(Z)-N-methyl-N-styrylbenzamide and its use in the preparation of anti-inflammatory medicaments

Abstract

The application discloses (Z)-N-methyl-N-phenylstyrylbenzamide and application thereof in preparation of anti-inflammatory drugs. The chemical structure of (Z)-N-methyl-N-phenylstyrylbenzamide is shown as formula (a). (Z)-N-methyl-N-phenylstyrylbenzamide has an anti-inflammatory effect, which provides a new leading compound for treating respiratory system diseases such as lung injury and chronic obstructive pulmonary disease.

Description

Technical fields:

[0001] This invention belongs to the field of biomedicine, specifically relating to (Z)-N-methyl-N-styrenebenzamide, its anti-inflammatory properties, and its role in treating respiratory diseases such as lung injury and chronic obstructive pulmonary disease. Background technology:

[0002] Pneumonia refers to inflammation of the distal lungs, namely the interstitium, alveolar spaces, and terminal airways. The main causative factors are pathogenic microorganisms such as bacteria, parasites, fungi, and viruses, or factors like chemical allergies and radiation. Clinical symptoms of pneumonia primarily include cough, fever, hemoptysis, or expectoration, often accompanied by shortness of breath or chest tightness. Etiologically, pneumonia can be classified into viral pneumonia, fungal pneumonia, bacterial pneumonia, mycoplasma pneumonia, physical and chemical pneumonia, allergic pneumonia, pneumonia caused by other pathogens, and immune-mediated pneumonia. Bacterial pneumonia is the most common, accounting for over 70% of pneumonia cases in adults. When pathogens invade lung tissue, signaling pathways such as mitogen-activated protein kinase (MAPK) and nuclear factor-κB are activated, triggering an inflammatory response in response to the immune system. During the inflammatory response, immune cells such as alveolar macrophages, neutrophils, and lymphocytes are activated by signal transduction pathways and release inflammatory mediators such as tumor necrosis factor, interleukins, interferons, growth factors, and chemokines. These mediators, including TNF-α, IL-6, IFN-γ, IL-1β, IL-12, and IL-18, provide feedback regulation to the inflammatory response and signal transduction pathways. Pulmonary inflammation is a significant component of many acute and chronic respiratory diseases such as COPD, asthma, and ARDS. It can lead to symptoms such as excessive mucus secretion, airway obstruction, emphysema, and pulmonary edema, as well as complications in other organs and tissues, and even systemic diseases, which can be life-threatening in severe cases. Summary of the Invention:

[0003] The purpose of this invention is to provide (Z)-N-methyl-N-styrenebenzamide with anti-inflammatory effects.

[0004] The chemical structure of the (Z)-N-methyl-N-styrenebenzamide is shown in (a):

[0005]

[0006] This invention has shown through experiments that (Z)-N-methyl-N-styrenebenzamide can effectively inhibit the expression levels of IL-6 and IL-1β in a dose-dependent manner, and has good activity in reducing inflammatory factor indicators. It can be used as a drug for treating respiratory diseases such as lung injury and chronic obstructive pulmonary disease.

[0007] Therefore, a second object of the present invention is to provide the use of (Z)-N-methyl-N-styrenebenzamide or a salt thereof in the preparation of anti-inflammatory drugs.

[0008] Preferably, the anti-inflammatory drug is a drug for treating respiratory diseases.

[0009] Preferably, the anti-inflammatory drug is a drug for treating lung injury or chronic obstructive pulmonary disease.

[0010] A third objective of this invention is to provide an anti-inflammatory drug containing (Z)-N-methyl-N-styrenebenzamide or a salt thereof as an active ingredient.

[0011] Due to its unique structural characteristics, compound (Z)-N-methyl-N-styrenebenzamide has several mechanisms for combating pneumonia, including: regulating signaling pathways such as MAPK and NF-κB, affecting the accumulation of inflammatory cells in the lungs, inhibiting the release of cytokines, and reducing the production of pro-inflammatory mediators, thereby suppressing the occurrence and development of inflammatory responses.

