Compounds having anti-organ fibrosis activity and uses thereof

Abstract

The application discloses a compound with anti-organ fibrosis activity and application thereof, and relates to the technical field of drug synthesis. It is found by the application that a small molecule compound ZLYH-24 has anti-fibrosis activity, and derivatives (indolin-2-ketone compounds) obtained by functional group modification, conjugated structure adjustment and the like of the small molecule compound also have anti-organ fibrosis activity. The indolin-2-ketone derivatives provided by the application can be applied to the preparation of anti-organ fibrosis drugs. Pharmacokinetic data show that the compound is prepared into a nanoparticle dosage form, so that the oral compliance can be improved, and the relative bioavailability can be improved by 2.73 times. The application covers a compound preparation method, a drug preparation and a medical use.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical synthesis technology, and in particular to compounds with anti-organ fibrosis activity and their applications. Background Technology

[0002] Renal fibrosis is a common pathological change in the progression of various chronic kidney diseases. It is characterized by excessive accumulation of extracellular matrix, abnormal activation of fibroblasts, and collagen deposition in the renal interstitium and peritubular tissues, ultimately leading to gradual loss of kidney function. Similarly, idiopathic pulmonary fibrosis (IPF) is a chronic, progressive fibrotic interstitial lung disease of unknown cause, characterized by abnormal repair of alveolar epithelial cells, formation of fibroblast foci, and excessive deposition of extracellular matrix, resulting in lung tissue structural destruction and impaired gas exchange. Currently, clinical treatments for renal fibrosis are limited, mainly focusing on controlling the primary disease (such as diabetes and hypertension) and using immunosuppressants. However, these measures are unlikely to effectively block or reverse the fibrotic process. Therefore, the development of novel drugs with clear anti-fibrotic activity has become an urgent research need.

[0003] According to the International Society of Nephrology (ISN) 2023 Global Kidney Health Atlas, the prevalence of chronic kidney disease (CKD) in Southeast Asia (including China) is as high as 11.04%, with a related mortality rate of 2.5%. Furthermore, according to a 2024 report by the World Health Organization, CKD has become the ninth leading cause of death globally, with deaths increasing by approximately 95% between 2000 and 2021. In the field of interstitial kidney disease (IPF), epidemiological data shows an upward trend in its global prevalence. A cohort study based on the English population showed that the incidence of IPF increased by 25% between 2008 and 2018, with a mortality rate of 7.9 per 100,000 person-years in 2018, a 53% increase compared to ten years prior. Globally, IPF affects approximately 3 million people, with about 130,000 patients in the United States, and a median survival of only 35 months after diagnosis. For the elderly, especially men aged 60-70, smoking history is the strongest associated risk factor for the disease. Currently, the main drugs used clinically to delay renal fibrosis include angiotensin II receptor antagonists (such as losartan), angiotensin-converting enzyme inhibitors (such as benazepril), immunosuppressants (such as cyclosporine), glucocorticoids, and statins (such as atorvastatin). However, their indications are mostly concentrated in hypertension, heart failure, or cardiovascular diseases, with anti-renal fibrosis only being a secondary use. Furthermore, some of these drugs have nephrotoxicity and other significant adverse reactions. Regarding pulmonary fibrosis, the currently recommended anti-fibrotic drugs pirfenidone (a TGF-β inhibitor) and nintedanib (a triple tyrosine kinase inhibitor) can delay the decline in forced vital capacity (FVC), but real-world data show that the rate of acute exacerbations and mortality remains high, and 10%–20% of patients discontinue medication due to adverse events such as gastrointestinal reactions and rashes. Therefore, there is an urgent clinical need to develop specific anti-fibrotic drugs that combine clear efficacy with good safety.

[0004] Against this backdrop, the American cockroach (Periplaneta americana L.), a traditional Chinese medicine, has gradually come into the research spotlight. This insect has a long history of medicinal use, first recorded in the *Shennong Bencao Jing* (Shennong's Classic of Materia Medica). It is cold in nature and salty in taste, traditionally believed to have the effects of promoting blood circulation, eliminating masses, detoxifying, and healing wounds, and is often used to treat gastric ulcers, carbuncles, tissue sclerosis, and diseases caused by blood stasis. Modern pharmacological studies have shown that the American cockroach is rich in various bioactive components, including amino acids, polypeptides, and nucleosides, with free amino acids accounting for as much as 43.17% and essential amino acids accounting for 35.37%. In recent years, its extracts (such as Kangfuxin Liquid and Xiaozheng Yigan Tablets) have been widely used in clinical fields such as burn healing, anti-ulcer, and anti-tumor treatment. Notably, multiple studies suggest that American cockroach extracts have anti-inflammatory, antioxidant, immunomodulatory, and anti-fibrotic potential, and can alleviate fibrotic lesions of the lungs, liver, and kidneys through mechanisms such as inhibiting fibroblast activation, regulating cytokine expression, and reducing extracellular matrix deposition.

[0005] Although the American cockroach (Periplaneta americana) shows promising potential in combating organ fibrosis, its pharmacodynamic material basis and mechanism of action remain unclear. Existing formulations are mostly crude extracts with complex compositions, making quality control and in-depth mechanistic studies difficult, thus limiting their development into modern innovative drugs. Therefore, it is necessary to develop novel anti-fibrotic drugs from the American cockroach. Any discovery of such drugs, based on a phenotype-based drug discovery model, would promise significant future market and clinical applications. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide indoline-2-one compounds with anti-organ fibrosis activity and their applications.

[0007] To address the above problems, the present invention proposes the following technical solution: This invention provides an indoline-2-one compound with anti-organ fibrosis activity, the structure of which is shown in general formula (I) below: , In formula (I), R1 is selected from one of an unsubstituted or at least monosubstituted benzene ring, an unsubstituted or at least monosubstituted six- or five-membered aromatic heterocycle, or an unsubstituted or at least monosubstituted aromatic heterocycle. R2 may be selected from one of the following: hydroxyl, O-alkyl of hydroxyl, O-acyl of hydroxyl, O-tert-butoxycarbonyl of hydroxyl, O-silyl of hydroxyl, amino, N-alkyl of amino, N-acyl of amino, N-tert-butoxycarbonyl of amino, thio, S-alkyl of thio, S-acyl of thio, or different halogen substituents.

[0008] Furthermore, in equation (I), It is either a trans double bond or a cis double bond.

[0009] Furthermore, the indoline-2-one compounds include compound ZLYH-24 and its derivatives, the structural formula of which is as follows: .

[0010] Furthermore, the structural formula of the indoline-2-one compound is at least one of the following formulas Z1-Z37: .

[0011] The present invention also provides a method for synthesizing the indoline-2-one compounds with anti-organ fibrosis activity, the chemical reaction formula of which is as follows: ; R1 is selected from one of an unsubstituted or at least monosubstituted benzene ring, an unsubstituted or at least monosubstituted six- or five-membered aromatic heterocycle, or an unsubstituted or at least monosubstituted aromatic heterocycle. R2 may be selected from one of the following: hydroxyl, O-alkyl of hydroxyl, O-acyl substituent of hydroxyl, O-tert-butoxycarbonyl substituent of hydroxyl, O-silyl substituent of hydroxyl, amino, N-alkyl of amino, N-acyl substituent of amino, N-tert-butoxycarbonyl substituent of amino, thio, S-alkyl of thio, S-acyl of thio, or different halogen substituents. It is either a trans double bond or a cis double bond.

[0012] Furthermore, the synthesis method provided by the present invention specifically includes the following steps: mixing two reaction raw materials in a molar ratio of 1:1 and dissolving them in an ethanol solvent, adding 4-methylpiperidine in the same molar ratio, heating to 85-95°C under reflux, and reacting for 4-6 hours to obtain the target product.

[0013] Furthermore, after obtaining the target compound, the process also includes removing the solvent by vacuum distillation of the reaction material, extracting with ethyl acetate, washing the organic phase three times with saturated brine, drying the organic phase with anhydrous magnesium sulfate, and purifying the sample by silica gel column chromatography; or precipitating the solid from the reaction solution in an ice bath, filtering and collecting the solid, washing it with ice-cold ethanol, and if the purity is insufficient, performing column chromatography for further purification.

[0014] The reaction process can be monitored by thin-layer chromatography, and the reaction time can be adjusted according to the degree of reaction of the starting materials.

