group, compound NR-521 group, compound NE-521 group.

[0120] Drug administration: A mouse model of pulmonary fibrosis was induced by a single intratracheal infusion of bleomycin (2.5 mg / kg). Treatment began on day 7 after modeling and the mice were euthanized on day 28 to end the experiment.

[0121] Specific administration methods for each group: Solvent control group: A single intratracheal infusion of normal saline was administered, followed by an equal volume of solvent (5% DMSO + 40% PEG-300 + 5% Tween-80 + 50% normal saline) starting on day 7, administered via tail vein injection twice a week until day 28.

[0122] Model group: Modeling was achieved by a single intratracheal infusion of bleomycin, without any other therapeutic drugs or solvents.

[0123] Nintedanib group: A single intratracheal infusion of bleomycin was administered to establish the bleomycin model, and the date of the infusion was recorded as day 1. Nintedanib (60 mg / kg / day) was started on day 7 after model establishment and administered orally (po) once daily until day 28.

[0124] Pirfenidone group: A single intratracheal infusion of bleomycin was administered to establish the model, and the date of the bleomycin infusion was recorded as day 1. Pirfenidone (200 mg / kg / day) was started on day 7 after model establishment and administered orally (po) once daily until day 28.

[0125] The following groups underwent bleomycin modeling with a single intratracheal infusion: DPI group, compound 521 group, compound PR-DPI group, compound PE-DPI group, compound PR-521 group, compound PE-521 group, compound NR-DPI group, compound NE-DPI group, compound NR-521 group, and compound NE-521 group. The infusion time was recorded as day 1. Starting on day 7 post-modeling, each group received the drug (1 mg / kg) via tail vein injection twice weekly until day 28.

[0126] Evaluation indicators: (1) Weight measurement: At the end of the experiment, the weight of each mouse was measured and recorded using an electronic balance.

[0127] (2) Lung function test: After anesthetizing the mice, endotracheal intubation was performed and the tube was tightly connected to the small animal pulmonary function instrument. The instrument’s built-in standardized test program was used to accurately measure and read the dynamic compliance of the lungs (Cdyn), which reflects the elasticity of the lung tissue, and the forced vital capacity (FVC), which represents the maximum expiratory capacity, thereby objectively quantifying the respiratory mechanics of the lungs.

[0128] (3) Histopathological analysis: Mouse lung tissue was immediately fixed with 4% paraformaldehyde solution, then dehydrated, cleared, and embedded in paraffin. The embedded tissue blocks were sectioned and stained with H&E (hematoxylin-eosin) to observe the overall tissue morphology and structure, and Sirius red staining was performed simultaneously to specifically mark collagen fibers. The degree of fibrosis was semi-quantitatively assessed using the Ashcroft score, and the area of ​​the positive region (red) in the Sirius red stained sections was quantified using image analysis software to accurately calculate the proportion of collagen deposition.

[0129] (4) Detection of fibrosis proteins: Mouse lung tissue was collected, homogenized with RIPA lysis buffer at low temperature, and centrifuged to extract total protein. After quantification of protein concentration using the BCA method, SDS-PAGE electrophoresis was performed to separate the proteins and transfer them to an NC membrane. After blocking with 5% skim milk, primary antibodies and corresponding secondary antibodies against Fibronectin, Collagen I, α-SMA, Vimentin, and the internal control protein GAPDH were added sequentially for incubation. Finally, ECL chemiluminescence imaging was used. The gray values ​​of the target band and the internal control band were analyzed using ImageJ software. The relative expression level of each fibrosis marker protein was expressed as the ratio of target protein to GAPDH, and semi-quantitative analysis was performed.

[0130] (5) Expression of inflammatory cytokines mRNA: Total RNA was extracted from mouse lung tissue using the Trizol method, and its concentration and purity were determined. A suitable amount of high-quality RNA was used to synthesize first-strand cDNA using a reverse transcription kit. Using this cDNA as a template, amplification was performed on a real-time quantitative PCR instrument (qPCR) using specific primers targeting the TGF-β and TNF-α genes. Using GAPDH as an internal control, the relative expression levels of each inflammatory cytokine mRNA were analyzed using the 2^(-ΔΔCt) calculation method.

