Application of P2Y6R-targeted quinoline derivative in preparation of acute lung injury treatment and anti-inflammatory drugs

Abstract

The invention provides an application of a quinoline derivative as shown in a formula (1) in preparation of drugs for treating acute lung injury and resisting inflammation. It is found for the first time that the quinoline derivative has the function of a P2Y6R antagonist, has an obvious antagonistic effect on P2Y6R-related inflammation and can be used for preparing P2Y6R-related anti-inflammatory drugs.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of pharmaceutical chemistry, and particularly relates to application of a quinoline derivative targeting P2Y6R in preparation of drugs for treating acute lung injury and anti-inflammation. BACKGROUND

[0002] The purinergic receptor family is mainly divided into two types: P1 adenosine and P2 nucleotide, wherein the P2 family is divided into two subfamilies: ligand-gated ion channel type receptors (P2X receptors) and G protein-coupled receptors (P2Y receptors). The P2Y receptor family has eight subtypes (P2Y1R, P2Y2R, P2Y4R, P2Y6R, P2Y11R, P2Y12R, P2Y13R and P2Y14R). 11 R, P2Y 12 R, P2Y 13 R and P2Y 14 R).

[0003] P2Y6R is a member of the eight subtypes of the receptor family, which has a typical seven-transmembrane structure, belongs to the G protein-coupled receptor family, and belongs to the G protein-coupled nucleotide receptor that activates the phospholipase C signaling pathway. P2Y6R is widely involved in a series of human diseases, including atherosclerosis and other cardiovascular diseases. P2Y6R can be selectively activated by UDP, enhance the expression of IP3, and induce the continuous increase of intracellular Ca 2+ P2Y6R is widely expressed on stromal cells such as endothelial cells (Esc) and smooth muscle cells (SMCs), and the functional expression of P2Y6R is also found on white blood cells including macrophages and neutrophils.

[0004] According to previous studies, P2Y6R and vascular inflammation are related to each other. Under the stimulation of TNF-a or LPS, P2Y6 receptors on vascular endothelial cells are selectively up-regulated, and not other P2Y or P2X receptors. P2Y6 receptor activates the NF-κB pathway to induce the expression of interleukin-8 (IL-8) and vascular cell adhesion molecule-1, and promotes the occurrence of vascular inflammatory response. In a P2Y6 gene knockout mouse model, the response of smooth muscle cells, vascular endothelial cells and the like to UDP is significantly weakened. Therefore, the development of antagonists for P2Y6R can achieve the effect of treating inflammation, and also has a wide application prospect in the treatment of diabetes, cancer and the like. SUMMARY

[0005] In order to improve the above technical problems, the present application provides application of a quinoline derivative in preparation of drugs for treating P2Y6R related acute lung injury and anti-inflammation.

[0006] The present application provides application of a quinoline derivative or a pharmaceutically acceptable salt thereof shown in formula (1) in preparation of drugs for preventing and / or treating acute lung injury and anti-inflammation.

[0007]

[0008] According to an embodiment of the present application, the acute lung injury is P2Y6R-related acute lung injury.

[0009] In another aspect, the present application provides a use of the quinoline derivative represented by formula (1) or a pharmaceutically acceptable salt thereof in the preparation of a P2Y6R antagonist.

[0010] According to an embodiment of the present application, the pharmaceutically acceptable salt is selected from one or more of hydrochloride, phosphate, sulfate, acetate, maleate, citrate, benzenesulfonate, methylbenzenesulfonate, fumarate and tartrate of the quinoline derivative represented by formula (1).

[0011] Advantages

[0012] Compared with the prior art, the present application provides a use of the quinoline derivative in the preparation of a drug for treating acute lung injury and anti-inflammation. The present application first discovers that the quinoline derivative has the function of a P2Y6R antagonist, has obvious antagonistic effect on P2Y6R-related inflammation, and can be used as a P2Y6R-related anti-inflammatory drug.

[0013] The present application has measured the biological activity of the quinoline derivative represented by formula (1), found that it has obvious antagonistic activity on P2Y6R, and can obviously reduce the expression of IL-1β, IL-6 and TNF-α inflammatory factors in the LSP-induced RAW264.7 cell culture supernatant. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 The results of cell thermal migration assay between the quinoline derivative and P2Y6R.