[0015] The present invention also provides the use of the aforementioned indoline-2-one compounds with antifibrotic activity, or pharmaceutically acceptable salts thereof, in the preparation of antifibrotic drugs.

[0016] Furthermore, the anti-fibrotic treatment specifically includes anti-renal fibrosis, which is caused by various chronic kidney diseases, diabetic nephropathy, etc.; and pulmonary fibrosis, namely idiopathic fibrosis and secondary fibrosis.

[0017] The present invention also provides an antifibrotic pharmaceutical composition comprising one or more of the aforementioned indoline-2-one compounds or their metabolites, prodrugs, and solvates.

[0018] Furthermore, the antifibrotic pharmaceutical composition also includes a pharmaceutically acceptable carrier or excipient.

[0019] Furthermore, the dosage form of the antifibrotic drug includes tablets, capsules, nanoemulsions, or injections.

[0020] Furthermore, the antifibrotic drug composition is a nanoemulsion or nanoparticle formulation.

[0021] Compared with the prior art, the technical effects achieved by the present invention include: This invention provides small molecule compounds with anti-organ fibrosis activity, and further obtains indoline-2-one derivatives with the same anti-fibrosis activity through structural modification. The indoline-2-one derivatives provided by this invention possess a novel anti-fibrosis mechanism by targeting the PARN-regulated TGF-β / Smad signaling pathway, which is beneficial for the development of anti-organ fibrosis drugs.

[0022] The indoline-2-one derivatives provided by this invention can be used to prepare anti-fibrotic drugs. Attached Figure Description

[0023] Figure 1 The results show the screening results of the anti-renal fibrosis activity of the indoline-2-one derivatives of this invention.

[0024] Figure 2To verify the anti-renal fibrosis efficacy of ZLYH-24 in in vitro TGF-β1-induced NRK-52e and NRK-49F cell models, in vivo unilateral ureteral obstruction (UUO) model, adenine-induced model, and folic acid (FA)-induced model; A shows the cytotoxicity of compound ZLYH-24 on rat fibroblasts (NRK-49F) and rat renal tubular epithelial cells (NRK-52E) at concentrations of 5-40 μM; B shows the anti-fibrotic efficacy of compound ZLYH-24 on in vitro NRK-49F and NRK-52E cells at a concentration of 10 μM; C shows the efficacy of ZLYH-24 in treating the in vivo ureteral obstruction (UUO) renal fibrosis model; D shows the efficacy of ZLYH-24 in treating the in vivo folic acid (FA) renal fibrosis model; E shows the efficacy of ZLYH-24 in treating the in vivo adenine (Adenine) renal fibrosis model.

[0025] Figure 3 To investigate the effects of compound ZLYH-24 on the TGF-β / Smad signaling pathway using in vitro TGF-β1-induced NRK-52e and NRK-49F cell models and in vivo UUO and FA fibrotic mouse models; A shows the validation results of ZLYH-24 on the TGF-β / Smad signaling pathway in in vitro NRK-52e and NRK-49F cell models; B shows the validation results of ZLYH-24 on the TGF-β / Smad signaling pathway in in vivo UUO and FA fibrotic mouse models.

[0026] Figure 4 To screen and verify the target proteins of ZLYH-24 in the fight against renal fibrosis using DARTS, SPR, siRNA interference, and molecular docking techniques; A: DARTS experiment combined with proteomics analysis to screen proteins strongly associated with ZLYH-24's anti-renal fibrosis effect; B: siRNA experiment to verify whether the ZLYH-24 compound binds to the PARN protein; C: SPR experiment to verify the binding affinity between the compound and the target protein; D: siRNA experiment to verify the relationship between the target protein PARN and the TGF-β / Smad signaling pathway; E: molecular docking to simulate the binding of the compound ZLYH-24 to the target protein PARN.

[0027] Figure 5 To investigate the therapeutic effect of ZLYH-24 on pulmonary fibrosis using a bleomycin-induced mouse model of pulmonary fibrosis.

[0028] Figure 6 The results show the acute toxicity evaluation of compound ZLYH-24 in mice; where A is the flowchart of the acute toxicity experiment design; B is the body weight change curve of mice in each group of the acute toxicity experiment of compound ZLYH-24; and C is the summary table of organ coefficients of mice in each group of the acute toxicity experiment.

[0029] Figure 7 The results show the subacute toxicity evaluation of compound ZLYH-24 in rats; A is the experimental design of subacute toxicity of compound ZLYH-24; B is the curve of changes in body weight and daily intake of rats in each group of experiments for subacute toxicity of compound ZLYH-24; C is the comparison of blood biochemical and hematological parameters of rats in each group of subacute toxicity experiments of compound ZLYH-24; D is the organ coefficient of rats in each group of subacute toxicity experiments of compound ZLYH-24; E is the HE staining of histopathological sections of rat tissues in each group of subacute toxicity experiments of compound ZLYH-24. The results show that the compound is essentially non-toxic to rats.

[0030] Figure 8 The results are from the HPLC determination of the content of compound ZLYH-24.

[0031] Figure 9 The pharmacokinetic results of different dosage forms of compound ZLYH-24 are shown; where A is the blood concentration-time curve of ZLYH-24 nanoparticles, ZLYH-24 nanoemulsions and ZLYH-24 suspension; B is the blood concentration-time curve of ZLYH24 solution-iv.

[0032] It should be noted that the scale bar for each pathological slide image is 200μm. Detailed Implementation

[0033] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the embodiments described below are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0034] All chemicals and reagents used in the following examples were purchased from market suppliers and used directly without further purification; the temperatures mentioned are in Celsius; and 200-300 mesh silica gel was used for column chromatography.

[0035] In this invention, pharmaceutically acceptable salts refer to salts that can be prepared by conventional chemical methods and are non-toxic to mammals such as humans. The salts of the compounds provided herein can be derived from inorganic or organic acids and bases. Examples of acids include, but are not limited to, hydrochloric acid, sulfuric acid, hydrobromic acid, aminosulfonic acid, phosphoric acid, nitric acid, acetic acid, propionic acid, succinic acid, glycolic acid, stearic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, pyric acid, maleic acid, hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, p-aminobenzenesulfonic acid, 2-acetoxybenzoic acid, fumaric acid, toluenesulfonic acid, methanesulfonic acid, ethanedisulfonic acid, oxalic acid, hydroxyethylsulfonic acid, and trifluoroacetic acid. Examples of bases include, but are not limited to, lithium salts, ammonium salts, calcium salts, copper salts, iron salts, ferrous salts, lithium salts, magnesium salts, manganese salts, manganese salts, potassium salts, sodium salts, zinc salts, primary amine salts, secondary amine salts, and tertiary amine salts. For pharmaceutically acceptable salts and their preparation, please refer to the description in J. Pharm. Sci. 1977, 66, 1-19. Furthermore, the compounds of this invention also include internal salts or zwitterions generated in the in vivo environment.

[0036] This invention provides indoline-2-one compounds with anti-fibrotic activity, the structure of which is shown in general formula (I): , In formula (I), R1 is selected from one of an unsubstituted or at least monosubstituted benzene ring, an unsubstituted or at least monosubstituted six- or five-membered aromatic heterocycle, or an unsubstituted or at least monosubstituted aromatic heterocycle. R2 can be selected from one of the following: hydroxyl group, O-alkyl group of hydroxyl group, O-acyl group of hydroxyl group, O-tert-butoxycarbonyl group of hydroxyl group, O-silyl group of hydroxyl group, amino group, N-alkyl group of amino group, N-acyl group of amino group, N-tert-butoxycarbonyl group of amino group, thio group, S-alkyl group of thio group, S-acyl group of thio group, or different halogen substituents. Examples are given below: Example 1: Synthesis of (E)-5-fluoro-3-(quinoline-6-methylene)indol-2-one (compound Z24, or compound ZLYH-24), the synthetic route is as follows: .

[0037] 3.5 g of 5-fluoroindol-2-one and 3.7 g of quinoline-6-carboxaldehyde were mixed and dissolved in 120 mL of ethanol, and then 2.72 mL of 4-methylpiperidine was added. The mixture was refluxed and stirred at 90 °C for 5 h. After the reaction was completed, the reaction solution was cooled to precipitate, the precipitate was directly filtered, and the precipitate was repeatedly washed with ice-cold ethanol until the color lightened. The precipitate was dried under reduced pressure to obtain 4.6 g of yellow solid product, namely compound ZLYH-24, with a yield of 68% and a purity greater than 95%.