[0012] The (Z)-N-methyl-N-styrenebenzamide described in this invention has anti-inflammatory effects, which provides a new lead compound for the development of treatments for respiratory diseases such as lung injury and chronic obstructive pulmonary disease. Attached Figure Description

[0013] Figure 1 : Expression levels of IL-6 and IL-1β. Detailed Implementation

[0014] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0015] Example 1: Synthesis of Compound 2096

[0016] The synthesis route is as follows:

[0017]

[0018] Synthesis steps:

[0019] Commercially available carboxylic acid 5 (5 mmol) was dissolved in dichloromethane (10 mL), the temperature was lowered to 0 °C, oxaloyl chloride (0.43 mL, 5 mmol) was added, and two drops of DMF were added as a catalyst. The reaction was maintained at 0 °C for 0.5 h, then allowed to return to room temperature for 2 h, and concentrated to approximately 2.5 mL. Then, 5 mL of dichloromethane was added, the temperature was lowered to 0 °C, and excess ammonia water was added dropwise. After the addition was complete, the reaction was allowed to return to room temperature for 1 h. The mixture was allowed to stand and separated. The aqueous layer was extracted with dichloromethane (3 × 10 mL), and the organic phases were combined. The organic phase was washed once with 10 mL of saturated sodium chloride and dried over anhydrous sodium sulfate. The mixture was concentrated and subjected to column chromatography, eluented with petroleum ether and acetone (v / v = 2:1), to give a white powdery solid 6 (3.4 mmol).

[0020] Under an argon atmosphere, iodine (2.80 g, 11 mmol) and anhydrous morpholine (2.62 mL, 30 mmol) were dissolved in anhydrous benzene (12 mL) and stirred thoroughly at room temperature for 30 min. Then, phenylacetylene 7 (2.25 g, 10 mmol) was slowly added dropwise to the system. The reaction mixture was then placed in an oil bath at 45 °C and reacted for 24 h. After removing the oil bath and allowing it to cool naturally to room temperature, the solid was filtered through diatomaceous earth. The filter residue was washed with ether (3 × 20 mL). The filtrate was collected and washed successively with saturated ammonium chloride solution (20 mL), saturated sodium bicarbonate aqueous solution (20 mL), and saturated sodium chloride solution (20 mL). The combined organic phases were dried over anhydrous sodium sulfate for 30 min. After filtering off the solid, the organic solvent was removed by rotary evaporation of the organic phase. The crude product was purified by silica gel column chromatography. The eluent was petroleum ether, yielding a yellow oily liquid compound 8 (2.23 g, 9.8 mmol), with a yield of 98%.

[0021] Compound 8 (2.23 g, 9.8 mmol) was added to a mixed solution of THF (20 mL) and water (20 mL), followed by the sequential addition of p-toluenesulfonyl hydrazine (3.65 g, 19.6 mmol) and anhydrous sodium acetate (2.41 g, 29.4 mmol). The mixture was refluxed overnight. After cooling to room temperature, the reaction was quenched by adding saturated ammonium chloride solution in an ice-water bath. The mixture was extracted with diethyl ether (3 × 20 mL), and the organic phase was washed once with saturated sodium chloride solution (20 mL) and dehydrated with anhydrous sodium sulfate for 30 min. After filtering off the solid, the organic phase was removed by rotary evaporation to remove the organic solvent. The crude product was purified by silica gel column chromatography. The eluent was petroleum ether, yielding a colorless oily liquid 9 (1.31 g, 5.68 mmol), in 58% yield.

[0022] In an argon atmosphere, compound 6 (1.2 mmol), activated CuI (9.5 mg, 0.05 mmol), and Cs₂CO₃ (407 mg, 1.25 mmol) were added sequentially to a Schlenk tube. Compound 9 (230 mg, 1 mmol) was dissolved in anhydrous THF (5 mL) and added dropwise to the reaction system. Then, N,N'-dimethylethylenediamine (10.8 μL, 0.1 mmol) was added dropwise using a microsyringe. The Schlenk tube was sealed, and the reaction was carried out in an oil bath at 60 °C, monitored by TLC. After the reactants were completely consumed, the oil bath was removed, and the mixture was allowed to return to room temperature. The solids were filtered off using diatomaceous earth, and the mixture was washed with ethyl acetate (30 mL). The organic solvent was removed by rotary evaporation of the organic phase, and the crude product was purified by silica gel column chromatography. The eluent was petroleum ether and ethyl acetate (v / v = 5:1), yielding a pale yellow oil, 10.

