Inhibitors of hdac6 and methods of making and using the same in anti-tumor and pulmonary fibrosis

Abstract

The application belongs to the field of chemical medicine, and particularly relates to an HDAC6 inhibitor and a preparation method and use thereof in anti-tumor and pulmonary fibrosis. The technical problem to be solved by the application is to provide a new compound having the function of an HDAC6 inhibitor and further having the effect of anti-pulmonary fibrosis. The new compound provided by the application has the structure shown in formula I. The compound provided by the application has the activity of anti-pulmonary fibrosis and the activity of anti-tumor, and has a good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of chemical medicine, specifically relating to HDAC6 inhibitors, their preparation methods, and their uses in anti-tumor and pulmonary fibrosis treatment. Background Technology

[0002] Histone deacetylases (HDACs) and tumor necrosis factor-related apoptosis-inducing ligands (TRAILs) are two highly promising targets in cancer and pulmonary fibrosis. HDACs are enzymes that remove acetyl groups from histones. Histone acetylation promotes chromatin relaxation and gene transcription, while deacetylation has the opposite effect, inhibiting gene transcription. In tumor cells, HDAC activity is typically high and closely related to the biological behaviors of cancer cells, such as proliferation, metastasis, and malignant transformation. Multiple studies have shown that HDACs (histone deacetylases inhibitors) can inhibit tumor cell proliferation and induce apoptosis through various mechanisms, making them important therapeutic agents for cancer. TRAILs are cytokines that induce tumor cell apoptosis. By binding to death receptors, they trigger a series of responses, including endoplasmic reticulum stress, intracellular calcium regulation, and a decrease in mitochondrial membrane potential, ultimately leading to tumor cell apoptosis. TRAIL primarily induces tumor cell apoptosis by activating TRAIL receptors on the surface of tumor cells, including DR4 and DR5 receptors.

[0003] Pulmonary fibrosis is a chronic inflammatory disease characterized by alveolar and bronchial wall fibrosis and impaired lung function. Studies have shown that hepatocellular carcinoma cells (HDACs) play a crucial role in the pathogenesis of pulmonary fibrosis. HDACs can inhibit the expression of inflammatory factors, cytokines, and matrix metalloproteinases, thereby slowing the progression of pulmonary fibrosis and improving lung function. The occurrence of pulmonary fibrosis is often associated with abnormal HDAC expression. Research has found that HDAC6 protein expression is abnormally increased in idiopathic pulmonary fibrosis tissues, suggesting that HDAC6 may be an important promoting factor for idiopathic pulmonary fibrosis. The mechanism of action of TRAILs in pulmonary fibrosis remains unclear. Some literature reports that TRAILs may participate in the occurrence and development of pulmonary fibrosis by regulating apoptosis, inflammatory responses, and fibrosis. Furthermore, TRAILs can also inhibit the fibrosis process by inducing fibroblast apoptosis, making them a potential therapeutic target for pulmonary fibrosis. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a new compound that also has the effect of anti-pulmonary fibrosis.

[0005] This invention provides a compound with HDAC6 inhibitory activity, the structural formula of which is shown in Formula I:

[0006]

[0007] Where R is X is C, O, S or N; R1 to R3 are independently -H, halogen, -CF3, C1 to C4 alkoxy, C1 to C4 alkyl or -NH3.

[0008] Preferably, R is X is C or N; R1 to R3 are independently -H, halogen, -CF3, C1 to C4 alkoxy, C1 to C4 alkyl or -NH3.

[0009] Further preferred, R is X is C, O, S or N; R1 to R3 are independently -H, halogen, -CF3, C1 to C4 alkoxy or C1 to C4 alkyl.

[0010] Optimal, R is X is C or N; R1 to R3 are independently -H, halogen, -CF3, C1 to C4 alkoxy or C1 to C4 alkyl.

[0011] The structural formula of the above HDAC6 / TRAIL dual-target compound is as follows:

[0012]

[0013] This invention also provides a method for preparing the above-mentioned HDAC6 / TRAIL dual-target compound, the preparation route of which is as follows:

[0014]

[0015] The preparation method of the above-mentioned HDAC6 / TRAIL dual-target compound comprises the following steps:

[0016] a. N-tert-butoxycarbonyl-4-piperidinone, NaH and dimethyl carbonate were dissolved in anhydrous THF (tetrahydrofuran) and reacted at 75-90℃ for 5-7 h. The reaction was monitored by TLC (thin-layer chromatography) to ensure complete reaction. The pH was adjusted to 7 with dilute hydrochloric acid, and the mixture was washed with ethyl acetate and saturated brine to separate the organic phase. The organic phase was then evaporated under reduced pressure to obtain intermediate 1.

[0017] b. Methyl 4-aminomethylbenzoate and concentrated sulfuric acid were dissolved in methanol and reacted at 100°C for 4-6 hours. After the reaction was complete by TLC monitoring, the pH was adjusted to 10 with sodium hydroxide solution, and then washed with ethyl acetate and saturated brine to separate the organic phase. The organic phase was then evaporated under reduced pressure to obtain intermediate 2.

[0018] c. Intermediate 2 and 2-methylthio-2-imidazoline hydroiodate were dissolved in anhydrous dioxane and reacted at 100°C for 5 h under nitrogen protection. After the reaction was completed by TLC monitoring, the mixture was washed with ethyl acetate and filtered to obtain intermediate 3.

[0019] d. Dissolve intermediate 2, intermediate 3 and sodium methoxide in methanol and react at 80°C for 3-5 h. After the reaction is complete as monitored by TLC, wash with dichloromethane and saturated brine, separate the organic phase, evaporate to dryness under reduced pressure, and then obtain intermediate 4 by silica gel column chromatography.

[0020] e. Dissolve intermediate 4 in dioxane hydrochloride, stir at room temperature for 1-4 hours, monitor the reaction by TLC until complete, and then evaporate to dryness under reduced pressure to obtain intermediate 5.

[0021] f. Dissolve intermediate 5, R-Br and cesium carbonate in anhydrous DMF (N,N-dimethylformamide), stir at room temperature for 2-4 h, monitor the reaction until complete by TLC, wash with ethyl acetate and saturated brine, separate the organic phase, evaporate to dryness under reduced pressure, and then obtain intermediate 6 by silica gel column chromatography.