[0038] ESIMS m / z291 [M + H] + ; 1 H NMR (500 MHz, DMSO- d 6) δ H 10.70 (s, 1H), 8.99 (dd, J = 4.2, 1.7 Hz, 1H), 8.47 (dd, J = 8.5, 1.7 Hz, 1H), 8.35 (d, J =2.0 Hz, 1H), 8.16 (d, J = 8.7 Hz, 1H), 8.03 (dd, J = 8.7, 2.0 Hz, 1H), 7.87(s, 1H), 7.62 (dd, J = 8.5, 4.2 Hz, 1H), 7.20 (dd, J = 9.3, 2.6 Hz, 1H), 7.11(brtd, J = 9.0, 2.6 Hz, 1H), 6.89 (dd, J = 8.5, 4.6 Hz, 1H); 13 C NMR (125 MHz, DMSO- d 6) δ C 168.5, 157.9, 156.4, 151.8, 147.9, 139.5, 136.6, 136.5, 132.3, 129.9, 129.5, 129.4, 128.2, 128.2, 127.8, 122.4, 121.7, 121.7, 116.8, 116.7, 111.0, 110.9, 109.6, 109.4. Example 2 ( E The synthesis of 5-chloro-3-(4-hydroxybenzyl)indol-2-one (compound Z7) is described below: .

[0039] 78 mg of 5-chloroindol-2-one and 57 mg of 4-hydroxybenzaldehyde were mixed and dissolved in 6 mL of ethanol, and then 55 μL of 4-methylpiperidine was added. The mixture was refluxed and stirred at 90 °C for 5 h. After the reaction was completed, the reaction solution was cooled to precipitate. The precipitate was filtered directly and washed repeatedly with ice-cold ethanol until the color lightened. The precipitate was dried under reduced pressure to give 88.5 mg of yellow solid product, namely compound Z7, with a yield of 70%.

[0040] ESIMS m / z 272 [M + H] + ; 1 H NMR (500 MHz, DMSO- d 6) δ H 10.70 (s, 1H), 8.44 (d, J = 8.4 Hz, 2H), 7.84 (s, 1H), 7.80 (d, J = 2.2 Hz, 1H), 7.19 (dd, J = 8.1, 2.2 Hz, 1H), 6.88 (d, J = 8.4 Hz, 2H), 6.81 (d, J = 8.1 Hz, 1H); 13 CNMR (125 MHz, DMSO- d 6) δ C 167.7, 161.1, 139.9, 139.1, 135.7, 135.7, 128.0, 127.7, 126.0, 125.7, 122.3, 119.5, 115.8, 115.8, 110.9. Example 3 ( E The synthesis of 5-hydroxy-3-(3-chloro-4-hydroxybenzyl)indol-2-one (compound Z14) is described below: .

[0041] 69 mg of 5-hydroxyindole-2-one and 72 mg of 3-chloro-4-hydroxybenzaldehyde were mixed and dissolved in 5 mL of ethanol, and then 54 μL of 4-methylpiperidine was added. The mixture was refluxed and stirred at 90 °C for 4 h. After the reaction was completed, the reaction solution was cooled to precipitate. The precipitate was filtered directly and washed repeatedly with ice-cold ethanol until the color lightened. The precipitate was dried under reduced pressure to give 86.3 mg of yellow solid product, namely compound Z14, with a yield of 65%.

[0042] ESIMS m / z 288 [M + H] + ; 1 H NMR (500 MHz, DMSO- d 6) δ H 10.93 (brs, 1H),10.29 (s, 1H), 8.99 (brs, 1H), 8.83 (d, J = 2.1 Hz, 1H), 8.15 (dd, J = 8.6,2.1 Hz, 1H), 7.55 (s, 1H), 7.07 (d, J = 2.0 Hz, 1H), 7.03 (d, J = 8.6 Hz, 1H), 6.61 (m, 2H); 13 C NMR (125 MHz, DMSO- d 6) δ C 167.5, 155.3, 152.2, 135.4, 133.7, 133.3, 132.9, 126.7, 126.2, 125.2, 119.5, 116.1, 115.2, 109.7, 106.8. Example 4 ( E The synthesis of 5-hydroxy-3-(3-hydroxy-4-chlorobenzyl)indol-2-one (compound Z15) was carried out via the following route: .

[0043] 158 mg of 5-hydroxyindole-2-one and 165 mg of 3-hydroxy-4-chlorobenzaldehyde were mixed and dissolved in 12 mL of ethanol, and then 124 μL of 4-methylpiperidine was added. The mixture was refluxed and stirred at 90 °C for 4 h. After the reaction was completed, the reaction solution was cooled to precipitate. The precipitate was filtered directly and washed repeatedly with ice-cold ethanol until the color lightened. The precipitate was dried under reduced pressure to give 198.7 mg of yellow solid product, namely compound Z15, with a yield of 65%.

[0044] ESIMS m / z 288 [M + H] + ; 1 H NMR (500 MHz, DMSO- d 6) δ H 10.29 (s, 1H), 7.45 (d, J= 8.3 Hz, 1H), 7.44 (s, 1H), 7.25 (d, J = 2.0 Hz, 1H), 7.06 (m,2H), 6.65 (m, 2H); 13 C NMR (125 MHz, DMSO- d 6) δ C 168.6, 153.4, 151.9, 135.4, 134.5, 134.4, 130.2, 128.7, 121.4, 120.9, 120.8, 116.9, 116.7, 110.6, 110.3. Example 5 ( E The synthesis of 5-hydroxy-3-(1-H-benzimidazol-6-methylene)indol-2-one (compound Z16) is described below: .

[0045] 13 mg of 5-hydroxyindole-2-one and 13 mg of 1-H-benzimidazole-6-carboxaldehyde were mixed and dissolved in 2 mL of ethanol, and then 10 μL of 4-methylpiperidine was added. The mixture was refluxed and stirred at 90 °C for 4 h. After the reaction was completed, the reaction solution was cooled to precipitate. The precipitate was filtered directly and washed repeatedly with ice-cold ethanol until the color lightened. The precipitate was dried under reduced pressure to give 15.8 mg of yellow solid product, namely compound Z16, with a yield of 65%.

[0046] ESIMS m / z 278 [M + H] + ; 1 H NMR (500 MHz, DMSO- d 6) δ H 12.73 (brs, 1H),10.27 (s, 1H), 8.98 (brs, 1H), 8.37 (s, 1H), 7.96 (brs, 1H), 7.74 (s, 1H),7.56 (brd, J = 8.0 Hz, 1H), 7.20 (d, J = 1.8 Hz, 1H), 6.67 (m, 2H); 13 C NMR (125 MHz, DMSO- d 6) δ C169.5, 152.2, 144.2, 137.6, 135.6, 127.2, 124.7, 122.4, 116.8, 110.8, 110.1. Example 6 ( E The synthesis of 5-fluoro-3-(3,4-dimethoxybenzyl)indol-2-one (compound Z18) was carried out via the following route: .

[0047] 88 mg of 5-fluoroindol-2-one and 97 mg of 3,4-dimethoxybenzaldehyde were mixed and dissolved in 10 mL of ethanol, and then 69 μL of 4-methylpiperidine was added. The mixture was refluxed and stirred at 90 °C for 5 h. After the reaction was completed, the reaction solution was cooled to precipitate, the precipitate was directly filtered, and the precipitate was repeatedly washed with ice-cold ethanol until the color lightened. The precipitate was dried under reduced pressure to give 174 mg of yellow solid product, namely compound Z18, with a yield of 80%.

[0048] ESIMS m / z 300 [M + H] + ; 1 H NMR (500 MHz, DMSO- d 6) δ H 10.57 (s, 1H), 8.71 (dd, J = 2.0 Hz, 1H), 7.83 (dd, J = 8.1, 2.0 Hz, 1H), 7.82 (s, 1H), 7.59(dd, J = 9.3, 2.6 Hz, 1H), 7.08 (d, J = 8.1 Hz, 1H), 6.99 (br td, J = 9.0, 2.6 Hz, 1H), 6.80 (dd, J = 8.5, 4.6 Hz, 1H), 3.85 (s, 1H), 3.84 (s, 1H); 13 CNMR (125 MHz, DMSO- d 6) δ C168.0, 159.4, 157.5, 151.9, 148.5, 139.6, 136.8, 128.4, 127.5, 127.5, 124.0, 124.0, 115.5, 114.8, 114.6, 111.6, 110.4, 110.3, 107.2, 107.0, 56.1, 55.8. Example 7 ( E The synthesis of 5-fluoro-3-(3-methoxy-4-hydroxybenzyl)indol-2-one (compound Z19) is described below: .

