P-hydroxybenzyl substituted tryptophan derivatives, and pharmaceutical composition and application thereof

Abstract

The invention belongs to the technical field of medicines, and discloses hydroxybenzyl substituted tryptophan derivatives as well as a pharmaceutical composition and application thereof. Specifically disclosed are NO release inhibitory activity of p-hydroxybenzyl substituted tryptophan compounds. Experimental results prove that the compound 1 has an obvious NO release inhibition effect and is expected to become a medicine for treating inflammation-related diseases.

Description

Technical Field

[0001] This invention relates to p-hydroxybenzyl-substituted tryptophan derivatives synthesized using a simple method, and the application of their pharmaceutical salts in the preparation of drugs for the prevention and treatment of inflammation. It belongs to the field of pharmaceutical technology. Background Technology

[0002] Gastrodia elata Blume, also known as Chijianzhi, Duyaozhi, Limu, Helicao, Shencao, Guiduyou, Mupu, Mingtianma, Dingfengcao, Bailongpi, Chijian, etc., is a perennial symbiotic plant belonging to the genus Gastrodia R.Br. of the Orchidaceae family. Its dried tubers are used medicinally. It is mainly produced in Yunnan, Sichuan, Shaanxi, and Guizhou provinces. The Flora of China records approximately 20 species of Gastrodia R.Br., distributed from East Asia, Malaysia to Oceania, with 13 species found in my country. Among them, Gastrodia elata Bl. is distributed from Northeast China to Tibet. [1] .

[0003] Gastrodia elata rhizome, a precious traditional Chinese medicine, was first recorded in the *Shennong Bencao Jing* (Shennong's Classic of Materia Medica) and listed as a superior-grade herb. It is pungent, warm, and non-toxic. Modern pharmacological studies have shown that the pharmacological activities of Gastrodia elata extract and its main chemical components include enhancing immunity and anti-inflammatory effects. Studies have shown that Gastrodia elata injection can significantly enhance the phagocytic function of mouse macrophages and serum lysozyme capacity. The values ​​in the hemolytic plaque test, IRFC test, and anti-SRBC test were all significantly higher than the control group, indicating that Gastrodia elata injection enhances both non-specific immunity and cellular and humoral immunity in mice. 25mg of Gastrodia elata injection can increase the DTH response in mice. [2] Repeated administration significantly increased spleen weight in rats, suggesting that it has a promoting effect on immune function. [3] Over the years, scholars both at home and abroad have conducted relatively systematic research on the chemical composition of Gastrodia elata, revealing that it contains phenols and their glycosides, citric acid and its benzyl esters, nitrogen-containing compounds, sterols, and polysaccharide compounds. [4-15] It contains various types of components, with phenols and their glycosides being the main components, especially gastrodin, which has the highest content.

[16] Meanwhile, related studies have also shown that gastrodin is the main active ingredient in Gastrodia elata, and it has been artificially synthesized. [17,18] In addition, a series of structural modification studies were conducted, resulting in the synthesis of many derivatives, such as acetylgastrodin, vanillin, and fluorinated gastrodin. [19-21] wait.

[0004] Anti-inflammatory drugs are widely used in clinical practice, both prescription and over-the-counter. Currently, the most widely used anti-inflammatory drugs are traditional nonsteroidal anti-inflammatory drugs (NSAIDs), with aspirin, acetaminophen, and ibuprofen being representative examples. However, traditional NSAIDs work by inhibiting cyclooxygenase II (COXII), thereby suppressing the synthesis of inflammatory prostaglandins. But during their action, COXII is also inhibited, leading to a significant reduction in the synthesis of physiological prostaglandins, causing gastrointestinal side effects such as gastrointestinal mucosal erosion and ulcers. In severe cases, patients may experience coagulation disorders and kidney toxicity. Furthermore, hematologic and cardiovascular symptoms are also common adverse reactions during the use of anti-inflammatory and analgesic drugs, seriously affecting patient safety and greatly limiting the clinical application of traditional anti-inflammatory and analgesic drugs. Therefore, there is an urgent need to develop new anti-inflammatory drugs with fewer side effects and a wider range of clinical applications.