[0023] The entire reaction was conducted under an argon atmosphere, requiring strict anhydrous and oxygen-free conditions. Compound 10 (0.5 mmol) was dissolved in anhydrous DMF (2.5 mL), and 60% pure NaH (40 mg, 1 mmol) was slowly added at 0°C. After maintaining the reaction at 0°C for 0.5 h, iodomethane (46.7 μL, 0.75 mmol) was slowly added dropwise. The reaction was allowed to return to room temperature naturally, and TLC was used for monitoring. After the starting materials were consumed (approximately 3 h), the reaction was quenched by slowly adding water (10 mL) at 0°C. The reaction mixture was extracted with diethyl ether (3 × 10 mL), the organic phases were combined, washed with saturated sodium chloride solution (1 × 10 mL), and the organic phase was dehydrated with anhydrous sodium sulfate for 0.5 h. The organic solvent was removed by rotary evaporation, and the crude product was purified by silica gel column chromatography. The eluent was petroleum ether and ethyl acetate (v / v = 10:1), yielding product 2 (compound 2096). 79%yield.Colorless oil.1H NMR(400MHz, CDCl3)δ:7.37-7.28(m,5H),7.24(d,J=7.7Hz,1H),7.13(d,J=7.6Hz,1H),7.07-7.03(m,1H) ,6.95(dd,J=8.5,2.6Hz,1H),6.32(d,J=8.9Hz,1H),5.87(d,J=8.9Hz,1H),3.76(s,3H),3.11(s,3H).13C NMR(100MHz, CDCl3)δ:171.3,159.3,137.0,135.2,131.2,129.1,128.7(2×C ),128.6(2×C),127.7,120.7,119.8,116.8,113.3,55.4,35.5.HRMS(ESI)m / z calculated for C17H18O2N+[M+H]+268.1332,found268.1332.

[0024] The chemical structural formula of product 2 (compound 2096) is shown below:

[0025]

[0026] Example 2: Study on the anti-inflammatory activity of product 2 (compound 2096)

[0027] The expression level of cellular inflammatory factors was inhibited by compound 2096 of the present invention using the conventional qPCR method.

[0028] I. Cell Culture

[0029] Mouse monocyte-macrophage RAW264.7 cells were cultured in DMEM high-glucose medium supplemented with 10% (v / v) FBS, 100 U / mL penicillin, and 100 μg / mL streptomycin. Culture conditions were 37°C and 5% CO2. Cells were passaged when they reached 80% confluence.

[0030] II. Cell Drug Delivery and Induction of Cellular Inflammation

[0031] The experiment used cells in the logarithmic growth phase, with cells at a density of 4*102 5 / wells were seeded in 12-well plates and cultured at 37°C and 5% CO2 until 70% growth was achieved. Groups were set up: blank control group, LPS stimulation group (Vehicle), positive control group (1 μM dexamethasone, Dex), and drug-treated groups (20, 40, and 80 μM compound 2096). The culture medium was carefully removed. Fresh complete culture medium containing the compound was added to the positive control and drug-treated groups, while an equal volume of DMSO was added to the blank control and LPS stimulation groups. After 1 hour, except for the blank stimulation group, 1 μg / mL LPS was added to all groups for 4 hours to induce cell inflammation.

[0032] III. RNA Extraction and qPCR Assay

[0033] 1. After completely discarding the culture medium from the cultured cells, add 0.5 mL of RA2 from the RNA extraction kit to each well for lysis (the cells can be frozen at -80℃ overnight for better lysis results, or they can be lysed at 4℃ for 10 min and then collected).