[0049] 77 mg of 5-fluoroindol-2-one and 77 mg of 3-methoxy-4-hydroxybenzaldehyde were mixed and dissolved in 6 mL of ethanol, and then 60 μL of 4-methylpiperidine was added. The mixture was refluxed and stirred at 90 °C for 5 h. After the reaction was completed, the reaction solution was cooled to precipitate, the precipitate was filtered directly, and the precipitate was repeatedly washed with ice-cold ethanol until the color became light. The precipitate was dried under reduced pressure to give 112.9 mg of yellow solid product, namely compound Z19, with a yield of 78%.

[0050] ESIMS m / z 286 [M + H] + ; 1 H NMR (500 MHz, DMSO- d 6) δ H 10.54 (s, 1H), 8.71 (dd, J = 2.0 Hz, 1H), 7.78 (s, 1H), 7.75 (dd, J = 8.1, 2.0 Hz, 1H), 7.58(dd, J = 9.3, 2.6 Hz, 1H), 6.98 (br td, J = 9.0, 2.6 Hz, 1H), 6.88 (d, J =8.1 Hz, 1H), 6.80 (dd, J = 8.5, 4.6 Hz, 1H), 3.86 (s, 1H); 13 C NMR (125 MHz, DMSO- d 6) δ C168.0, 159.3, 157.5, 150.7, 147.5, 140.1, 136.6, 128.9, 127.8, 127.7, 126.4, 122.9, 116.4, 115.7, 114.5, 114.3, 110.3, 110.2, 106.9, 106.7, 55.9. Example 8 ( E The synthesis of 5-fluoro-3-(3-chloro-4-hydroxybenzyl)indol-2-one (compound Z20) was carried out via the following route: .

[0051] 92 mg of 5-fluoroindol-2-one and 95 mg of 3-chloro-4-hydroxybenzaldehyde were mixed and dissolved in 10 mL of ethanol, and then 72 μL of 4-methylpiperidine was added. The mixture was refluxed and stirred at 90 °C for 4 h. After the reaction was completed, the reaction solution was cooled to precipitate, the precipitate was directly filtered, and the precipitate was repeatedly washed with ice-cold ethanol until the color became light. The precipitate was dried under reduced pressure to give 126.8 mg of yellow solid product, namely compound Z20, with a yield of 72%.

[0052] ESIMS m / z 290 [M + H] + ; 1 H NMR (500 MHz, DMSO- d 6) δ H 10.98 (brs, 1H),10.60 (s, 1H), 7.74 (d, J = 2.0 Hz, 1H), 7.60 (dd, J = 8.1, 2.0 Hz, 1H), 7.59(s, 1H), 7.32 (dd, J = 9.3, 2.6 Hz, 1H), 7.14 (d, J = 8.1 Hz, 1H), 7.08 (brtd, J = 9.0, 2.6 Hz, 1H), 6.86 (dd, J = 8.5, 4.6 Hz, 1H); 13 C NMR (125 MHz, DMSO- d 6) δ C169.2, 158.6, 156.7, 155.4, 139.5, 137.1, 132.1, 130.2, 126.4, 126.3, 122.5, 122.4, 120.6, 117.3, 116.6, 116.4, 111.2, 111.2, 109.7, 109.5. Example 9 ( E The synthesis of 5-fluoro-3-(1,4-benzodioxane-6-methylene)indol-2-one (compound Z23) is described below: 66 mg of 5-fluoroindol-2-one and 72 mg of 1,4-benzodioxane-6-carboxaldehyde were mixed and dissolved in 6 mL of ethanol, and then 52 μL of 4-methylpiperidine was added. The mixture was refluxed and stirred at 90 °C for 4 h. After the reaction was completed, the reaction solution was cooled to precipitate. The precipitate was filtered directly and washed repeatedly with ice-cold ethanol until the color lightened. The precipitate was dried under reduced pressure to give 104.3 mg of yellow solid product, namely compound Z23, with a yield of 80%.

[0053] ESIMS m / z 298 [M + H] + ; 1 H NMR (500 MHz, DMSO- d 6) δ H 7.59 (s, 1H), 7.37 (dd, J = 9.3, 2.6 Hz, 1H), 7.25 (m, 2H), 7.08 (br td, J = 9.0, 2.6 Hz, 1H), 7.03 (d, J = 8.1 Hz, 1H), 6.86 (dd, J = 8.5, 4.6 Hz, 1H); 13 C NMR (125MHz, DMSO- d 6) δ C 169.3, 158.5, 156.7, 145.8, 143.9, 139.6, 137.9, 127.4, 126.2, 123.7, 122.5, 118.8, 118.0, 116.6, 116.4, 111.2, 111.1, 109.7, 109.5. Example 10 ( EThe synthesis of 5-fluoro-3-(1-H-benzimidazol-6-methylene)indol-2-one (compound Z26) is described below: .

[0054] 0.50 g of 5-fluoroindol-2-one and 0.48 g of 1H-benzimidazole-6-carboxaldehyde were mixed and dissolved in 5 mL of ethanol, and then 390 μL of 4-methylpiperidine was added. The mixture was refluxed and stirred at 90 °C for 4 h. After the reaction was completed, the reaction solution was cooled to precipitate. The precipitate was directly filtered and repeatedly washed with ice-cold ethanol until the color lightened. The precipitate was dried under reduced pressure to give 0.55 g of yellow solid product, namely compound Z26, with a yield of 60%.

[0055] ESIMS m / z 280 [M + H] + ; 1 H NMR (600 MHz, DMSO- d 6) δ H 12.76 (brs, 1H),10.63 (s, 1H), 8.38 (s, 1H), 8.00 (brs, 1H), 7.87 (s, 1H), 7.75 (brd, J = 8.2Hz, 1H), 7.58 (brd, J = 8.3 Hz, 1H), 7.41 (dd, J = 9.5, 2.7 Hz, 1H), 7.07(td, J = 8.9, 2.6 Hz, 1H), 6.87 (dd, J = 8.5, 4.7 Hz, 1H); 13 C NMR (150 MHz, DMSO- d 6) δ C 168.9, 157.9, 156.4, 144.1, 139.3, 139.1, 125.7, 123.8, 122.3, 122.2, 116.1, 115.9, 110.7, 110.7, 109.0, 108.9. Example 11 ( E The synthesis of 5-hydroxy-3-(3,4-bis(ethoxymethoxy))indol-2-one (compound Z30) was carried out via the following synthetic route: .

