Application of P2Y14R targeted benzoate derivative in preparation of acute lung injury treatment and anti-inflammatory drugs

Abstract

The invention provides an application of a 3-substituted-5-acylamino benzoate derivative shown in a formula (1) in preparation of drugs for treating acute lung injury and resisting inflammation. The invention discovers that the 3-substituted-5-acylamino benzoate derivative has the function of a P2Y14R antagonist for the first time, has an obvious antagonistic effect on P2Y14R related inflammation, and can be used for preparing the P2Y14R related anti-inflammatory drugs.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of pharmaceutical chemistry, and particularly relates to a 3-substituted-5-amido benzoate derivative targeting P2Y 14 R in preparation of drugs for treating acute lung injury and anti-inflammatory drugs. BACKGROUND

[0002] The human immune system is divided into innate immune system and acquired immune system, which plays a crucial role in resisting invasion of foreign bacteria and diseases. When the human body is injured by external factors, some special tissues or organs release extracellular nucleotides. The purine nucleotides in the extracellular nucleotides play a role in regulating the human immune system by binding to the cell surface purinergic receptors. The purinergic receptors are mainly divided into P1 and P2 receptors, and the P2 receptors are further divided into ligand-gated ion channel type receptors (P2XR) and protein-coupled type receptors (P2YR). There are two subtypes under the P2Y receptor, in which P2Y1, P2Y2, P2Y4, P2Y6 and P2Y12 belong to P2Y1-like receptors, and P2Y11, P2Y12, P2Y13 and P2Y14 belong to P2Y-like receptors. 11 12 13 14 12

[0003] P2Y 14 R is widely expressed in most tissues and organs, such as placenta, spleen, bone marrow, thymus, stomach, intestine, adipose tissue and brain, and is also prominent in the immune system. The specific physiological function of P2Y 14 R remains to be confirmed, and experimental observation and its significant expression in immune cells show that it is involved in immune and inflammatory responses and is related to the progression of some organic pathological diseases. P2Y 14 R can be specifically activated by related UDP sugars such as uridine diphosphate glucose (UDP-Glc), and can inhibit adenylyl cyclase through Gi protein to reduce the production of intracellular cAMP, and promote the migration and recruitment of macrophages and neutrophils, the release of mast cell mediators, pro-inflammatory cytokines and chemokines, and the hypersensitivity of microglia. According to previous studies, P2Y 14 R is related to acute gouty arthritis, acute kidney injury and other inflammatory diseases, such as the deletion of P2Y 14 R can significantly destroy the synovial histopathological changes of the MSU-induced acute gouty arthritis model in rats and reduce the number of macrophage pyroptosis on the NLRP3 street, and alleviate the symptoms of arthritis; or in the kidney ischemia / reperfusion injury model, the selective antagonist is used to block P2Y 14 ​​​​​R can alleviate kidney inflammation and proximal tubular injury, and improve renal dysfunction caused by renal ischemia / reperfusion injury. Therefore, the development of antagonists against P2Y 14 Developing antagonists against P2Y SUMMARY

[0004] To improve the above technical problems, the present application provides a 3-substituted-5-amido benzoate derivative in the preparation of a drug for treating P2Y 14 R related acute lung injury and anti-inflammatory drugs.

[0005] The present application provides a benzoate derivative represented by formula (1) or a pharmaceutically acceptable salt thereof in the preparation of a drug for preventing and / or treating acute lung injury and anti-inflammatory drugs.

[0006]

[0007] According to an embodiment of the present application, the acute lung injury is P2Y 14 R related acute lung injury.

[0008] In another aspect, the present application provides a benzoate derivative represented by formula (1) or a pharmaceutically acceptable salt thereof in the preparation of a P2Y 14 R antagonist.

[0009] 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 benzoate derivative represented by formula (1).

[0010] Advantages

[0011] Compared with the prior art, the present application provides the use of a 3-substituted-5-amido benzoate derivative in the preparation of a drug for treating acute lung injury and anti-inflammatory drugs. The present application first discovers that the benzoate derivative has the function of a P2Y 14 R antagonist, has obvious antagonistic effect on P2Y 14 R related inflammation, and can be used for preparing a P2Y 14 R related anti-inflammatory drug.

