A phytosterol hydrogen sulfide donor type derivative, and a preparation method and application thereof

Abstract

The application belongs to the technical field of natural medicine development, and particularly relates to a phytostanol hydrogen sulfide donor type derivative, a preparation method and application thereof. The phytostanol hydrogen sulfide donor derivative provided by the application is prepared by using phytostanol as a leading compound, modifying a hydrogen sulfide donor on a hydroxyl group at C3 of the phytostanol by means of esterification and condensation reaction. The phytostanol hydrogen sulfide donor derivative provided by the application can slowly release hydrogen sulfide in vivo and in vitro, has significant anti-inflammatory activity, and has no gastrointestinal side effects. The preparation method of the phytostanol hydrogen sulfide donor derivative has the advantages of mild reaction conditions, short reaction time, simple operation, high yield and the like.

Description

Technical Field

[0001] This invention belongs to the field of natural drug development technology, specifically relating to a phytosterol hydrogen sulfide donor derivative and its preparation method and application. Background Technology

[0002] Phytosterols are an important component of plant cell membranes and are widely found in most plants in nature. Phytosterols are a class of physiologically active natural products with a cyclopentane-perhydrophenanthrene backbone (also known as the sterol nucleus). To date, more than 250 phytosterols have been identified from various plants, among which β-sitosterol (BS), stigmasterol (SS), and campesterol (CS) are relatively common. Phytosterol compounds have similar chemical structures, differing only in the number and position of double bonds and the length of side chains. Phytosterols typically exist in plants in the form of free sterols, fatty acid sterol esters, hydroxycinnamate sterol esters, sterol glycosides, and acylated sterol glycosides (He Wen-sen, Zhu Han-yue, Chen Zhen-yu. J. Agric. Food Chem. 2018, 66, 3047-3062). Currently, the main sources of phytosterols include deodorized distillates, a byproduct of vegetable oil extraction, floating oil residues from industrial production, and fermentation waste from the brewing industry. High-purity phytosterols can be obtained by enriching unsaponifiables during the saponification and refining process of vegetable oils, followed by separation.

[0003] Phytosterols, as natural products derived from plants, are widely available. Various plants contain phytosterols, and the methods and technologies for extracting them from plants are becoming increasingly mature. A large amount of phytosterol resources remain to be developed. Currently, besides being used as a major ingredient in food supplements, phytosterols are also used in the pharmaceutical and cosmetic industries. As natural products derived from plants, phytosterols have a structure similar to that of cholesterol from animal sources and possess a wide range of physiological activities in vivo. Early research mainly focused on the cholesterol-lowering effect of phytosterols, generally believed to be due to competition with cholesterol to reduce cholesterol absorption. In recent years, with in-depth research, it has been found that phytosterols can lower the level of low-density lipoprotein in the blood, thereby reducing the risk of atherosclerosis; see published literature Simin Feng, Zhuqing Dai, Anna Liu, et al. FoodFunct., 2017, 8, 4179-4186; Bei-xian Zhou, Jing Li, Xiao-li Liang, et al. Acta Pharmacologica Sinica, 2020, 41, 1178-1196. β-Sitosterol and stigmasterol also possess anti-inflammatory activity similar to hydrocortisone and antipyretic and analgesic effects similar to aspirin. Furthermore, because phytosterols can cross the blood-brain barrier, they have potential therapeutic effects in animal models of neuroinflammation, neurodegenerative diseases, and the progression of various central nervous system diseases (Tim Vanmierlo, et al. Progress in Lipid Research. 2015, 58, 26-39). Phytosterols cannot be synthesized by the human body and can only be obtained through food sources or supplements. Numerous animal and clinical studies have shown that phytosterol intake between 1.5g and 3g can reduce LDL cholesterol by approximately 10% to 20%. In addition, existing technologies have confirmed that phytosterols have anti-inflammatory effects and, as anti-inflammatory drugs, have the advantage of low gastrointestinal irritation. However, as shown in the published literature (Bei-xian Zhou, Jing Li, Xiao-li Liang, et al. Acta Pharmacologica Sinica, 2020, 41, 1178-1196), phytosterols only exhibit a highly significant anti-inflammatory correlation at a dosage of 200 mg / kg. Therefore, currently, using phytosterols directly as anti-inflammatory drugs presents the problem of low activity and the need for large doses to achieve significant anti-inflammatory levels.The abundant natural resources of phytosterols need to be fully and cost-effectively developed and utilized. Existing technologies have proposed some solutions to the problem of low activity of phytosterols. For example, patent document CN113621670A discloses the use of esterification reaction of phytosterols and oleic acid to obtain phytosterol esters that have the same function as phytosterols but have better solubility. Furthermore, phytosterol ester fermentation with yeast can produce phytosterol ester ferments with higher anti-inflammatory activity. However, there is still a certain gap between its anti-inflammatory activity and that of positive nonsteroidal anti-inflammatory drugs, and this method of resource utilization is costly. Summary of the Invention