[0056] 15 mg of 5-hydroxyindole-2-one and 25 mg of 3,4-bis(ethoxymethoxy)benzaldehyde were mixed and dissolved in 2 mL of ethanol, and then 12 μL of 4-methylpiperidine was added. The mixture was refluxed and stirred at 90 °C for 4 h. After the reaction was completed, the reaction solution was cooled to precipitate. The precipitate was filtered directly and washed repeatedly with ice-cold ethanol until the color lightened. The precipitate was dried under reduced pressure to give 24.9 mg of a yellow solid product, namely compound Z30, with a yield of 65%. ESIMS m / z 386 [M + H] + ; 1 H NMR (600 MHz, DMSO- d 6) δ H 10.25 (s, 1H), 8.96 (s, 1H), 7.49 (s, 1H), 7.46 (d, J = 2.1 Hz, 1H), 7.32 (dd, J = 8.5, 2.1 Hz, 1H), 7.24 (d, J = 8.5 Hz, 1H), 7.19 (d, J =2.3 Hz, 1H), 6.68 (d, J = 8.3 Hz, 1H), 6.65 (dd, J = 8.3, 2.3 Hz, 1H), 5.32(s, 2H), 5.27 (s, 2H), 3.71 (m, 4H), 1.16 (t, J = 7.0 Hz, 3H), 1.12 (t, J =7.0 Hz, 3H); 13 C NMR (150 MHz, DMSO- d 6) δ C 168.9, 151.9, 148.3, 146.8, 135.4, 135.2, 128.1, 127.0, 124.1, 121.7, 117.6, 116.4, 116.3, 110.4, 109.9, 93.6, 93.3, 64.0, 64.0, 15.0, 14.9. Experiment 1: Screening of in vitro antifibrotic activity of indoline-2-one compounds provided in this invention. The frozen NRK-52e cells were quickly extracted from the liquid nitrogen tank and gently thawed in a 37°C water bath. After drying, the cryovials were sprayed with 75% alcohol. The completely thawed NRK-52e cell cryovials were then quickly transferred to a clean bench. DMEM medium (complete medium) containing 10% fetal bovine serum (FBS) was preheated in a 37°C water bath. A 15 mL sterile centrifuge tube was taken, and 3.0 mL of complete medium was added. The thawed cell suspension was then transferred to the centrifuge tube. The cells were centrifuged at 1000 rpm for 3 minutes, the supernatant was discarded, and 3 mL of complete medium was added again to resuspend and mix well. The mixture was then transferred to a culture dish and placed in a cell culture incubator to observe the cell condition after attachment. The cells were observed to be in normal condition. Once the cell density reached approximately 90%, 2 mL of 0.25% trypsin digest was added for 2 minutes, followed by 2.5 mL of complete medium to stop the digestion. After collecting and centrifuging to resuspend the cells and re-counting them, dilute them sequentially with complete culture medium according to the experimental requirements. In 12-well plates, divide the cells at a ratio of 1 × 10⁶ cells / well. 5 After seeding cells at a density of 1 cell / well, the cells were incubated at 37 °C in a 5% CO2 incubator. Once the cells reached 60%-70% confluence, they were starved in blank medium (serum-free medium) for 6 hours. Then, the medium was replaced with medium containing 10 ng / μL TGF-β1 and 2% FBS to induce fibrosis. Compound Z1-Z37 (working concentration 20 μmol / L) was added simultaneously. After incubation for 48 hours, all remaining medium was removed, and the cells were washed with PBS. 100 μL of pre-chilled RIPA lysis buffer (containing 1% protease inhibitor and 1% phosphatase inhibitor) was added to each well. Lysis was performed on ice for 10 min, and the cells were scraped off and the suspension transferred to 1.5 mL EP tubes. The cells were incubated at 4 °C for 15 min for complete lysis. Centrifuge at 4 ℃ and 12000 rpm for 10 minutes, retain the supernatant and transfer it to a new centrifuge tube. Determine the protein concentration using a BCA kit, add loading buffer, heat to denature, and finally perform a Western blotting experiment.

[0057] Prepare SDS-PAGE polyacrylamide gels, and prepare the necessary separating and concentrating gels for the experiment. Place the gels in the electrophoresis tank, taking care not to tear the sample wells of the polyacrylamide gel when removing the comb. After removing the comb, fill the assembled tank with electrophoresis buffer. Add 30 μg of protein sample to each well. After turning on the instrument, perform electrophoresis at a constant voltage of 80 V for 20-30 min until the sample moves from the concentrating gel to the separating gel. Adjust the voltage to 130 V and continue electrophoresis at a constant voltage for 1 hour until the leading bromophenol blue indicator reaches the bottom of the gel. Prepare a PDVF membrane of appropriate size beforehand, activate it in anhydrous methanol for 30 seconds, and then equilibrate it in pre-cooled transfer buffer. Place the (anodine) sponge-filter paper-membrane-gel-filter paper-sponge (cathode) in the transfer clamp in the following order, remove all air bubbles, clamp the transfer clamp, and place it in the transfer tank. Fill the tank with pre-cooled transfer buffer and perform transfer at a constant current of 250 mA at low temperature for 2 hours. After transfer, wash with TBST, then transfer the membrane to a 5% skim milk solution (dissolved in TBST) and block on a shaker at room temperature for 1 hour. After blocking, wash away excess skim milk with TBST, then place in the target antibody working solution (prepared and stored according to the target antibody instructions) and incubate overnight at 4 °C. The next day, recover the primary antibody, wash with TBST for 10 min, repeat 3 times, then pour in the corresponding genus-derived secondary antibody working solution (prepared and stored according to the target antibody instructions) and incubate on a shaker at room temperature for 1 hour. After primary and secondary antibody treatment, wash the target band three times with TBST, 10 minutes each time on a shaker. Prepare the developing solution according to the instructions, immerse the membrane in the developing solution for 1 minute, and then place it in the developing apparatus for development and photography.

[0058] The results are as follows Figure 1 As shown, the initial screening results indicated that compounds Z8, Z10, Z20, Z26, Z28, and Z30 were cytotoxic, while compound Z25 was insoluble. Experimental results showed that compounds Z2, Z11, Z18, Z23, Z24, Z26, Z32, Z33, and Z34 significantly reduced fibrosis markers such as Fibronectin, CTGF, and Collagen I. Based on anti-fibrotic effects and solubility, compound Z24 (ZLYH-24) performed best.

[0059] Experiment 2: Detection of the in vivo and in vitro anti-renal fibrosis activity of compound ZLYH-24 Logarithmically growing NRK-52e and NRK-49F cells were centrifuged, resuspended, and counted. The cell suspension was serially diluted with complete culture medium and seeded at a density of 6000 cells / well in 96-well plates. The plates were incubated for 12 hours for synchronization. Then, medium containing 2% FBS was used, and the drug stock solution was diluted with DMSO to obtain a drug concentration gradient for ZLYH-24. The solution was added and immediately mixed (avoiding contact with cells). Three replicates were set up for each drug concentration, and the cells were then cultured again. After 48 hours, the original culture medium was discarded, and CCK8 working solution was added. The cells were incubated for approximately 1 hour, and the optical density (OD) value at 450 nm was measured using a cell standard to determine cell viability. See [link to relevant documentation]. Figure 2 As shown in A, the ZLYH-24 compound is non-toxic to both NRK-52e and NRK-49F within the concentration range of 5-40 μM.

[0060] After cells (NRK-49F, NRK-52e) reached 60%-70% confluence, they were synchronized with serum-free medium for 12 hours, then incubated with 2% FBS medium supplemented with ZLYH-24 for 1 hour, followed by induction with TGF-β1. After 48 hours, cells were lysed, proteins were extracted, quantified, denatured, and then the expression of CTGF, α-SMA, Collagen-I, and Fibronectin was detected by Western blotting. The results showed that... Figure 2 ZLYH-24 (B in the formula) can effectively reduce the expression level of fibrotic proteins.

[0061] UUO model mice: Male 6-8 week old C57BL / 6J mice were randomly divided into four groups: sham surgery group, UUO model group, UUO-D1 group (administration started on the day after UUO surgery), and UUO-D7 group (administration started on the 8th day after UUO surgery), with 6 mice in each group. Except for the Control group, mice in the other groups were general anesthetized, fixed, and the left ureter was bluntly dissected through a dorsal incision on the left side. The ureter was ligated once distally and once proximally below the kidney, and then sutured. In the Sham group, only a dorsal incision was made without ligation, and the inner and outer skin incisions were sutured. UUO-D1 group: ZLYH-24 25 mg / kg was administered by gavage once daily, starting on day 1 of UUO modeling. UUO-D7 group: ZLYH-24 was administered by gavage once daily, starting on day 8 of UUO modeling. UUO model group: The corresponding volume of the solution was administered by gavage once daily, starting on day 1 of UUO modeling. On the 14th day after surgery, the mice were euthanized by cervical dislocation, and the left kidney tissue was harvested. The kidney tissue was divided into three equal parts. The middle part was fixed at room temperature in 4% paraformaldehyde or tissue fixative for staining of pathological sections, and the remaining parts were stored at -80 ℃ for later use.

[0062] Folic acid (FA) model mice: Male 6-8 week old C57BL / 6J mice were randomly divided into four groups: Control group, FA model group, FA-low-dose group, FA-medium-dose group, and FA-high-dose group, with 6 mice in each group. 100 mg of folic acid was weighed and dissolved in 5 mL of 0.3M NaHCO3 (pH=7.4) to prepare a working solution of 20 mg / mL. Except for the Control group, the mice in the other groups were intraperitoneally injected with folic acid (250 mg / kg). FA-low-dose group: ZLYH-24 12.5 mg / kg was administered by gavage to each mouse daily; FA-medium-dose group: ZLYH-24 25 mg / kg was administered by gavage to each mouse daily; FA-high-dose group: ZLYH-24 50 mg / kg was administered by gavage to each mouse daily; the Control group was given the corresponding volume of solvent daily. On day 28 after modeling, the mice were sacrificed by cervical dislocation, and the left kidney tissue was harvested. The kidney tissue was divided into three equal parts. The middle part was fixed at room temperature in 4% paraformaldehyde or tissue fixative for staining of pathological sections, and the remaining parts were stored at -80 ℃ for later use.