[0015] Figure 2 The quinoline derivative reduces LPS-induced lung inflammation in mice. (A) H&E staining of lung sections. (B) IL-1β, (C) IL-6, (D) TNF-α, (E) MPO protein levels in lung tissues after different treatments (n=6). Each column represents the average SEM of three independent experiments. **p<0.01, ***p<0.001 compared with the LPS only group. DETAILED DESCRIPTION

[0016] The technical solutions of the present application will be further described in detail below in combination with specific examples. It should be understood that the following examples are only illustratively described and explained the present application, and should not be interpreted as limiting the scope of protection of the present application. Any technology realized based on the above description of the present application is covered within the scope of protection intended by the present application.

[0017] The starting materials and reagents used in the following examples are commercially available or can be prepared by known methods unless otherwise stated.

[0018] In order to further illustrate the present application, the application of the quinoline derivatives provided by the present application in the preparation of drugs for preventing and / or treating P2Y6R related acute lung injury and anti-inflammatory drugs is described in detail below in conjunction with examples.

[0019] Preparation Example 1

[0020] The preparation method and characterization data of the quinoline derivative represented by formula (1) are as follows:

[0021]

[0022] Compound 1 (2.0 mmol, 314 mg, 1.0 equiv) and compound 2 (2.0 mmol, 18 mg, 1.0 equiv) were placed in a 25 mL round-bottom flask and dissolved in 4 mL of ethanol solvent. 1 mL of sodium hydroxide aqueous solution (10% by mass) was added to the reaction system. After 0.5 h, TLC detection showed that the reaction was complete, and a large amount of precipitate was generated in the system. The precipitate was filtered out and washed with ethanol to obtain 200 mg of compound 3. The yield was 40%.

[0023] Compound 3 (0.8 mmol, 200 mg, 1.0 equiv) was dissolved in 3 mL of ethanol and placed in a 25 mL round-bottom flask. Hydrazine hydrate (4 mmol, 200 mg, 5.0 equiv) was added to the system. After refluxing for 0.5 h, TLC detection showed that the reaction was complete. After the reaction cooled, the ethanol was removed by reduced pressure distillation, and 30 mL*3 of ethyl acetate and 40 mL of saturated brine were used for extraction. The organic phase was dried with sodium sulfate and then distilled under reduced pressure to obtain 200 mg of compound 4. The yield was 96%. Compound 4 was directly used in the next step without purification.

[0024] Compound 4 (0.76 mmol, 200 mg, 1.0 equiv) and compound 5 (1.14 mmol, 114 mg, 1.5 equiv) were placed in a 25 mL round-bottom flask, and 4 mL of chloroform was added. After refluxing for 0.5 h, TLC detection showed that the reaction was complete, and a large amount of precipitate was generated in the reaction system. After the system cooled, the precipitate was filtered out and dried to obtain the crude product. Purification by slurry with ethyl acetate obtained 121 mg of pure compound represented by formula (1). The yield was 44%.

[0025]

[0026] 1H NMR (400 MHz, DMSO-d6) δ 12.14 (s, 1H), 11.62 (s, 1H), 8.85 - 8.78 (m, 1H), 8.09 (s, 1H), 7.99 (dd, J = 18.5, 8.2 Hz, 2H), 7.74 (t, J = 7.4 Hz, 1H), 7.60 (t, J = 7.4 Hz, 1H), 7.00 (s, 1H), 6.54 (s, 1H), 6.20 - 6.12 (m, 1H), 5.73 (dd, J = 11.6, 4.4 Hz, 1H), 3.87 (dd, J = 17.8, 11.8 Hz, 1H), 3.23 (dd, J = 17.8, 4.5 Hz, 1H), 3.09 (dt, J = 16.5, 6.8 Hz, 1H), 2.91 (dt, J = 16.9, 6.5 Hz, 1H). 13 C NMR (101 MHz, DMSO) δ 174.49, 169.07, 149.96, 148.70, 147.35, 135.42, 132.31, 129.81, 129.07, 128.54, 127.89, 127.29, 123.81, 123.18, 113.25, 109.75, 57.40, 42.29, 29.11, 29.01. HRMS (ESI+) m / z calcd for C 20 H 18 N4O3[M+H] + 363.1457, found 363.1454.