[0022] g. Dissolve hydroxylamine hydrochloride and potassium hydroxide in methanol, stir at room temperature for 20-40 min, then filter and collect the filtrate; add the above filtrate to the mixture of intermediate 6 and sodium methoxide, stir at room temperature for 2-5 h, and monitor the reaction for completeness by TLC; adjust the pH to 6 with dilute hydrochloric acid, evaporate to dryness under reduced pressure, and then pass through a C2... 18 Compound I was obtained by two reverse-phase separations.

[0023] Where R is X is C, O, S or N; R1 to R3 are independently -H, halogen, -CF3, C1 to C4 alkoxy, C1 to C4 alkyl or -NH3.

[0024] In the preparation method of the above compound, the molar ratio of N-tert-butoxycarbonyl-4-piperidinone, NaH, and dimethyl carbonate in step a is 1:2:1-2. The concentration of the dilute hydrochloric acid is 1 mol / L.

[0025] In the preparation method of the above compound, in the ethyl acetate / water mixture described in step b, the volume ratio of ethyl acetate to water is 3:2.

[0026] In the preparation method of the above compound, the molar ratio of intermediate 2 and 2-methylthio-2-imidazoline hydroiodate in step c is 1:1.

[0027] In the preparation method of the above compounds, the molar ratio of intermediate 3, intermediate 2, and sodium methoxide in step d is 2-3:4-5:8-9. The volume ratio of the eluent used in the silica gel column chromatography is dichloromethane:methanol = 60:1.

[0028] In the preparation method of the above compound, the molar concentration of dioxane hydrochloride in step e is 4 mol / L.

[0029] In the preparation method of the above compound, the molar ratio of intermediate 5, R-Br, and cesium carbonate in step f is 1–2:1–2:3–4. The volume ratio of the eluent used in the silica gel column chromatography is dichloromethane:methanol = 30:1. Wherein, R is… X is C, O, S or N; R1 to R3 are independently -H, halogen, -CF3, C1 to C4 alkoxy, C1 to C4 alkyl or -NH3.

[0030] In the above preparation method, the molar ratio of hydroxylamine hydrochloride, potassium hydroxide, intermediate 6, and sodium methoxide in step g is 100:100:3 to 4:100. The concentration of the dilute hydrochloric acid is 1 mol / L.

[0031] This invention also provides the use of the above-mentioned compounds in the preparation of HDAC6 inhibitors.

[0032] The present invention also provides the use of the above-mentioned compound in the preparation of a medicament for treating fibrosis. Further, the fibrosis is pulmonary fibrosis.

[0033] The present invention also provides the use of the above-mentioned compound in the preparation of a drug for treating tumors. Further, the tumor is myeloma.

[0034] The present invention also provides a drug for treating pulmonary fibrosis or tumors, which is prepared by adding pharmaceutically acceptable excipients or auxiliary ingredients to the above-mentioned compounds. Furthermore, the dosage form of the above-mentioned drug may be an oral preparation or an injection.

[0035] The novel compound provided by this invention is a highly selective HDAC6 inhibitor, exhibiting inhibitory effects on both NIH / 3T3 and HPF fibroblasts; it also demonstrates good antitumor activity against RPMI 8226 cells, showing good pharmacological activity in target validation and activity concentration studies. In in vivo pharmacodynamic evaluation experiments, it slowed the progression of pulmonary fibrosis in a mouse-induced pulmonary fibrosis model, primarily by reducing lung tissue inflammation and collagen deposition in the lungs. Furthermore, it improved weight loss during pulmonary fibrosis and showed good safety during continuous treatment. Attached Figure Description

[0036] Figure 1 The preferred compound provided by this invention affects the cell state (40×) of HPF cells after 24 hours of treatment.

[0037] Figure 2 The preferred compound provided by this invention affects the cell state (40×) of HPF cells after 48 hours of treatment.

[0038] Figure 3 (A) Compound LT-1433-303 upregulated the expression of Ac-α-Tubulin and Ac-H4; (B) Quantitative analysis results of target proteins (compared with the control group, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001).

[0039] Figure 4 H&E, Masson, and Sirius red staining results of lung tissue pathological sections from mice in each experimental group.

[0040] Figure 5 Daily weight changes in a pulmonary fibrosis model.

[0041] Figure 6 Organ coefficient results: (A) Heart, (B) Liver, (C) Spleen, (D) Kidney (Compared with the control group, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001).

[0042] Figure 7 Results of liver function, kidney function, and blood lipid biochemical indicators.

[0043] Figure 8 H&E staining results of major organ histopathology (200×).

[0044] Figure 9 Products e, h, k 1 HNMR results.

[0045] Figure 10 Products h and k 13 C-NMR results. Detailed Implementation

[0046] This invention provides a new class of compounds with the structural formula shown in Formula I:

[0047]

[0048] Where R is X is C, O, S or N; R1 to R3 are independently -H, halogen, -CF3, C1 to C4 alkoxy, C1 to C4 alkyl or -NH3.

[0049] This invention has revealed that the novel compound possesses HDAC6 inhibitory properties and exhibits high selectivity in inhibiting HDAC6 activity. Subsequent cell experiments and in vivo pharmacodynamic studies have confirmed its anti-pulmonary fibrosis effect.

[0050] In addition, the compounds of this invention also have anti-tumor effects. These functions may also be related to the fact that the compounds of this invention also have TRAIL inhibitory effects.

[0051] The more specific preparation route of the compound with HDAC6 inhibition function of the present invention is shown below:

[0052] a. N-tert-butyloxycarbonyl-4-piperidinone, NaH, and dimethyl carbonate were dissolved in anhydrous THF (tetrahydrofuran) and reacted at 75–90 °C for 5–7 h. The reaction was monitored by TLC (thin-layer chromatography) to ensure complete reaction. The pH was adjusted to 7 with dilute hydrochloric acid, and the mixture was washed with ethyl acetate and saturated brine to separate the organic phase. The organic phase was then evaporated under reduced pressure to obtain intermediate 1. The molar ratio of N-tert-butyloxycarbonyl-4-piperidinone, NaH, and dimethyl carbonate was 1:2:1–2. The concentration of the dilute hydrochloric acid was 1 mol / L.

[0053] b. Methyl 4-aminomethylbenzoate and concentrated sulfuric acid were dissolved in methanol and reacted at 100°C for 4-6 hours. After the reaction was complete, the pH was adjusted to 10 with sodium hydroxide solution. The mixture was then washed with ethyl acetate and saturated brine to separate the organic phase. The organic phase was evaporated under reduced pressure to obtain intermediate 2. The volume ratio of ethyl acetate to water in the ethyl acetate / water mixture was 3:2.