[0063] Adenine mouse model method: Male 6-8 week old C57BL / 6J mice were randomly divided into four groups: Control group, Model group, Low-dose group (12.5 mg / kg), Medium-dose group (25 mg / kg), and High-dose group (50 mg / kg), with 10 mice in each group. 100 mg of adenine powder was dissolved in 10 mL of physiological saline. Except for the Control group, the mice in the other groups were gavaged with the adenine suspension daily. The blank control group was given the same volume of physiological saline. The groups that were administered the drug simultaneously with modeling were gavaged daily, while the blank control group and the model group were given the corresponding volumes of solvent. After 28 days of continuous gavage, the mice were sacrificed by cervical dislocation, and the left kidney tissue was harvested. The kidney tissue was divided into three equal parts. The middle part was fixed in 4% paraformaldehyde or tissue fixative at room temperature for pathological staining, and the remaining parts were stored at -80 ℃ for later use.

[0064] The results are as follows Figure 2 As shown in the CE diagram, ZLYH-24 reduced the expression levels of fibrosis-related proteins in both prophylactic and therapeutic administration. In the model group, glomeruli shrank, renal tubules dilated, Bowman's capsule walls thickened, and a large amount of collagen fibers were deposited in the interrenal spaces. After ZLYH-24 treatment, fibrosis protein levels decreased, renal interstitial inflammatory cells decreased, the degree of fibrosis lessened, some renal tubules returned to normal morphology, renal interstitial collagen fiber deposition decreased, and the area of ​​renal tubular atrophy decreased. The high-dose group showed a more significant therapeutic effect compared to the low-dose group. The activity of ZLYH-24 was reproduced in both the FA and adenine models. Therefore, ZLYH-24 exhibits strong anti-renal fibrosis activity both in vivo and in vitro.

[0065] Experiment 3 investigates the effects of compound ZLYH-24 on the classical TGF-β / Smad signaling pathway. NRK-52e and NRK-49F cells in the logarithmic growth phase were digested, centrifuged, and then processed at a concentration of 1×10⁻⁶ cells / cells. 5 Cells were seeded at a density of 1 cells / well in 12-well plates and incubated. After 24 hours, the medium was replaced with 4% serum after synchronization treatment, ZLYH-24 (10 μM) was added, and the plates were incubated for 1 hour. Then, TGF-β1 was added for induction for 2 hours. Cells were lysed, proteins were extracted, quantified, denatured, and then subjected to further detection.

[0066] Take 50-100 mg of kidney tissue (fresh, -80 ℃, or stored in liquid nitrogen are all acceptable), place it in a 2.0 mL EP tube, add 0.5 mL of RIRA lysis buffer (containing protease and phosphatase inhibitors), grind, and incubate on ice for 10 min for complete lysis. Centrifuge at 4 ℃ and 12000 rpm for 10 min, retain the supernatant and transfer it to a new EP tube, dilute it, and determine the protein concentration using a BCA kit. Add loading buffer, heat to denature, and perform subsequent detection.

[0067] The results are shown below. Figure 3 The small molecule compound ZLYH-24, containing components A and B, significantly inhibited Smad3 phosphorylation both in vitro and in vivo without affecting Smad2 phosphorylation levels. This demonstrates that ZYH-24 inhibits TGF-β / Smad pathway activation by specifically inhibiting Smad3 phosphorylation, thereby effectively preventing and treating renal fibrosis.

[0068] Experiment 4: Screening and Validation of Anti-renal Fibrosis Targets for ZLYH-24 Compound Drug affinity response-target stability assay (DARTS) is a label-free method for identifying and detecting the interaction between small molecules and their target proteins. The principle is that when a natural small molecule drug binds to a target protein, the physicochemical properties of the target protein change. The small molecule drug can protect the target protein, giving it resistance to protease hydrolysis. By combining this with quantitative proteomics to compare changes in the experimental and control groups, potential target proteins of the natural drug small molecule can be identified. This experiment uses DARTS technology, combined with siRNA interference, SPR, and molecular docking techniques, to explore the anti-fibrotic target of the compound ZLYH-24.

[0069] Kidney tissues from mice in the model group and the drug-treated group were excised and placed in centrifuge tubes. Lysis buffer was added, and the tissues were ground and lysed. After quantification using the BCA method, Pronase enzyme was added at a ratio of 1 μL / 1000 μg protein, and the mixture was digested at 37 °C for 30 minutes. The reaction was then terminated on ice. Loading buffer was added, and the mixture was heated to denature the samples. The samples were stored at -80 °C or used for subsequent assays.

[0070] Samples were separated by SDS-PAGE gel electrophoresis and detected by Coomassie brilliant blue staining. Bands were compared between the model group and the drug-treated group. Differentially enhanced protein bands due to drug protection were extracted from the drug-treated group, and the extracted gel strips were sent to a mass spectrometry platform for quantitative proteomics analysis. The analysis results, through molecular weight and abundance screening, identified and screened potential drug target proteins. The results are shown in [Figure number missing]. Figure 4 A in the middle.

[0071] siRNAs were designed based on the target protein sequences, and the siRNA sequences are shown in Table 1. NRK-52e cells were seeded in 12-well plates. After 12 hours of cell growth, the medium was replaced with 0.8 mL of blank (serum-free) medium. Following the transfection reagent instructions, 3 μL of siRNA working solution was added to each well, and the blank group was treated with an empty vector sequence. After 12 hours of transfection, the medium was replaced with ZLYH-24 (20 μM), and the cells were incubated for 1 hour. TGF-β1 was then added for induction for 48 hours. Cell proteins were extracted and subsequently analyzed. The results are shown in Table 1. Figure 4 In the case of B, when PARN (Poly(A)-specific ribonuclease) protein was downregulated, the anti-fibrotic activity of ZLYH-24 was weakened. At the same time, fibrosis-related ECM proteins Fibronectin, Collagen I, and α-SMA were also downregulated, suggesting that PARN is a target protein of ZLYH-24 and a key protein in the process of renal fibrosis.

[0072] Table 1. Parn siRNA sequence Surface plasmon resonance (SPR) experiments were performed using a CM5 chip on a Biacorex100 molecular interaction instrument. Since the coupling rate between PARN protein and the chip varied at different pH levels, the protein was dissolved in NaCH3COOH aqueous solutions at pH values ​​of 6, 5.5, 5, 4.5, 4, and 3.5 to achieve a target protein concentration of approximately 10 μg / mL. PBS was used as the buffer to couple the protein to the chip at different pH conditions. Screening revealed that a solution at pH 4.0 yielded the highest coupling amount of PARN protein. A 20 μg / mL concentration of PARN protein was then coupled to the chip on the instrument for further experiments. The instrument used a PBS solution containing 5% DMSO as a solvent to prepare and adjust the concentration of compound ZLYH-24 to 5 μM. The analyte concentration was then diluted to 2.5 μM, 1.25 μM, 0.625 μM, 0.3125 μM, 0.15625 μM, 0.078125 μM, and 0.0390625 μM with 5% DMSO buffer. Standard dissolution curves were plotted using 5.7%, 5.3%, 4.9%, and 4.5% DMSO buffer. The instrument was then started. Experimental results showed (…). Figure 4 (C in the original text), KD = 6.773 × 10 -8 M; This indicates that ZLYH-24 has a strong binding affinity to PARN protein.