[0034] 2. Collect cell lysates and extract total RNA according to the kit instructions, and measure its concentration using an ultra-micro UV-Vis spectrophotometer;

[0035] 3. RT-PCR (20 μL per system) includes 4 μL of 5x BUFFER, X μL of Total RNA (1 pg-1 μg, usually 200-400 ng), and μL of DEPC-H2O (16-X). After placing the tubes in an 8-tube series, label them with serial numbers, and place them in a PCR instrument at 50℃ for 15 min, 85℃ for 5 s, and 16℃ for ∝ for reverse transcription.

[0036] 4. Add 80 μL DEPC-H2O to the transcribed cDNA to a final volume of 100 μL, centrifuge to mix, and store at -20℃ for later use.

[0037] 5. Multiply (SYBR 10μL + DEPC-H2O 10μL + primer 0.5μL) by the number of samples, take 18μL / well into a 96-well plate, add 2μL cDNA to make a 20μL system, and centrifuge at 1200rpm for 1min to mix.

[0038] 6. Place the sample plate into the CFX Connect Real-Time System (real-time fluorescence quantitative PCR instrument) for detection. Perform the detection according to the program of (95℃ 2min, 95℃ 20s, 57℃ 20s, 72℃ 20s) for a total of 39 cycles, and 95℃ 1min, 55℃ 30s, 95℃ 30s. The instrument takes a total of 2.5 hours.

[0039] Data was saved and analyzed after 7.2 hours, using 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.

[0040] Primer design: This invention targets the IL-6 and IL-1β genes, using Actin as an internal reference gene. The target gene was searched in the NCBI gene database, and its sequence was viewed in GeneBank. Primer parameters were designed according to the detection requirements, as shown in Table 1 below.

[0041] Table 1

[0042]

[0043] cDNA synthesis: cDNA was synthesized using total RNA as a template using a 5x HiScript II Q Select RTSuperMix. Before use, each solution was vortexed to mix thoroughly, and the liquid remaining on the tube wall was collected after rapid centrifugation. The reaction system was prepared in an ice bath. After thawing the template RNA on ice, the reverse transcription reaction system was prepared according to Table 2.

[0044] Table 2

[0045]

[0046] The genome removal and reverse transcription reactions were performed in a Veriti 96well Thermal Cycler PCR instrument. The reaction program was: 50℃ for 15 min, 85℃ for 5 s. The reverse transcription products were stored at -20℃ for later use.

[0047] Real-time qPCR: The real-time qPCR reaction system was prepared as shown in Table 3. The primer sequences used in this experiment are shown in Table 1. The Actin gene was selected as an internal control. The primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0048] Table 3

[0049]

[0050] Real-time qPCR reactions were performed using a CFX Connect Real-Time System. The amplification program was: 95℃ for 2 min, followed by 40 cycles: 95℃ for 20 s, 57℃ for 20 s, and 72℃ for 20 s. Melting curves were obtained by increasing the temperature from 55℃ to 95℃. The results were automatically analyzed by the software, generating amplification curves and calculating Ct values. Two [analytes / components] were used. -ΔΔct The experimental results were analyzed, and the calculation formula is as follows: △Ct 目的基因 =Ct 目的基因 -Ct 内参基因 , △△Ct 目的基因 =△Ct 实验组目的基因 -△Ct 对照组目的基因 2 -ΔΔct The expression fold of the target gene in the experimental group relative to the control group is represented by the LPS stimulation group, which is normalized.

[0051] The results are as follows Figure 1 As shown:

[0052] The test results showed that compound 2096 effectively inhibited the expression levels of IL-6 and IL-1β in RAW264.7 macrophages in a dose-dependent manner. Figure 1 The above experimental results show that compound 2096 of the present invention has good activity in reducing inflammatory factor indicators and can be used in drug research for the treatment of respiratory diseases such as lung injury and chronic obstructive pulmonary disease.

Claims

1. The use of (Z)-N-methyl-N-styrenebenzamide or its salt in the preparation of an anti-inflammatory drug, wherein the anti-inflammatory drug is a drug for treating respiratory diseases; the chemical structure of the (Z)-N-methyl-N-styrenebenzamide is shown in (a): （a）。 2. The application according to claim 1, characterized in that, The anti-inflammatory drugs mentioned are used to treat lung injury and chronic obstructive pulmonary disease.

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