[0131] result: Figure 1 This is a graph showing the lung-targeted delivery efficiency of the single drug and prodrug of this invention. (Example) Figure 1 As shown, the distribution of each drug differed significantly in different organs. In the single-drug groups, nintedanib and pirfenidone showed wide systemic distribution but limited lung targeting. In contrast, DPI and 521 exhibited relatively high fluorescence intensity in lung tissue, indicating a certain lung targeting tendency. All prodrug groups showed significant improvement in lung targeting. The strongest CY3 fluorescence signal was detected in lung tissue homogenate, with significantly higher intensity than in non-target organs such as the heart, liver, and spleen. Prodrugs can significantly improve drug accumulation in the lungs, and their targeting efficiency is superior to that of single-drug components, achieving efficient and specific lung targeting.

[0132] Figure 2This is a diagram showing the effects of the single drug and prodrug of this invention on pulmonary fibrosis in mice; where (A) is an H&E staining diagram; (B) is a quantitative Ashcroft score diagram; (C) is a Sirius red staining diagram; and (D) is a quantitative collagen analysis diagram. Figure 2 As shown in A and 2C, compared with the model group, both monotherapy and prodrug therapy improved lung tissue pathological damage to varying degrees. Specifically, alveolar structure destruction was reduced, inflammatory cell infiltration was decreased, and septal thickening and collagen deposition were significantly inhibited. Figure 2 The quantitative results of the Ashcroft score of B confirmed that the fibrosis score of the prodrug group was significantly lower than that of the model group and the single-drug group. Figure 2 Quantitative collagen analysis of D further revealed that the prodrug most effectively reduced the Sirius red-positive area, indicating its optimal effect in reversing collagen deposition. In conclusion, the prodrug strategy demonstrated superior synergistic therapeutic efficacy compared to single-drug therapy in alleviating the pathological changes of pulmonary fibrosis in mice.

[0133] Figure 3 These are the expression diagrams of the single drug and prodrug of the present invention inhibiting fibrosis-related proteins; (A) is the expression diagram of the single drug and prodrug of the present invention inhibiting Fibronectin, Vimentin, Collagen I, and α-SMA; (B) is the expression diagram of Fibronectin; (C) is the expression diagram of Vimentin; (D) is the expression diagram of Collagen I; (E) is the expression diagram of α-SMA. Figure 3 As shown in Figure A, compared with the control group, the expression of four fibrosis marker proteins (Fibronectin, Collagen I, α-SMA, and Vimentin) in the lung tissue of the model group mice was significantly upregulated, indicating that the fibrosis model was successfully established. Each single-drug treatment could inhibit the expression of these proteins to some extent. However, the prodrug treatment group showed the most significant inhibitory effect, with the gray values ​​of each protein band significantly lower than those in the model group and the single-drug group. Figure 3 As shown in B-3E, the prodrug group was superior to the monotherapy group in downregulating the expression of all the above-mentioned fibrosis-related proteins, indicating that the combination therapy can synergistically inhibit myofibroblast activation and excessive extracellular matrix deposition, thereby more effectively delaying the progression of pulmonary fibrosis at the molecular level.

[0134] Figure 4 These are diagrams showing the effects of the single drug and prodrug of this invention on physiological function and inflammation; (A) is the effect on body weight; (B) is the effect on forced vital capacity; (C) is the effect on lung compliance; (D) is the expression of TNF-α in lung tissue; and (E) is the expression of TGF-β in lung tissue. Figure 4 As shown, the body weight of mice in the model group decreased significantly ( Figure 4 A), accompanied by a rapid deterioration in FVC and lung compliance (A), and a sharp decline in FVC and lung compliance ( Figure 4B, 4C), while the mRNA expression of TNF-α and TGF-β in lung tissue was significantly upregulated ( Figure 4 (D, 4E) successfully simulated the core characteristics of pulmonary fibrosis. All treatment groups showed varying degrees of improvement. Monotherapy partially halted weight loss, slightly improved lung function, and downregulated inflammatory cytokine expression. The prodrug group showed the most significant effect, with weight recovery approaching that of the control group, the greatest improvement in FVC and lung compliance, and the lowest suppression of TNF-α and TGF-β mRNA expression. These results fully demonstrate that the prodrug synergistically improves the overall condition and respiratory mechanics of fibrotic mice and inhibits pulmonary inflammatory responses, exhibiting a comprehensive therapeutic effect.