[0054] c. Intermediate 2 and 2-methylthio-2-imidazoline hydroiodate were dissolved in anhydrous dioxane and reacted at 100°C for 5 h under nitrogen protection. After the reaction was completed by TLC monitoring, the intermediate was washed with ethyl acetate and filtered to obtain intermediate 3. The molar ratio of intermediate 2 and 2-methylthio-2-imidazoline hydroiodate was 1:1.

[0055] d. Intermediate 2, intermediate 3, and sodium methoxide are dissolved in methanol and reacted at 80°C for 3–5 h. After the reaction is complete as monitored by TLC, the mixture is washed with dichloromethane and saturated brine, and the organic phase is separated and evaporated to dryness under reduced pressure. Then, intermediate 4 is obtained by silica gel column chromatography. The molar ratio of intermediate 3, intermediate 2, and sodium methoxide is 2–3:4–5:8–9. The volume ratio of the eluent used in the silica gel column chromatography is dichloromethane:methanol = 60:1.

[0056] e. Dissolve intermediate 4 in dioxane hydrochloride, stir at room temperature for 1-4 hours, monitor the reaction for completeness by TLC, and then evaporate to dryness under reduced pressure to obtain intermediate 5; the molar concentration of dioxane hydrochloride is 4 mol / L.

[0057] f. Intermediate 5, R-Br, and cesium carbonate were dissolved in anhydrous DMF (N,N-dimethylformamide) and reacted with stirring at room temperature for 2–4 h. After the reaction was complete as monitored by TLC, the mixture was washed with ethyl acetate and saturated brine, and the organic phase was separated and evaporated to dryness under reduced pressure. The organic phase was then subjected to silica gel column chromatography to obtain intermediate 6. The molar ratio of intermediate 5, R-Br, and cesium carbonate was 1–2:1–2:3–4. The volume ratio of the eluent used in the silica gel column chromatography was dichloromethane:methanol = 30:1.

[0058] g. Dissolve hydroxylamine hydrochloride and potassium hydroxide in methanol, stir at room temperature for 20-40 min, then filter and collect the filtrate; add the above filtrate to the mixture of intermediate 6 and sodium methoxide, stir at room temperature for 2-5 h, and monitor the reaction for completeness by TLC; adjust the pH to 6 with dilute hydrochloric acid, evaporate to dryness under reduced pressure, and then pass through a C2... 18 The HDAC6 / TRAIL dual-target compound, Formula I, was obtained by two reverse-phase separations; the molar ratio of hydroxylamine hydrochloride, potassium hydroxide, intermediate 6, and sodium methoxide was 100:100:3 to 4:100; and the concentration of the dilute hydrochloric acid was 1 mol / L.

[0059] Those skilled in the art can use the compounds of the present invention as the main raw material, and add pharmaceutically acceptable excipients or auxiliary ingredients to prepare drugs in various dosage forms.

[0060] Furthermore, the dosage form of the above-mentioned drugs may be an oral preparation or an injection.

[0061] The pharmaceutically acceptable adjuvant component described in this invention possesses certain physiological activities. However, the addition of this component does not alter the dominant role of the aforementioned pharmaceutical composition in the disease treatment process; rather, it merely exerts an adjuvant effect. These adjuvant effects are simply the utilization of the known activity of the component, and are a commonly used adjuvant therapy method in the pharmaceutical field. If the aforementioned adjuvant component is used in combination with the pharmaceutical composition of this invention, it should still fall within the scope of protection of this invention.

[0062] The present invention will be further illustrated by the following examples.

[0063] The details of the cells and experimental materials used in the embodiments of this invention are shown in the table below.

[0064] Table 1. Cell types and sources used in the embodiments of the present invention.

[0065] Cell Name Chinese name Cell source RPMI 8226 Peripheral blood B lymphocytes in multiple myeloma patients Wuhan Pronosei Life Science Technology Co., Ltd. NIH / 3T3 mouse embryonic fibroblasts Cell Bank of Chinese Academy of Sciences HPF Human lung fibroblasts Peking Union Medical College Cell Resource Center

[0066] Table 2 Experimental materials used in the embodiments of the present invention

[0067]

[0068] Example 1: Preparation of the compound with HDAC6 inhibition function of the present invention

[0069]

[0070] N-tert-Butoxycarbonyl-4-piperidinone (2 g, 10.04 mmol) was dissolved in anhydrous THF (20 mL). NaH (802.94 mg, 20.08 mmol) was slowly added under ice bath conditions. After stirring for 30 min, dimethyl carbonate (1.01 mL, 12.05 mmol) was added. The reaction was carried out at 85 °C for 6 h, and TLC was used to monitor the reaction until complete. After cooling, the pH was adjusted to 7 with 1 mol / L dilute hydrochloric acid under ice bath conditions. The mixture was diluted with ethyl acetate, washed with saturated brine, and the organic phase was separated. The organic phase was dried over anhydrous sodium sulfate and evaporated under reduced pressure to obtain intermediate 1 (2.58 g of yellow oil, 100% yield). The product was used directly in the next reaction without purification. ESI-MS m / z: 258.13 [M+H] + .

[0071]

[0072] Methyl 4-aminomethylbenzoate (2 g, 13.23 mmol) was dissolved in methanol (20 mL), and concentrated sulfuric acid (0.8 mL) was added dropwise at room temperature. The reaction was carried out at 100 °C for 5 h, and the reaction was monitored by TLC until complete. After the reaction system cooled, the solvent was evaporated under reduced pressure. The crude product was dissolved in ethyl acetate / water (24 mL / 16 mL), and the pH was adjusted to 10 with sodium hydroxide solution. The organic phase was separated, washed with saturated brine, dried over anhydrous sodium sulfate, and evaporated under reduced pressure to obtain intermediate 2 (1.64 g of pale yellow oil, yield 74.89%). The product was used directly in the next reaction without purification. ESI-MS m / z: 166.08 [M+H] + .