[0005] Previously, the research group isolated a series of p-hydroxybenzyl-substituted amino acids or peptides from Gastrodia elata. [22,23] Using this as a lead structure, structural derivatization studies were conducted. The compound in this application is a derivative of a p-hydroxybenzyl-substituted amino acid obtained from Gastrodia elata, a traditional and precious Chinese medicinal herb, and exhibits significant anti-inflammatory effects.

[0006] References:

[0007] [1] Editorial Committee of Flora of China, Chinese Academy of Sciences. Flora of China, Vol. 18. [M]. Beijing: Science Press, 1999: 29.

[0008] [2] Chen Yimin, Xu Jingyun, Xu Shoulin. Effects of Gastrodia elata injection on immune function in mice [J]. Shanghai Journal of Immunology, 1988, (5): 337-338, 336.

[0009] [3] Yu Longshun, Xia Jingsheng. Study on the anti-inflammatory effect of Gastrodia elata [J]. Chinese Traditional and Herbal Drugs, 1989, 20(5):19-21.

[0010] [2] Li Juan, Tong Jing'an, Chen Cheng, et al. Treatment of 46 cases of anterior ischemic optic neuropathy with TCM syndrome differentiation combined with conventional Western medicine[J]. Shaanxi Journal of Traditional Chinese Medicine, 2007(10):1354-1355.

[0011] [3] Wu Zhengzhi, Li Ming, Li Yaofang, et al. Effects of Tiantai No. 1 on the activity of the central cholinergic system in a spontaneous senile dementia model (English) [J]. Chinese Journal of Clinical Rehabilitation, 2006(35):163-165.

[0012] [4] Editorial Board of Chinese Materia Medica, State Administration of Traditional Chinese Medicine. Chinese Materia Medica [M]. Shanghai: Shanghai Science and Technology Press, 1999: 716-722.

[0013] [5] Feng Xiaozhang, Chen Yuwu, Yang Junshan. Study on chemical components of Gastrodia elata [J]. Acta Chimica Sinica, 1979, 37(03):175-182.

[0014] [6] Zhou Jun, Yang Yanbin, Yang Chongren. Neophenolic glycosides in Gastrodia elata: Gastrodin[J]. Chinese Science Bulletin, 1979, 24(07):335-336. [7]Taguchi, H., I. Yosioka, K. Yamasaki, et al. Studies on the Constituents of Gastrodia ElataBlume[J]. Chem. Pharm. Bull., 1981, 29(1):55-62.

[0015] [8] Zhou Jun, Pu Xiangyu, Yang Yanbin. Nine phenolic components of fresh Gastrodia elata [J]. Chinese Science Bulletin, 1981, 26(18): 1118-1120.

[0016] [9] Hao Xiaoyan, Tan Ninghua, Zhou Jun. Chemical constituents of Gastrodia elata produced in Guizhou [J]. Yunnan Botanical Research, 2000, 22(01):81-84.

[0017]

[10] Xiao Yongqing, Li Li, You Xiaolin. Study on chemical components of effective parts of Gastrodia elata (I) [J]. Chinese Journal of Traditional Chinese Medicine, 2002, 27(01):35-36.

[0018]

[11] Wang Li, Xiao Hongbin, Liang Xinmiao. Study on chemical components of Gastrodia elata (I) [J]. Chinese Traditional and Herbal Drugs, 2003, 34(07):584-585.

[0019]

[12] Huang Zhanbo, Song Dongmei, Chen Fakui. Study on chemical constituents of Gastrodia elata (I) [J]. Chinese Journal of Medicinal Chemistry, 2005, 15(04):227-229.

[0020]

[13] Sun Yanan. Study on isolation, purification and physicochemical properties of Gastrodia elata polysaccharide [D]. Southwest University, 2007.