[0135] Compared with the model group, all treatment groups showed varying degrees of reduction in fibrosis. The combination therapy group was significantly more effective than the single-drug group in alleviating weight loss, restoring lung function, improving lung tissue structure, reducing collagen deposition, and decreasing the expression of inflammatory factors. This invention successfully designed and synthesized a novel lung-targeting and microenvironment-responsive prodrug. In vitro and in vivo experiments have confirmed that this prodrug can efficiently target pulmonary fibrosis lesions, intelligently release the original drug at the lesion site, significantly enhance anti-fibrotic efficacy, and effectively reduce systemic toxicity, demonstrating significant clinical translational potential and application value.

[0136] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A prodrug compound for pulmonary fibrosis, characterized in that, The anti-pulmonary fibrosis prodrug compound comprises an active unit D, a lung-targeting unit T, and a linker unit L, and has the structure shown in general formula (I): TLD(I); D is selected from pirfenidone and nintedanib; T is a lung-targeting ligand; the lung-targeting ligand is selected from diphenyl iodide chloride and its analogue 521; the analogue 521 is a dinitro-substituted diphenyl iodide chloride. L represents the linker unit, L is PEG4, or L1-L2-L3; where L1 is PEG4, L2 is 5-hexynoic acid, and L3 is compound 2-6; the structural formula of compound 2-6 is: ; Preferably, the structural formulas of diphenyl iodide chloride and simulant 521 are respectively and .

2. The anti-pulmonary fibrosis prodrug compound as described in claim 1, characterized in that, The anti-pulmonary fibrosis prodrug compound is selected from: 。 3. The method for preparing the anti-pulmonary fibrosis prodrug compound according to any one of claims 1-2, characterized in that, Includes the following steps: (1) Synthesize carboxylated lung-targeting unit T; (2) The carboxylated lung-targeting unit T was modified and a breakable linker L was introduced into its molecule to obtain the intermediate TL; (3) In the presence of catalyst SmI2 / isopropanethiol, the TL from step (2) is coupled with the active drug D through a reaction to obtain the target prodrug compound TLD; (4) Purify the crude product; or, (a) Synthesizing carboxylated lung-targeting unit T; (b) The carboxylated lung-targeting unit T was modified and a breakable linker L1 was introduced onto its molecule to obtain intermediate T-L1; (c) Modify the active drug D by introducing breakable linkers L2 and L3 onto its molecule to obtain intermediate L2-L3-D; (d) In the presence of copper sulfate and sodium ascorbate, T-L1 from step (b) and L2-L3-D from step (c) are coupled by reaction to obtain the target prodrug compound T-L1-L2-L3-D; (e) Purify the crude product.