[0073]

[0074] Intermediate 2 (1.64 g, 9.95 mmol) and 2-methylthio-2-imidazoline hydroiodate (1.16 g, 9.95 mmol) were dissolved in anhydrous dioxane (25 mL). The mixture was purged with nitrogen three times and reacted at 100 °C for 5 h. The reaction was monitored by TLC until complete. After the reaction system cooled, the solvent was evaporated under reduced pressure, the mixture was washed with ethyl acetate, filtered, and the filter cake was collected. The filtrate was then evaporated to dryness, and the above steps were repeated twice to obtain intermediate 3 (1.43 g white solid, yield 61.64%). The product was used directly in the next reaction without purification. ESI-MS m / z: 234.12 [M+H] + .

[0075]

[0076] Intermediate 3 (650 mg, 2.79 mmol) was dissolved in methanol (8 mL), followed by the addition of intermediate 2 (1.15 g, 4.46 mmol) and sodium methoxide (451.61 mg, 8.36 mmol). The reaction was carried out at 80 °C for 4 h, and the reaction was monitored by TLC until complete. After cooling, the reaction solution was diluted with dichloromethane and washed with saturated brine. The organic phase was separated and evaporated to dryness under reduced pressure. The crude product was subjected to silica gel column chromatography (dichloromethane:methanol = 60:1) to obtain intermediate 4 (557 mg of pale yellow solid, yield 45.28%), ESI-MS m / z: 441.21 [M+H]. + .

[0077]

[0078] Intermediate 4 (557 mg, 1.26 mmol) was dissolved in 4 mol / L dioxane hydrochloride (5 mL), stirred at room temperature for 2 h, and the reaction was monitored by TLC until complete. The solution was evaporated under reduced pressure to obtain intermediate 5 (430.40 mg of brownish-yellow solid, 100% yield). ESI-MS m / z: 341.15 [M+H] + .

[0079]

[0080] Intermediate 5 (430.40 mg, 1.26 mmol) was dissolved in anhydrous DMF (5 mL), and benzyl bromide (165.21 mL, 1.39 mmol) and cesium carbonate (1.24 g, 3.79 mmol) were added sequentially. The mixture was stirred at room temperature for 3 h, and the reaction was monitored by TLC until complete. The reaction solution was diluted with ethyl acetate, washed with saturated brine, and the organic phase was separated and evaporated to dryness under reduced pressure. The crude product was subjected to silica gel column chromatography (dichloromethane:methanol = 30:1) to give intermediate 6a (170 mg of yellow solid, yield 31.23%). ESI-MS m / z: 431.20 [M+H] + .

[0081]

[0082] Hydroxylamine hydrochloride (823.19 mg, 11.85 mmol) was dissolved in methanol (10 mL), potassium hydroxide (664.65 mg, 11.85 mmol) was added, and the mixture was stirred at room temperature for 30 min, then filtered. The filtrate was added to intermediate 6 (170 mg, 394.88 μmol), followed by slow addition of sodium methoxide (639.99 mg, 11.85 mmol). The mixture was stirred at room temperature for 3 h, and the reaction was monitored by TLC until complete. The pH was adjusted to 6 with 1 mol / L dilute hydrochloric acid, and the mixture was evaporated to dryness under reduced pressure. The crude product was then subjected to C... 18The product was separated twice by reverse phase to give product a (26 mg of brown solid, yield 15.26%). ESI-MS m / z: 432.20 [M+H] + .

[0083] The preparation methods for other compounds of the present invention can refer to the preparation method of product a described above.

[0084] Table 3. Structural formulas and proton NMR data of the compounds of this invention.

[0085]

[0086]

[0087]

[0088] Biological experiment:

[0089] Example 2: HDAC1 and HDAC6 enzyme activity experiments of the compounds of the present invention

[0090] (1) Take 5 mL of HDAC buffer and dilute it with 45 mL of ultrapure water to a concentration equal to 1:1 as dilution buffer (25 mM Tris-HCl).

[0091] pH 8.0, 1mM MgCl2, 137mM NaCl, 2.7mM KCl), place on an ice pack for later use;

[0092] (2) Take 10 μL of each of the positive control reagents HDAC1 and HDAC6 and add them to 190 μL of dilution buffer. Place them on an ice box for later use.

[0093] (3) Take 50 μL of Trichostatin A stock solution and add 450 μL of dilution buffer for later use;

[0094] (4) The HDAC substrate solution and HDAC deacetylation standard were dissolved in DMSO for later use;

[0095] (5) Add 4 mL of dilution buffer to the colorimetric reagent, and then add 100 μL of Trichostatin A to prepare the colorimetric reagent for later use;

[0096] (6) Add 10 μL of the test compound (final concentration of 1 μM) and 140 μL of dilution buffer to the sample well. The final concentration of DMSO in the system is 1%.

[0097] (7) The concentrations of the positive control drug Chidamide were set to 1 nM, 3.3 nM, 10 nM, 33 nM, 100 nM, 333 nM, 1 μM, 3.3 μM, and 10 μM; the concentrations of ACY-1215 were set to 0.01 nM, 0.03 nM, 0.1 nM, 0.33 nM, and 1 μM.

[0098] nM, 3.3nM, 10nM, 33nM, 100nM;

[0099] (8) Add 150 μL of dilution buffer and 10 μL of the corresponding concentration of standard to each well of the deacetylation standard curve. HDAC1

[0100] Add 10 μL of HDAC1 and HDAC6 reagents and 140 μL of dilution buffer to the HDAC6 positive control well; add 10 μL of the test compound and 140 μL of dilution buffer to the sample and positive control wells. The final concentration of DMSO in the system is 1%.

[0101] (9) Add 10 μL of diluted Trichostatin A to the two positive control wells and the sample wells, while HDAC1 and HDAC6 are positive.

[0102] Add 10 μL of dilution buffer to the control wells and the sample wells that do not contain Trichostatin A;

[0103] (10) Add 10 μL of HDAC substrate to all wells, with a final substrate concentration of 200 μM per well, and incubate on a shaker at 37°C for 30 minutes.

[0104] (11) Add 40 μL of colorimetric reagent to all wells and incubate at room temperature for 15 minutes;

[0105] (12) Detect fluorescence using an ELISA reader at excitation wavelengths of 340-360 nm and emission wavelengths of 440-465 nm. Obtain a deacetylation standard curve based on the experimental results. Substitute the sample values ​​into the standard curve to obtain the corrected fluorescence values, and calculate using the formula.