[0021]

[14] Guo Meihua, Zhang Xinjian, Yang Lijie, et al. Study on extraction process of effective components of Gastrodia elata [J]. Chinese Journal of Drugs and Clinical Use, 2010, 10(2):175-176.

[0022]

[15] Liu Xingjie, Yang Yi. Study on components of Gastrodia elata (a traditional Chinese medicine) I. Extraction and identification of vanillin [J]. Journal of Shanghai First Medical College, 1958(S1):67-68.

[0023]

[16] Yin Min. Effects of different processing methods on the content of gastrodin in Gastrodia elata [J]. Journal of Chinese Medicine Information, 2010, 17(7):49-50.

[0024]

[17] Chen Zaicheng, Sun Changjun, Wang Yibin, et al. Synthesis of gastrodin intermediate by phase transfer catalysis [J]. Pharmaceutical Industry, 1988, 19(08): 345, 374.

[0025]

[18] Li Chunqiu, Wu Junming, Chen Yao. Application of phase transfer catalysis in the synthesis of gastrodin [J]. Journal of Yangzhou Teachers College (Natural Science Edition), 1983(02):57-58.

[0026]

[19] Zhou Jun, Yang Yanbin, Yang Chongren. Chemical research on Gastrodia elata II. Synthesis of gastrodin and its analogues [J]. Acta Chimica Sinica, 1980(02):162-166.

[0027]

[20] Huang Weiyuan, Qian Zhaohui. Synthesis of fluorinated gastrodin [J]. Acta Chimica Sinica, 1987(12):1175-1179.

[0028]

[21] Yang Yan. Study on the changes in chemical composition of five medicinal plants during growth and the synthesis of gastrodin aglycone derivatives [D]. Northwest University, 2008.

[0029]

[22] Guo, QL; Wang, YN; Lin, S.; et al. 4-Hydroxybenzyl-substituted aminoacid derivatives from Gastrodia elata[J]. Acta Pharm. Sin. B, 2015, 5(4): 350-357.

[0030]

[23] Guo, QL; Wang, YN; Zhu, CG; et al. 4-Hydroxybenzyl-substitutedglutathione derivatives from Gastrodia elata[J]. J. Asian Nat. Prod. Res., 2015, 17(5): 439-454.

[0031] The p-hydroxybenzyl-substituted tryptophan derivatives involved in this invention have not yet been isolated from animals, plants, or microorganisms or prepared by chemical synthesis or biosynthesis by other research groups; moreover, no one has reported that the compounds designed in this patent have anti-inflammatory functions. Summary of the Invention

[0032] The technical problem to be solved by the present invention is to provide a class of hydroxybenzyl-substituted tryptophan derivatives, their pharmaceutical compositions and uses.

[0033] To solve the technical problem of this invention, the present invention provides the following technical solution:

[0034] The first aspect of the present invention is to provide a compound as shown in general formula (I).

[0035] Specifically, a compound as shown in formula (I) and its pharmaceutically acceptable salt:

[0036] A compound of formula (I) and its pharmaceutically acceptable salt:

[0037]

[0038] The configuration of compound (I) is selected from R or S.

[0039] In addition, the use of the compound of formula (I) and its pharmacodynamically acceptable salt in the preparation of medicaments for the prevention and / or treatment and / or relief of inflammation is also provided:

[0040] The second aspect of the present invention is to provide a method for preparing the compound described in the first aspect.

[0041] Dissolve 1 equivalent of L- or D-tryptophan in an aqueous NaOH solution and stir magnetically until completely dissolved. Slowly add 1.0–1.5 equivalents of a solution of p-hydroxybenzaldehyde in ethanol or methanol, stirring at room temperature for 2–4 hours. Add 2–4 equivalents of NaBH4 in portions at 0°C, monitoring the reaction by TLC. After the reaction is complete, extract with ethyl acetate 2–4 times, collect the aqueous layer, and adjust the pH to 4–5 with dilute hydrochloric acid. Let stand at 0–5°C, filter, and wash with ethanol to obtain a white powder product.

