A PDE4 inhibitor, its preparation method and application

Abstract

This invention discloses a PDE4 inhibitor, its preparation method, and its applications, belonging to the field of pharmaceutical technology. Targeting PDE4, this invention designed and synthesized 36 novel PDE4 inhibitors. The synthesized PDE4 inhibitors exhibit significant inhibitory effects on PDE4, and most compounds possess anti-inflammatory activity. Compound 11b has been shown to have therapeutic effects on pulmonary fibrosis.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a PDE4 inhibitor, its preparation method, and its application. Background Technology

[0002] Idiopathic pulmonary fibrosis (IPF) is a fatal chronic interstitial lung disease characterized by alveolar structural destruction and excessive collagen deposition in the pulmonary interstitium. Patients typically survive only 2-3 years after diagnosis, earning it the nickname "the cancer that isn't cancer." Currently approved drugs such as pirfenidone and nintedanib can only slow disease progression, not reverse fibrosis, and have side effects such as gastrointestinal reactions and liver damage. Significant unmet needs remain in clinical treatment.

[0003] In recent years, research on the pathogenesis of IPF has confirmed that the "vicious cycle of inflammation-fibrosis" formed by abnormal activation of inflammatory pathways and excessive activation of fibroblasts is the core driving force of disease progression. Epithelial-mesenchymal transition (EMT), as a key pathway for fibroblast activation, significantly promotes collagen deposition in the pulmonary interstitium through abnormal activation. Among these pathways, dysfunction of the cyclic adenosine monophosphate (cAMP) signaling pathway is considered a key regulatory node. Decreased cAMP levels significantly enhance the release of pro-inflammatory factors, myofibroblast differentiation, and the EMT process. Phosphodiesterase 4 (PDE4), as an enzyme that specifically hydrolyzes cAMP, is highly expressed in pulmonary fibroblasts and is upregulated with the progression of fibrosis, making it a potential target for IPF treatment. Summary of the Invention

[0004] The purpose of this invention is to provide a PDE4 inhibitor, its preparation method, and its application. Targeting PDE4, this invention designs and synthesizes 36 novel PDE4 inhibitors.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] One of the technical solutions of this invention is to provide a PDE4 inhibitor, the structural formula of which is shown in any of the following structures:

[0007]

[0008]

[0009]

[0010]

[0011]

[0012]

[0013]

[0014]

[0015]

[0016]

[0017] .

[0018] The second technical solution of the present invention provides a method for preparing the above-mentioned PDE4 inhibitor, comprising the following steps:

[0019] The general structural formulas for compounds 5a~5n are shown in formula V: ;

[0020] The preparation process of the compound shown in Formula V is as follows:

[0021]

[0022] Step a includes: reacting compound I with ethyl difluorochloroacetate and sodium carbonate at 80°C for 12 h to obtain compound II;

[0023] Step b includes: reacting compound II with potassium carbonate and bromomethylcyclopropane at 80°C for 8-12 hours to obtain compound III;

[0024] Step c includes: reacting compound III with sodium hydroxide and 30wt% hydrogen peroxide solution at 50°C for 1 hour to obtain compound IV;

[0025] Step d includes: reacting compound IV with R1-OH at 20~50℃ for 4~8h to obtain compound V;

[0026] The general structural formulas corresponding to compounds 8a to 8d are shown in formula VIII: ;

[0027] The preparation process of the compound shown in Formula VIII is as follows:

[0028]

[0029] Step e includes: reacting compound III with a boron tetrahydrofuran complex at room temperature for 4 hours to obtain compound VI;

[0030] Step f includes: stirring the compound of formula IV with carbon tetrabromide and triphenylphosphine at room temperature for 8-12 hours to obtain the compound of formula VII;

[0031] Step g includes: reacting compound VII with R2-OH at 80°C for 10-12 h to obtain compound VIII;

[0032] The general structural formulas of compounds 11a~11j are shown in Formula XⅠ: ;

[0033] The preparation process of the compound shown in Formula XⅠ is as follows:

[0034]

[0035] Step h includes: reacting compound III with methyl 3,4-diaminobenzoate at 80°C for 10-14 h to obtain compound IX;

[0036] Step i includes: hydrolyzing compound IX at 20~50℃ for 2~6h to obtain compound X;

[0037] Step j includes: reacting compound X with R3-OH at 20~50℃ for 4~8h to obtain compound XⅠ;

[0038] The preparation process of compound 14a is as follows:

[0039]

[0040] Step k includes: reacting compound X with a borate tetrahydrofuran complex at room temperature for 4 hours to obtain compound XII;

[0041] Step 1 includes: stirring compound XII with carbon tetrabromide and triphenylphosphine at room temperature for 8-12 hours to obtain compound XIII;

[0042] Step m includes: reacting compound XIII with R4-OH at 80°C with stirring for 10-14 h to obtain compound 14a;

[0043] The general structural formulas of compounds 8e~8k are shown in formula XⅣ: ;

[0044] The preparation process of the compound shown in Formula XIV is as follows:

[0045]

[0046] Step n includes: reacting compound 8d with R4-NH2 at room temperature for 4-8 hours to obtain compound XⅣ.

[0047] The third technical solution of the present invention is to provide a pharmaceutically acceptable salt of the above-mentioned PDE4 inhibitor.

[0048] The fourth technical solution of the present invention provides the application of the above-mentioned PDE4 inhibitor in the preparation of drugs for treating idiopathic pulmonary fibrosis.

[0049] Fifth technical solution of the present invention: providing the application of a pharmaceutically acceptable salt of the above-mentioned PDE4 inhibitor in the preparation of a drug for treating idiopathic pulmonary fibrosis.

[0050] The beneficial technical effects of the present invention are as follows:

[0051] Targeting PDE4, this invention designed and synthesized 36 novel PDE4 inhibitors. The synthesized PDE4 inhibitors exhibit significant inhibitory effects on PDE4, and most compounds possess anti-inflammatory activity. Compound 11b has been shown to have therapeutic effects on pulmonary fibrosis. Attached Figure Description

[0052] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0053] Figure 1 The effects of the compounds in Experiment 2 on the mRNA expression levels of inflammatory factors IL-6 (A), TNF-α (B), and IL-1β (C) in RAW 264.7 cells were investigated.

[0054] Figure 2 The IC50 of compound 11b in Experiment 2 against the inflammatory factor IL-6 in RAW 264.7 cells. 50 (A) and compound 14a's IC50 of IL-6 inflammatory cytokine in RAW 264.7 cells 50 (B).

[0055] Figure 3 The anti-fibrotic ability of compounds 11b and 14a in the scratch assay of A549 cells in Experiment Example 3 is shown in Figure (A) and the statistical results are shown in Figure (B).

[0056] Figure 4 This is a statistical result of the number of cells in the bronchoalveolar lavage fluid of the mouse model of pulmonary fibrosis in Experiment Example 4.

[0057] Figure 5 This is a Giemsa staining image of cells in the bronchoalveolar lavage fluid from the mouse model of pulmonary fibrosis in Experiment Example 4.

[0058] Figure 6 The levels of IL-6 (A) and TNF-α (B) inflammatory factors in the BALF supernatant of the pulmonary fibrosis mouse model in Experiment Example 4.

[0059] Figure 7The expression levels of IL-6 (A), TNF-α (B), and IL-1β (C) mRNA in the lung tissue of the pulmonary fibrosis mouse model in Experiment Example 4.

[0060] Figure 8 HE staining image of lung tissue from the pulmonary fibrosis mouse model in Experiment Example 4.

[0061] Figure 9 Masson staining image of lung tissue from the pulmonary fibrosis mouse model in Experiment Example 4. Detailed Implementation

[0062] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention. It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the present invention.

[0063] It should be noted that any aspects not described in detail in this invention are conventional practices in the field and are not the focus of this invention.

[0064] Furthermore, regarding the numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, are also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0065] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar to or equivalent to those described herein may be used in the implementation or testing of this invention.

[0066] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0067] Unless otherwise specified, room temperature in this invention refers to a temperature of 20±10℃.

[0068] Example 1

[0069] The synthetic routes for compounds 5a~5n are as follows:

[0070]

[0071] Intermediates 2, 3, and 4 mentioned in the route have all been reported. This invention will simply describe their synthesis steps and will not mention their characterization details again.

[0072] Synthesis of intermediate 2:

[0073] Weigh 3.3 g of starting material 1 (21.6 mmol, 1.0 equiv), add 1.1 g of sodium carbonate (26 mmol, 1.2 equiv), add N,N-dimethylformamide (DMF, 10 mL), and finally add 3.4 g of ethyl difluorochloroacetate (26.0 mmol, 1.2 equiv). React at 80 °C for 12 h. After the reaction is complete, extract three times with ethyl acetate and wash the organic phase with saturated sodium chloride solution. Concentrate the organic phase and perform rapid column chromatography to obtain 1.6 g of intermediate 2, with a yield of 37%.

[0074] Synthesis of intermediate 3:

[0075] 3.1 g of compound 2 (15.0 mmol, 1.0 equiv) was weighed, and 4.2 g of potassium carbonate (30.0 mmol, 2.0 equiv) was added. DMF (5 mL) was then added, followed by 2.49 g of bromomethylcyclopropane (18.0 mmol, 1.2 equiv). The reaction was carried out at 80 °C for 5 h until complete. The mixture was extracted three times with ethyl acetate and washed with saturated sodium chloride solution. The organic phase was concentrated and subjected to rapid column chromatography to give 2.2 g of intermediate 3 in 75% yield.

[0076] Synthesis of intermediate 4:

[0077] Sodium hydroxide (15.9 g, 396.8 mmol, 8.0 equiv) and 30 wt% hydrogen peroxide solution (248.0 mmol, 5.0 equiv) were added to a mixed solution of intermediate 3 (12.0 g, 49.6 mmol, 1.0 equiv) (methanol / water, V / V = 2:1). The reaction mixture was heated to 50 °C and stirred for 1 h. After the reaction was complete, the mixture was cooled to room temperature and concentrated under reduced pressure. The system was adjusted to acidity (pH = 6) with dilute hydrochloric acid. The precipitated solid was filtered and dried to obtain intermediate 4 in 91% yield.

[0078] Synthesis of compound 5a:

[0079] 100 mg of compound 4 (0.39 mmol, 1.0 equiv) was weighed, and 89 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 0.49 mmol, 1.2 equiv) and 12 mg of 4-dimethylaminopyridine (DMAP, 0.1 mmol, 0.25 equiv) were added. Anhydrous dichloromethane (DCM, 3 mL) was then added as solvent, followed by 107 mg of 2-trifluoromethyl-4-hydroxy-8-fluoroquinoline (0.49 mmol, 1.2 equiv). The reaction was carried out at room temperature. After the reaction was complete, the mixture was extracted three times with ethyl acetate and washed with saturated sodium chloride solution. The organic phase was concentrated and subjected to rapid column chromatography to give compound 5a in 90% yield.

[0080]

[0081] The obtained compound 5a was identified by nuclear magnetic resonance spectroscopy, and the identification results were as follows:

[0082] 1 H NMR (500 MHz, CDCl3) δ 7.93 (dd, J = 8.5, 2.0 Hz, 1H), 7.86 (s,1H), 7.84 (s, 1H), 7.81 (d, J = 2.0 Hz, 1H), 7.65 (td, J = 8.0, 5.0 Hz, 1H),7.58–7.55 (m, 1H), 7.36 (d, J = 8.0 Hz, 1H), 6.81 (s, 1H), 4.00 (d, J = 7.0Hz, 2H), 1.40–1.29 (m, 1H), 0.73–0.67 (m, 2H), 0.43–0.39 (m, 2H). 13 C NMR (126MHz, CDCl3) δ 162.7, 158.3, 155.8, 150.7, 148.9, 145.5, 139.6, 129.1, 125.7,124.7, 123.9, 122.2, 120.9, 117.1, 115.9, 115.7, 115.6, 110.5, 74.4, 10.0,3.3 (2×C).

[0083] Synthesis of compound 5b:

[0084] 100 mg of compound 4 (0.39 mmol, 1.0 equiv) was weighed, and 89 mg of EDCI (0.49 mmol, 1.2 equiv) and 12 mg of DMAP (0.1 mmol, 0.25 equiv) were added. Then, anhydrous DCM (3 mL) was added as solvent, followed by 95 mg of 6,7-methoxy-4-hydroxyquinoline (0.49 mmol, 1.2 equiv). The reaction was carried out under ambient conditions. After the reaction was complete, the mixture was extracted three times with ethyl acetate and washed with saturated sodium chloride solution. The organic phase was concentrated and subjected to rapid column chromatography to give compound 5b in 85% yield.

[0085]

[0086] The obtained compound 5b was identified by nuclear magnetic resonance spectroscopy, and the identification results were as follows:

[0087] 1 H NMR (600 MHz, DMSO-d6) δ 8.74 (t, J = 4.8 Hz, 1H), 7.93 (dd, J =8.4, 2.4 Hz, 1H), 7.87 (d, J = 2.4 Hz, 1H), 7.47 (d, J = 3.6 Hz, 1H), 7.45(d, J = 8.4 Hz, 1H), 7.40 (d, J = 4.8 Hz, 1H), 7.34 (s, 1H), 7.21 (d, J = 9.6Hz, 1H), 4.04 (d, J = 7.2 Hz, 2H), 3.96 (s, 3H), 3.87 (s, 3H), 1.34–1.23 (m,1H), 0.64–0.56 (m, 2H), 0.42–0.35 (m, 2H). 13 C NMR (151 MHz, DMSO-d6) δ 163.5,153.2, 152.9, 150.5, 150.2, 148.9, 147.3, 145.1, 126.4, 123.9, 120.7, 117.5,116.8, 115.8, 112.5, 108.4, 99.2, 73.9, 56.3, 56.1, 10.4, 3.6. (2×C).

[0088] Synthesis of compound 5c:

[0089] 100 mg of compound 4 (0.39 mmol, 1.0 equiv) was weighed, and 89 mg of EDCI (0.49 mmol, 1.2 equiv) and 12 mg of DMAP (0.1 mmol, 0.25 equiv) were added. Then, anhydrous DCM (3 mL) was added as solvent, followed by 130 mg of 5,7-dichloro-4-hydroxy-2-(trifluoromethyl)quinoline (0.49 mmol, 1.2 equiv). The reaction was carried out at room temperature. After the reaction was complete, the mixture was extracted three times with ethyl acetate and washed with saturated sodium chloride solution. The organic phase was concentrated and subjected to rapid column chromatography to give compound 5c in 88% yield.