4. The preparation method according to claim 3, characterized in that, Carboxylated lung-targeting unit T is DPI-COOH or 521-COOH; Preferably, the synthesis method of DPI-COOH is as follows: Compound a was prepared by reacting 1,2-diiodobenzene with ethyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxoboronyl-2-yl)benzoate in the presence of a palladium catalyst and a base. Compound a is hydrolyzed under alkaline conditions to give compound b; Compound b was reacted in the presence of mCPBA and TfOH to prepare DPI-COOH; Preferably, the molar ratio of 1,2-diiodobenzene to ethyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxoboronyl-2-yl)benzoate is 1-3:1; the reaction temperature is 65-75 °C; the reaction time is 10-15 h; and the solvent is a mixed solution of THF and water. Preferably, the hydrolysis reaction temperature is 45-55 ℃; Preferably, the molar ratio of compound b to mCPBA is 1:1.3-1.8; the molar ratio of compound b to TfOH is 1:25-35; the reaction temperature is 20-30 °C; and the reaction time is 1-3 h. The synthetic route is as follows: 。 Preferably, the synthesis method of 521-COOH is as follows: Compound 1-1 was prepared by reacting 1,2-diiodo-3-nitrobenzene with pinene 4-methoxycarbonyl-2-nitrobenzeneboronic acid in the presence of a palladium catalyst and a base. Compound 1-1 was hydrolyzed under alkaline conditions to prepare compound 1-2; Compounds 1-2 were reacted in the presence of mCPBA and TfOH to prepare 521-COOH; the synthetic route is as follows: ； Preferably, the molar ratio of 1,2-diiodo-3-nitrobenzene to pinene 4-methoxycarbonyl-2-nitrobenzeneboronic acid pinene ester is 1-3:1; the reaction temperature is 65-75 °C; the reaction time is 10-15 h; and the solvent is a mixed solution of THF and H2O. Preferably, the hydrolysis reaction temperature is 45-55 ℃; Preferably, the molar ratio of compound 1-2 to mCPBA is 1:1.3-1.8; the molar ratio of compound 1-2 to TfOH is 1:25-35; the reaction temperature is 20-30 °C; and the reaction time is 1-3 h.

5. The preparation method according to claim 3, characterized in that, In step (2), L is PEG4; and / or, in step (b), L1 is PEG4; in step (c), L2 is 5-hexyneic acid; L3 is compound 2-6; the structural formula of compound 2-6 is: ; Preferably, the synthesis method of compounds 2-6 is as follows: Compound 2-1 was prepared by oxidation of 2,6-dimethyl-1-nitrobenzene under alkaline conditions. Compound 2-1 was dissolved in THF and reacted with borane-THF solution to prepare compound 2-2; Compound 2-2 was reacted with hydrazine hydrate in methanol in the presence of a palladium on carbon catalyst to prepare compound 2-3; Compound 2-3 and imidazole were dissolved in a solvent and reacted with TBSCl to prepare compound 2-4; Compound 2-5 was prepared by reacting compound 2-4 and compound 2-4-1 under alkaline conditions; Compound 2-5 was reacted with p-toluenesulfonic acid to prepare compound 2-6; the synthetic route is as follows: Preferably, the oxidant used in the oxidation reaction is potassium permanganate; the molar ratio of potassium permanganate to 2,6-dimethyl-1-nitrobenzene is 3.5-4.5:1; Preferably, the molar ratio of compound 2-2 to hydrazine hydrate is 1:2.5-3.5; the reaction temperature is 65-75 °C; and the reaction time is 6-10 h. Preferably, the molar ratio of compound 2-3 to imidazole is 1:3.5-4.5; the molar ratio of compound 2-3 to TBSCl is 1:2.5-3.

5. Preferably, the molar ratio of compound 2-4 to compound 2-4-1 is 1:0.9-1.1; the reaction solvent for compounds 2-4 and 2-4-1 is toluene; the reaction temperature is 100-120 °C; and the reaction time is 10-15 h. Preferably, the molar ratio of compounds 2-5 to p-toluenesulfonic acid is 1:1.5-2.5; the reaction temperature is 20-30 °C; and the reaction time is 1-3 h.