[0042] A third aspect of the present invention is to provide a pharmaceutical composition comprising a compound of general formula (I) as an active ingredient and a carrier commonly used in the pharmaceutical field.

[0043] Typically, the pharmaceutical compositions of this invention contain 0.1-95% by weight of the compounds of this invention.

[0044] Pharmaceutical compositions of the compounds of the present invention can be prepared according to methods known in the art. For this purpose, if desired, the compounds of the present invention can be combined with one or more solid or liquid pharmaceutical excipients and / or adjuvants to formulate suitable administration or dosage forms for use as human or veterinary medicine.

[0045] The compounds of the present invention or pharmaceutical compositions containing them can be administered in unit dose form via enteral or non-enteric routes, such as oral, intramuscular, subcutaneous, nasal, oral mucosa, skin, peritoneum or rectum, with oral administration being preferred.

[0046] The compounds of this invention or pharmaceutical compositions containing them can be administered by injection. Injection includes intravenous injection, intramuscular injection, subcutaneous injection, and intradermal injection.

[0047] Dosage forms can be liquid or solid. Liquid dosage forms include true solutions, colloids, microparticles, emulsions, and suspensions. Other dosage forms include tablets, capsules, pellets, aerosols, pills, powders, solutions, suspensions, emulsions, granules, suppositories, and lyophilized powder injections.

[0048] The extracts or compounds of this invention can be formulated into ordinary preparations, sustained-release preparations, controlled-release preparations, targeted preparations, and various microparticle delivery systems.

[0049] To formulate unit-dosage dosage forms into tablets, a wide variety of carriers known in the art can be used. Examples of carriers include diluents and absorbents such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose, and aluminum silicate; humectants and binders such as water, glycerin, polyethylene glycol, ethanol, propanol, starch paste, dextrin, syrup, honey, glucose solution, gum arabic paste, gelatin paste, sodium carboxymethyl cellulose, shellac, methylcellulose, potassium phosphate, and polyvinylpyrrolidone; disintegrants such as dried starch, alginate, agar powder, brown algae starch, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene sorbitol fatty acid ester, sodium dodecyl sulfate, methylcellulose, and ethylcellulose; disintegration inhibitors such as sucrose, tristearate, cocoa butter, and hydrogenated oil; absorption promoters such as quaternary ammonium salts and sodium dodecyl sulfate; and lubricants such as talc, silica, corn starch, stearates, boric acid, liquid paraffin, and polyethylene glycol. Tablets can also be further processed into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or bilayer and multilayer tablets.

[0050] For example, various carriers known in the art can be widely used to formulate the drug delivery unit into pills. Examples of carriers include diluents and absorbents such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, polyvinylpyrrolidone, gelucire, kaolin, talc, etc.; binders such as gum arabic, tragacanth, gelatin, ethanol, honey, liquid sugar, rice paste, or flour paste, etc.; and disintegrants such as agar powder, dried starch, alginate, sodium dodecyl sulfate, methylcellulose, ethylcellulose, etc.

[0051] For example, to formulate the drug delivery unit into a capsule, the active ingredient, the extract or compound of the present invention, is mixed with the various carriers described above, and the resulting mixture is placed in a hard gelatin capsule or a soft capsule. Alternatively, the active ingredient, the compound of the present invention, can be formulated as a microcapsule, suspended in an aqueous medium to form a suspension, or filled into a hard capsule or formulated as an injectable preparation for use.

[0052] For example, the extracts or compounds of this invention can be formulated into injectable preparations, such as solutions, suspension solutions, emulsions, and lyophilized powders for injection. These preparations can be aqueous or non-aqueous and may contain one or more pharmacodynamically acceptable carriers, diluents, binders, lubricants, preservatives, surfactants, or dispersants. Diluents may be selected from water, ethanol, polyethylene glycol, 1,3-propanediol, ethoxylated isostearyl alcohol, polyoxyethylene isostearyl alcohol, polyoxyethylene sorbitan fatty acid esters, etc. Furthermore, to prepare isotonic injections, appropriate amounts of sodium chloride, glucose, or glycerol can be added to the injectable formulation. In addition, conventional solubilizers, buffers, pH adjusters, etc., may also be added. These excipients are commonly used in the art.