[0106] HDAC enzyme activity;

[0107] (13) The inhibition rates of the test compounds on the activities of HDAC1 and HDAC6 enzymes were calculated. Using GraphPad Prism 9 software, the nonlinear regression method of Dose-response-Inhibition inhibitor vs. normalized response was selected for linear fitting to obtain the IC50 of the positive drug. 50 value.

[0108] Table 4. Inhibition rate of the compounds of the present invention on HDAC1 enzyme activity

[0109]

[0110]

[0111] Table 5. Inhibition rate of the compounds of the present invention on HDAC6 enzyme activity.

[0112] compound Inhibition rate (%) compound Inhibition rate (%) LT-1400-736 97.2 LT-1433-306 95.1 LT-1400-742 95.4 LT-1433-314 94.7 LT-1400-743 93.8 LT-1433-315 91.3 LT-1433-302 97.1 LT-1433-316 94.1 LT-1433-303 94.8 LT-1433-317 96.2 LT-1433-304 93.6 ACY-1215 98.5

[0113] The results are shown in Tables 4 and 5. The compounds provided by this invention generally showed low inhibition rates against HDAC1 enzyme activity at a single concentration of 1 μM, but all showed inhibition rates against HDAC6 enzyme activity of over 90%. The results indicate that the compounds provided by this invention have high selectivity for HDAC6.

[0114] Example 3: RPMI 8226, NIH / 3T3, and HPF cell IC 50 Value determination

[0115] I. The compound provided by this invention is a highly selective inhibitor targeting HDAC6. To verify its pharmacological activity, human multiple myeloma RPMI 8226 cells were initially selected for cell proliferation experiments to determine its cellular activity IC50 in tumor treatment. 50 Value. Cell culture methods:

[0116] (1) NIH / 3T3: DMEM complete medium containing 10% FBS and 1% P / S, placed in a cell culture incubator, cultured at 37℃ and 5% CO2.

[0117] (2) RPMI 8226: RPMI 1640 complete medium containing 10% FBS and 1% P / S, placed in a cell culture incubator, cultured at 37℃ and 5% CO2.

[0118] (3) HPF: FM complete medium contains 2% FBS, 1% FGS-acf and 1% P / S. It is placed in a cell culture incubator and cultured under the conditions of 37℃ and 5% CO2.

[0119] Table 6 shows the IC50 of the compounds of the present invention on RPMI 8226 cells. 50 value

[0120]

[0121]

[0122] The results are shown in Table 6. The compounds provided by this invention have good inhibitory activity against RPMI 8226 cells. The five compounds with the best activity are LT-1400-742, LT-1433-303, LT-1400-736, LT-1400-743, and LT-1433-317, with IC50 values ​​of [missing information]. 50 The values ​​were 0.10 μM, 0.44 μM, 0.62 μM, 1.06 μM, and 1.08 μM, respectively. The activity of these compounds was superior to or comparable to that of the positive control drug ACY-1215.

[0123] II. To further investigate the effects of this series of compounds on pulmonary fibrosis, the IC50 values ​​of their activity in the treatment of pulmonary fibrosis were determined using mouse embryonic fibroblast NIH / 3T3 cells. Cell proliferation experiment:

[0124] (1) Take cells in the logarithmic growth phase, process them according to the cell culture method, centrifuge them and add a certain amount of culture medium to make a cell suspension. Use a cell counting chamber to count the number of cells in the prepared cell suspension, calculate the required amount of cells and prepare a plate-laying cell suspension for later use.

[0125] (2) Adherent cells NIH / 3T3 and HPF were treated at 5×10 3 Cells / well, suspension cells RPMI 8226 at 1×10⁻⁶ 4 Cells per well, seeded into 96-well plates. Adjust cell concentration with freshly prepared complete culture medium, and seal the edges of the 96-well plates with 100 μL of PBS.

[0126] (3) Gently pipette the prepared cell suspension to a sterile sample loading trough. Use an 8-channel pipette to add 100 μL of the cell suspension to each well of a 96-well plate. Add 100 μL of cell-free complete culture medium to the blank control group. Incubate overnight at 37°C with 5% CO2.

[0127] (4) After 24 hours of culture, the concentration of the test compound was prepared using a 96-well deep-well sample plate in a clean bench. The old culture medium was discarded from the adherent cells, and 100 μL of complete culture medium containing the drug was added to each well. The final concentrations of the compound and the positive control (Chidamide, ACY-1215) were 0 μM, 0.1 μM, 0.3 μM, 1 μM, 3 μM, 10 μM, and 30 μM, respectively. The blank control group consisted of 100 μL of complete culture medium containing 0.01% DMSO, and the negative control group consisted of 100 μL of complete culture medium containing 0.01% DMSO and cells, but without the test compound (three replicates were set for each single concentration). The cells were cultured at 37°C and 5% CO2 for 24 hours.

[0128] (5) After 24 hours, add 10 μL of CCK8 reagent to each well, and incubate at 37°C for 1-4 hours in the dark with 20 μL of CCK8 reagent in each well of suspended cells at 5% CO2.

[0129] (6) Use an ELISA reader to detect the absorbance value of each well at a wavelength of 450 nm;

[0130] (7) Calculate the cell inhibition rate according to the following formula: Inhibition rate % = (OD value of control group - OD value of drug-treated group) ÷ (OD value of control group - OD value of blank group) × 100.

[0131] Table 7. IC50 of the compounds of the present invention on NIH / 3T3 cells 50 Value (Note: "—" indicates not detected)

[0132]

[0133]

[0134] The results are shown in Table 7. Within the selected concentration range, the software did not simulate the IC50 values ​​for some compounds. 50 Compounds with good inhibitory activity against the growth and proliferation of NIH / 3T3 cells include LT-1400-743, LT-1433-304, LT-1433-303, and LT-1400-736, with IC50 values... 50 The values ​​were 0.09 μM, 0.10 μM, 0.89 μM, and 1.00 μM, respectively.

[0135] The cell proliferation experiments of the two cell types above showed that the compound provided by this invention exhibited a good inhibitory effect on HDAC6 and showed good activity in NIH / 3T3 cells. Therefore, different cell types may have different sensitivities to HDAC6 inhibitors. In the future, compounds with better effects will be screened and their activity will be measured in human lung fibroblasts (HPF).