[0012] The present application determines the biological activity of the benzoate derivative represented by formula (1), and finds that it has obvious antagonistic activity on P2Y 14 R, and can obviously reduce the expression of IL-1β, IL-6 and TNF-α inflammatory factors in the culture supernatant of RAW264.7 cells induced by LPS. BRIEF DESCRIPTION OF DRAWINGS

[0013] Figure 1 The benzoate derivative (No. 45) and P2Y 14 The results of the cell thermal migration assay between the benzoate derivative (No. 45) and P2Y

[0014] Figure 2 The benzoate derivative (No. 45) attenuated 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 mean SEM of three independent experiments. **p < 0.01, ***p < 0.001 compared with the LPS only group. DETAILED DESCRIPTION

[0015] The technical solutions of the present disclosure 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 disclosure, and should not be interpreted as limiting the scope of protection of the present disclosure. Any technology realized based on the above description of the present disclosure is covered within the scope intended to be protected by the present disclosure.

[0016] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0017] To further illustrate the present application, the 3-substituted-5-amido benzoate derivative (compound No. 45 in the following figure) provided by the present application is described in detail below in combination with examples in the preparation of P2Y 14 R related acute lung injury and anti-inflammatory drugs.

[0018] Preparation Example 1

[0019] The preparation method and characterization data of the benzoate derivative represented by formula (1) are as follows:

[0020]

[0021] 5-nitro isophthalic acid monomethyl ester (540 mg, 2.4 mmol) and o-phenylenediamine (270 mg, 2.5 mmol) were placed in a 25 mL round-bottom flask, and an appropriate amount of tetrahydrofuran solvent was added to just dissolve the compounds, then the above system was placed in a nitrogen environment and stirred in an ice water bath for 10 minutes, then DCC (515 mg, 2.5 mmol) was added to the above reaction system using a syringe. The above system was gradually restored to room temperature for reaction, and the reaction was stopped after about 12 hours when no additional solids were precipitated in the system. After the reaction was completed, the reaction liquid was directly filtered, and the filtrate was collected. The filtrate was purified by vacuum distillation and column chromatography without additional treatment to obtain intermediate compound 1, a yellow solid, yield: 72%.1 H NMR (400 MHz, DMSO-d6) δ 10.22 (s, 1H), 9.10 - 9.03 (m, 1H), 8.93 (t, J = 1.4 Hz, 1H), 8.80 - 8.74 (m, 1H), 7.14 (dd, J = 7.8, 1.3 Hz, 1H), 7.04 - 6.97 (m, 1H), 6.82 - 6.75 (m, 1H), 6.60 (td, J = 7.7, 1.2 Hz, 1H), 5.05 (s, 2H), 3.97 (s, 3H).

[0022] After intermediate compound 1 (315 mg, 1 mmol) was dissolved in 5 mL glacial acetic acid, the system was brought to reflux for 12 hours. After TLC detection of the end of the reaction, the reaction system was diluted with a large amount of saturated sodium bicarbonate solution, and the aqueous phase was extracted with ethyl acetate (20 mL x 3), and the organic phase was dried with anhydrous sodium sulfate, and then distilled under reduced pressure to obtain the crude product intermediate compound 2, white solid, yield: 60%. Intermediate compound 2 was directly used in the next step without purification. 1 H NMR (400 MHz, DMSO-d6) δ 13.36 (s, 1H), 9.10 (s, 1H), 9.00 (s, 1H), 8.51 (s, 1H), 7.75 - 7.44 (m, 2H), 7.29 - 7.19 (m, 2H), 3.93 (s, 3H). After intermediate compound 2 (2.5 mmol, 743 mg) and reduced iron powder (15 mmol, 840 mg) were mixed and added to 5 mL glacial acetic acid, the system was then brought to room temperature for reaction, and the reaction was stopped when the system became viscous. After dilution with 50 mL ethyl acetate and filtration to remove unreacted iron powder, the organic phase was extracted with water and saturated brine, respectively, and the organic phase was retained, and anhydrous sodium sulfate was added for drying. After the dried organic phase was distilled under reduced pressure to remove the solvent, intermediate compound 3 was purified by column chromatography, white solid, yield: 69%. 1 H NMR (400 MHz, DMSO-d6) δ 13.33 (s, 1H), δ 8.78 (s, 1H), 8.42 (s, 1H), 8.32 (s, 1H), 7.73 - 7.56 (m, 2H), 7.40 - 7.34 (m, 2H), 5.92 (s, 2H), 3.89 (s, 3H).