6. The method for preparing the anti-pulmonary fibrosis prodrug compound as described in claim 2, characterized in that, The synthesis method of the prodrug PR-DPI is as follows: Pirfenidone reacts with compounds 2-6, and after the reaction is complete, HCl solution is added to give compound 3-1; compound 3-1 reacts with 5-hexyneic acid to synthesize compound 3-2. Compound 3-3 was prepared by reacting DPI-COOH with 1-amino-11-azido-3,6,9-trioxaundecan; Compounds 3-3 and 3-2 were reacted in the presence of copper sulfate and sodium ascorbate to prepare the crude prodrug PR-DPI; the crude product was then purified; the synthetic route is as follows: Preferably, the molar ratio of pirfenidone to compounds 2-6 is 1:10-20; or, NaBH4 is added during the reaction of pirfenidone with compounds 2-6; the molar ratio of NaBH4 to pirfenidone is 1:1-3. Preferably, the HCl solution has a mass fraction of 10-20%; the reaction is carried out for 3-5 hours after adding the HCl solution; or the reaction temperature is 50-70 °C. Preferably, the molar ratio of compound 3-1 to 5-hexyneic acid in the reaction of compound 3-1 and 5-hexyneic acid is 1:1-1.2; or, the condensing agent is DMAP and DCC; the molar ratio of DMAP and DCC is 1:9-11. Preferably, the molar ratio of amino-11-azido-3,6,9-trioxaundecan to DPI-COOH is 1:1.2-1.8; or, the condensing agent is HATU and DIPEA; the molar ratio of HATU and DIPEA is 1:2-4. Preferably, the molar ratio of compound 3-3 to compound 3-2 is 1:0.9-1.1; or, the molar ratio of copper sulfate to compound 3-2 is 0.3-0.4:1; or, the molar ratio of sodium ascorbate to compound 3-2 is 1.3-1.7:1; or, the reaction temperature is 65-75℃ and the reaction time is 15-20 h. And / or, the synthesis method of the prodrug molecule PE-DPI is as follows: Compounds 3-4 were prepared by condensation reaction of 15-amino-4,7,10,13-tetraoxapentadecanal with DPI-COOH. The prodrug molecule PE-DPI was prepared by reacting pirfenidone with compound 3-4; the synthetic route is as follows: Preferably, the molar ratio of 15-amino-4,7,10,13-tetraoxapentadecanal to DPI-COOH is 1:1.3-1.7; or, the condensing agents are HATU and DIPEA; the molar ratio of 15-amino-4,7,10,13-tetraoxapentadecanal to HATU is 1:0.9-1.1; the molar ratio of 15-amino-4,7,10,13-tetraoxapentadecanal to DIPEA is 1:2.5-3.5; or, the condensation reaction temperature is 20-30 °C, and the reaction time is 20-25 h. Preferably, during the reaction of pirfenidone with compound 3-4, the molar ratio of pirfenidone to compound 3-4 is 1:1.3-1.5; or, during the reaction of pirfenidone with compound 3-4, the catalyst SmI2 / isopropanethiol is also added. And / or, the synthesis method of the prodrug molecule NR-DPI is as follows: Nydanib reacted with compounds 2-6, and after the reaction was complete, HCl solution was added to react again to prepare compound 4-1. Compound 4-1 was condensed with 5-hexyneic acid to prepare compound 4-2; Compounds 3-3 and 4-2 were reacted in the presence of copper sulfate and sodium ascorbate to prepare the prodrug molecule NR-DPI; the synthetic route is as follows: Preferably, the molar ratio of nintedanib to compound 2-6 is 1:1-2; or, the reaction temperature of nintedanib with compound 2-6 is 15-25 °C, and the reaction time is 2-3 h; or, NaBH4 is added during the reaction of nintedanib with compound 2-6; the molar ratio of NaBH4 to compound 2-6 is 0.01:8-10. Preferably, the HCl solution has a mass fraction of 10-20%; the reaction is carried out for 3-5 hours after adding the HCl solution; or the reaction temperature is 50-70 °C. Preferably, the molar ratio of compound 4-1 to 5-hexyneic acid is 1:1-1.2; the condensing agents in the condensation reaction are DMAP and DCC; or, the molar ratio of DMAP and DCC is 1-1.5:

9. Preferably, the molar ratio of compound 3-3 to compound 4-2 is 1:0.9-1.1; the molar ratio of compound 4-2 to copper sulfate is 4-6:1; and the molar ratio of compound 4-2 to sodium ascorbate is 1:1-2. And / or, the synthesis method of the prodrug molecule NE-DPI is as follows: Nintedanib was reacted with compound 3-4 to prepare the prodrug molecule NE-DPI; the synthetic route is as follows: Preferably, the molar ratio of nintedanib to compound 3-4 is 1:1.3-1.5; or, the catalyst SmI2 / isopropanethiol is added during the reaction of nintedanib with compound 3-4. And / or, the synthesis method of the prodrug molecule PR-521 is as follows: Compound 5-1 was prepared by condensation reaction of 1-amino-11-azido-3,6,9-trioxaundecane with 521-COOH. Compound 5-1 and compound 3-2 were reacted in the presence of copper sulfate and sodium ascorbate to prepare the prodrug molecule PR-521; the synthetic route is as follows: Preferably, the molar ratio of 1-amino-11-azido-3,6,9-trioxaundecan to 521-COOH is 1:1.3-1.7; or, the condensing agents in the condensation reaction are HATU and DIPEA; the molar ratio of HATU to 521-COOH is 1:1.3-1.7; and the molar ratio of 521-COOH to DIPEA is 1:1-3. Preferably, the molar ratio of compound 5-1 to compound 3-2 is 1:0.9-1.1; the molar ratio of compound 3-2 to copper sulfate is 4-6:1; and the molar ratio of compound 3-2 to sodium ascorbate is 1:1.3-1.