[0136] Finally, LT-1400-736, LT-1433-302, and LT-1433-303 were screened and their activity was measured in HPF cells. The results are shown in Table 8. Of the three compounds, LT-1433-303 showed the best inhibitory effect on HPF growth and proliferation, with an IC50 concentration of [missing value]. 50 The value was 29.38 μM, which was lower than that of the two positive drugs.

[0137] Table 8. IC50 of the preferred compounds of the present invention on HPF cells. 50 value

[0138] compound <![CDATA[IC 50 (μM)]]> compound <![CDATA[IC 50 (μM)]]> LT-1400-736 49.68 Chidamide 45.64 LT-1433-302 49.91 ACY-1215 35.10 LT-1433-303 29.38

[0139] Example 4: The effect of the preferred compound on HPF cell proliferation and its good target activity:

[0140] I. HDAC6 is the main deacetylase of tubulin, and α-Tubulin is a major acetylated protein and also one of the substrates for HDAC6. Inhibition of HDAC6 enzyme activity specifically upregulates Ac-α-Tubulin expression while having little effect on Ac-H3 expression; this is one of the key differences between HDAC6 inhibitors and other HDAC inhibitors. Preferred compounds were tested for HPF cell proliferation.

[0141] (1) The method is the same as in Example 3, using compounds (LT-1400-736, LT-1433-302, LT-1433-303), IIc 1 The concentrations of the positive control drug (Chidamide, ACY-1215) were 0 μM, 5 μM, 10 μM, 20 μM, 40 μM, and 80 μM.

[0142] (2) The cell morphology at 40 μM and 80 μM concentrations was photographed at 24 hours and 48 hours.

[0143] Cell morphology of HPF cells after 24 hours of treatment is as follows Figure 1 As shown, observation of cell state revealed that LT-1433-303 at concentrations of 40 μM and 80 μM had a good inhibitory effect on cell growth and proliferation; cell morphology after 48 hours was as follows. Figure 2 As shown, LT-1433-303 exhibited good inhibitory effects on cell growth and proliferation at a concentration of 40 μM, and its inhibitory effect on cell growth and proliferation at a concentration of 80 μM was comparable to that of the two positive control drugs. The effect of compound IIc was slightly weaker than that of LT-1433-303 based on cell state. The structural formula of compound IIc is:

[0144]

[0145] II. Investigation of Target Effects and Active Concentration:

[0146] (1) Take RPMI 8226 cells in the logarithmic growth phase, process the cells according to the cell passage method, centrifuge, add a certain amount of culture medium to make a cell suspension, count the number of cells in the prepared cell suspension with a cell counting chamber, calculate the required amount of cells, and prepare a plate-laying cell suspension for later use.

[0147] (2) Cells at 5 × 10 5 One cell / well, plated in a 6-well plate, and added medicine after 24 hours of incubation;

[0148] (3) The final concentrations of compound LT-1433-303 were 0.25, 0.5, and 1 μM, and the final concentrations of compound IIc and positive control drugs (Chidamide, ACY-1215) were 1 μM.

[0149] (4) Discard the old culture medium, add 2 mL of complete culture medium containing the drug to the drug treatment group, and add 2 mL of complete culture medium containing 0.01% DMSO to the control group, and act for 24 hours;

[0150] (5) After 24 hours of treatment, protein extraction and sample preparation were performed, and finally, a protein immunoblotting experiment was conducted.

[0151] (6) Western blot assay:

[0152] a) Add 1 mL of PBS to each well of the 6-well plate and wash twice. Use a pipette to remove the PBS and place the plate on ice. Mix PMSF (100 mM) and RIPA at a ratio of 1:99 to prepare a lysis buffer for later use.

[0153] b) Add 50 μL of lysis buffer to each well, shake the plate to cover the cell surface with lysis buffer, and place on ice for 15 minutes for lysis, shaking the 6-well plate a few times during the process; after 10 minutes, scrape off the cells and lysis buffer, transfer them to a 1.5 mL centrifuge tube, and place on ice for lysis for 5 minutes.

[0154] c) Completely lyse the cells using an ultrasonic cell disruptor, place them on ice, and sonicate under the following conditions (20 Hz, sonicate for 10 seconds, pause for 10 seconds, repeat 3 times) at 4°C, 12000 rpm for 15 minutes. Separate the supernatant into a 1.5 mL centrifuge tube and place it on ice.

[0155] d) Add the protein sample to be tested to a 96-well plate, and add 2 μL of the sample to 18 μL of PBS to dilute it 10 times;

[0156] e) Two standard curves are used, with three replicates for each sample;

[0157] f) Add the BSA standard (2 mg / mL) to the corresponding wells of the 96-well plate as shown in Table 9;

[0158] Table 9 BCA Quantitative Sampling Table

[0159] Kong Hao 1 2 3 4 5 6 7 8 BSA standard addition amount (μL) 0 1.25 2.5 5 7.5 10 15 20 PBS addition volume (μL) 20 18.75 17.5 15 12.5 10 5 0 Final BSA concentration (mg / mL) 0 0.125 0.25 0.5 0.75 1 1.5 2 Total volume (μL) 20 20 20 20 20 20 20 20

[0160] g) Prepare the colorimetric working solution, add 200 μL to each well, mix thoroughly, incubate at 37°C for 30 minutes, and cool to room temperature;

[0161] h) Measure the absorbance of the samples and standard curve wells at a wavelength of 562 nm using an enzyme-linked immunosorbent assay (ELISA) reader;

[0162] i) Plot a standard curve and calculate the protein concentration in the sample;

[0163] j) Calculate the concentration of the protein to be tested, and normalize the concentration and loading volume based on the amount of protein loaded.

[0164] k) Add 1 μL of SDS-PAGE protein loading buffer to every 4 μL of protein sample, mix well, and boil in a 100°C metal bath for 5 minutes.

[0165] (7) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)

[0166] a) Prepare the required concentration gel for 1.5 mm using the PAGE gel rapid preparation kit: Mix 3.5 mL each of the lower gel solution and lower gel buffer thoroughly. Add 70 μL of modified coagulant to the mixture and mix well. Pour the mixture into the gel preparation glass plate and fill it with isopropanol. Press the gel and let it solidify for 30 minutes. Then, pour off the upper layer of isopropanol and blot dry. Mix 1.5 mL each of the upper gel solution and upper gel buffer thoroughly. Add 30 μL of modified coagulant to the mixture and pour the mixture into the gel preparation glass plate. Insert the electrophoresis gel preparation comb and let it solidify for 20 minutes to prepare the SDS-PAGE gel.