[0004] The purpose of this invention is to provide a class of phytosterol hydrogen sulfide donor derivatives, which, as anti-inflammatory drugs, have better anti-inflammatory activity than phytosterols extracted from natural plants, and can achieve the same level of anti-inflammatory activity as positive non-steroidal anti-inflammatory drugs without significant gastrointestinal damage.

[0005] The phytosterol hydrogen sulfide donor derivative provided by this invention is prepared by combining a hydrogen sulfide-releasing structural unit, namely a hydrogen sulfide donor, into the structure of a physiologically active natural product, phytosterol, thereby producing a synergistic effect to enhance the anti-inflammatory activity of phytosterol. Specifically, the phytosterol hydrogen sulfide donor derivative is prepared by modifying and combining a hydrogen sulfide donor with the C3 hydroxyl group of the phytosterol using esterification and condensation reactions, with phytosterol as the lead compound. The phytosterol hydrogen sulfide donor derivative can slowly release H2S and has superior activity compared to natural phytosterol.

[0006] Hydrogen sulfide (H2S) has long been considered a significant air and water pollutant, and excessive inhalation can affect the normal function of various organs such as the lungs, brain, and kidneys. In 1996, Abe and Kimura published a paper, Abe K, Kimura H, *The Journal of Neuroscience: the official journal of the Society for Neuroscience*, 1996, 16(3), 1066-71, which discovered the endogenous production pathway and signal transduction capacity of H2S in the mammalian brain, proving that H2S is the third gaseous signaling molecule after nitric oxide and carbon monoxide. Numerous studies have shown that H2S levels are usually lower than normal in disease states; therefore, timely supplementation of H2S to restore its normal level may be a strategy for treating diseases. However, as a gaseous molecule, H2S is difficult to directly apply as a supplement to the human body. Early studies also used sodium hydrosulfide as a hydrogen sulfide donor, but sodium hydrosulfide dissolves in water and can release large amounts of hydrogen sulfide gas in a short time, which is not conducive to the human body's utilization of hydrogen sulfide and is prone to toxicity. Given the drawbacks of directly using H2S gas and sodium hydrosulfide, various inorganic and organic compounds capable of slowly releasing hydrogen sulfide have been developed in existing technologies. Currently, commonly used H2S donors are DTT-type compounds, namely 1,2-dithia-3-thionone cyclopentene compounds, among which representative examples are ADT-OCH3 (5-(p-methoxyphenyl)-1,2-dithiocyclopentyl-4-ene-3-thionone) and ADT-OH (5-(p-hydroxyphenyl)-1,2-dithiocyclopentyl-4-ene-3-thionone).

[0007] To achieve the above objectives, the present invention provides the following technical solution: a phytosterol hydrogen sulfide donor derivative, the chemical structure of which is shown below:

[0008]

[0009] Among them, R 1 Selected from one of the following A, B, or C groups:

[0010]

[0011] When R 1 When the derivative is group A, it is a stigmasterol derivative SSD; R 1 When the derivative is a B group, it is a β-sitosterol derivative BSD; R 1 When the derivative is a C group, it is a campesterol derivative CSD;

[0012] R 2 Independently selected from one of the following groups:

[0013]

[0014] R 3 Independently selected from one of the following groups:

[0015]

[0016] The phytosterol hydrogen sulfide donor derivative provided by this invention introduces hydrogen sulfide donor structural units into phytosterols, thereby releasing hydrogen sulfide while retaining the activity of phytosterols and achieving the effect of enhancing their anti-inflammatory effects.

[0017] This invention also provides a method for preparing phytosterol hydrogen sulfide donor derivatives, and the preparation method of derivative I is shown in the following route:

[0018]

[0019] Among them, R 4 It can be -CH2CH2-, -CH2-, (cis)-CH=CH-, (trans)-CH=CH-, -O-, -NHCH2-, or o-diphenyl.

[0020] When R 3 for Derivative I can also be prepared using the following route:

[0021]

[0022] Where X is either -Cl or -Br.