[0090]

[0091] The obtained compound 5c was identified by nuclear magnetic resonance spectroscopy, and the identification results were as follows:

[0092] 1 H NMR (500 MHz, CDCl3) δ 8.24 (d, J = 2.1 Hz, 1H), 7.89 (dd, J = 8.4,2.0 Hz, 1H), 7.80 (d, J = 2.0 Hz, 1H), 7.72 (d, J = 2.1 Hz, 1H), 7.59 (s,1H), 7.33 (d, J = 8.3 Hz, 1H), 6.80 (s, 1H), 3.98 (d, J = 7.0 Hz, 2H), 1.38 –1.29 (m, 1H), 0.72 – 0.67 (m, 2H), 0.42 – 0.38 (m, 2H). 13 C NMR (126 MHz, CDCl3) δ 163.3, 156.2, 150.7, 150.5, 150.3, 150.0, 145.3, 136.6, 132.1,129.1, 126.1, 124.3, 122.0, 120.6, 120.2, 115.9, 115.6, 112.8, 74.2, 10.0,3.3 (2×C).

[0093] Synthesis of compound 5d:

[0094] 100 mg of compound 4 (0.39 mmol, 1.0 equiv) was weighed, and 89 mg of EDCI (0.49 mmol, 1.2 equiv) and 12 mg of DMAP (0.1 mmol, 0.25 equiv) were added. Then, anhydrous DCM (3 mL) was added as solvent, followed by 106 mg of 6-trifluoromethoxyquinoline-4-ol (0.49 mmol, 1.2 equiv). The reaction was carried out at room temperature. After the reaction was complete, the mixture was extracted three times with ethyl acetate and washed with saturated sodium chloride solution. The organic phase was concentrated and subjected to rapid column chromatography to give compound 5d in 82% yield.

[0095]

[0096] The obtained compound 5d was identified by nuclear magnetic resonance spectroscopy, and the identification results were as follows:

[0097] 1 H NMR (600 MHz, DMSO-d6) δ 9.08 (d, J = 4.8 Hz, 1H), 8.28 (d, J = 9.6Hz, 1H), 8.00 (s, 1H), 7.93 (dd, J = 8.4, 2.4 Hz, 1H), 7.87 (dd, J = 6.0, 2.4Hz, 2H), 7.74 (d, J = 4.8 Hz, 1H), 7.46 (d, J = 8.4 Hz, 1H), 7.35 (s, 1H), 4.04 (d, J = 7.2 Hz, 2H), 1.32–1.26 (m, 1H), 0.62–0.58 (m, 2H), 0.40–0.37 (m,2H). 13 C NMR (151 MHz, DMSO-d6) δ 163.3, 154.2, 152.7, 150.1, 148.2, 147.0,145.2, 132.6, 126.0, 124.8, 124.1, 122.9, 120.7, 120.5, 116.8, 115.9, 115.3,113.2, 73.9, 10.3, 3.5 (2×C).

[0098] Synthesis of compound 5e:

[0099] 100 mg of compound 4 (0.39 mmol, 1.0 equiv) was weighed, and 89 mg of EDCI (0.49 mmol, 1.2 equiv) and 12 mg of DMAP (0.1 mmol, 0.25 equiv) were added. Then, anhydrous DCM (3 mL) was added as solvent, followed by 99 mg of 4-hydroxy-8-trifluoromethylquinoline (0.49 mmol, 1.2 equiv). The reaction was carried out at room temperature. After the reaction was complete, the mixture was extracted three times with ethyl acetate and washed with saturated sodium chloride solution. The organic phase was concentrated and subjected to rapid column chromatography to give compound 5e in 78% yield.

[0100]

[0101] The obtained compound 5e was identified by nuclear magnetic resonance spectroscopy, and the identification results were as follows:

[0102] 1 H NMR (400 MHz, DMSO-d6) δ 9.16 (d, J = 4.8 Hz, 1H), 8.35 (dd, J =8.4, 1.2 Hz, 1H), 8.27 (d, J = 8.0 Hz, 1H), 7.94 (dd, J = 8.4, 2.0 Hz, 1H),7.86 (d, J = 2.0 Hz, 1H), 7.82–7.75 (m, 2H), 7.45 (d, J = 8.4 Hz, 1H), 7.36(s, 1H), 4.03 (d, J = 7.2 Hz, 2H), 1.34–1.24 (m, 1H), 0.63–0.57 (m, 2H),0.40–0.36 (m, 2H). 13 C NMR (101 MHz, DMSO-d6) δ 163.4, 154.6, 152.9, 150.2,145.7, 145.3, 129.6, 127.1, 126.9, 126.5, 125.8, 125.0, 124.1, 123.1, 120.6,116.8, 115.8, 115.5, 73.9, 10.3, 3.5 (2×C).

[0103] Synthesis of compound 5f:

[0104] 100 mg of compound 4 (0.39 mmol, 1.0 equiv) was weighed, and 89 mg of EDCI (0.49 mmol, 1.2 equiv) and 12 mg of DMAP (0.1 mmol, 0.25 equiv) were added. Then, anhydrous DCM (3 mL) was added as solvent, followed by 84 mg of 4-hydroxy-8-chloroquinoline (0.49 mmol, 1.2 equiv). The reaction was carried out at room temperature. After the reaction was complete, the mixture was extracted three times with ethyl acetate and washed with saturated sodium chloride solution. The organic phase was concentrated and subjected to rapid column chromatography to give compound 5f in 75% yield.

[0105]

[0106] The obtained compound 5f was identified by nuclear magnetic resonance spectroscopy, and the identification results were as follows:

[0107] 1 H NMR (600 MHz, DMSO-d6) δ 9.02 (d, J = 4.8 Hz, 1H), 8.15 (d, J = 9.0Hz, 1H), 8.06 (d, J = 2.4 Hz, 1H), 7.93 (dd, J = 8.4, 2.4 Hz, 1H), 7.86 (dt,J = 5.4, 2.4 Hz, 2H), 7.66 (d, J = 4.8 Hz, 1H), 7.45 (d, J = 8.4 Hz, 1H), 7.35 (s, 1H), 4.03 (d, J = 7.2 Hz, 2H), 1.32–1.24 (m, 1H), 0.63–0.59 (m, 2H), 0.40–0.36 (m, 2H). 13 C NMR (151 MHz, DMSO-d6) δ 163.4, 153.5, 152.3, 150.1,148.3, 145.2, 132.5, 131.9, 131.5, 126.0, 124.1, 123.3, 120.8, 120.7, 116.8,116.0, 115.3, 73.9, 10.4, 3.6 (2×C).

[0108] Synthesis of 5g of compound:

[0109] 100 mg of compound 4 (0.39 mmol, 1.0 equiv) was weighed, and 89 mg of EDCI (0.49 mmol, 1.2 equiv) and 12 mg of DMAP (0.1 mmol, 0.25 equiv) were added. Then, anhydrous DCM (3 mL) was added as solvent, followed by 104 mg of 6-bromo-4-hydroxyquinoline (0.49 mmol, 1.2 equiv). The reaction was carried out at room temperature. After the reaction was complete, the mixture was extracted three times with ethyl acetate and washed with saturated sodium chloride solution. The organic phase was concentrated and subjected to rapid column chromatography to give 5 g of compound, in 85% yield.

[0110]

[0111] The obtained compound (5g) was identified using nuclear magnetic resonance spectroscopy, and the results were as follows:

[0112] 1 H NMR (600 MHz, DMSO-d6) δ 9.04 (d, J = 4.8 Hz, 1H), 8.23 ​​(d, J = 2.4Hz, 1H), 8.08 (d, J = 9.0 Hz, 1H), 7.99 (dd, J = 9.0, 2.4 Hz, 1H), 7.93 (dd,J = 8.4, 1,8 Hz, 1H), 7.87 (d, J = 2.4 Hz, 1H), 7.67 (d, J = 4.8 Hz, 1H), 7.46 (d, J = 8.4 Hz, 1H), 7.35 (s, 1H), 4.05 (d, J = 7.2 Hz, 2H), 1.32–1.26(m, 1H), 0.62–0.58 (m, 2H), 0.40–0.38 (m, 2H). 13 C NMR (151 MHz, DMSO-d6) δ163.4, 153.4, 152.4, 150.2, 148.5, 145.2, 134.1, 131.9, 126.0, 124.1, 124.1,123.8, 121.1, 120.7, 116.8, 116.0, 115.3, 73.9, 10.4, 3.6 (2×C).

[0113] Synthesis of compound 5h:

[0114] 100 mg of compound 4 (0.39 mmol, 1.0 equiv) was weighed, and 89 mg of EDCI (0.49 mmol, 1.2 equiv) and 12 mg of DMAP (0.1 mmol, 0.25 equiv) were added. Then, anhydrous DCM (3 mL) was added as solvent, followed by 81 mg of 4-hydroxy-7-methoxyquinoline (0.49 mmol, 1.2 equiv). The reaction was carried out at room temperature. After the reaction was complete, the mixture was extracted three times with ethyl acetate and washed with saturated sodium chloride solution. The organic phase was concentrated and subjected to rapid column chromatography to obtain compound 5 h in 80% yield.

[0115]

[0116] The obtained compound was identified by nuclear magnetic resonance spectroscopy after 5 hours. The identification results were as follows:

[0117] 1 H NMR (600 MHz, DMSO-d6) δ 8.90 (d, J = 4.8 Hz, 1H), 7.93–7.91 (m,1H), 7.89 (s, 1H), 7.84 (d, J = 2.4 Hz, 1H), 7.50 (d, J = 2.4 Hz, 1H), 7.45(d, J = 8.4 Hz, 1H), 7.42 (d, J = 4.8 Hz, 1H), 7.35 (s,1H), 7.30 (dd, J =9.0, 2.4 Hz, 1H), 4.03 (d, J = 6.8 Hz, 2H), 3.95 (s, 3H), 1.32–1.25 (m, 1H), 0.62–0.57 (m, 2H), 0.40–0.37 (m, 2H). 13 C NMR (151 MHz, DMSO-d6) δ 163.5,161.2, 154.4, 152.1, 151.9, 150.2, 145.2, 126.1, 123.9, 123.1, 120.7, 120.6,117.2, 116.8, 115.8, 112.4, 108.0, 73.9, 56.1, 10.4, 3.5 (2×C).

[0118] Synthesis of compound 5i:

[0119] 100 mg of compound 4 (0.39 mmol, 1.0 equiv) was weighed, and 89 mg of EDCI (0.49 mmol, 1.2 equiv) and 12 mg of DMAP (0.1 mmol, 0.25 equiv) were added. Then, anhydrous DCM (3 mL) was added as solvent, followed by 131 mg of 2,8-bis(trifluoromethyl)-4-hydroxyquinoline (0.49 mmol, 1.2 equiv). The reaction was carried out at room temperature. After the reaction was complete, the mixture was extracted three times with ethyl acetate and washed with saturated sodium chloride solution. The organic phase was concentrated and subjected to rapid column chromatography to give compound 5i in 91% yield.

[0120]

[0121] The obtained compound 5i was identified by nuclear magnetic resonance spectroscopy, and the identification results were as follows:

[0122] 1 H NMR (500 MHz, CDCl3) δ 8.26 (dd, J = 8.5, 1.5 Hz, 1H), 8.22 (d, J =7.0 Hz, 1H), 7.94 (dd, J = 8.5, 2.0 Hz, 1H), 7.85 (s, 1H), 7.82 (d, J = 2.0Hz, 1H), 7.75 (t, J = 8.0 Hz, 1H), 7.37 (d, J = 8.5 Hz, 1H), 6.82 (s, 1H), 4.00 (d, J = 7.0 Hz, 2H), 1.38–1.32 (m, 1H), 0.75–0.67 (m, 2H), 0.43–0.40 (m, 2H). 13 C NMR (126 MHz, CDCl3) δ 162.7, 155.9, 150.7, 149.3, 145.5, 145.3,129.8, 129.2, 127.7, 125.6, 123.9, 123.7, 123.0, 122.2, 122.2, 119.7, 115.9,115.6, 110.5, 74.4, 10.0, 3.3 (2×C).

[0123] Synthesis of compound 5j:

[0124] 100 mg of compound 4 (0.39 mmol, 1.0 equiv) was weighed, and 89 mg of EDCI (0.49 mmol, 1.2 equiv) and 12 mg of DMAP (0.1 mmol, 0.25 equiv) were added. Then, anhydrous DCM (3 mL) was added as solvent, followed by 101 mg of ethyl 4-hydroxyquinoline-3-carboxylate (0.49 mmol, 1.2 equiv). The reaction was carried out at room temperature. After the reaction was complete, the mixture was extracted three times with ethyl acetate and washed with saturated sodium chloride solution. The organic phase was concentrated and subjected to rapid column chromatography to give compound 5j in 78% yield.

[0125]

[0126] The obtained compound 5j was identified by nuclear magnetic resonance spectroscopy, and the identification results were as follows:

[0127] 1 H NMR (500 MHz, CDCl3) δ 8.38 (d, J = 8.5 Hz, 1H), 8.15 (s, 1H), 8.00 (dd, J = 8.5, 1.5 Hz, 1H), 7.94 (dd, J = 8.0, 2.0 Hz, 1H), 7.87–7.79 (m, 2H),7.69–7.65 (m, 1H), 7.35 (d, J = 8.5 Hz, 1H), 6.80 (s, 1H), 4.57 (q, J = 7.0Hz, 2H), 3.99 (d, J = 7.0 Hz, 2H), 1.49 (t, J = 7.0 Hz, 3H), 1.39–1.29 (m,1H), 0.73–0.65 (m, 2H), 0.43–0.38 (m, 2H). 13 C NMR (126 MHz, CDCl3) δ 164.8,163.1, 155.3, 150.6, 149.4, 149.1, 145.2, 131.1, 130.9, 129.0, 126.3, 123.8,123.5, 122.1, 121.1, 115.8, 115.6, 113.5, 74.3, 62.5, 14.4, 10.0, 3.3 (2×C).