[0167] b) Prepare electrophoresis and transfer buffer: Dilute the 10× electrophoresis and transfer buffer to 1× for later use;

[0168] c) Sample loading: First, add 4 μL of tricolor pre-stained protein marker, then add 30 μg of each protein sample to the gel wells in sequence;

[0169] d) Electrophoresis at a constant voltage of 80V. After electrophoresis to the separating gel, adjust the parameters to 100V. Stop electrophoresis when the protein marker and bromophenol blue indicator reach the bottom.

[0170] e) Cut a PVDF membrane to the same size as the adhesive, immerse it in a methanol solution for 20-30 seconds to activate it, and then transfer it to the transfer solution for later use;

[0171] f) Wet transfer: After placing the sandwich transfer plate consisting of filter paper, membrane and gel in sequence to prevent air bubbles, clamp the transfer plate and place it in the transfer instrument containing transfer buffer. Set the constant current to 260mA and the transfer time to 60-90 minutes according to the protein molecular weight.

[0172] g) After the transfer is complete, remove the PVDF membrane, wash it once in TBST solution, and then transfer the membrane to the rapid blocking solution and block it on a shaker for 15-20 minutes at room temperature.

[0173] h) Wash the PVDF membrane once with TBST solution, cut the target protein at the possible location according to the protein molecular weight region indicated by the protein Maker, put different bands into the hybridization band, pour in the corresponding primary antibody dilution solution, and incubate overnight at 4°C.

[0174] i) Place the strip in TBST solution and wash it three times on a shaker for eight minutes each time;

[0175] j) Place the strip in the working solution of the second antibody corresponding to the same species prepared with TBST solution, incubate slowly on a shaker at room temperature for 1 hour, place the strip in TBST solution, and wash it 3 times on a shaker for 8 minutes each time;

[0176] k) After ECL development, imaging was performed using an iBright 1500 instrument, and the results were analyzed using iBright Analysis Software, Version 5.0.0.

[0177] The results are as follows Figure 3 As shown, when the concentrations of compound LT-1433-303 were 0.25, 0.5, and 1 μM, the expression levels of Ac-α-Tubulin and Ac-H4 proteins increased in a concentration gradient, while the effect on Ac-H3 protein was relatively small. Compared with the control group, when the concentration of compound LT-1433-303 was 1 μM, it significantly upregulated the expression of Ac-α-Tubulin and Ac-H4 proteins in RPMI 8226 cells (P<0.0001), although the upregulation intensity was slightly weaker than that of the positive control drug ACY-1215. Compound IIc, as an HDAC1 inhibitor, had little effect on the expression of Ac-Ac-α-Tubulin and Ac-H4 proteins in RPMI 8226 cells. The results indicate that compound LT-1433-303 is an HDAC6 inhibitor with good target activity.

[0178] Example 5: In vivo pharmacodynamics of compound LT-1433-303IPF model:

[0179] I. Effects of compound LT-1433-303IPF on the progression of pulmonary fibrosis in mice

[0180] (1) Select C57BL / 6J mice, 6-8 weeks old, male, with a weight range of 18-22g;

[0181] (2) The animals were randomly divided into groups according to their body weight: blank control group, model group, drug treatment group (25, 50 mg / kg), and positive control group (ACY-1215 50 mg / kg), with 6 animals in each group;

[0182] (3) Except for the blank control group, all other groups were given bleomycin (2 mg / kg) via intratracheal infusion at a volume of 50 μL / 10 liters.

[0183] g, idiopathic pulmonary fibrosis model was established, and the blank control group was given the same volume of physiological saline;

[0184] (4) The solvent for the compound is 40% PEG300, 5% Tween 80, and 55% Saline. The compound is prepared every three days and then sonicated after vortexing.

[0185] 10 minutes;

[0186] (5) The drug was administered on the first day after the model was established. After the body weight was recorded, the volume of the drug was calculated according to the relationship between body weight and dosage. The volume of the drug was 0.1 mL / 10 g. The blank control group and the model group were administered the drug via gavage with the solvent.

[0187] (6) After 14 days of continuous administration, blood was drawn from the eyeballs, and the lungs were photographed and recorded. Cardiopulmonary perfusion was performed, and the lungs were fixed with 4% paraformaldehyde general tissue fixative. Subsequently, H&E, Mason and Sirius red staining were performed.

[0188] The degree of pulmonary fibrosis in mice was assessed based on histopathological staining, and the results were as follows: Figure 4 As shown, observations of H&E, Masson, and Sirius red staining results revealed that the lung tissue of the control group mice was structurally intact, with normal alveolar morphology and no obvious inflammatory areas. In the model group mice, the lung interstitium was thickened, the alveolar cavities were narrowed, the structure was irregular, and varying degrees of inflammatory cell infiltration were observed. Compared with the lung tissue of the model group mice, LT-1433-303 reduced inflammatory cell infiltration in the mouse lung tissue in a concentration gradient-dependent manner, with the 50 mg / kg dose showing the most significant reduction. Masson and Sirius red staining results indicated that LT-1433-303 could slow the progression of pulmonary fibrosis in mice and reduce the occurrence of inflammation in the mouse lung tissue.

[0189] II. Preliminary Study on the In Vivo Safety of LT-1433-303

[0190] Currently, bleomycin is commonly used to establish pulmonary fibrosis models in mice. The induced pulmonary fibrosis process is similar to clinical pathology, but it also causes weight loss and decreased activity levels in the mice. When investigating the therapeutic effect of the HDAC6 inhibitor LT-1433-303 on the progression of pulmonary fibrosis, its in vivo safety also needs to be investigated.

[0191] During the 14-day treatment period, the body weight and activity level of mice were recorded daily. At the end of the experiment, serum and major organs were collected for relevant indicator testing. Figure 5As shown, compared with the control group, the mice in the model group began to experience a continuous decrease in body weight starting from the third day after modeling. All treatment groups also showed a decrease in body weight, while the mice in the LT-1433-303 50mg / kg treatment group experienced the smallest decrease in body weight. The mice in the positive control group (ACY-1215 and LT-1433-303 25mg / kg treatment groups) both experienced varying degrees of body weight decrease, but their body weight began to increase starting from the 9th day after administration. This indicates that HDAC6 inhibitors have the effect of slowing down the decrease in body weight in the bleomycin-induced mouse pulmonary fibrosis model.