[0073] Log-grown NRK-52 cells were transfected with PARN-siRNA for 12 hours, then the culture medium was changed and ZLYH-24 (20 μM) was added. The cells were incubated for 1 hour, followed by induction with TGF-β1 for 2 hours. Cell proteins were extracted and TGF-β / Smad pathway proteins were detected. Results are shown below. Figure 4 Downregulation of D. PARN protein weakened the inhibitory effect of ZLYH-24 on Smad3 phosphorylation. Molecular docking results are as follows: Figure 4 As shown in Figure E, ZLYH-24 has the potential to bind to PARN proteins. ZLYH-24 forms a stable hydrogen bond network with SER-467 (2.78 Å), THE-334 (2.91 Å), and ARG-419 (2.99 Å). Furthermore, hydrophobic interactions with residues such as MET-418 and TRP-468 further solidify the formation of the ligand-receptor complex. This suggests that ZLYH-24 plays a role in preventing and treating renal fibrosis by targeting PARN to inhibit Smad3 phosphorylation and suppressing TGF-β / Smad pathway activation.

[0074] Experiment 5: Detection of the anti-pulmonary fibrosis activity of ZLYH-24 in a model Male C57BL / 6J mice aged 6-8 weeks were randomly divided into four groups: Control group, Model group (bleomycin: BLM), Positive control group (pirfenidone), ZLYH-24 low-dose group, and ZLYH-24 high-dose group. After anesthesia, mice were fixed on a worktable, and the trachea was exposed by blunt dissection of the neck skin. The Control group received saline infusion, while the other groups received bleomycin infusion. Immediately after infusion, the mice were upright and their backs were gently patted to ensure even distribution of the drug in both lungs. Drug administration began on the second day after model establishment: Positive control group: 200 mg / kg pirfenidone (PFD) per mouse per day by gavage; Low-dose group: 25 mg / kg ZLYH-24 per mouse per day by gavage; High-dose group: 50 mg / kg ZLYH-24 per mouse per day by gavage. Mice were sacrificed on day 28 after model establishment by myelination. The left lung was removed and fixed at room temperature in 4% paraformaldehyde or tissue fixative for pathological staining. The remaining tissue was stored at -80 °C for later use.

[0075] See results Figure 5 Compared with the control group, the model group mice showed alveolar atrophy, septal thickening, significant inflammatory cell infiltration, and significantly increased collagen fiber deposition in the lung tissue. ZLYH-24 treatment improved lung tissue lesions to a certain extent, and the level of fibrosis-related proteins was significantly downregulated. The effect was dose-dependent, with the high-dose ZLYH-24 (50 mg / kg) group being superior to the pirfenidone group.

[0076] Experiment 6: In vivo toxicity evaluation of compound ZLYH-24 in animals Acute toxicity test: 6-8 week old C57BL / 6J mice were selected as experimental subjects; a control group and a maximum dose group (2 g / kg) were set up, with 10 mice in each group, half male and half female; weight changes were recorded, and the mice's behavior, mental state, and mortality were observed; on day 14, the mice were euthanized by cervical dislocation, and all organs were collected. The organ coefficients were calculated to determine the toxicity of ZLYH-24 in vivo. The results are shown below. Figure 6 The organ coefficient and body weight curve showed no significant difference, indicating that the ZLYH-24 compound is non-toxic at a dose of 2 g / kg.

[0077] Subacute toxicity test ( Figure 7 (A) SD rats were selected as the research subjects, with 12 rats in each group, half male and half female. Three doses were set: 100 mg / kg, 250 mg / kg, and 500 mg / kg, administered by gavage daily. Changes in rat body weight and daily intake were recorded. Rats were sacrificed on day 30, and blood and organs were collected. Hematological parameters, blood biochemical indicators, and organ coefficients were measured, and pathological staining was performed. These results were used as detection indicators. The experimental results are shown in […]. Figure 7In the study, compound ZLYH-24 showed no significant toxicity in rats at all three doses, with organ coefficients and hematological parameters remaining essentially normal. Pathological examination of liver and kidney sections revealed no significant differences between the treated and control groups, further demonstrating the lack of significant toxicity of compound ZLYH-24 in vivo. Furthermore, the uric acid content decreased with increasing concentration, suggesting that compound ZLYH-24 may have some efficacy against diseases causing abnormal uric acid levels.

[0078] Example 12: Content determination and nanoform preparation of compound ZLYH-24 obtained in Example 1. The chromatographic conditions are as follows: Column: CE553-C18-CB (5 μm 100Å 250 mm × 4.6 mm); Mobile phase: Acetonitrile / water (60 / 40); Flow rate: 1 mL / min; Detection wavelength: 400 nm; Injection volume: 20 µL; Column temperature: 25℃.

[0079] See results Figure 8 The chromatographic peak of compound ZLYH-24 was a single peak without interference from impurity peaks, with a retention time of 4.25 min. Furthermore, the methanol solution did not interfere with the elution of ZLYH-24, indicating that the chromatographic method has good specificity.

[0080] Accurately weigh 2.5 mg of compound ZLYH-24 into a 5 mL volumetric flask, add chromatographic methanol, sonicate to fully dissolve the drug, shake well, and dilute to 5 mL to obtain a stock solution with a concentration of 500 µg / mL. Take the above stock solution and dilute it sequentially with chromatographic methanol to 250 µg / mL, 100 µg / mL, 50 µg / mL, 25 µg / mL, 10 µg / mL, 5 µg / mL, 1 µg / mL, and 0.1 µg / mL. Then, inject 20 µL of each concentration sample sequentially. Perform linear regression of peak area (Y) against drug concentration (X) to obtain the standard curve equation. The standard curve equation for ZLYH-24 by HPLC is y = 0.2143x + 0.4228, R0. 2 = 0.9997, with a linear range of 0.1 µg / mL to 500 µg / mL.

[0081] Preparation of ZLYH-24 drug nanoparticles Preparation method: The preparation was carried out using a media grinding method, specifically as follows: 25 mg of TPGS was weighed into a vial and ultrasonically dispersed with 4.5 mL of deionized water. 75 mg of ZLYH-24 was added and ultrasonically dispersed again. 10 g of zirconia beads and a magnetic stir bar were added, and the mixture was stirred magnetically for 5 h (300 rpm). After 5 h, the entire drug solution was transferred out, and the zirconia beads were rinsed with 0.5 mL of deionized water until colorless. The rinsed beads were then combined with the transferred drug solution. The mixture was ultrasonicated for 15 min under ice bath conditions using a 300 W probe (on: 3 s, off: 2 s) to obtain ZLYH-24 drug nanoparticles.

[0082] Preparation of ZLYH-24 drug nanoemulsion Lipophile WL 1349 and Labrasol were used as the oil phase, and TPGS aqueous solution (1 mg / mL) was used as the emulsifier.

[0083] Preparation method: Weigh 5 mg of ZLYH-24 and dissolve it in 100 mg of Lipophile WL 1349 and 25 mg of Labrasol. Dissolve by sonication. Add 2 mg of TPGS to 2 mL of water to form the aqueous phase (1 mg / mL) and sonicate at 45℃. Add the aqueous phase to the oil phase. Sonicate at 250 W for 5 min (5 s on, 5 s off), then homogenize at 1560 bar at room temperature and high pressure for 15 cycles to prepare the emulsion.

[0084] The oral bioavailability of two nano-formulations in rats was studied to verify their effectiveness in improving the oral absorption of ZLYH-24. The results are shown in [Figure Number]. Figure 9Rats weighing approximately 200 g were deeply anesthetized by intraperitoneal injection of 1% sodium pentobarbital (0.4 mL / 100 g). After successful anesthesia, the rats were fixed in a supine position on the operating table. A longitudinal incision of approximately 0.5 cm was made upwards from the middle of the left clavicle, and the subcutaneous tissue was bluntly dissected to expose the jugular vein (characterized by a slight pulsation at the thoracic inlet, with a diameter of approximately 2-4 mm). The distal end was ligated. A small incision was made in the jugular vein using ophthalmic scissors, and a silicone tube was inserted from the incision towards the heart, approximately 2.5 cm in length. The silicone tube was ligated and secured to the vessel at the ligation point. The remaining portion of the silicone tube was passed subcutaneously to the back and exited, where it was ligated again. The back and neck wounds were sutured with a suture needle. 0.2 mL of 1% heparin was injected through the silicone tube in the back, and a stainless steel plug was inserted to prevent clotting and bleeding. ZLYH-24 suspension (suspended in 0.5% CMC-Na), ZLYH-24 nanoparticles, ZLYH-24 nanoemulsion, and ZLYH-24 solution-iv (iv. dissolved in 10% DMSO, 40% polyoxyethylene castor oil, and 50% physiological saline) were administered to 9 rats per group at a dose of 25 mg / kg. 50 μL of rat plasma was collected and 200 μL of a mixture of [acetonitrile / methanol (8:2 / v,v)] + 0.1% formic acid containing an internal standard solution (concentration 5 ng / mL) was added for precipitation. The mixture was vortexed for approximately 3 min, centrifuged at 13000 rpm for 10 min at 4℃, and the supernatant was quantitatively aspirated for LC-MS / MS analysis. Simultaneously, a standard curve was prepared using blank plasma.