[0023] As a preferred embodiment of the present invention, R 4 -CH2CH2-, R 3 for The preparation method of the phytosterol hydrogen sulfide donor derivative is as follows: Succinic anhydride is dissolved in a first solvent, and then an acid-binding agent and phytosterol are added sequentially to carry out a substitution reaction. After the reaction is completed, the first reaction solution is purified to obtain phytosterol succinate monoester. The phytosterol succinate monoester and ADT-OH are dissolved in a second solvent, and a condensing agent is added to carry out a condensation reaction. The second reaction solution is purified to obtain the phytosterol hydrogen sulfide donor derivative. The molar ratio of succinic anhydride, acid-binding agent, and phytosterol is (3-6):(6-12):1, more preferably 4:9:1. The first solvent is any one of dichloromethane, tetrahydrofuran, and dimethylformamide. The acid-binding agent is triethylamine or pyridine. The temperature of the substitution reaction is room temperature (25℃-60℃), and the reaction time is 6-24 hours.

[0024] The molar ratio of the phytosterol succinate monoester to the ADT-OH reactant is (0.9–1.1):(0.9–1.1), and more preferably 1:1; the second solvent is dichloromethane or tetrahydrofuran; the condensing agent is a mixture of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDCI) and 4-dimethylaminopyridine (DMAP), or a mixture of dicyclohexylcarbodiimide (DCC) and DMAP; the condensation reaction temperature is room temperature (25°C–60°C), and the reaction time is 0.5–4 hours.

[0025] Preferably, the purification method for the first reaction solution and the second reaction solution is silica gel column chromatography.

[0026] The present invention provides a pharmaceutical composition comprising one or a mixture of two or three of the compounds described in the present invention, or a pharmaceutically acceptable salt thereof, as an active ingredient, and a pharmaceutically acceptable carrier or excipient.

[0027] The present invention also provides the use of the pharmaceutically acceptable salts of the said compounds or the above-described pharmaceutical compositions in the preparation of anti-inflammatory drugs.

[0028] The present invention also provides the use of the pharmaceutically acceptable salts of the said compounds or the above-described pharmaceutical compositions in the preparation of drugs for the prevention or treatment of tumors.

[0029] The present invention also provides the use of the said compound or a pharmaceutically acceptable salt thereof or the above pharmaceutical composition in the preparation of a pain-relieving medicament.

[0030] The present invention also provides the use of the said compound or a pharmaceutically acceptable salt thereof or the above-described pharmaceutical composition in the preparation of a medicament for treating cardiovascular and cerebrovascular diseases.

[0031] The present invention also provides the use of the pharmaceutically acceptable salts of the said compounds or the above-described pharmaceutical compositions in the preparation of medicaments for treating nervous system diseases.

[0032] The compounds of this invention can be used, but are not limited to, for treating inflammation or for treating inflammation-related diseases, such as as antipyretic, analgesic, and anti-inflammatory drugs. For example, the compounds of this invention can be used to treat arthritis, including but not limited to rheumatoid arthritis, osteoarthritis, rheumatic arthritis, gouty arthritis, and lupus syndrome. They can also be used to treat skin-related inflammations such as psoriasis, eczema, burns, and dermatitis. Furthermore, they can be used to treat gastrointestinal disorders such as inflammatory bowel disease, Crohn's disease, gastritis, irritable bowel syndrome, and ulcerative colitis. The compounds of this invention can be used to treat asthma, bronchitis, bursitis, and tenosynovitis. They can also be used for the prevention or treatment of cancer, such as colorectal cancer.

[0033] It can also be used for inflammation in certain diseases, including but not limited to vascular diseases, migraines, nodular arteritis, thyroiditis, aplastic anemia, sclerosis, rheumatic fever, diabetes, myasthenia gravis, gingivitis, nephritis, allergies, post-traumatic swelling, and myocardial ischemia. It can also be used for lung inflammation, acute lung injury, acute respiratory distress syndrome, various infectious (e.g., bacterial, viral, mycoplasmal, fungal, and parasitic) pneumonias, physical and chemical (e.g., radioactive, inhaled lipid-based) pneumonias, allergic (e.g., allergic and rheumatic) pneumonias, chronic obstructive pulmonary disease, and endotoxin shock. It is also used for inflammation related to hemorrhoids, spasmodic anal pain, and rectal fissures; liver, gallbladder, and / or biliary tract diseases, such as cholangitis, sclerosing cholangitis, primary biliary cirrhosis and cholecystitis, and appendicitis. It can also be used to treat central nervous system diseases, such as Alzheimer's disease, Parkinson's disease, atherosclerosis, and central nervous system damage caused by stroke, ischemia, or trauma. In addition to its use in treating human diseases, it can also be used on other mammals, including pigs, cattle, horses, sheep, cats, dogs, and rats.