[0128] Synthesis of compound 5k:

[0129] 100 mg of compound 4 (0.39 mmol, 1.0 equiv) was weighed, and 89 mg of EDCI (0.49 mmol, 1.2 equiv) and 12 mg of DMAP (0.1 mmol, 0.25 equiv) were added. Then, anhydrous DCM (3 mL) was added as solvent, followed by 76 mg of 4-hydroxy-6-fluoroquinoline (0.49 mmol, 1.2 equiv). The reaction was carried out at room temperature. After the reaction was complete, the mixture was extracted three times with ethyl acetate and washed with saturated sodium chloride solution. The organic phase was concentrated and subjected to rapid column chromatography to give compound 5K in 75% yield.

[0130]

[0131] The obtained compound 5k was identified using nuclear magnetic resonance spectroscopy, and the identification results were as follows:

[0132] 1 H NMR (400 MHz, DMSO-d6) δ 8.99 (dt, J = 4.8, 2.0Hz, 1H), 8.19–8.24(m, 1H), 7.93 (dd, J = 8.4, 2.0 Hz, 1H), 7.86 (q, J = 2.0 Hz, 1H), 7.80–7.75(m, 2H), 7.66 (dt, J = 4.8, 2.0 Hz, 1H), 7.48–7.41 (m, 1H), 7.37–7.32 (m,1H), 4.04 (dt, J = 7.2, 2.0 Hz, 2H), 1.34–1.25 (m, 1H), 0.62–0.57 (m, 2H), 0.42–0.35 (m, 2H). 13 C NMR (101 MHz, DMSO-d6) δ 163.4, 161.9, 159.4, 154.0,151.3, 150.1, 132.8, 147.1, 145.2, 126.0, 124.1, 123.3, 120.6, 119.4, 116.8,115.9, 115.1, 105.7, 73.9, 10.4, 3.6 (2×C).

[0133] Synthesis of compound 5l:

[0134] 100 mg of compound 4 (0.39 mmol, 1.0 equiv) was weighed, and 89 mg of EDCI (0.49 mmol, 1.2 equiv) and 12 mg of DMAP (0.1 mmol, 0.25 equiv) were added. Then, anhydrous DCM (3 mL) was added as solvent, followed by 82 mg of 6-fluoro-2-methylquinoline-4-ol (0.49 mmol, 1.2 equiv). The reaction was carried out at room temperature. After the reaction was complete, the mixture was extracted three times with ethyl acetate and washed with saturated sodium chloride solution. The organic phase was concentrated and subjected to rapid column chromatography to give compound 5l in 83% yield.

[0135]

[0136] The obtained compound 5l was identified by nuclear magnetic resonance spectroscopy, and the identification results were as follows:

[0137] 1 H NMR (600 MHz, DMSO-d6) δ 8.09 (dd, J = 9.0, 5.4 Hz, 1H), 7.91 (dd,J = 8.4, 2.4 Hz, 1H), 7.87–7.82 (m, 1H), 7.74–7.66 (m, 2H), 7.54 (d, J = 1.8Hz, 1H), 7.44 (d, J = 8.4 Hz, 1H), 7.35 (s, 1H), 4.03 (d, J = 7.2 Hz, 2H), 2.69 (s, 3H), 1.31–1.27 (m, 1H), 0.63–0.57 (m, 2H), 0.40–0.36 (m, 2H). 13 C NMR(151 MHz, DMSO-d6) δ 163.5, 161.0, 159.8, 159.3, 154.1, 150.1, 146.6, 145.2,131.9, 126.0, 124.0, 121.6, 120.6, 116.8, 115.9, 115.6, 105.6, 73.9, 25.3,10.4, 3.5 (2×C).

[0138] Synthesis of compound 5m:

[0139] 100 mg of compound 4 (0.39 mmol, 1.0 equiv) was weighed, and 89 mg of EDCI (0.49 mmol, 1.2 equiv) and 12 mg of DMAP (0.1 mmol, 0.25 equiv) were added. Then, anhydrous DCM (3 mL) was added as solvent, followed by 106 mg of 2-trifluoromethyl-4-hydroxy-6-methylquinoline (0.49 mmol, 1.2 equiv). The reaction was carried out at room temperature. After the reaction was complete, the mixture was extracted three times with ethyl acetate and washed with saturated sodium chloride solution. The organic phase was concentrated and subjected to rapid column chromatography to give compound 5m in 85% yield.

[0140]

[0141] The obtained compound 5m was identified by nuclear magnetic resonance spectroscopy, and the identification results were as follows:

[0142] 1 H NMR (600 MHz, DMSO-d6) δ 8.17 (d, J = 9.0 Hz, 1H), 8.11 (s, 1H), 7.94 (dd, J = 8.4, 1.8 Hz, 1H), 7.93–7.91 (m, 1H), 7.87 (d, J = 2.4 Hz, 1H),7.85 (dd, J = 9.0, 1.8 Hz, 1H), 7.47 (d, J = 8.4 Hz, 1H), 7.36 (s, 1H), 4.04(d, J = 7.2 Hz, 2H), 2.55 (s, 3H), 1.33–1.26 (m, 1H), 0.64–0.59 (m, 2H), 0.42–0.37 (m, 2H). 13 C NMR (151 MHz, DMSO-d6) δ 163.4, 155.8, 150.2, 147.4,146.7, 145.3, 140.6, 134.9, 129.8, 125.9, 124.2, 123.4, 121.8, 120.7, 120.6,116.8, 116.0, 110.8, 73.9, 21.9, 10.4, 3.6 (2×C).

[0143] Synthesis of compound 5n:

[0144] 100 mg of compound 4 (0.39 mmol, 1.0 equiv) was weighed, and 89 mg of EDCI (0.49 mmol, 1.2 equiv) and 12 mg of DMAP (0.1 mmol, 0.25 equiv) were added. Then, anhydrous DCM (3 mL) was added as solvent, followed by 94 mg of ethyl 4-hydroxyquinoline-3-carboxylate (0.49 mmol, 1.2 equiv). The reaction was carried out at room temperature. After the reaction was complete, the mixture was extracted three times with ethyl acetate and washed with saturated sodium chloride solution. The organic phase was concentrated and subjected to rapid column chromatography to give compound 5n in 88% yield.

[0145]

[0146] The obtained compound 5n was identified by nuclear magnetic resonance spectroscopy, and the identification results were as follows:

[0147] 1 H NMR (600 MHz, DMSO-d6) δ 8.27 (d, J = 8.4 Hz, 1H), 8.18 (s, 1H), 8.11 (dd, J = 8.4, 1.8 Hz, 1H), 7.98–7.95 (m, 1H), 7.93 (dd, J = 8.4, 2.4 Hz,1H), 7.87 (d, J = 2.4 Hz, 1H), 7.83–7.80 (m, 1H), 7.45 (d, J = 8.4 Hz, 1H),7.36 (s, 1H), 4.04 (d, J = 7.2 Hz, 2H), 3.98 (s, 3H), 1.33–1.24 (m, 1H), 0.64–0.57 (m, 2H), 0.40–0.37 (m, 2H). 13 C NMR (151 MHz, DMSO-d6) δ 165.2,163.4, 155.4, 150.2, 149.0, 148.9, 145.2, 132.0, 130.6, 130.1, 126.0, 124.1,123.4, 122.0, 120.7, 116.8, 115.9, 113.9, 73.9, 53.3, 10.4, 3.6 (2×C).

[0148] Example 2

[0149] The synthetic route for compounds 8a~8k is as follows:

[0150]

[0151] Intermediates 6 and 7 mentioned in the route have been reported. This invention will simply describe their synthesis steps and will not mention their characterization details again.

[0152] Synthesis of intermediate 6:

[0153] Compound 4 (10.8 mmol, 1.0 equiv) was dissolved in tetrahydrofuran (THF). After stirring in an ice bath at 0°C for 10 min, a borane-tetrahydrofuran complex (12.96 mmol, 1.2 equiv) was added. The mixture was purged with nitrogen three times, and the reaction was allowed to proceed to room temperature for 4 h. After the reaction was complete, the mixture was quenched with 3M sodium hydroxide solution and 30 wt% H₂O₂, extracted three times with ethyl acetate, and washed with saturated sodium chloride solution. The organic phase was concentrated and subjected to rapid column chromatography to give intermediate 6 in 87% yield.

[0154] Synthesis of intermediate 7:

[0155] 1.8 g of compound 6 (7.4 mmol, 1.0 equiv) was dissolved in anhydrous DCM, and triphenylphosphine (8.9 mmol, 1.2 equiv) and carbon tetrabromide (8.9 mmol, 1.2 equiv) were added. The reaction was carried out at 0 °C to room temperature for 4 h. After the reaction was completed, the mixture was extracted three times with ethyl acetate and washed with saturated sodium chloride solution. The organic phase was concentrated and subjected to rapid column chromatography to give intermediate 7 in 91% yield.

[0156] Synthesis of compound 8a:

[0157] 100 mg of compound 7 (0.3 mmol, 1.0 equiv) was dissolved in DMF, and 213 mg of cesium carbonate (0.6 mmol, 2.0 equiv) and 91 mg of 2-trifluoromethyl-4-hydroxy-8-fluoroquinoline (0.4 mmol, 1.2 equiv) were added. The reaction was carried out at 80 °C for 4 h. After the reaction was completed, the mixture was extracted three times with ethyl acetate and washed with saturated sodium chloride solution. The organic phase was concentrated and subjected to rapid column chromatography to give compound 8a in 83% yield.

[0158]

[0159] The obtained compound 8a was identified by nuclear magnetic resonance spectroscopy, and the identification results were as follows:

[0160] 1H NMR (400 MHz, DMSO-d6) δ 8.04 (dd, J = 7.6, 1.6 Hz, 1H), 7.80–7.68(m, 2H), 7.65 (s, 1H), 7.39 (d, J = 2.0 Hz, 1H), 7.25 (d, J = 8.0 Hz, 1H),7.19 (dd, J = 8.4, 2.0 Hz, 1H), 5.51 (s, 2H), 3.93 (d, J = 7.2 Hz, 2H), 1.31–1.23 (m, 1H), 0.60–0.55 (m, 2H), 0.37–0.33 (m, 2H). 13 C NMR (101 MHz, DMSO-d6)δ 163.1, 157.7, 150.4, 148.6, 140.3, 138.1, 134.3, 128.8, 123.5, 121.8,121.8, 121.1, 118.4, 117.2, 116.2, 115.1, 99.6, 73.5, 71.2, 10.4, 3.5 (2×C).

[0161] Synthesis of compound 8b:

[0162] 100 mg of compound 7 (0.3 mmol, 1.0 equiv) was dissolved in DMF, and 213 mg of cesium carbonate (0.6 mmol, 2.0 equiv) and 80 mg of 6,7-methoxy-4-hydroxyquinoline (0.4 mmol, 1.2 equiv) were added. The reaction was carried out at 80 °C for 4 h. After the reaction was completed, the mixture was extracted three times with ethyl acetate and washed with saturated sodium chloride solution. The organic phase was concentrated and subjected to rapid column chromatography to give compound 8b in 83% yield.

[0163]

[0164] The obtained compound 8b was identified by nuclear magnetic resonance spectroscopy, and the identification results were as follows:

[0165] 1H NMR (400 MHz, DMSO-d6) δ 8.10 (d, J = 7.6 Hz, 1H), 7.53 (s, 1H), 7.21 (d, J = 1.2 Hz, 1H), 7.13 (d, J = 8.4 Hz, 1H), 7.03 (s, 1H), 7.00 (s,1H), 6.78 (dd, J = 8.4, 2.0 Hz, 1H), 6.06 (d, J = 7.8 Hz, 1H), 5.47 (s, 2H), 3.84 (d, J = 6.8 Hz, 2H), 3.81 (s, 3H), 3.78 (s, 3H), 1.24–1.17 (m, 1H),0.58–0.52 (m, 2H), 0.33–0.29 (m, 2H). 13 C NMR (101 MHz, DMSO-d6) δ 175.7,153.1, 150.5, 146.9, 144.0, 139.7, 135.8, 135.8, 122.0, 121.4, 119.4, 117.2,113.9, 108.7, 105.7, 99.6, 73.5, 56.3, 55.9, 55.2, 10.4, 3.5 (2×C).

[0166] Synthesis of compound 8c:

[0167] 100 mg of compound 7 (0.3 mmol, 1.0 equiv) was dissolved in DMF, and 213 mg of cesium carbonate (0.6 mmol, 2.0 equiv) and 110 mg of 5,7-dichloro-4-hydroxy-2-(trifluoromethyl)quinoline (0.4 mmol, 1.2 equiv) were added. The reaction was carried out at 80 °C for 4 h. After the reaction was completed, the mixture was extracted three times with ethyl acetate and washed with saturated sodium chloride solution. The organic phase was concentrated and subjected to rapid column chromatography to give compound 8c in 83% yield.

[0168]

[0169] The obtained compound 8c was identified by nuclear magnetic resonance spectroscopy, and the identification results were as follows:

[0170] 1H NMR (600 MHz, DMSO-d6) δ 8.16 (s, 1H), 7.92 (s, 1H), 7.63 (s, 1H), 7.40 (s, 1H), 7.23 (d, J = 7.2 Hz, 1H), 7.16 (d, J = 8.4 Hz, 1H), 7.11(s,1H), 5.50 (s, 2H), 3.90 (d, J = 7.2 Hz, 2H), 1.22–1.29 (m, 1H), 0.58 (d, J =7.8 Hz, 2H), 0.34 (d, J = 4.8 Hz, 2H). 13 C NMR (151 MHz, DMSO-d6) δ 164.2,150.2, 149.8, 140.0, 135.6, 134.0, 131.2, 130.3, 128.4, 122.4, 121.6, 120.7,120.6, 118.3, 117.2, 114.7, 100.7, 73.5, 71.8, 10.4, 3.5 (2×C).

[0171] Synthesis of compound 8d:

[0172] 100 mg of compound 7 (0.3 mmol, 1.0 equiv) was dissolved in DMF, and 213 mg of cesium carbonate (0.6 mmol, 2.0 equiv) and 74 mg of quinoline 4-hydroxy-2-carboxylic acid (0.4 mmol, 1.2 equiv) were added. The reaction was carried out at 80 °C for 4 h. After the reaction was completed, the mixture was extracted three times with ethyl acetate and washed with saturated sodium chloride solution. The organic phase was concentrated and subjected to rapid column chromatography to give compound 8d in 83% yield.