[0053] In addition, colorants, preservatives, flavorings, tasters, sweeteners or other materials may be added to pharmaceutical preparations if necessary.

[0054] To achieve the purpose of medication and enhance the therapeutic effect, the drug or drug composition of the present invention can be administered using any known method of administration.

[0055] The dosage of the compounds and pharmaceutical compositions of this invention depends on many factors, such as the nature and severity of the disease to be prevented or treated, the sex, age, weight, personality, and individual response of the patient or animal, the route of administration, frequency of administration, and therapeutic purpose. Therefore, the therapeutic dosage of this invention can vary widely. Generally speaking, the dosage of the pharmaceutical components used in this invention is well known to those skilled in the art. The actual amount of drug contained in the final formulation of the compound composition according to this invention can be appropriately adjusted to achieve the required therapeutic dose and fulfill the preventive or therapeutic purpose of this invention. The appropriate daily dosage range for the compounds of this invention is 0.001-150 mg / kg body weight, preferably 0.01-100 mg / kg body weight, more preferably 0.01-60 mg / kg body weight, and most preferably 0.1-10 mg / kg body weight. The above dosages can be administered in a single dose or divided into several doses, such as two, three, or four doses. This is limited by the clinical experience of the dispensing physician and the administration regimen, including the use of other treatment methods.

[0056] The total dose required for each treatment can be divided into multiple administrations or administered as a single dose. The compounds and compositions of the present invention can be taken alone or in combination with other therapeutic or symptomatic drugs, with dosage adjustments.

[0057] The fourth aspect of the present invention is to provide the use of the compound as shown in general formula (I) in the preparation of a medicament for inflammatory diseases.

[0058] The inventors have discovered that the compound (I) of the present invention and its pharmaceutically acceptable salt have significant anti-inflammatory effects. Therefore, the compound (I) of the present invention and its pharmaceutically acceptable salt, on the other hand, relate to methods for treating and improving inflammation-related diseases. The methods include administering a therapeutically effective amount of the compound (I) or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, to a patient requiring treatment.

[0059] No publicly reported compounds (I) or pharmaceutically acceptable salts have been found.

[0060] Beneficial Technical Effects: During their research on the active components of the traditional Chinese medicine Gastrodia elata, the inventors of this invention isolated a series of p-hydroxybenzyl-substituted amino acids or peptides, and synthesized their derivatives using these as lead structures. Anti-inflammatory pharmacological experiments were conducted on the compounds of this invention. Inflammation is a complex biological process that plays a crucial role in various human diseases. The latest theories of inflammatory responses suggest that pathogens or non-infectious factors can activate endothelial cells in the body, releasing large amounts of inflammatory mediators and pain-inducing substances, including histamine, prostaglandin PGE2, leukotrienes, and nitric oxide (NO); and by activating specific receptors on the surface of immune cells, releasing inflammatory cytokines, including tumor necrosis factor-α (TNF-α), interleukins, and interferons. Therefore, the release of various inflammatory mediators and inflammatory factors is an important cause of inflammatory responses.

[0061] Lipopolysaccharide (LPS)-induced macrophages are one of the most commonly used in vitro models in inflammation research, characterized by low requirements for model establishment, ease of technique mastery, high reliability, and good reproducibility. LPS activates the immune system, especially macrophages, by releasing various pro-inflammatory mediators. Macrophages, in turn, recognize LPS, activating downstream intracellular signaling cascades, leading to the production of several inflammatory mediators and cytokines (including NO), thereby mediating inflammation. Therefore, in this study, an LPS-stimulated Raw 264.7 macrophage model was used to detect the anti-inflammatory activity of compounds.