[0192] To investigate the extent of organ damage in mice caused by continuous in vivo administration of HDAC6 inhibitors such as LT-1433-303, major organs of mice were harvested and weighed at the experimental endpoint, and organ coefficients were calculated. The results are as follows: Figure 6 As shown, compared with the control group, there were no significant changes in the organ coefficients of each drug administration group, indicating that it did not damage the organs and had good safety in vivo.

[0193] Example 6: Blood Biochemistry Detection

[0194] (1) When the animal experiment reached the end of the experiment, blood was collected from the eyeballs of mice in each experimental group;

[0195] (2) Centrifuge the collected blood at 3500 rpm for 10 minutes, and store the supernatant serum at -80℃;

[0196] (3) After diluting each group of serum with distilled water at a ratio of 1:1, liver function indicators ALT, AST, TP, kidney function indicator UA, and blood lipid indicators HDLC and LDLC were detected on a blood biochemistry instrument.

[0197] Blood biochemistry test results as follows Figure 7 As shown, compared with the control group, the serum AST level in the model group increased significantly, while the serum AST level in each treatment group decreased compared with the model group, but did not decrease to the same level as the control group. The serum HDLC level in the model group and each treatment group decreased slightly. The results show that bleomycin induces pulmonary fibrosis and causes mild liver damage, manifested as an increase in serum AST level; HDAC6 inhibitors may have a slight effect on blood lipid levels.

[0198] Example 7: Histopathological staining:

[0199] (1) Preparation of paraffin slices:

[0200] a) After the tissue has been fixed for 48 hours, it is removed from the fixative. The tissue at the target site is trimmed and smoothed in a fume hood, and the trimmed tissue is placed in an embedding frame.

[0201] b) Place the collected tissue in tap water and rinse for 20 minutes;

[0202] c) Place the embedding frame containing the tissue into the basket of the dehydrator and dehydrate it with alcohol in a gradient manner in the dehydrator;

[0203] d) Embed the paraffin-impregnated tissue in an embedding machine. After the paraffin solidifies, remove the paraffin block from the mold and trim it. e) Place the trimmed paraffin block in a paraffin microtome and section it to a thickness of 4 μm. Bake the sections at 60℃ for 30 minutes.

[0204] After 2 hours, remove the slices and store them at room temperature for later use.

[0205] (2) Dewaxing and hydration of paraffin sections:

[0206] a) Dewax the product sequentially in environmentally friendly dewaxing agent (1), environmentally friendly dewaxing agent (2), and environmentally friendly dewaxing agent (3) for 10 minutes each, then rinse with anhydrous ethanol, 95% ethanol, 85% ethanol, and 75% ethanol for 5 minutes each. Rinse with tap water for 1 minute.

[0207] b) Stain with hematoxylin (Harris) solution for 4 minutes, then wash with tap water for 2 minutes until no excess staining solution is removed from the slide.

[0208] c) Differentiate with 0.8% hydrochloric acid alcohol for 2 seconds, rinse with tap water, or use lithium carbonate aqueous solution to return to blue, then wash with water for 2 minutes; d) Dye with eosin dye solution (alcohol soluble) for 20 seconds, no need to wash with water, directly add 95% ethanol to adjust the color for 5 seconds, then add anhydrous ethanol (1) and anhydrous ethanol (2) to dehydrate for 2 minutes.

[0209] e) Environmentally friendly transparent agent for transparency, sealing, and microscopic inspection.

[0210] (3) Masson staining:

[0211] a) Soak the slices in Bouin's solution overnight, and rinse them thoroughly with running water the next day;

[0212] b) Stain with iron hematoxylin for 5-10 minutes, then rinse with running water;

[0213] c) Differentiate with 0.8%-1% hydrochloric acid and alcohol, rinse with running water for 2 minutes, then use lithium carbonate to blue for 3 seconds, and rinse with running water;

[0214] d) Stain with Ponceau S acid fuchsin solution for 5-10 minutes, then rinse with running water;

[0215] e) Treat with phosphomolybdic acid solution for about 5 minutes, do not wash with water, and directly counterstain with aniline blue solution for 5 minutes;

[0216] f) Treat with 1% glacial acetic acid for 1 minute, then dehydrate repeatedly with 95% alcohol;

[0217] g) Mounting slides for microscopic examination.

[0218] (4) Sirius red staining:

[0219] a) Stain with Harris hematoxylin for 5-10 minutes, then wash with distilled water;

[0220] b) Stain with Sirius red saturated picric acid solution for 15-30 minutes;

[0221] c) Differentiation and dehydration of anhydrous ethanol;

[0222] d) Microscopic examination of the mounting slide.

[0223] like Figure 8 As shown, the histopathological staining results of the major organs showed that the tissue structure of each organ was normal, and no organic lesions were found in the organs of the mice in the high-dose administration group, indicating that compound LT-1433-303 did not damage the major organs of mice and had good safety.

[0224] The compounds provided in this invention are highly selective HDAC6 inhibitors, exhibiting good antitumor activity against RPMI 8226 cells. Compounds, represented by LT-1433-303, demonstrated good pharmacological activity in target validation and activity concentration studies, and showed inhibitory effects on both NIH / 3T3 and HPF fibroblasts. In in vitro pharmacodynamic evaluation experiments, LT-1433-303 slowed the progression of pulmonary fibrosis in a bleomycin-induced mouse pulmonary fibrosis model, primarily by reducing lung inflammation and collagen deposition in the lungs. Furthermore, the series II compound LT-1433-303 also improved weight loss during pulmonary fibrosis and showed good safety during continuous treatment.

[0225] References:

[0226] 1. Hao Gui, Zan Hu, Kang Yang, Jingkun Huang, Yichao Wu, Quanwei Chen, RanWei, Pengfei Wang, Hui Wang, Hongmei Li, Yadong Chen, Tao Lu, Yuqin Yao, Design and synthesis of TRAIL expression HDAC inhibitors based on ONC201 to promote apoptosis of colorectal cancer, European Journal of Medicinal Chemistry 240(2002)114484.

Claims

1. A compound with the following structural formula: or .

2. Use of the compound of claim 1 in the preparation of an anti-pulmonary fibrosis drug.

3. A drug for treating pulmonary fibrosis, characterized in that: It is prepared by adding pharmaceutically acceptable excipients or auxiliary ingredients to the compound of claim 1.

Images