[0173]

[0174] The obtained compound 8d was identified by nuclear magnetic resonance spectroscopy, and the identification results were as follows:

[0175] 1H NMR (400 MHz, DMSO-d6) δ 8.71 (d, J = 8.4 Hz, 1H), 8.30 (d, J = 8.4Hz, 1H), 7.98-7.92 (m, 2H), 7.76 (t, J = 7.6 Hz, 1H), 7.43 (s, 1H), 7.25 (d,J = 8.0 Hz, 1H), 7.21 (d, J = 8.4 Hz, 1H), 7.12 (s, 1H), 5.57 (s, 2H), 3.95(d, J = 6.8 Hz, 2H), 1.30–1.25 (m, 1H), 0.60–0.56 (m, 2H), 0.38–0.35 (m, 2H),OH (not found). 13 C NMR (101 MHz, DMSO-d6) δ 166.2, 163.9, 153.5, 150.5, 145.4,140.1, 134.7, 131.8, 129.0, 128.1, 122.1, 122.0, 121.8, 120.7, 117.3, 114.8,100.8, 73.5, 70.6, 10.5, 3.5 (2×C).

[0176] Synthesis of compound 8e:

[0177] 100 mg of compound 8d (0.24 mmol, 1.0 equiv) was weighed, and 55 mg of EDCI (0.29 mmol, 1.2 equiv) and 7 mg of DMAP (0.06 mmol, 0.25 equiv) were added. Then, anhydrous DCM (3 mL) was added as solvent, followed by 43 mg of 6-aminobenzothiazole (0.29 mmol, 1.2 equiv). The reaction was carried out at room temperature. After the reaction was complete, the mixture was extracted three times with ethyl acetate and washed with saturated sodium chloride solution. The organic phase was concentrated and subjected to rapid column chromatography to give compound 8e in 75% yield.

[0178]

[0179] The obtained compound 8e was identified by nuclear magnetic resonance spectroscopy, and the identification results were as follows:

[0180] 1H NMR (400 MHz, DMSO-d6) δ 10.98 (s, 1H), 9.34 (s, 1H), 8.82 (d, J =2.0 Hz, 1H), 8.27 (d, J = 8.4 Hz, 1H), 8.22 (d, J = 8.4 Hz, 1H), 8.12 (d, J =8.8 Hz, 1H), 8.05 (dd, J = 8.8, 2.0 Hz, 1H), 7.92 (t, J = 7.6 Hz, 1H), 7.83(s, 1H), 7.72 (t, J = 7.6 Hz, 1H), 7.41 (d, J = 2.0 Hz, 1H), 7.25 (d, J = 8.4Hz, 1H), 7.20 (dd, J = 8.4, 2.0 Hz, 1H), 7.11 (s, 1H), 5.51 (s, 2H), 3.95 (d,J = 6.8 Hz, 2H), 1.33–1.24 (m, 1H), 0.60–0.55 (m, 2H), 0.41–0.30 (m, 2H). 13 CNMR (101 MHz, DMSO-d6) δ 163.3, 162.6, 155.6, 151.9, 150.4, 150.1, 147.5,140.1, 136.5, 134.9, 134.6, 131.4, 129.6, 128.2, 123.4, 122.3, 122.0, 121.8,120.7, 120.4, 117.2, 114.8, 113.4, 99.7, 73.5, 70.3, 10.4, 3.5 (2×C).

[0181] Synthesis of compound 8f:

[0182] 100 mg of compound 8d (0.24 mmol, 1.0 equiv) was weighed, and 55 mg of EDCI (0.29 mmol, 1.2 equiv) and 7 mg of DMAP (0.06 mmol, 0.25 equiv) were added. Then, anhydrous DCM (3 mL) was added as solvent, followed by 33 mg of 4-aminomethyltetrahydropyran (0.29 mmol, 1.2 equiv). The reaction was carried out at room temperature. After the reaction was complete, the mixture was extracted three times with ethyl acetate and washed with saturated sodium chloride solution. The organic phase was concentrated and subjected to rapid column chromatography to give compound 8f in 75% yield.

[0183]

[0184] The obtained compound 8f was identified by nuclear magnetic resonance spectroscopy, and the identification results were as follows:

[0185] 1 H NMR (400 MHz, DMSO-d6) δ 8.94 (t, J = 6.4 Hz, 1H), 8.23 ​​(dd, J =8.4, 1.6 Hz, 1H), 8.08 (d, J = 8.4 Hz, 1H), 7.87–7.83 (m, 1H), 7.73 (s, 1H),7.69–7.64 (m, 1H), 7.38 (d, J = 2.0 Hz, 1H), 7.24 (d, J = 8.4 Hz, 1H), 7.17(dd, J = 8.4, 2.0 Hz, 1H), 7.11 (s, 1H), 5.46 (s, 2H), 3.93 (d, J = 6.8 Hz,2H), 3.87–3.82 (m, 2H), 3.30–3.23 (m, 4H), 1.92–1.83 (m, 1H), 1.60–1.56 (m,2H), 1.29–1.26 (m, 1H), 1.26–1.21 (m, 2H), 0.59–0.55 (m, 2H), 0.37–0.33 (m, 2H). 13 C NMR (101 MHz, DMSO-d6) δ 164.6, 162.3, 152.2, 150.4, 147.6, 140.0,134.9, 131.2, 129.5, 127.7, 122.2, 121.8, 120.6, 119.8, 117.2, 114.7, 99.6,73.5, 70.2, 67.2 (2×C), 45.1, 35.4, 30.9 (2×C), 10.4, 3.5 (2×C).

[0186] Synthesis of 8g of compound:

[0187] 100 mg of compound 8d (0.24 mmol, 1.0 equiv) was weighed, and 55 mg of EDCI (0.29 mmol, 1.2 equiv) and 7 mg of DMAP (0.06 mmol, 0.25 equiv) were added. Then, anhydrous DCM (3 mL) was added as solvent, followed by 31 mg of 4-methylaminopyridine (0.29 mmol, 1.2 equiv). The reaction was carried out at room temperature. After the reaction was complete, the mixture was extracted three times with ethyl acetate and washed with saturated sodium chloride solution. The organic phase was concentrated and subjected to rapid column chromatography to give 8 g of compound, in 75% yield.

[0188]

[0189] The obtained compound (8g) was identified using nuclear magnetic resonance spectroscopy. The identification results were as follows:

[0190] 1 H NMR (400 MHz, DMSO-d6) δ 9.59 (t, J = 6.4 Hz, 1H), 8.66–8.56 (m,1H), 8.46 (dt, J = 4.8, 1.2 Hz, 1H), 8.23 ​​(d, J = 8.4 Hz, 1H), 8.08 (d, J =8.4 Hz, 1H), 7.85 (tt, J = 8.4, 1.2 Hz, 1H), 7.81 –7.76 (m, 1H), 7.74 (s,1H), 7.67 (tt, J = 6.8, 1.2 Hz, 1H), 7.40–7.33 (m, 2H), 7.24 (d, J = 8.0 Hz,1H), 7.20–7.14 (m, 1H), 7.11 (s, 1H), 5.46 (s, 2H), 4.58 (d, J = 6.4 Hz, 2H), 3.92 (d, J = 6.8 Hz, 2H), 1.29–1.22 (m, 1H), 0.59–0.54 (m, 2H), 0.36–0.30 (m, 2H). 13C NMR (101 MHz, DMSO-d6) δ 164.8, 162.3, 152.0, 150.4, 149.5, 148.6,147.7, 140.0, 135.8, 135.5, 134.9, 131.3, 129.4, 127.8, 124.0, 122.3, 121.9,121.8, 120.6, 117.2, 114.7, 99.7, 73.5, 70.2, 40.8, 10.4, 3.5 (2×C).

[0191] Synthesis of compound 8h:

[0192] 100 mg of compound 8d (0.24 mmol, 1.0 equiv) was weighed, and 55 mg of EDCI (0.29 mmol, 1.2 equiv) and 7 mg of DMAP (0.06 mmol, 0.25 equiv) were added. Then, anhydrous DCM (3 mL) was added as solvent, followed by 40 mg of 2-methoxy-5-(aminomethyl)pyridine (0.29 mmol, 1.2 equiv). The reaction was carried out at room temperature. After the reaction was complete, the mixture was extracted three times with ethyl acetate and washed with saturated sodium chloride solution. The organic phase was concentrated and subjected to rapid column chromatography to give compound 8h in 75% yield.

[0193]

[0194] The obtained compound was identified by nuclear magnetic resonance spectroscopy after 8 hours. The identification results were as follows:

[0195] 1H NMR (400 MHz, DMSO-d6) δ 9.47 (t, J = 6.4 Hz, 1H), 8.22 (d, J = 8.4Hz, 1H), 8.18 (t, J = 1.6 Hz, 1H), 8.07 (d, J = 8.4 Hz, 1H), 7.87–7.83 (m,1H), 7.75–7.72 (m, 2H), 7.69–7.65 (m, 1H), 7.37 (t, J = 3.2 Hz, 1H), 7.24 (d,J = 8.0 Hz, 1H), 7.18–7.15 (m, 1H), 7.11 (s, 1H), 6.79 (d, J = 8.4 Hz, 1H),5.45 (s, 2H), 4.49 (d, J = 6.4 Hz, 2H), 3.92 (d, J = 7.4 Hz, 2H), 3.82 (s,3H), 1.30–1.24 (m, 1H), 0.59–0.54 (m, 2H), 0.36–0.32 (m, 2H). 13 C NMR (101MHz, DMSO-d6) δ 164.6, 163.2, 162.3, 152.1, 150.4, 147.7, 146.5, 140.0,139.6, 134.9, 131.2, 129.4, 128.5, 127.8, 122.2, 121.8, 121.8, 120.6, 119.8,117.2, 114.6, 110.7, 99.7, 73.5, 70.2, 53.6, 10.4, 3.5 (2×C).

[0196] Synthesis of compound 8i:

[0197] 100 mg of compound 8d (0.24 mmol, 1.0 equiv) was weighed, and 55 mg of EDCI (0.29 mmol, 1.2 equiv) and 7 mg of DMAP (0.06 mmol, 0.25 equiv) were added. Then, anhydrous DCM (3 mL) was added as solvent, followed by 29 mg of 2-aminothiazole (0.29 mmol, 1.2 equiv). The reaction was carried out at room temperature. After the reaction was complete, the mixture was extracted three times with ethyl acetate and washed with saturated sodium chloride solution. The organic phase was concentrated and subjected to rapid column chromatography to give compound 8i in 75% yield.

[0198]

[0199] The obtained compound 8i was identified by nuclear magnetic resonance spectroscopy, and the identification results were as follows:

[0200] 1 H NMR (400 MHz, DMSO-d6) δ 12.14 (s, 1H), 8.26 (dd, J = 8.4, 1.6 Hz,1H), 8.20 (d, J = 8.4 Hz, 1H), 7.93–7.88 (m, 1H), 7.81 (s, 1H), 7.75–7.71 (m,1H), 7.62 (d, J = 3.6 Hz, 1H), 7.41 (d, J = 2.0 Hz, 1H), 7.39 (d, J = 3,6 Hz,1H), 7.25 (d, J = 8.4 Hz, 1H), 7.19 (dd, J = 8.4, 2.0 Hz, 1H), 5.51 (s, 2H), 3.94 (d, J = 7.2 Hz, 2H), 1.33–1.24 (m, 1H), 0.61–0.54 (m, 2H), 0.39–0.33 (m, 2H). 13 C NMR (101 MHz, DMSO-d6) δ 163.1, 162.7, 157.8, 150.4, 149.8, 147.6,138.7, 134.8, 131.6, 129.8, 128.5, 122.3, 122.2, 121.8, 120.7, 117.2, 115.0,114.8, 99.9, 73.5, 70.4, 10.4, 3.5 (2×C).

[0201] Synthesis of compound 8j:

[0202] 100 mg of compound 8d (0.24 mmol, 1.0 equiv) was weighed, and 55 mg of EDCI (0.29 mmol, 1.2 equiv) and 7 mg of DMAP (0.06 mmol, 0.25 equiv) were added. Then, anhydrous DCM (3 mL) was added as solvent, and finally 27 mg of aniline (0.29 mmol, 1.2 equiv) was added. The reaction was carried out at room temperature. After the reaction was complete, the mixture was extracted three times with ethyl acetate and washed with saturated sodium chloride solution. The organic phase was concentrated and subjected to rapid column chromatography to give compound 8j in 75% yield.

[0203]

[0204] The obtained compound 8j was identified by nuclear magnetic resonance spectroscopy, and the identification results were as follows:

[0205] 1 H NMR (400 MHz, DMSO-d6) δ 10.73 (s, 1H), 8.26 (dd, J = 8.4, 1.6 Hz,1H), 8.23–8.18 (m, 1H), 7.98–7.94 (m, 2H), 7.93–7.89 (m, 1H), 7.82 (s, 1H),7.74–7.70 (m, 1H), 7.44–7.40 (m, 3H), 7.25 (d, J = 8.0 Hz, 1H), 7.20 (dd, J =8.4, 2.0 Hz, 1H), 7.18–7.14 (m, 1H), 7.1 (s, 1H), 5.50 (s, 2H), 3.94 (d, J =7.2 Hz, 2H), 1.31–1.24 (m, 1H), 0.60–0.55 (m, 2H), 0.37–0.34 (m, 2H). 13 C NMR(101 MHz, DMSO-d6) δ 163.1, 162.6, 152.0, 150.4, 147.5, 140.0, 138.7, 134.9,131.4, 129.6, 129.3 (2×C), 128.1, 124.5, 122.3, 122.0, 121.8, 120.7 (2×C),120.7, 117.2, 114.7, 99.7, 73.5, 70.3, 10.4, 3.5 (2×C).

[0206] Synthesis of compound 8k:

[0207] 100 mg of compound 8d (0.24 mmol, 1.0 equiv) was weighed, and 55 mg of EDCI (0.29 mmol, 1.2 equiv) and 7 mg of DMAP (0.06 mmol, 0.25 equiv) were added. Then, anhydrous DCM (3 mL) was added as solvent, and finally 32 mg of 1-methyl-1H-pyrazol-3-yl)methylamine (0.29 mmol, 1.2 equiv) was added. The reaction was carried out at room temperature. After the reaction was complete, the mixture was extracted three times with ethyl acetate and washed with saturated sodium chloride solution. The organic phase was concentrated and subjected to rapid column chromatography to give compound 8k in 75% yield.