[0027] Example 1

[0028] In vitro antagonistic activity test of compounds on P2Y6 receptor

[0029] CHO-hP2Y6R cells were added to each well of an assay plate (Greiner-781090). Cells were treated with quinoline derivatives shown in formula (1) and then stimulated with P2Y6R agonist UDP sodium salt. Ca2+flux was then detected by FLIPR Calcium 6 assay kit (Molecular Devices) and the results were analyzed using the Prism software. 2+ The data was analyzed using Prism and the curve fitting equation "log (antagonist) vs. response variable slope".

[0030] The results of the experiment are shown in Table 1:

[0031] Table 1. Results of in vitro antagonistic activity test of compounds on P2Y6 receptor

[0032]

[0033] Example 2

[0034] Thermal shift assay

[0035] To screen the temperature, P2Y6 stable expressing cells were collected in 1 ml PBS. The cell suspension was divided into 14 different PCR tubes, incubated with the compound (10 μM) or DMSO for 3 h in a constant temperature shaker, then heated in a PCR machine at different temperatures for 10 min, after centrifugation the supernatant was discarded, and the cells were lysed with lysis buffer. The total protein extract was denatured in 100 °C bath. Finally, the proteins were analyzed by immunoblotting. To screen the concentration, equal amount of cells were incubated with different concentrations of the compound (0, 10 -3 , 10 -2 , 10 -1 , 100 and 101 pM) for 3 h in a constant temperature shaker, heated in a PCR machine at 57 °C for 10 min, and the supernatant containing protein lysate was collected. Subsequently, Western blot analysis was performed for detection, and the results are shown in Figure 1 . The above results demonstrate that the interaction between the compound and P2Y6R is very strong and effective.

[0036] Example 3

[0037] In vivo pharmacodynamics study of the compound in LSP-induced acute lung injury model in mice

[0038] To further understand the anti-inflammatory effect of the compound, a mouse ALI model was used. Inflammatory factors that play an important role in the occurrence and progression of ALI include three cytokines: IL-1β, IL-6 and TNF-α. To verify the effectiveness of the compound, we measured the levels of IL-1β, IL-6 and TNF-α in BALF. The pre-treatment of the compound significantly reduced the levels of BALF cytokines induced by LPS. As shown in Figure 2 A-C. To examine the effect of the compound on neutrophil infiltration in the lung, we measured the concentration of MPO in the lung tissue of LPS-challenged mice Figure 2 D). The data showed that the compound inhibited the increase in MPO concentration in LPS-induced mice. The above results suggest that the compound can effectively inhibit neutrophil infiltration in the lung tissue of ALI mice and reduce the production of pro-inflammatory cytokines.

[0039] Alveolar septum thickening, tissue destruction, interstitial edema, inflammatory infiltration are the pathological changes of LPS-induced lung tissue, as shown in Figure 2 E. The pathological changes induced by LPS were significantly reduced in the compound group. No obvious lesions were observed in the lung parenchyma, and the alveolar structure was relatively intact, with only mild congestion in the alveolar septum.

[0040] The above has exemplarily described the embodiments of the technical scheme of the present disclosure. It should be understood that the protection scope of the present disclosure is not limited to the above embodiments. Any modification, equivalent replacement, improvement, etc. made by those skilled in the art within the spirit and principle of the present disclosure shall be included in the protection scope of the claims of the present application.

Claims

1. Use of a quinoline derivative represented by formula (1) or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for preventing and / or treating acute lung injury and anti-inflammatory: ###0001### Formula (1) wherein the pharmaceutically acceptable salt is selected from one or more of hydrochloride, phosphate, sulfate, acetate, maleate, citrate, besylate, methylbesylate, fumarate and tartrate of the quinoline derivative represented by formula (1).

2. Use according to claim 1, characterized in that, The acute lung injury is P2Y6R-associated acute lung injury.

3. Use according to claim 1 or 2, characterized in that, 4. Use of a quinoline derivative represented by formula (1) or a pharmaceutically acceptable salt thereof in the manufacture of a P2Y6R antagonist: ###0001### Formula (1) wherein the pharmaceutically acceptable salt is selected from one or more of hydrochloride, phosphate, sulfate, acetate, maleate, citrate, besylate, methylbesylate, fumarate and tartrate of the quinoline derivative represented by formula (1). ​

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