[0034] Doctors or patients may adjust the dosage of the compounds of this invention according to their specific circumstances or disease state, as long as the dosage is within an effective and safe range. This is readily apparent to those skilled in the art. Generally, the most suitable dosage level should be 1 mg to 1000 mg / kg, preferably 5-100 mg / kg, but this can be varied depending on the patient's weight, condition, and sensitivity to the drug, as well as the type of drug formulation chosen and the time and interval of administration. Sometimes, dosage levels below the lower limit or above the upper limit of the above range may also be used, as long as no side effects affecting treatment are produced.

[0035] The compounds of this invention can be formulated into various pharmaceutical dosage forms, the properties of which depend on the route of administration. These pharmaceutical compositions can be prepared using conventional methods with compatible, pharmaceutically acceptable excipients or carriers. These dosage forms can be tablets, capsules, granules, sprays, transdermal patches, lozenges, tablets, syrups, powders, gels, suppositories, etc., and can be prepared as temporary solutions, injectable formulations, rectal, ocular, vaginal preparations, etc., with preferred routes of administration being oral and rectal administration.

[0036] When intended for oral administration, various excipients such as microcrystalline cellulose, sodium citrate, calcium carbonate, dicalcium phosphate, and glycine, as well as various disintegrants such as starch, sodium alginate, and certain complex silicates, and granular binders such as polyvinylpyrrolidone, sucrose, gelatin, and gum arabic can be used. Furthermore, lubricants such as magnesium stearate, sodium lauryl sulfate, and talc can be used for tableting; similar types of solid compositions can also use fillers in capsules, with preferred substances including lactose or milk sugar and high molecular weight polyethylene glycol. When an aqueous suspension is desired for oral administration, the active ingredient can be combined with sweeteners or flavoring agents, coloring agents, and emulsifiers and / or suspending agents, and with diluents such as water, ethanol, propylene glycol, glycerin, and various combinations thereof.

[0037] Dosage forms can be designed for slow release, controlled release, immediate release, delayed release, or targeted delayed release. The definitions of these terms are well known to those skilled in the art.

[0038] When used parenterally, sesame or peanut oil or aqueous propylene glycol solutions of the active compound can be used. If necessary, the aqueous solution should be appropriately buffered, preferably with a pH greater than 8. The liquid diluent should be isotonic, and the aqueous solution should be suitable for intravenous administration. All these solutions are prepared under aseptic conditions using standard pharmaceutical techniques known to those skilled in the art.

[0039] The stigmasterol hydrogen sulfide donor derivative provided by this invention exhibits good anti-inflammatory effects without significant gastrointestinal damage. Furthermore, the release of a suitable amount of hydrogen sulfide from this stigmasterol hydrogen sulfide donor derivative reduces the occurrence of cardiovascular events. Therefore, the stigmasterol hydrogen sulfide donor derivative provided by this invention can avoid gastrointestinal damage and / or adverse cardiovascular events or other toxic side effects caused by clinically used nonsteroidal anti-inflammatory drugs (NSAIDs), and has the potential to replace existing clinical medications, showing great application prospects. Compared with other existing methods for enhancing the activity of phytosterols, this method has the advantages of mild reaction conditions, short reaction time, simple operation, and high yield. Attached Figure Description

[0040] Figure 1 The infrared spectrum of the stigmasterol hydrogen sulfide donor derivative prepared in Example 1;

[0041] Figure 2 The stigmasterol hydrogen sulfide donor derivative prepared in Example 1 1 H NMR spectrum;

[0042] Figure 3 The stigmasterol hydrogen sulfide donor derivative prepared in Example 1 13 C NMR spectrum;

[0043] Figure 4 The effect of stigmasterol hydrogen sulfide derivatives on xylene-induced ear swelling in mice;

[0044] Figure 5 The effect of a single dose of stigmasterol hydrogen sulfide derivative on the gastrointestinal tract of rats;

[0045] Figure 6 Effects of continuous administration of stigmasterol hydrogen sulfide derivatives on the gastrointestinal tract of rats. Detailed Implementation

[0046] The present invention will be described in detail below with reference to embodiments, but this does not imply any adverse limitation on the invention. The present invention has been described in detail, and specific embodiments thereof have been disclosed. It will be apparent to those skilled in the art that various changes and modifications can be made to the specific embodiments of the present invention without departing from the spirit and scope thereof.

[0047] Unless otherwise specified, all equipment used in this embodiment is conventional experimental equipment, and all materials and reagents used are commercially available unless otherwise specified. The experimental methods are also conventional experimental methods unless otherwise specified.