[0208]

[0209] The obtained compound 8k was identified by nuclear magnetic resonance spectroscopy, and the identification results were as follows:

[0210] 1 H NMR (400 MHz, DMSO-d6) δ 10.62 (s, 1H), 8.24 (d, J = 8.4 Hz, 1H), 8.16 (d, J = 8.4 Hz, 1H), 7.88 (t, J = 8.0 Hz, 1H), 7.78 (s, 1H), 7.73–7.64(m, 2H), 7.41–7.35 (m, 1H), 7.24 (d, J = 8.0 Hz, 1H), 7.22–7.15 (m, 1H), 7.11(s, 1H), 6.67 (d, J = 2.4 Hz, 1H), 5.49 (s, 2H), 3.93 (d, J = 6.8 Hz, 2H), 3.81 (s, 3H), 1.30–1.23 (m, 1H), 0.59–0.55 (m, 2H), 0.37–0.33 (m, 2H). 13 C NMR(101 MHz, DMSO-d6) δ 162.7, 161.7, 151.0, 150.4, 147.5, 146.2, 140.1, 134.8,132.1, 131.4, 129.6, 128.1, 122.2, 122.0, 121.8, 120.6, 117.2, 114.7, 99.4,96.9, 73.5, 70.3, 38.9, 10.4, 3.5 (2×C).

[0211] Example 3

[0212] The synthetic routes for compounds 11a~11j are as follows:

[0213]

[0214] Intermediates 9 and 10 mentioned in the route have been reported. This invention will simply describe their synthesis steps and will not mention their characterization details again.

[0215] Synthesis of intermediate 9:

[0216] 2.7 g of intermediate 3 (11.2 mmol, 1.0 equiv) was dissolved in DMF, followed by the addition of 2.6 g of sodium metabisulfite (13.4 mmol, 1.2 equiv), and finally 2 g of methyl 2,3-diaminobenzoate (11.2 mmol, 1.0 equiv). The reaction was carried out at 80 °C for 8 h. After the reaction was complete, the mixture was extracted three times with ethyl acetate and washed with saturated sodium chloride solution. The organic phase was concentrated and subjected to rapid column chromatography to give intermediate 9 in 84% yield.

[0217] Synthesis of intermediate 10:

[0218] 3.0 g of intermediate 9 (7.7 mmol, 1.0 equiv) was weighed and dissolved in an aqueous solution of acetic acid (acetic acid:water, V / V = 3:1). 1.48 g of lithium hydroxide (LiOH, 30.8 mmol, 4.0 equiv) was added, and the reaction was carried out at room temperature for 8 h. After the reaction was complete, the pH was adjusted to 6 with dilute hydrochloric acid, and a white solid precipitated by filtration was obtained. The solid was dried under vacuum to give intermediate 10, with a yield of 97%.

[0219] Synthesis of compound 11a:

[0220] 100 mg of compound 10 (0.26 mmol, 1.0 equiv) was weighed, and 61 mg of EDCI (0.3 mmol, 1.2 equiv) and 8 mg of DMAP (0.06 mmol, 0.25 equiv) were added. Then, anhydrous DCM (3 mL) was added as solvent, followed by 74 mg of 2-trifluoromethyl-4-hydroxy-8-fluoroquinoline (0.33 mmol, 1.2 equiv). The reaction was carried out at room temperature. After the reaction was complete, the mixture was extracted three times with ethyl acetate and washed with saturated sodium chloride solution. The organic phase was concentrated and subjected to rapid column chromatography to give compound 11a in 75% yield.

[0221]

[0222] The obtained compound 11a was identified by nuclear magnetic resonance spectroscopy, and the identification results were as follows:

[0223] 1H NMR (600 MHz, DMSO-d6) δ 12.68 (s, 1H), 8.40 (s, 1H), 8.01–7.93 (m,3H), 7.87–7.81 (m, 2H), 7.67–7.60 (m, 2H), 7.46 (t, J = 7.8 Hz, 1H), 7.35 (s,1H), 7.21 (s, 1H), 3.97 (s, 2H), 1.31–1.25 (m, 1H), 0.60–0.57 (m, 2H), 0.3(s, 2H). 13 C NMR (151 MHz, DMSO-d6) δ 163.8, 158.6, 156.8, 156.6, 153.5,150.4, 147.8, 142.0, 138.9, 130.4, 128.0, 127.6, 126.3, 125.1, 123.5, 122.5,121.8, 121.4, 118.7, 117.1, 116.6, 115.9, 115.4, 113.8, 101.4, 73.7, 10.4,3.5 (2×C). minor conformer: 1 H NMR (600 MHz, DMSO-d6) δ 13.44 (s, 1H), 8.90(s, 1H), 8.20–8.10 (m, 3H), 7.87–7.81 (m, 2H), 7.67–7.60 (m, 2H), 7.46 (t, J= 7.8 Hz, 1H), 7.35 (s, 1H), 7.20 (s, 1H), 4.04 (d, J = 7.2 Hz, 2H), 1.35–1.32 (m, 1H), 0.63–0.59 (m, 2H), 0.42–0.40 (m, 2H). 13 C NMR (151 MHz, DMSO-d6)δ 163.8, 158.6, 156.9, 156.6, 153.5, 150.4, 147.8, 142.0, 138.9, 130.4,128.0, 127.6, 126.3, 125.1, 123.5, 122.5, 121.8, 121.4, 118.7, 117.1, 116.6,115.9, 115.4, 113.8, 101.4, 73.7, 10.5, 3.5 (2×C).

[0224] Synthesis of compound 11b:

[0225] 100 mg of compound 10 (0.26 mmol, 1.0 equiv) was weighed, and 61 mg of EDCI (0.3 mmol, 1.2 equiv) and 8 mg of DMAP (0.06 mmol, 0.25 equiv) were added. Then, anhydrous DCM (3 mL) was added as solvent, followed by 66 mg of 6,7-methoxy-4-hydroxyquinoline (0.33 mmol, 1.2 equiv). The reaction was carried out at room temperature. After the reaction was complete, the mixture was extracted three times with ethyl acetate and washed with saturated sodium chloride solution. The organic phase was concentrated and subjected to rapid column chromatography to give compound 11b in 75% yield.

[0226]

[0227] The obtained compound 11b was identified by nuclear magnetic resonance spectroscopy, and the identification results were as follows:

[0228] 1 H NMR (600 MHz, DMSO-d6) δ 12.74 (s, 1H), 8.78 (s, 1H), 8.1 (d, J =8.4 Hz, 1H), 8.98–7.82 (m, 3H), 7.50–7.47 (m, 3H), 7.35–7.29 (m, 2H), 7.21(s, 1H), 3.97 (d, J = 3.0 Hz, 2H), 3.82 (s, 6H), 1.30–1.26 (m, 1H), 0.60–0.57(m, 2H), 0.38–0.37 (m, 2H). 13 C NMR (151 MHz, DMSO-d6) δ 163.5, 153.2, 153.1,150.5, 150.3, 148.9, 147.4, 145.4, 141.9, 135.6, 128.0, 126.0, 122.5, 121.3,121.0, 119.9, 119.3, 117.9, 117.1, 113.9, 113.1, 112.9, 108.4, 99.4, 73.7,56.3, 56.1, 10.4, 3.5 (2×C). minor conformer: 1H NMR (600 MHz, DMSO-d6) δ13.48 (s, 1H), 8.79 (s, 1H), 8.24 (d, J = 7.8 Hz, 1H), 8.06–8.04 (m, 3H),7.53–7.50 (m, 3H), 7.40–7.35 (m, 2H), 7.23 (s, 1H), 4.01 (d, J = 7.2 Hz, 2H), 3.66 (s, 6H), 1.24–1.19 (m, 1H), 0.53–0.50 (m, 2H), 0.24–0.22 (m, 2H). 13 C NMR(151 MHz, DMSO-d6) δ 164.2, 153.5, 153.1, 150.5, 150.3, 149.0, 147.4, 143.4,141.9, 137.1, 128.0, 125.8, 122.9, 121.6, 121.0, 119.9, 119.3, 117.7, 117.1,113.9, 113.1, 112.4, 108.3, 100.5, 73.6, 56.2, 55.8, 10.3, 3.4 (2×C).

[0229] Synthesis of compound 11c:

[0230] 100 mg of compound 10 (0.26 mmol, 1.0 equiv) was weighed, and 61 mg of EDCI (0.3 mmol, 1.2 equiv) and 8 mg of DMAP (0.06 mmol, 0.25 equiv) were added. Then, anhydrous DCM (3 mL) was added as solvent, followed by 90 mg of 5,7-dichloro-4-hydroxy-2-(trifluoromethyl)quinoline (0.33 mmol, 1.2 equiv). The reaction was carried out at room temperature. After the reaction was complete, the mixture was extracted three times with ethyl acetate and washed with saturated sodium chloride solution. The organic phase was concentrated and subjected to rapid column chromatography to give compound 11c in 75% yield.

[0231]

[0232] The obtained compound 11c was identified by nuclear magnetic resonance spectroscopy, and the identification results were as follows:

[0233] 1H NMR (600 MHz, DMSO-d6) δ 12.66 (s, 1H), 8.49–8.39 (m, 2H), 8.20–8.10 (m, 3H), 8.02 (s, 1H), 7.95 (d, J = 9.0 Hz, 1H), 7.50 (t, J = 7.8 Hz,1H), 7.33 (s, 1H), 7.20 (d, J = 1.8 Hz, 1H), 4.01 (d, J = 6.6 Hz, 2H), 1.31–1.26 (m, 1H), 0.60–0.57 (m, 2H), 0.38–0.35 (m, 2H). 13 C NMR (151 MHz, DMSO-d6)δ 163.5, 157.0, 153.5, 150.3, 149.5, 145.4, 141.9, 136.5, 135.5, 132.7,129.1, 129.1, 127.9, 126.5, 126.4, 122.5, 122.1, 121.3, 121.1, 120.8, 120.3,117.0, 114.6, 114.0, 112.8, 73.7, 10.4, 3.5 (2×C).

[0234] Synthesis of compound 11d:

[0235] 100 mg of compound 10 (0.26 mmol, 1.0 equiv) was weighed, and 61 mg of EDCI (0.3 mmol, 1.2 equiv) and 8 mg of DMAP (0.06 mmol, 0.25 equiv) were added. Then, anhydrous DCM (3 mL) was added as solvent, followed by 73 mg of 6-trifluoromethoxyquinoline-4-ol (0.33 mmol, 1.2 equiv). The reaction was carried out at room temperature. After the reaction was complete, the mixture was extracted three times with ethyl acetate and washed with saturated sodium chloride solution. The organic phase was concentrated and subjected to rapid column chromatography to give compound 11d in 75% yield.

[0236]

[0237] The obtained compound 11d was identified by nuclear magnetic resonance spectroscopy, and the identification results were as follows:

[0238] 1H NMR (600 MHz, CDCl3) δ 10.74 (s, 1H), 8.96 (d, J = 4.8 Hz, 1H), 8.21 (dd, J = 12.6, 8.4 Hz, 2H), 8.14 (d, J = 7.8 Hz, 1H), 7.85 (s, 1H), 7.74(s, 1H), 7.61 (dd, J = 9.0, 3.0 Hz, 1H), 7.50–7.45 (m, 3H), 7.20 (d, J = 8.4Hz, 1H), 3.94 (d, J = 7.2 Hz, 2H), 1.32–1.22 (m, 1H), 0.65–0.62 (m, 2H), 0.36–0.32 (m, 2H). 13 C NMR (151 MHz, CDCl3) δ 164.0, 153.8, 152.4, 151.2,151.1, 148.2, 147.5, 145.1, 142.3, 135.9, 132.1, 127.3, 126.7, 125.5, 124.2,122.8, 122.7, 122.6, 120.5, 119.0, 115.9, 113.8, 112.8, 111.9, 111.4, 74.2,10.0, 3.2 (2×C).

[0239] Synthesis of compound 11e:

[0240] 100 mg of compound 10 (0.26 mmol, 1.0 equiv) was weighed, and 61 mg of EDCI (0.3 mmol, 1.2 equiv) and 8 mg of DMAP (0.06 mmol, 0.25 equiv) were added. Then, anhydrous DCM (3 mL) was added as solvent, followed by 68 mg of 4-hydroxy-8-trifluoromethylquinoline (0.33 mmol, 1.2 equiv). The reaction was carried out at room temperature. After the reaction was complete, the mixture was extracted three times with ethyl acetate and washed with saturated sodium chloride solution. The organic phase was concentrated and subjected to rapid column chromatography to give compound 11e in 75% yield.

[0241]

[0242] The obtained compound 11e was identified by nuclear magnetic resonance spectroscopy, and the identification results were as follows:

[0243] 1H NMR (600 MHz, CDCl3) δ 10.73 (s, 1H), 9.10 (d, J = 4.8 Hz, 1H), 8.26 (dd, J = 16.2, 8.4 Hz, 2H), 8.13 (dd, J = 13.8, 8.4 Hz, 2H), 7.75 (s,1H), 7.62 (t, J = 7.8 Hz, 1H), 7.53 (d, J = 4.8 Hz, 1H), 7.46 (t, J = 7.2 Hz,2H), 7.20 (d, J = 8.4 Hz, 1H), 3.94 (d, J = 7.2 Hz, 2H), 1.31–1.27 (m, 1H),0.65–0.63 (m, 2H), 0.35–0.33 (m, 2H). 13 C NMR (151 MHz, CDCl3) δ 164.1, 154.1,152.4, 151.8, 151.1, 146.4, 145.1, 142.3, 135.9, 128.8, 128.2, 127.3, 126.7,125.9, 125.7, 125.5, 123.8, 123.1, 122.7, 122.5, 119.0, 115.9, 114.0, 112.7,111.4, 74.2, 10.0, 3.2 (2×C).

[0244] Synthesis of compound 11f:

[0245] 100 mg of compound 10 (0.26 mmol, 1.0 equiv) was weighed, and 61 mg of EDCI (0.3 mmol, 1.2 equiv) and 8 mg of DMAP (0.06 mmol, 0.25 equiv) were added. Then, anhydrous DCM (3 mL) was added as solvent, and finally 57 mg of 4-hydroxy-8-chloroquinoline (0.33 mmol, 1.2 equiv) was added. The reaction was carried out at room temperature. After the reaction was complete, the mixture was extracted three times with ethyl acetate and washed with saturated sodium chloride solution. The organic phase was concentrated and subjected to rapid column chromatography to give compound 11f in 75% yield.