[0023] Intermediate compound 3 (80 mg, 0.3 mmol), 4-methylbenzeneacetic acid (50 mg, 0.33 mmol), EDCI (86 mg, 0.45 mmol), HOBt (61 mg, 0.45 mmol) and DIPEA (156 μL, 0.9 mmol) were dissolved in 3 mL of DMF, and the system was then allowed to react at room temperature for 6 hours. The reaction was stopped after most of the raw material was detected by TLC. The system was diluted with 50 mL of water, and the aqueous phase was extracted with ethyl acetate (20 mL x 3), and then the organic phase was combined and dried over anhydrous sodium sulfate. After the organic phase was dried, the solvent was removed by reduced pressure distillation, and the final compound 45 was purified by column chromatography to obtain a white solid, with a yield of 52%. Melting point 148°C. 1 H NMR (400 MHz, DMSO-d6) δ 13.19 (s, 1H), 10.59 (s, 1H), 8.79 (s, 1H), 8.45 (s, 1H), 8.33 (s, 1H), 7.71-7.51 (m, 2H), 7.26 (d, J = 7.9 Hz, 2H), 7.22 (dd, J = 5.9, 3.0 Hz, 2H), 7.14 (d, J = 7.9 Hz, 2H), 3.92 (s, 3H), 3.65 (s, 2H), 2.27 (s, 3H). 13 C NMR (101 MHz, DMSO-d6) δ 170.53, 165.87, 149.58, 140.91, 136.52, 136.16, 133.07, 131.55, 129.53, 129.37, 128.65, 124.53, 122.75, 122.57, 122.17, 115.25, 53.05, 43.37, 21.14. HRMS (ESI+) m / z calcd for C 24 H 21 N3O3[M+H] + 400.1661, found, 400.1658.

[0024] Example 1

[0025] Compound P2Y 14 Receptor in vitro antagonistic activity test

[0026] The previously constructed human P2Y 14 R stable-transfected HEK293 cells were cultured in DMEM medium containing 10% fetal bovine serum, 1% non-essential amino acids, 100 U / mL penicillin, 100 μg / mL streptomycin, 2 mM L-glutamine and 3.7 g / L NaHCO3, and 6-well culture plates were inoculated before the experiment, with an inoculation density of 5 x 10 5The cells were cultured in serum-free medium for 12 h before the experiment, and then 1 μM of the compound was added to each well. After 30 min, 10 μM of UDP was added, and the cells were incubated for 12 h. The samples were collected and used to determine the intracellular IP3 content.

[0027] The 3-phosphoinositide (IP3) enzyme-linked immunosorbent assay kit used a competitive ELISA method. In the experiment, IP3 in the sample or standard competed with the coated IP3 for the binding sites on the biotin-labeled anti-IP3 monoclonal antibody. The free components were washed away. Horseradish peroxidase-labeled avidin was added, and the biotin and avidin specifically combined to form an immune complex. The free components were washed away. Color developing substrate (TMB) was added, and the TMB turned blue under the catalysis of horseradish peroxidase. After the addition of a stop solution, the TMB turned yellow. The OD value was measured at 450 nm using a microplate reader. The IP3 concentration was inversely proportional to the OD450 value. The IP3 concentration in the sample was calculated by drawing a standard curve. The average OD value of each group of duplicate wells was calculated. The standard curve of the four-parameter logistic function was drawn on a double logarithmic coordinate paper with the concentration as the abscissa and the OD value as the ordinate. The IP3 concentration in the sample was calculated by the standard curve. The experiment was repeated three times, and the average value was calculated and used to calculate the P2Y 14 IC 50 The experimental results are shown in Table 1.