[0062] The inventors discovered that the compounds provided by this invention exhibit excellent anti-inflammatory activity in the inflammatory response induced by lipopolysaccharide (LPS)-stimulated macrophages. Pharmacological experiments show that the compounds of this invention have a good protective effect against the inflammatory response induced by LPS and can be used to prepare drugs for the prevention and / or treatment of inflammatory responses. They belong to a novel lead compound in the process of anti-inflammatory drug development.

[0063] The preferred inflammatory diseases are selected from a variety of common inflammatory reactions, such as respiratory system inflammation, musculoskeletal system inflammation, skin inflammation, and autoimmune diseases. Attached Figure Description

[0064] Figure 1 NO release inhibition of compound 1

[0065] Figure 2 HRESIMS diagram of compound 1

[0066] Figure 3 Compound 1 1 H NMR image

[0067] Figure 4 HRESIMS diagram of compound 2

[0068] Figure 5Compound 2 1 H NMR image Detailed Implementation

[0069] The following experimental examples further illustrate the present invention, but do not limit the invention in any way.

[0070] Example 1: The preparation process of compound (I) is as follows:

[0071] 10 mmol of L- or D-tryptophan was dissolved in a 2 mol / L NaOH aqueous solution and stirred magnetically until completely dissolved. A 12 mmol solution of p-hydroxybenzaldehyde in ethanol was slowly added dropwise, and the mixture was stirred at room temperature for 3 hours. 0.12 g of NaBH4 was added in portions at 0 °C, and the reaction was monitored by TLC (ethyl acetate:ethanol:water = 4:2:1). After the reaction was complete, the mixture was extracted three times with ethyl acetate, and the aqueous layer was collected. The pH was adjusted to 4–5 with 2 mol / L dilute hydrochloric acid, at which point a large amount of white solid precipitated. The mixture was allowed to stand at 0–5 °C, filtered, and washed with ethanol to obtain a white powdery product.

[0072] N-p-hydroxybenzyl-L-tryptophan (compound 1): white amorphous powder; 1 H NMR (DMSO-d6, 400MHz): δ H 10.86(1H,d,J=2.8Hz,NH),7.49(1H,d,J=8.0Hz,H-7),7.33(1H,d,J=8.4Hz,H-4),7.15(1H,d,J=2.8Hz,H -2),7.05(1H,t,J=8.0Hz,H-6),7.04(2H,d,J=8.4Hz,H-2′ / 6′),6.96(1H,dd,J=8.0,7.6Hz,H-5),6.67(2H ,d,J=8.4Hz,H-3′ / 5′),3.69(1H,d,J=13.2Hz,H-7′a),3.64(1H,d,J=13.2Hz,H-7′b),3.39(1H,dd,J=8.8 ,7.0Hz,H-9),3.17(1H,dd,J=15.2,7.0Hz,H-8a),3.03(1H,dd,J=15.2,8.8Hz,H-8b); (+)-HR-ESI-MS:m / z 311.13913[M+H] + (C 18 H 19 Calculated value of O3N2, 311.13902).

[0073] N-p-hydroxybenzyl-D-tryptophan (compound 2): white amorphous powder; 1 H NMR (DMSO-d6, 400MHz): δH 10.91(1H,d,J=2.4Hz,NH),7.52(1H,d,J=7.6Hz,H-7),7.34(1H,d,J=8.0Hz,H-4),7.17(1H,d,J=2.4Hz,H -2),7.05(1H,t,J=8.0Hz,H-6),7.04(2H,d,J=8.4Hz,H-2′ / 6′),6.95(1H,dd,J=8.0,7.6Hz,H-5),6.69(2H ,d,J=8.4Hz,H-3′ / 5′),3.73(1H,d,J=12.0Hz,H-7′a),3.69(1H,d,J=12.0Hz,H-7′b),3.39(1H,dd,J=8.8 ,7.0Hz,H-9),3.20(1H,dd,J=15.2,7.0Hz,H-8a),3.07(1H,dd,J=15.2,8.8Hz,H-8b); (+)-HR-ESI-MS:m / z 311.1398[M+H] + (C 18 H 19 Calculated value of O3N2, 311.1390).