[0246]

[0247] The obtained compound 11f was identified by nuclear magnetic resonance spectroscopy, and the identification results were as follows:

[0248] 1H NMR (600 MHz, DMSO-d6) δ 12.69 (s, 1H), 9.06–8.99 (m, 1H), 8.22–8.13 (m, 3H), 8.04–7.95 (m, 3H), 7.88–7.76 (m, 2H), 7.49-7.45 (m, 1H), 7.39–7.32 (m, 1H), 7.21 (s, 1H), 4.00 (d, J =6.6 Hz, 2H), 1.30–1.24 (m, 1H), 0.60–0.58 (m, 2H), 0.38–0.35 (m, 2H). 13 C NMR (151 MHz, DMSO-d6) δ 163.3, 153.6,152.3, 150.3, 148.3, 145.4, 143.3, 141.9, 135.6, 132.5, 131.8, 131.4, 128.0,126.2, 123.7, 122.4, 121.6, 121.2, 120.2, 118.1, 117.1, 115.3, 113.9, 112.7,73.7, 10.4, 3.5 (2×C). minor conformer: 1 H NMR (600 MHz, DMSO-d6) δ 13.50 (s,1H), 9.06–8.99 (m, 1H), 8.22–8.13 (m, 3H), 8.04–7.95 (m, 3H), 7.88–7.76 (m,2H), 7.49-7.45 (m, 1H), 7.39–7.32 (m, 1H), 7.24 (s, 1H), 3.92 (d, J =6.6 Hz,2H), 1.30–1.24 (m, 1H), 0.56–0.54 (m, 2H), 0.28–0.27 (m, 2H). 13 C NMR (151MHz, DMSO-d6) δ 164.0, 153.4, 152.3, 150.5, 148.4, 145.4, 143.3, 142.0,137.1, 132.3, 131.7, 131.4, 128.0, 126.3, 123.3, 122.8, 122.1, 121.6, 120.2,118.8, 117.1, 115.3, 114.5, 113.0, 73.6, 10.3, 3.5 (2×C).

[0249] Synthesis of compound 11g:

[0250] 100 mg of compound 10 (0.26 mmol, 1.0 equiv) was weighed, and 61 mg of EDCI (0.3 mmol, 1.2 equiv) and 8 mg of DMAP (0.06 mmol, 0.25 equiv) were added. Then, anhydrous DCM (3 mL) was added as solvent, followed by 56 mg of 4-hydroxy-7-methoxyquinoline (0.33 mmol, 1.2 equiv). The reaction was carried out at room temperature. After the reaction was complete, the mixture was extracted three times with ethyl acetate and washed with saturated sodium chloride solution. The organic phase was concentrated and subjected to rapid column chromatography to give 11 g of compound 10, in 75% yield.

[0251]

[0252] The obtained compound 11g was identified by nuclear magnetic resonance spectroscopy, and the identification results were as follows:

[0253] 1 H NMR (600 MHz, DMSO-d6) δ 12.70 (s, 1H), 8.94–8.78 (m, 1H), 8.22–7.89 (m, 5H), 7.61–7.42 (m, 3H), 7.35–7.29 (m, 2H), 7.21 (s, 1H), 4.00 (d, J= 6.6 Hz, 2H), 3.96 (s, 3H), 1.29–1.26 (m, 1H), 0.59–0.58 (m, 2H), 0.37–0.30 (m, 2H). 13 C NMR (151 MHz, DMSO-d6) 163.5, 161.2, 154.5, 153.5, 152.0, 150.3,145.4, 141.9, 135.6, 128.0, 126.0, 123.4, 122.5, 121.3, 121.1, 120.5, 119.9,117.9, 117.5, 117.1, 114.0, 112.8, 112.6, 107.9, 73.7, 56.1, 10.4, 3.5 (2×C). minor conformer: 1H NMR (600 MHz, DMSO-d6) δ 12.70 (s, 1H), 8.94–8.78 (m,1H), 8.22–7.89 (m, 5H), 7.61–7.42 (m, 3H), 7.35–7.29 (m, 2H), 7.21 (s, 1H), 4.00 (d, J = 6.6 Hz, 2H), 3.96 (s, 3H), 1.29–1.26 (m, 1H), 0.59–0.58 (m, 2H), 0.37–0.30 (m, 2H). 13 C NMR (151 MHz, DMSO-d6) 164.1, 161.2, 154.5, 153.1,151.9, 150.6, 143.3, 142.0, 137.0, 128.1, 126.1, 124.3, 122.8, 121.6, 121.1,120.3, 119.1, 117.9, 117.3, 117.1, 114.0, 113.0, 111.8, 107.8, 73.7, 56.1,10.4, 3.5 (2×C).

[0254] Synthesis of compound 11h:

[0255] 100 mg of compound 10 (0.26 mmol, 1.0 equiv) was weighed, and 61 mg of EDCI (0.3 mmol, 1.2 equiv) and 8 mg of DMAP (0.06 mmol, 0.25 equiv) were added. Then, 3 mL of anhydrous DCM was added, followed by 52 mg of 4-hydroxy-6-fluoroquinoline (0.33 mmol, 1.2 equiv). The reaction was carried out at room temperature. After the reaction was complete, the mixture was extracted three times with ethyl acetate and washed with saturated sodium chloride solution. The organic phase was concentrated and subjected to rapid column chromatography to give compound 11h, in 75% yield.

[0256]

[0257] The obtained compound was identified by nuclear magnetic resonance spectroscopy after 11 hours. The identification results were as follows:

[0258] 1H NMR (600 MHz, CDCl3) δ 10.77 (s, 1H), 8.90 (d, J = 4.8 Hz, 1H), 8.24 (d, J = 7.8 Hz, 1H), 8.20–8.08 (m, 2H), 7.76 (s, 1H), 7.63 (dd, J = 9.0,3.0 Hz, 1H), 7.55–7.40 (m, 4H), 7.22 (d, J = 8.4 Hz, 1H), 6.72 (s, 1H), 3.96(d, J = 7.8 Hz, 2H), 1.33–1.29 (m, 1H), 0.67–0.64 (m, 2H), 0.37–0.34 (m, 2H). 13 C NMR (151 MHz, CDCl3) δ 164.1, 161.7, 160.0, 153.5, 152.3, 151.1, 150.0,147.2, 145.0, 142.3, 135.8, 132.4, 127.3, 126.6, 125.5, 123.2, 122.6, 120.6,119.1, 115.9, 113.7, 112.8, 111.5, 105.0, 74.2, 10.0, 3.2 (2×C).

[0259] Synthesis of compound 11i:

[0260] 100 mg of compound 10 (0.26 mmol, 1.0 equiv) was weighed, and 61 mg of EDCI (0.3 mmol, 1.2 equiv) and 8 mg of DMAP (0.06 mmol, 0.25 equiv) were added. Then, anhydrous DCM (3 mL) was added as solvent, followed by 73 mg of 2-trifluoromethyl-4-hydroxy-6-methylquinoline (0.33 mmol, 1.2 equiv). The reaction was carried out at room temperature. After the reaction was complete, the mixture was extracted three times with ethyl acetate and washed with saturated sodium chloride solution. The organic phase was concentrated and subjected to rapid column chromatography to give compound 11i in 75% yield.

[0261]

[0262] The obtained compound 11i was identified by nuclear magnetic resonance spectroscopy, and the identification results were as follows:

[0263] 1H NMR (600 MHz, DMSO-d6) δ 12.71 (s, 1H), 8.28–8.15 (m, 3H), 8.04 (s,2H), 7.96 (d, J = 8.4 Hz, 1H), 7.86 (dd, J = 9.0, 2.4 Hz, 1H), 7.50 (t, J =7.8 Hz, 1H), 7.34 (s, 1H), 7.21 (s, 1H), 4.00 (s, 2H), 2.54 (s, 3H), 1.30–1.25 (h, J = 6.6 Hz, 1H), 0.58–0.57 (m, 2H), 0.37–0.29 (m, 2H). 13 C NMR (151MHz, DMSO-d6) δ 163.2, 155.9, 153.5, 150.3, 147.5, 146.7, 145.3, 141.9,140.5, 135.7, 134.8, 129.8, 127.9, 126.3, 123.6, 122.8, 122.5, 121.3, 121.1,120.8, 118.6, 117.1, 113.9, 112.6, 110.9, 73.7, 55.4, 10.4, 3.5 (2×C).

[0264] Synthesis of compound 11j:

[0265] 100 mg of compound 10 (0.26 mmol, 1.0 equiv) was weighed, and 61 mg of EDCI (0.3 mmol, 1.2 equiv) and 8 mg of DMAP (0.06 mmol, 0.25 equiv) were added. Then, anhydrous DCM (3 mL) was added as solvent, followed by 65 mg of ethyl 4-hydroxyquinoline-3-carboxylate (0.33 mmol, 1.2 equiv). The reaction was carried out at room temperature. After the reaction was complete, the mixture was extracted three times with ethyl acetate and washed with saturated sodium chloride solution. The organic phase was concentrated and subjected to rapid column chromatography to give compound 11j in 75% yield.

[0266]

[0267] The obtained compound 11j was identified by nuclear magnetic resonance spectroscopy, and the identification results were as follows:

[0268] 11H NMR (600 MHz, DMSO-d6) δ 12.72 (s, 1H), 8.25–8.13 (m, 4H), 8.04–7.95 (m, 4H), 7.82–7.80 (m, 1H), 7.47–7.43 (m, 1H), 7.32 (s, 1H), 7.21 (s,1H), 4.00–3.95 (m, 5H), 1.30–1.26 (m, 1H), 0.60–0.58 (m, 2H), 0.38–0.36 (m,2H). 13 13C NMR (151 MHz, DMSO-d6) δ 165.3, 163.2, 155.5, 153.5, 150.3, 149.1,148.8, 145.4, 141.9, 135.6, 131.9, 130.5, 130.0, 127.9, 126.3, 126.2, 123.6,122.5, 122.2, 121.3, 121.1, 118.8, 117.1, 114.0, 112.7, 73.7, 53.4, 10.4, 3.5(2×C). minor conformer: 1 1H NMR (600 MHz, DMSO-d6) δ 13.76 (s, 1H), 9.07–8.25(m, 4H), 8.07–8.04 (m, 4H), 7.87–7.85 (m, 1H), 7.49–7.47 (m, 1H), 7.33 (s,1H), 7.24 (s, 1H), 4.01–4.00 (m, 5H), 1.30–1.26 (m, 1H), 0.57–0.54 (m, 2H),0.31–0.30 (m, 2H). 13 13C NMR (151 MHz, DMSO-d6) δ 165.4, 163.9, 155.6, 153.2,150.6, 149.1, 148.8, 143.3, 142.0, 137.1, 131.8, 130.3, 129.7, 128.2, 126.3,126.2, 123.3, 122.7, 121.7, 121.1, 120.0, 118.5, 117.1, 113.2, 112.9, 73.8,53.3, 10.4, 3.5 (2×C).

[0269] Example 4

[0270] The synthetic route for compound 14a is as follows:

[0271] Synthesis of intermediate 12:

[0272] Weigh 2.8 g of lithium aluminum hydride (LiAlH4, 0.07 mol, 5.0 equiv) into a two-necked flask. After purging with nitrogen three times, add anhydrous tetrahydrofuran (40 mL). Bring the mixture to 0°C on ice, and slowly add a tetrahydrofuran solution of compound 9 (5.8 g, 0.014 mol, 1.0 equiv). After reacting for 1 h, allow to return to room temperature and react for 8 h. Once the reaction is complete, quench with water and 10 wt% sodium hydroxide solution. Extract three times with ethyl acetate and wash the organic phase with saturated sodium chloride solution. Concentrate the organic phase and perform rapid column chromatography to give intermediate 12 in 67% yield.

[0273]

[0274] The obtained intermediate 12 was identified by nuclear magnetic resonance spectroscopy, and the identification results were as follows:

[0275] 1 H NMR (500 MHz, CD3OD) δ 7.9 (d, J = 2.0 Hz, 1H), 7.8–7.7 (m, 1H), 7.5 (d, J = 8.0 Hz, 1H), 7.4–7.1 (m, 3H), 6.9 (s, 1H), 5.0 (s, 2H), 4.0 (d, J =7.0 Hz, 2H), 1.4 –1.2 (m, 1H), 0.7–0.6 (m, 2H), 0.4 (t, J = 5.0 Hz, 2H).

[0276] Synthesis of intermediate 13:

[0277] 1.34 g of compound 12 (3.71 mol, 1.0 equiv) was weighed, and triphenylphosphine (2.5 g, 7.42 mol, 2.0 equiv) was added. Anhydrous dichloromethane was added, and the mixture was brought to 0°C on ice. Then, carbon tetrabromide (1.46 g, 5.57 mol, 1.5 equiv) was slowly added. The mixture was allowed to return to room temperature, and the reaction was allowed to proceed for 8 h. After the reaction was complete, the reaction was quenched with saturated sodium sulfite. The mixture was extracted three times with ethyl acetate, and the organic phase was washed with saturated sodium chloride solution. The organic phase was concentrated and subjected to rapid column chromatography to give intermediate 13 in 58% yield.

[0278]

[0279] The obtained intermediate 13 was identified by nuclear magnetic resonance spectroscopy, and the identification results were as follows:

[0280] 1 H NMR (500 MHz, CD3OD) δ 7.55 (s, 2H), 7.44 (d, J = 8.0 Hz, 1H), 7.29(d, J = 8.0 Hz, 1H), 7.19–7.12 (m, 1H), 6.97 (d, J = 8.5 Hz, 1H), 5.30 (d, J= 14.5 Hz, 2H), 4.04 (d, J = 7.0 Hz, 2H), 1.45–1.31 (m, 1H), 0.76–0.65 (m,2H), 0.46 (dt, J = 6.0, 4.5 Hz, 2H).

[0281] Synthesis of compound 14a:

[0282] 100 mg of compound 13 (0.26 mmol, 1.1 equiv) was weighed and added to 105 mg of potassium carbonate (K₂CO₃, 0.3 mmol, 1.5 equiv) and 8 mg of 6,7-methoxy-4-hydroxyquinoline (0.06 mmol, 0.25 equiv). Anhydrous DMF (3 mL) was then added, and the mixture was heated to 80 °C. After the reaction was complete, the mixture was extracted three times with ethyl acetate and washed with saturated sodium chloride solution. The organic phase was concentrated and subjected to rapid column chromatography to give compound 14a in 92% yield.