[0028] Table 1. In vitro antagonistic activity of the compounds against P2Y 14 Test results of the in vitro antagonistic activity of the compounds against P2Y

[0029]

[0030] Example 2

[0031] Thermal shift experiment

[0032] RAW264.7 cells were cultured and grown in 100 mm culture dishes. Compound 45 (40 μM) was added to the cell culture dishes and incubated for 1-2 hours. After incubation with the small molecule, the culture dishes were transferred to a thermal cycler or a preheated heating block and placed at a temperature range (50°C, 53°C, 56°C, 59°C, 62°C, 65°C, 68°C) for 10 minutes. Then, the cells were freeze-thawed in liquid nitrogen and a 37°C water bath for 5 times to lyse the cells. Then, the cells were centrifuged at 10000 rpm and 4°C for 10 minutes to remove the cell debris, and the supernatant containing the protein lysate was collected. To screen the concentration, the same number of cells were incubated with different concentrations of 45 (0, 10 -2 , 10 -1 , 10 0 , 10 1 and 102 pM) incubated for 30 min in a thermocycler and heated at 54 °C for 10 min in a PCR machine. Experiments were performed as described above.

[0033] Results are shown in Figure 1 Figure 6. The above results demonstrate that the interaction ability between compound 45 and P2Y 14 R is very strong and effective.

[0034] Example 3

[0035] In vivo pharmacodynamic study of compounds in LPS-induced acute lung injury model in mice

[0036] To test the in vivo anti-inflammatory activity of the compounds, LPS-induced acute lung injury model in mice was used, and the compounds were added for intervention treatment, and the related indicators such as inflammatory factors were detected. The overproduction of inflammatory cytokines induced by LPS leads to coagulation, endothelial damage and microvascular leakage, resulting in lung organ dysfunction and injury. Dexamethasone, a clinical glucocorticoid, was used as a positive control anti-inflammatory agent. Histological analysis showed that in the lung tissue of mice induced by LPS, there were obvious pulmonary edema, pulmonary hyperemia, neutrophil infiltration and alveolar wall thickening, as shown in Figure 2 A. After treatment with compound 45, the inflammatory response in lung tissue induced by LPS was significantly reduced. Specifically, a low dose of compound 45 (2 mg / kg) could significantly reduce the number of neutrophils, and increasing the dose (6 mg / kg) did not cause substantial damage to the lung, and the alveolar structure remained relatively intact, as shown in Figure 2 A. After treatment with compound 45, the inflammatory response in lung tissue induced by LPS was significantly reduced. Specifically, a low dose of compound 45 (2 mg / kg) could significantly reduce the number of neutrophils, and increasing the dose (6 mg / kg) did not cause substantial damage to the lung, and the alveolar structure remained relatively intact, as shown in

[0037] In addition, important inflammatory factors that play a role in the development of acute lung injury and disease progression, including three cytokines IL-6, TNF-a and IL-β, were significantly increased in LPS-induced cell culture supernatant, and the levels of the above cytokines were significantly reduced after the addition of compound 45, as shown in Figure 2 B-D. Myeloperoxidase (MPO) is a neutrophil enzyme that promotes oxidative stress in many inflammatory diseases and serves as a marker of neutrophil accumulation within tissues. To test the effect of compound 45 on neutrophil lung infiltration, the expression level of MPO in lung tissue was also tested, Figure 2 E shows that treatment of mice with compound 45 at a dose of 2 and 6 mg / kg significantly reduced the level of MPO in lung tissue induced by LPS. The above results show that compound 45 can reduce the infiltration of immune cells, the content of inflammatory cytokines and the pathological changes in the lung in the LPS-induced acute lung injury model, showing a protective effect on lung inflammation.

[0038] 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. The use of the benzoic acid derivative of formula (1) or a pharmaceutically acceptable salt thereof in the preparation of drugs for the prevention and / or treatment of acute lung injury and anti-inflammatory purposes:

2. The application according to claim 1, characterized in that, The pharmaceutically acceptable salt is selected from one or more of the hydrochloride, phosphate, sulfate, acetate, maleate, citrate, benzenesulfonate, methylbenzenesulfonate, fumarate and tartrate of the benzoic acid derivative shown in formula (1).

3. The application according to claim 1 or 2, characterized in that, The acute lung injury was P2Y. 14 R-related acute lung injury.

4. The benzoic acid derivative shown in formula (1) or its pharmaceutically acceptable salt in the preparation of P2Y 14 Applications of R-antagonists:

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