7. Preferably, the reaction temperature of compound 5-1 with compound 3-2 is 65-75 °C; the reaction time is 15-20 h. And / or, the synthesis method of the prodrug molecule PE-521 is as follows: Compound 5-2 was prepared by the condensation reaction of 15-amino-4,7,10,13-tetraoxapentadecanal with 521-COOH. The prodrug molecule PE-521 was prepared by reacting pirfenidone with compound 5-2; the synthetic route is as follows: Preferably, the molar ratio of 15-amino-4,7,10,13-tetraoxapentadecanal to 521-COOH is 1:1.3-1.7; or, the condensing agents in the condensation reaction are HATU and DIPEA; the molar ratio of 15-amino-4,7,10,13-tetraoxapentadecanal to HATU is 1:0.9-1.1; the molar ratio of 15-amino-4,7,10,13-tetraoxapentadecanal to DIPEA is 1:2.6-3.2; Preferably, the molar ratio of pirfenidone to compound 5-2 is 1:1.2-1.5; or, the catalyst SmI2 / isopropanethiol is added during the reaction of pirfenidone with compound 5-2. And / or, the synthesis method of the prodrug molecule NR-521 is as follows: Compound 5-3 was prepared by the condensation reaction of 1-amino-11-azido-3,6,9-trioxaundecane with 521-COOH. Compounds 4-2 and 5-3 were hardened in the presence of copper sulfate and sodium ascorbate to prepare the prodrug molecule NR-521; the synthetic route is as follows: Preferably, the molar ratio of 1-amino-11-azido-3,6,9-trioxaundecan to 521-COOH is 1:1.3-1.7; or, the condensing agent in the condensation reaction is HATU and DIPEA; the molar ratio of 1-amino-11-azido-3,6,9-trioxaundecan to HATU is 1:0.8-1.2; the molar ratio of 1-amino-11-azido-3,6,9-trioxaundecan to DIPEA is 1:2.5-3.2; Preferably, the molar ratio of compound 4-2 to compound 5-3 is 1:0.9-1.1; the molar ratio of compound 4-2 to copper sulfate is 3-6:1; and the molar ratio of compound 4-2 to sodium ascorbate is 1:1-2. And / or, the synthesis method of the prodrug molecule NE-521 is as follows: NE-521 was prepared by reacting nintedanib with compounds 3-4; the synthetic route is as follows: Preferably, the molar ratio of nintedanib to compound 3-4 is 1:1.3-1.5; or, the catalyst SmI2 / isopropanethiol is added during the reaction of nintedanib with compound 3-4.

7. A pharmaceutical composition, characterized in that, It includes the anti-pulmonary fibrosis prodrug compound of claim 1 or a pharmaceutically acceptable salt thereof.

8. The pharmaceutical composition according to claim 7, characterized in that, The composition also includes one or more pharmaceutically acceptable carriers, diluents, or excipients.

9. The use of the anti-pulmonary fibrosis prodrug compound of claim 1 or the pharmaceutical composition of claim 7 in the preparation of a medicament for the prevention and / or treatment of pulmonary fibrosis and related diseases.

10. The application as described in claim 9, characterized in that, The pulmonary fibrosis includes idiopathic pulmonary fibrosis, silicosis, radiation-induced pulmonary fibrosis, and drug-induced pulmonary fibrosis.

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