[0283]

[0284] The obtained compound 14a was identified by nuclear magnetic resonance spectroscopy, and the identification results were as follows:

[0285] 1H NMR (600 MHz, DMSO-d6) δ 8.55 (d, J = 5.0 Hz, 1H), 7.65–7.58 (m,2H), 7.49 (dd, J = 9.0, 1.0 Hz, 1H), 7.43 (dd, J = 9.5, 1.0 Hz, 2H), 7.30 (d,J = 9.0 Hz, 1H), 7.25 (s, 1H), 7.17 (d, J = 9.0 Hz, 1H), 6.84–6.76 (m, 1H), 5.46 (s, 1H), 3.99 (d, J = 4.5 Hz, 2H), 3.89 (d, J = 13.5 Hz, 4H), 1.39–1.30(m, 1H), 0.7–0.6 (m, 2H), 0.4 (t, J = 5.0 Hz, 2H). 13 C NMR (151 MHz, DMSO-d6)δ 159.0, 151.7, 151.2, 150.9, 150.9, 148.8, 148.1, 146.2, 143.4, 143.3,143.2, 139.5, 137.5, 129.0, 124.4, 124.1, 123.4, 121.5, 118.4, 117.6, 117.6,117.4, 116.3, 114.2, 114.1, 113.8, 107.6, 104.5, 104.5, 73.4, 68.9, 56.7,56.1, 11.8, 3.5 (2×C).

[0286] Experimental Example 1

[0287] Activity tests of each compound against PDE4

[0288] The compounds of the present invention were subjected to biological tests using the following methods:

[0289] 1. Preparation of reaction buffer and reaction termination solution (reagents are shown in Table 1)

[0290] (1) Preparation of 1x reaction buffer

[0291] Dilute the IMAP reaction buffer (provided with the IMAP FP IPP Explorer Kit) containing 0.1% BSA (5×) to a 1x reaction buffer containing 1 mM DTT.

[0292] (2) Preparation of reaction termination solution

[0293] Prepare the reaction termination solution by mixing Progressive Binding Buffer A (5×), Progressive Binding Buffer B (5×), and Progressive Binding Reagent (provided with the IMAP FP IPP Explorer Kit) according to the instructions for use.

[0294] 2. Compound preparation

[0295] (1) Compound dilution

[0296] Prepare a solution of the compound to a final concentration 100-fold. Use an automated micropipette (Precision PRC384U) to serially dilute the compound to the set number of concentration points as follows: For example, for a 5-fold dilution, add 50 μL of the initial concentration of the compound in DMSO to well A2 of an Echo 384 plate, and add 40 μL of 100% DMSO to wells A3-A11. Take 10 μL of the compound from well A2 and add it to well A3, mix well, and repeat the 5-fold dilution process for 10 concentration points. Add 40 μL of 100% DMSO to wells A1 and A12.

[0297] (2) Transfer the compound to a 384 reaction plate

[0298] Using an Echo550 instrument, 200 nL of the compound was transferred from the diluted Echo384-well plate to a 384-well reaction plate. 200 nL of 100% DMSO was transferred to both the negative and positive controls.

[0299] 3. Enzymatic reactions

[0300] (1) Prepare a 2x enzyme solution

[0301] Add PDE4 to 1 volume of reaction buffer to form 2 volume enzyme solution (final PDE4 concentration: 0.00625 μg / mL).

[0302] (2) Prepare 2 times the amount of substrate solution

[0303] For enzyme PDE4, FAM-labeled cAMP was added to 1 volume of reaction buffer to form 2 volumes of substrate solution (FAM-cAMP final concentration: 0.1 μM).

[0304] (3) Add enzyme solution to 384-well plate

[0305] Add 10 μL of 2-fold enzyme solution to each well of a 384-well reaction plate. For control wells without enzyme activity, replace the enzyme solution with 10 μL of 1-fold reaction buffer. Centrifuge at 1000 rpm for 1 min and incubate at room temperature for 15 min.

[0306] (4) Add substrate solution to 384-well plate to start enzyme reaction.

[0307] Add 10 μL of twice the amount of substrate solution to each well of a 384-well reaction plate. Centrifuge at 1000 rpm for 1 min. React at 25 °C for 30 min.

[0308] (5) Termination of enzyme reaction

[0309] Add 60 μL of reaction termination solution to each well of the 384-well reaction plate to terminate the reaction, and incubate at room temperature with shaking at 600 rpm in the dark for 60 min.

[0310] 4. Use EnVision to read and calculate data.

[0311] Readings using EnVision.

[0312] 5. Inhibition rate calculation and IC 50 Curve Fitting

[0313] Data was copied from EnVision, where the maximum value represents the reading of the DMSO control and the minimum value represents the reading of the enzyme-free control. Inhibition rate (%) = (maximum value - sample value) / (maximum value - minimum value) × 100%.

[0314] Import the data into MS Excel and fit the IC using XLFit Excel add-in version 5.4.0.8. 50 value;

[0315] Fitting formula: Y = Bottom + (Top - Bottom) / (1 + (IC) 50 / X)^HillSlope).

[0316] Table 1 Reagent Information

[0317]

[0318] The inhibitory effect of each compound prepared in the examples on PDE4 was determined according to the above method, and the results are shown in Table 2.

[0319] Table 2. Inhibition rate or IC50 of each compound against PDE4 50 value

[0320]

[0321] Experimental Example 2

[0322] Anti-inflammatory activity tests of various compounds

[0323] 1. Cell culture

[0324] Mouse monocyte-macrophage Raw 264.7 cells were cultured in DMEM high-glucose medium (Gibco, catalog number: 11995065) supplemented with 10% (v / v) FBS (Gibco, catalog number: 10270-106), 100 U / mL penicillin, and 100 μg / mL streptomycin (Gibco, catalog number: 15140122). The culture conditions were 37 ℃ and 5% CO2. Cells were passaged when they reached 80% confluence.

[0325] Human alveolar basal epithelial cells (A549) for lung cancer were cultured in DMEM high-glucose medium (Gibco, catalog number: 11995065) supplemented with 10% (v / v) FBS (Gibco, catalog number: 10270-106), 100 U / mL penicillin, and 100 μg / mL streptomycin (Gibco, catalog number: 15140122). The culture conditions were 37 ℃ and 5% CO2. Cells were passaged when they reached 80% confluence.

[0326] 2. Detection of the expression levels of inflammatory factors by compounds

[0327] 2.1. An assay was performed to inhibit the expression levels of cellular inflammatory factors IL-6, IL-1β, and TNF-α.

[0328] RAW 264.7 cells were grown at a rate of 2 × 10⁻⁶. 5 Cells were seeded at a density of 1 mL / well in 12-well plates. When cell coverage reached approximately 80%, the positive control drug (dexamethasone, final concentration 1 μM) and the candidate compound (DMSO solvent control, candidate compound 10 μM) were added. After 1 h, LPS was added at a final concentration of 100 ng / mL. LPS stimulation continued for 4 h.

[0329] (1) RNA extraction and qPCR assay

[0330] After completely discarding the culture medium from the cultured cells, add 0.5 mL of RL from the RNA extraction kit to each well for lysis; collect the cell lysate and extract Total RNA according to the kit instructions (Vazyme, catalog number: RC112), and measure its concentration using an ultra-micro UV-Vis spectrophotometer.

[0331] (2) Reverse transcription reaction

[0332] A 20 μL reverse transcription system was prepared, including 4 μL of 5×Buffer, X μL of Total RNA (500 ng), and μL of DEPC-H2O (16-X). The mixture was placed in 8-tube sets, labeled, and placed in a PCR instrument at 50 ℃ for 15 min and 85 ℃ for 5 s for reverse transcription.

[0333] (3) Quantitative fluorescence

[0334] Add 80 μL DEPC-H2O to the transcribed cDNA to a final volume of 100 μL, centrifuge to mix, and store at -20℃. Take 18 μL of the mixture (SYBR 10 μL + DEPC-H2O 10 μL + primer 0.5 μL) multiplied by the number of samples and transfer it to a 96-well plate. Add 2 μL of cDNA to make a 20 μL mixture, centrifuge at 1200 rpm for 1 min to mix. Place the sample plate in a CFX Connect Real-Time System for detection, cycling 39 times (95 ℃, 2 min; 95 ℃, 20 s; 57 ℃, 20 s; 72 ℃, 20 s), followed by a cycle of 95 ℃, 1 min; 55 ℃, 30 s; 95 ℃, 30 s.

[0335] (4) Analysis

[0336] Use 2 -ΔΔCt The experimental results were analyzed using the following formulas: △Ct target gene = Ct target gene - Ct internal reference gene; △△Ct target gene = △Ct experimental group target gene - △Ct control group target gene. 2 -ΔΔCt This indicates the fold increase in the expression of the target gene in the experimental group compared to the control group.

[0337] The primers are shown in Table 3 below.

[0338] Table 3 Primer Sequences

[0339]

[0340] The effects of each compound on the mRNA expression levels of inflammatory factors IL-6 (A), TNF-α (B), and IL-1β (C) in RAW 264.7 cells are as follows: Figure 1 As shown in the figure. The inflammation screening results showed that most of the compounds could significantly inhibit the expression levels of inflammatory factors TNF-α, IL-1β, and IL-6, and had in vitro anti-inflammatory activity.

[0341] 3. Anti-inflammatory activity of the compound IC 50

[0342] 3.1. The expression levels of the cellular inflammatory factor IL-6 were inhibited by compounds 11b and 14a using the conventional ELISA method.

[0343] The experiment used cells in the logarithmic growth phase, with cells at a rate of 2 × 10⁻⁶. 5 / wells were seeded in 12-well plates and cultured at 37 ℃ and 5% CO2 until 70% growth was achieved. Groups were set up: a blank stimulation control group (Control), an LPS stimulation group, and drug-treated groups (compound 11b and compound 14a). The culture medium was carefully removed. Fresh complete culture medium containing the compounds was added to the positive control group (dexamethasone Dex) and the drug-treated groups, while an equal volume of DMSO was added to the blank stimulation control group and the LPS stimulation group. After 1 h, except for the blank stimulation control group, 1 μg / mL LPS was added to each well for 4 h to induce cell inflammation. The plates were then removed, and ELISA experiments were performed according to the kit instructions.

[0344] Collect the culture medium supernatant: Collect the supernatant into a 1.5 mL EP tube, centrifuge at 1000 rpm for 10 min, and use the supernatant for ELISA detection of cytokines.

[0345] The secretion of IL-6 in the culture medium supernatant was detected using an enzyme-linked immunosorbent assay (ELISA) kit (Jianglai, catalog number: IL-6: JL20268). The procedure was performed according to the kit's instructions, as follows:

[0346] (1) Reagent preparation

[0347] ① Remove from the refrigerator and allow to equilibrate to room temperature for 20 min. ② Dilute the washing buffer (20×) with double-distilled water to 1× to prepare the required washing buffer. ③ Add the standard diluent to one bottle of standard according to the volume indicated on the standard label and incubate at room temperature for 15 min. ④ Take five clean 1.5 mL centrifuge tubes, pre-add 250 μL of standard diluent to each tube, and perform serial dilutions of the standard to obtain six standard concentrations: 1000, 500, 250, 125, 62.5, and 31.25 pg / mL. Finally, add the diluted standard to the wells of the pre-coated plate sequentially, adding the standard diluent directly as the 0 pg / mL concentration, for a total of seven standard concentrations. ⑤ Add 300 μL to each well, and repeat the washing process after approximately 15-30 seconds. Wash the plate five times in total, and then pat it dry on paper.

[0348] (2) Operation steps

[0349] ① Calculate the number of pre-coated strips required for one experiment, and take out the required strips and place them in a 96-well frame.

[0350] ② Add the sample or standard of different concentrations to the corresponding well at a rate of 100 μL / well, seal the reaction well with a sealing film (transparent), and incubate at room temperature for 120 min.

[0351] ③ Wash the board 5 times, and pat it dry on thick absorbent paper on the last wash.

[0352] ④ Add 100 μL of biotinylated antibody to each well, seal the wells with a clear sealing film, and incubate at room temperature for 60 min.

[0353] ⑤ Wash the board 5 times, and pat it dry on thick absorbent paper on the last wash.

[0354] ⑥ Add 100 μL of horseradish peroxidase-labeled streptavidin per well. Seal the reaction wells with white sealing film and incubate at room temperature in the dark for 20 min.

[0355] ⑦ Wash the board 5 times, and pat it dry on thick absorbent paper on the last wash.

[0356] ⑧ Add 100 μL of TMB solution as a colorimetric reagent per well, seal the wells with white sealing film, and incubate at room temperature in the dark for 20 min.

[0357] ⑨ Add 50 μL of stop solution per well, mix well, and immediately measure the expression level of IL-6.

[0358] Compound 11b's IC50 of IL-6, an inflammatory factor, in RAW 264.7 cells 50 (A) and compound 14a's IC50 of IL-6 inflammatory cytokine in RAW 264.7 cells 50 (B) See Figure 2 . Figure 2 The results showed that, compared with the LPS group, compound 11b could downregulate the secretion level of the inflammatory factor IL-6 (IC50). 50 =6.584 μM), compound 14a can downregulate the secretion level of the inflammatory factor IL-6 (IC50 = 6.584 μM). 50 =1.285 μM), and it was downregulated in a dose-dependent manner.

[0359] Experimental Example 3

[0360] Tests of the ability of compounds to inhibit cell migration

[0361] A549 cells were used at a rate of 5 × 10 5TGF-β1 (1 μg / mL) was seeded at a density of 1 mL / well in 6-well plates. After the cells had filled all the wells, a line was drawn in the wells using a pipette tip. Except for the control group, TGF-β1 (1 μg / mL) was added to the other groups. Positive control drug (pirfenidone (PFD): 10 μM) and candidate compounds (candidate compound 11b: 7 μM, candidate compounds 14a: 1, 5 μM) were added. Cell migration was observed under a microscope 24 h after streaking.

[0362] The antifibrotic abilities of compounds 11b and 14a in A549 cell scratch assays (A) and their statistical results (B) are shown in the figure. Figure 3 . Figure 3 The results showed that A549 cells were stimulated with different compounds for 24 h before cell migration was photographed. Compared with the control group, the scratch marks in the TGF-β group were almost closed, and compounds 11b and 14a significantly inhibited cell proliferation.

[0363] Test Example 4

[0364] Evaluation of the anti-fibrotic efficacy of the compound.