[0085] The plasma concentration-time curves of the two ZLYH-24 nano-formulations and ZLYH-24 suspension are as follows: Figure 9 As shown on the left, the blood concentration-time curve of ZLYH-24 solution-iv is as follows. Figure 9 (Right) As shown. Pharmacokinetic parameters in rats are shown in Table 2. The results show that the plasma concentration of ZLYH-24 in the ZLYH-24 nanoparticle group was significantly higher than that in the suspension group, and the Cmax of the ZLYH-24 nanoparticle group was the highest among all experimental groups, at 36.502±17.122 ng / mL. Compared with the suspension group, the Tmax of both nanoparticle formulations was relatively prolonged, with the Tmax values ​​of the nanoparticle group and the nanoemulsion group being 1.25 times and 1.22 times that of the suspension group, respectively. The AUC0→∞ of the ZLYH-24 nanoparticle group was 383.672±203.822 ng / (mL*h), which was 2.74 times that of the ZLYH-24 suspension group. However, the ZLYH-24 nanoemulsion group showed no improvement compared to the ZLYH-24 suspension group.

[0086] The half-life (t1 / 2) of ZLYH-24 solution after intravenous injection was 24.67±12.12 h. Unlike intravenous injection, ZLYH-24 suspension, nanoemulsion, and nanoparticles were administered non-intravenously (Tmax 2.72 h, 4.31 h, and 4.20 h, respectively), and their half-lives were affected by both absorption and elimination. Specifically, the half-life of the suspension was 18.93±28.455 h, the nanoemulsion was 10.452±5.925 h, and the nanoparticles were 11.016±2.988 h.

[0087] In non-intravenous formulations, ZLYH-24 nanoparticles significantly improved drug exposure (AUC0₋∞ reached 383.67 ng / (mL·h), relative bioavailability 72.79%, Cmax 36.50 ng / mL, all significantly better than suspensions and nanoemulsions), while their half-life (approximately 11 h) was significantly shorter than that of suspensions (approximately 18.9 h).

[0088] The conclusion is that preparing ZLYH-24 into nanoparticles significantly improves oral bioavailability and blood drug concentration while effectively shortening the blood half-life (compared to suspension). Pharmacokinetic characteristics indicate that this compound has drug-like properties.

[0089] Absolute bioavailability (based on AUC) of ZLYH-24 nanoparticles, ZLYH-24 suspension, and ZLYH-24 nanoemulsion. 0→∞ The calculated bioavailability (CBA) values ​​were 72.79%, 26.59%, and 24.21%, respectively. The relative bioavailability of ZLYH-24 nanoparticles and ZLYH-24 nanoemulsions were 273.76% and 91.05% of that of the oral ZLYH-24 suspension, respectively. The bioavailability of the ZLYH-24 nanoparticle group was 2.73 times that of the ZLYH-24 suspension group, indicating that the oral bioavailability of ZLYH-24 nanoparticles was significantly improved.

[0090] Table 2. Pharmacokinetic parameters of ZLYH-24 in each treatment group (n=9, mean ± SD) Note: (*P<0.05; ***P<0.001 vs ZLYH24 solution -iv) (&P<0.05 vs ZLYH 24 suspension).

[0091] In summary, we believe that ZLYH-24 is the world's first anti-fibrotic molecule targeting PARN. PARN directly affects the post-transcriptional expression of key factors in the TGF-β / Smad pathway (such as Smad3) by regulating mRNA stability, providing a novel intervention point for anti-fibrotic therapy.

[0092] Compared with traditional direct TGF-β inhibitors, ZLYH-24 achieves "precise intervention" by targeting PARN, avoiding the immune side effects caused by systemic TGF-β inhibition and improving safety.

[0093] The indoline-2-one compounds provided by this invention possess novel structures derived from natural sources and formed through structural optimization. They exhibit a novel anti-fibrotic mechanism by targeting PARN to regulate the TGF-β / Smad signaling pathway (specifically inhibiting Smad3 phosphorylation), making them a first-in-class innovative drug with a novel structure and mechanism. Given that the TGF-β / Smad signaling pathway is a major pathological mechanism of organ fibrosis, these compounds hold promise for use in the preparation of drugs for the treatment of organ fibrosis. Experimental data also indicate that these compounds have the potential to treat pulmonary fibrosis, including idiopathic interstitial pulmonary fibrosis, a rare disease.

[0094] In the above embodiments, the descriptions of each embodiment have different focuses. For parts that are not described in detail in a certain embodiment, please refer to the relevant descriptions in other embodiments.

[0095] The above description describes specific embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any person skilled in the art can easily conceive of various equivalent modifications or substitutions within the technical scope disclosed in the present invention, and these modifications or substitutions should all be covered within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. An indoline-2-one compound with anti-organ fibrosis activity, characterized in that, The structure of the indoline-2-one compounds is shown in the following general formula (I): ， In formula (I), R1 is selected from one of an unsubstituted or at least monosubstituted benzene ring, an unsubstituted or at least monosubstituted six- or five-membered aromatic heterocycle, or an unsubstituted or at least monosubstituted aromatic heterocycle. R2 may be selected from one of the following: hydroxyl, O-alkyl of hydroxyl, O-acyl of hydroxyl, O-tert-butoxycarbonyl of hydroxyl, O-silyl of hydroxyl, amino, N-alkyl of amino, N-acyl of amino, N-tert-butoxycarbonyl of amino, thio, S-alkyl of thio, S-acyl of thio, or different halogen substituents.

2. The indoline-2-one compound with organ anti-fibrotic activity as described in claim 1, characterized in that, In formula (I), It is either a trans double bond or a cis double bond.

3. The indoline-2-one compound with anti-organ fibrosis activity as described in claim 1, characterized in that, The indoline-2-one compounds mentioned above are compound ZLYH-24 and its derivatives. The structural formula of compound ZLYH-24 is as follows: 。 4. The indoline-2-one compound with anti-organ fibrosis activity as described in claim 1, characterized in that, The structural formula of the indoline-2-one compound is selected from at least one of formulas Z1-Z37: 。 5. A method for synthesizing an indoline-2-one compound with anti-organ fibrosis activity as described in any one of claims 1-4, characterized in that, The chemical reaction formula is as follows: ； R1 is selected from one of an unsubstituted or at least monosubstituted benzene ring, an unsubstituted or at least monosubstituted six- or five-membered aromatic heterocycle, or an unsubstituted or at least monosubstituted aromatic heterocycle. R2 may be selected from one of the following: hydroxyl, O-alkyl of hydroxyl, O-acyl of hydroxyl, O-tert-butoxycarbonyl of hydroxyl, O-silyl of hydroxyl, amino, N-alkyl of amino, N-acyl of amino, N-tert-butoxycarbonyl of amino, thio, S-alkyl of thio, S-acyl of thio, or different halogen substituents. It is either a trans double bond or a cis double bond.

6. The synthesis method as described in claim 5, characterized in that, Specifically, the following steps are included: The two reactants were mixed in a 1:1 molar ratio and dissolved in ethanol. An equimolar amount of 4-methylpiperidine was added, and the mixture was heated to 85-95°C and refluxed for 4-6 hours to obtain the target product.

7. The use of an indoline-2-one compound having anti-organ fibrosis activity as described in any one of claims 1-4, or a pharmaceutically acceptable salt thereof, in the preparation of an anti-fibrotic medicament.

8. The application as described in claim 7, characterized in that, The anti-fibrotic agents include those for renal fibrosis, idiopathic pulmonary fibrosis, and secondary pulmonary fibrosis.

9. An antifibrotic pharmaceutical composition, characterized in that, Includes an indoline-2-one compound with anti-organ fibrosis activity as described in any one of claims 1-4, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient.

10. The antifibrotic pharmaceutical composition according to claim 9, characterized in that, The antifibrotic drug is in the form of a nanoemulsion or nanoparticles.

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