[0074] Example 2: LPS-stimulated Raw 264.7 macrophage assay to detect the anti-inflammatory ability of compounds

[0075] Experimental objective: To induce an inflammatory response in Raw 264.7 macrophages by treating them with LPS, and to detect the NO content in the cell supernatant after treatment with different drugs, in order to screen effective anti-inflammatory agents.

[0076] Experimental Methods: Frozen cells were quickly removed from liquid nitrogen and placed in a 37°C water bath, gently shaken to thaw, and 3 mL of preheated to room temperature complete culture medium (DMEM + 10% fetal bovine serum + 1% penicillin-streptomycin mixture) was added. After centrifugation at 800g for 3 min, the supernatant was discarded, and the cells were resuspended and transferred to a culture dish containing fresh complete culture medium. The cells were then incubated at 37°C in a CO2 incubator. In this experiment, revived cells were considered stable after two passages before being used in the experiment. Raw264.7 cells are semi-adherent cells, appearing round at rest, with several cells clustered in a "grape-like" shape. Cells were passaged when they reached approximately 90% confluence. The culture medium was discarded, fresh complete culture medium was added, and the cells were detached by pipetting. The cell suspension was collected in a centrifuge tube and centrifuged at 800g for 3 min. Discard the supernatant and collect the cell pellet. Add 1 mL of culture medium to resuspend the cell pellet. Gently pipette to disperse the cell pellet into single cells. Inoculate the cells into culture dishes at a ratio of 1:3 and incubate in a CO2 incubator at 37°C.

[0077] With ρ = 5 x 10 5Cells were evenly seeded into 96-well plates and incubated at 37°C in a CO2 incubator for 17-24 hours before proceeding with the next steps. The cell culture medium was then replaced with inoculation medium containing 1% fetal bovine serum (FBS) and starved for 4 hours. After 4 hours, the cell culture medium was replaced with maintenance medium containing 8% FBS, and 10 μL / well of the drug was added for 2 hours of treatment. Then, 1 μg / mL of lipopolysaccharide (LPS) was added for 12 hours of stimulation. The cell culture medium was collected for subsequent experiments.

[0078] NO content was determined using the Griess reagent method: 0.1% sulfanilamide solution and 0.1% NED solution were prepared and brought to room temperature. 0.1M sodium nitrite was diluted to 100μM with complete culture medium, and then serially diluted 1:2 to obtain eight standard concentrations. 50μL / well of sample and standard solutions were added to each well of a 96-well plate. 50μL / well of 1% sulfanilamide solution was added to each well, and the plate was incubated at room temperature in the dark for 5 min. 50μL / well of 0.1% NED solution was added to each well; the color reaction should appear immediately, and the plate was incubated at room temperature in the dark for 5 min. The absorbance at 525nm was recorded. A linear fit was performed on the absorbance of the standard solutions to obtain the linear equation C = a*A525 + b between the concentration C and the absorbance A525. The NO concentration in each sample was then calculated by substituting the sample absorbance into the equation.

[0079] Experimental results: Compound 1 reduced NO production in LPS-stimulated Raw264.7 macrophages, suggesting a significant anti-inflammatory effect. Specific results are shown in Table 1 and... Figure 1 .

[0080] Table 1. NO release inhibition effect of compounds

[0081]

Claims

1. A compound as shown in formula (I) and a pharmaceutically acceptable salt thereof: in, The configuration of compound (I) is selected from R or S.

2. The compound according to claim 1 and its pharmaceutically acceptable salt, characterized in that, The compounds are selected from the following group:

3. The compound according to claim 1 and its pharmaceutically acceptable salt, wherein, The pharmaceutically acceptable salt is selected from the salt of compound (I) with an organic or inorganic acid.

4. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the compound of any one of claims 1-2, its pharmaceutically acceptable salt, and a pharmaceutically acceptable carrier or excipient.

5. The use of the compound of any one of claims 1-2 and its pharmaceutically acceptable salt or the pharmaceutical composition of claim 3 in the preparation of a medicament for the prevention or treatment of inflammation.

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