[0365] 1. Bleomycin-induced pulmonary fibrosis mouse model

[0366] ① Animal groups: Control (8 animals), PF (8 animals), PFD (8 animals), 11b-L (8 animals), 11b-M (8 animals), 11b-H (8 animals).

[0367] ② Modeling method: Male C57 / BL6 mice, weighing 18-22 g, were acclimatized for 7 days. General conditions of the mice, including diet, body weight, and mental state, were observed. A mouse model of pulmonary fibrosis was established by intratracheal infusion of 5 mg / kg bleomycin (Beyotime, catalog number: ST1450).

[0368] ③ Animal administration: Animals were administered low (11b-L, 10 mg / kg), medium (11b-M, 50 mg / kg), and high (11b-H, 100 mg / kg) doses of compound 11b via gavage. The positive control group received pirfenidone (PFD, 100 mg / kg). The control and model groups (PF) were administered an equal volume of physiological saline via gavage. The administration groups received the drug via gavage once daily for 21 days starting from the beginning of the experiment.

[0369] ④ Animal sampling: Mice were injected intraperitoneally with 0.2 mL of 0.6% sodium pentobarbital, and blood was drawn from the orbital cavity. The mice died from spinal dislocation. After cardiac lavage, some lung tissue was taken and fixed in paraformaldehyde. Some lung tissue was stored at -80℃. Bronchoalveolar lavage fluid (BALF) was stored at -20℃.

[0370] 2. Tissue testing

[0371] 2.1. BALF Detection

[0372] (1) Cell count

[0373] The total number of cells in mouse bronchoalveolar lavage fluid was measured using a cell counter, and the results are as follows: Figure 4 As shown. Figure 4 The results showed that, compared to the Control group, the number of cells in the BALF of mice in the PF group increased; compared to the PF group, each treatment group could effectively reduce the number of cells in the BALF and had anti-inflammatory activity.

[0374] (2) Giemsa staining

[0375] ① Add 70% ethanol and fix for 10 min.

[0376] ② Add an appropriate amount of the prepared modified Giemsa staining working solution (1X) to the smear sample and stain for 45 min.

[0377] ③Wash thoroughly from one side with distilled water, and after drying, observe and photograph under a microscope.

[0378] Giemsa staining results as follows Figure 5 As shown. Figure 5 The results showed that, compared with the control group, the number of neutrophils in the BALF of mice in the PF group increased, and each treatment group could effectively reduce neutrophils and had anti-inflammatory activity.

[0379] 2.2. Expression of inflammatory factors in BALF

[0380] Collect BALF supernatant: Collect BALF into a 1.5 mL EP tube, centrifuge at 1000 rpm for 10 min, and use the supernatant for ELISA detection of cytokines.

[0381] The secretion of IL-6 and TNF-α in the supernatant was detected using an enzyme-linked immunosorbent assay (ELISA) kit (Jianglai, catalog number: IL-6: JL20268, TNF-α: JL20484). The procedure was performed according to the kit's instructions, as follows:

[0382] (1) Reagent preparation

[0383] ① Remove from the refrigerator and allow to equilibrate to room temperature for 20 min. ② Dilute the washing buffer (20×) with double-distilled water to 1× to prepare the required washing buffer. ③ Add the standard diluent to one bottle of standard according to the volume indicated on the standard label and incubate at room temperature for 15 min. ④ Take five clean 1.5 mL centrifuge tubes, pre-add 250 μL of standard diluent to each tube, and perform serial dilutions of the standard to obtain six standard concentrations: 1000, 500, 250, 125, 62.5, and 31.25 pg / mL. Finally, add the diluted standard to the wells of the pre-coated plate sequentially, adding the standard diluent directly as the 0 pg / mL concentration, for a total of seven standard concentrations. ⑤ Add 300 μL to each well, and repeat the washing process after approximately 15-30 seconds. Wash the plate five times in total, and then pat it dry on paper.

[0384] (2) Operation steps

[0385] ① Calculate the number of pre-coated strips required for one experiment, and take out the required strips and place them in a 96-well frame.

[0386] ② Add the sample or standard of different concentrations to the corresponding well at a rate of 100 μL / well, seal the reaction well with a sealing film (transparent), and incubate at room temperature for 120 min.

[0387] ③ Wash the board 5 times, and pat it dry on thick absorbent paper on the last wash.

[0388] ④ Add 100 μL of biotinylated antibody to each well, seal the wells with a clear sealing film, and incubate at room temperature for 60 min.

[0389] ⑤ Wash the board 5 times, and pat it dry on thick absorbent paper on the last wash.

[0390] ⑥ Add 100 μL of horseradish peroxidase-labeled streptavidin per well. Seal the reaction wells with white sealing film and incubate at room temperature in the dark for 20 min.

[0391] ⑦ Wash the board 5 times, and pat it dry on thick absorbent paper on the last wash.

[0392] ⑧ Add 100 μL of TMB solution as a colorimetric reagent per well, seal the wells with white sealing film, and incubate at room temperature in the dark for 20 min.

[0393] ⑨ Add 50 μL of stop solution per well, mix well, and immediately measure the expression levels of IL-6 and TNF-α.

[0394] The levels of IL-6 (A) and TNF-α (B) inflammatory factors in the BALF supernatant of a mouse model of pulmonary fibrosis are as follows: Figure 6As shown (compared with the Control group, *** indicates P < 0.001; compared with the PF group, # indicates P < 0.05, ## indicates P < 0.01, and ### indicates P < 0.001). Figure 6 The results showed that, compared with the PF group, compound 11b could downregulate the levels of inflammatory factors IL-6 and TNF-α in a dose-dependent manner, and had anti-inflammatory activity.

[0395] 2.3. Expression of inflammatory factors in lung tissue

[0396] (1) RNA extraction and qPCR assay

[0397] Weigh mouse lung tissue and add 0.5 mL of RL from the RNA extraction kit to each 10 mg tissue for lysis; collect tissue lysates and extract total RNA according to the kit instructions (Vazyme, catalog number: RC112), and measure its concentration using an ultra-micro UV-Vis spectrophotometer;

[0398] (2) Reverse transcription reaction

[0399] A 20 μL reverse transcription system was prepared, including 4 μL of 5×Buffer, X μL of Total RNA (500 ng), and μL of DEPC-H2O (16-X). The mixture was placed in 8-tube sets, labeled, and placed in a PCR instrument at 50 ℃ for 15 min and 85 ℃ for 5 s for reverse transcription.

[0400] (3) Quantitative fluorescence

[0401] Add 80 μL DEPC-H2O to the transcribed cDNA to a final volume of 100 μL, centrifuge to mix, and store at -20℃. Take 18 μL of the mixture (SYBR 10 μL + DEPC-H2O 10 μL + primer 0.5 μL) multiplied by the number of samples and transfer it to a 96-well plate. Add 2 μL of cDNA to make a 20 μL mixture, centrifuge at 1200 rpm for 1 min to mix. Place the sample plate in a CFX Connect Real-Time System for detection, cycling 39 times (95 ℃, 2 min; 95 ℃, 20 s; 57 ℃, 20 s; 72 ℃, 20 s), followed by a cycle of 95 ℃, 1 min; 55 ℃, 30 s; 95 ℃, 30 s.

[0402] (4) Analysis

[0403] Use 2 -ΔΔCtThe experimental results were analyzed using the following formulas: △Ct target gene = Ct target gene - Ct internal reference gene; △△Ct target gene = △Ct experimental group target gene - △Ct control group target gene. 2 -ΔΔCt This indicates the fold increase in the expression of the target gene in the experimental group compared to the control group.

[0404] The primers are shown in Table 1.

[0405] The expression levels of IL-6 (A), TNF-α (B), and IL-1β (C) mRNA in lung tissue of a mouse model of pulmonary fibrosis are as follows: Figure 7 As shown (compared with the Control group, * indicates P < 0.05, *** indicates P < 0.001; compared with the PF group, # indicates P < 0.05, ## indicates P < 0.01, ### indicates P < 0.001). Figure 7 The results showed that, compared with the control group, the expression of inflammatory factors in mice in the PF group was significantly increased, and compound 11b was able to partially reverse this condition, demonstrating anti-inflammatory activity.

[0406] 2.4. Pathological examination

[0407] (1) Tissue fixation, dehydration and embedding

[0408] ① Fix the mouse tissues in 10% neutral formaldehyde solution for 24 h.

[0409] ② Place the trimmed tissue block in an embedding cassette and wash with running water for 24 hours to completely remove residual formaldehyde.

[0410] ③ Perform graded alcohol dehydration on the tissue, with the following steps: 70% alcohol for 12 h, 80% alcohol for 1.5 h, 95% alcohol I for 45 min, 95% alcohol II for 30 min, 100% alcohol I for 25 min, and 100% alcohol II for 20 min.

[0411] ④ After dehydration, immerse the tissue in an alcohol / xylene (1:1, V / V) solution for 20 min.

[0412] ⑤ Soak the tissue in xylene I for 20 min, then soak it in xylene II for 10 min.

[0413] ⑥ Place the tissue into paraffin I and paraffin II, which have been preheated and melted in an oven at 60~65℃, for 1 hour each.

[0414] ⑦ Pour a small amount of embedding paraffin into the preheated metal embedding frame, place the skin tissue block inside, perpendicular to the embedding frame, with the bottom flat, pour in paraffin again, embed the tissue, and let it cool.

[0415] ⑧ Paraffin sectioning: Fix the tissue paraffin block onto a Leica microtome, sectioning to a thickness of 5 μm in a continuous manner. Use toothless forceps to immerse the sections in 40°C water for spreading, retrieve them with a glass slide, bake at 60°C for 2 hours, and then store them in a slide box at room temperature for later use.

[0416] (2) HE staining

[0417] ①Dewaxing and hydration: Placing paraffin sections of lung tissue into xylene I and xylene II for 15 min each, then into 100% ethanol I and ethanol II for 3 min each, 95% ethanol I and ethanol II for 3 min each, 80% ethanol for 3 min, and double-distilled water for 1 min.

[0418] ② Stain with hematoxylin for 15 min, then wash the slide with water to remove excess staining solution;

[0419] ③ Use 1% hydrochloric acid and ethanol (99 mL 70% ethanol + 1 mL concentrated hydrochloric acid) to separate colors for 3 seconds. Under a microscope, the cell nucleus and chromatin should be clearly visible.

[0420] ④ Rinse with running water for 15 minutes to restore blue color, then rinse with distilled water for 1 minute;

[0421] ⑤ Eosin for 2 min, rinse with running water for 1 min;

[0422] ⑥ Dehydrated with 80% and 100% ethanol for 2 seconds and 7 minutes, respectively;

[0423] ⑦ Xylene I and II, 5 min each;

[0424] ⑧ Mounting: Remove the slide from xylene II, drop neutral resin onto the tissue, gently cover with a coverslip, and allow to air dry naturally;

[0425] ⑨ Observation: Observe pathological changes under a microscope, take pictures, and analyze.

[0426] HE staining results are as follows Figure 8 As shown. Figure 8 The results showed that the lung structure of the PF group was damaged, with obvious inflammatory infiltration and collapse of alveolar spaces, and the PF group experienced some relief after administration of 11b.

[0427] (3) Masson staining

[0428] ①Dewaxing and hydration: Placing paraffin sections of lung tissue into xylene I and xylene II for 15 min each, then into 100% ethanol I and ethanol II for 3 min each, 95% ethanol I and ethanol II for 3 min each, 80% ethanol for 3 min, and double-distilled water for 1 min.

[0429] ② Stain with the prepared Weigert iron hematoxylin staining solution for 5-10 minutes;

[0430] ③ Differentiate in acidic ethanol solution for 5-15 seconds, then wash with water;

[0431] ④ Bluing solution returns to blue for 3-5 minutes, then wash with water and distilled water for 1 minute;

[0432] ⑤ Stain with Pompadour and magenta solution for 5-10 minutes;

[0433] ⑥ In the above operation, prepare a weak acid working solution according to the ratio of distilled water: weak acid solution, V / V=2:1, and wash with the weak acid working solution for 1 min;

[0434] ⑦ Wash with phosphomolybdic acid solution for 1-2 minutes. Then wash with the prepared weak acid working solution for 1 minute.

[0435] ⑧ Immerse directly in aniline blue staining solution for 1-2 minutes. Wash with prepared weak acid working solution for 1 minute;

[0436] ⑨ Rapid dehydration with 95% ethanol for 2-3 seconds, and dehydration with anhydrous ethanol three times, each time for 5-10 seconds;

[0437] ⑩ Apply xylene three times, each time for 1-2 minutes, and then seal with neutral resin.

[0438] Masson staining results are as follows: Figure 9 As shown. Figure 9 The results showed that mice in the Control group had a small amount of collagen fiber deposits around the trachea in their lung tissue, mice in the PF group had a large amount of blue collagen fiber deposits, indicating more severe pulmonary fibrosis. The blue collagen fiber deposits in the PFD and high-dose 11b groups were reduced to varying degrees, indicating a therapeutic effect on fibrosis.

[0439] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A PDE4 inhibitor, characterized in that, The structure is shown in any of the following structures: 。 2. A method for preparing the PDE4 inhibitor according to claim 1, characterized in that, Includes the following steps: The general structural formula corresponding to compound 11b is shown in Formula XⅠ: Where R3 is ; The preparation process of the compound shown in Formula XⅠ is as follows: Step h includes: reacting compound III with methyl 2,3-diaminobenzoate at 80°C for 10-14 h to obtain compound IX; Step i includes: hydrolyzing compound IX at 20~50℃ for 2~6h to obtain compound X; Step j includes: reacting compound X with R3-OH at 20~50℃ for 4~8h to obtain compound XⅠ; The preparation process of compound 14a is as follows: Step k includes: reacting compound X with a borate tetrahydrofuran complex at room temperature for 4 hours to obtain compound XII; Step 1 includes: stirring compound XII with carbon tetrabromide and triphenylphosphine at room temperature for 8-12 hours to obtain compound XIII; Step m includes: mixing the compound of formula XIII with... The reaction was stirred at 80°C for 10-14 h to obtain compound 14a.

3. A pharmaceutically acceptable salt of the PDE4 inhibitor of claim 1.

4. The use of the PDE4 inhibitor of claim 1 in the preparation of a drug for treating idiopathic pulmonary fibrosis.

5. The use of the pharmaceutically acceptable salt of the PDE4 inhibitor of claim 3 in the preparation of a medicament for treating idiopathic pulmonary fibrosis.

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