[0048] The main raw materials and reagents used in the following examples, comparative examples, and test cases are sourced from the following sources:

[0049] Stigmasterol, β-sitosterol, and campesterol were all purchased from Xi'an Muguo Biotechnology Co., Ltd.

[0050] Example 1

[0051] 1. Synthesis of 5-(4-hydroxyphenyl)-3H-1,2-dithiocyclopentyl-4-ene-3-thionone (ADT-OH)

[0052] (1) Synthesis of 5-(4-methoxyphenyl)-3H-1,2-dithiocyclopentyl-4-ene-3-thionone (ADT-OCH3)

[0053] Anethole (3.17 g, 0.021 mol), sulfur (4.70 g, 0.146 mol), and 30 mL of N,N-dimethylformamide were added to a 100 mL flask and heated under reflux with stirring for 6 h. After the reaction was complete, the mixture was poured into cold water, resulting in the precipitation of a large amount of precipitate. The precipitate was filtered, dried, and yielded an orange-yellow solid powder. Recrystallization from ethanol-water yielded orange-yellow crystals with a yield of 68% and a melting point of 110-111 °C. The theoretical value of ESI-MS m / z is C0. 10 H8OS3[M+H] + 240.97, measured value m / z 240.9812.

[0054] (2) Synthesis of 5-(4-hydroxyphenyl)-3H-1,2-dithiocyclopentyl-4-ene-3-thionone (ADT-OH)

[0055] ADT-OCH3 (20.6 g, 0.1 mol) and pyridine hydrochloride (46.0 g, 0.4 mol) were heated under reflux for 1 h, cooled, and the mixture was crushed, washed several times with water, filtered, and the filter cake was recrystallized from acetone-water with a yield of 80.0%, mp: 191-192 °C. Theoretical value of ESI-MS m / z: C9H6OS3[M+H] + 226.96, measured value m / z 226.9655.

[0056] 2. Synthesis of stigmasterol succinate monoester

[0057] Succinic anhydride (4.8 g, 0.048 mol) was placed in a 100 mL reaction flask and dissolved in 40 mL of dichloromethane by stirring. The reaction flask was placed on an ice bath, and triethylamine (15 mL, 0.108 mol) was slowly added dropwise to the reaction solution. After the addition was complete, stigmasterol (5.00 g, 0.012 mol) was added to the reaction solution in small amounts several times. After all the stigmasterol was added, the mixture was stirred for another ten minutes, and then the ice bath was removed. The reaction was carried out at room temperature (25 °C) for 24 h. After the reaction was completed, water and ethyl acetate were added to the reaction solution for extraction, and the organic layer was collected. The aqueous layer was extracted again with ethyl acetate, and the organic layers were combined. Anhydrous sodium sulfate was added, and the mixture was allowed to stand for 1 h. The mixture was filtered, and the organic solvent was recovered by rotary evaporation to obtain crude stigmasterol succinic acid monoester. The crude product was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate) to obtain a white solid with a yield of 80% and a melting point of 154-156 °C. The theoretical value of ESI-MS m / z is C. 33 H 52 O4[MH] - 511.3866, measured value 511.3826.

[0058] 3. Synthesis of stigmasterol hydrogen sulfide derivatives (SSD)

[0059] Take 1.53 g of stigmasterol succinate monoester (0.003 mol) and place it in a 100 mL reaction flask. Add 30 mL of dichloromethane to dissolve it. Then add EDCI (1.15 g, 0.006 mol), DMAP (0.037 g, 0.0003 mol), and ADT-OH (0.68 g, 0.003 mol) in sequence. Add 10 mL of dichloromethane to rinse the inner wall of the reaction flask. React at room temperature (25 °C) and monitor the reaction progress by TLC. After the reaction was completed for 4 hours, water extraction was performed. Glacial acetic acid was added to the reaction solution to adjust the pH to approximately 2, and the dichloromethane layer was collected. The aqueous layer was extracted again with dichloromethane. The dichloromethane solutions were combined, and anhydrous sodium sulfate was added to remove water. The mixture was allowed to stand for 1 hour. The organic solvent was recovered by rotary evaporation to obtain the crude product of stigmasterol hydrogen sulfide derivative. Purification was performed by silica gel column chromatography (eluent: petroleum ether / chloroform) to obtain a brownish-yellow solid with a yield of approximately 80% and a melting point of 179-181℃. The theoretical value of APCI-MS m / z is C0. 42 H 56 O4S3[M+H] + 721.3341, measured value 721.3418.

[0060] like Figure 1 The image shown is the infrared spectrum of the purified product, namely the stigmasterol hydrogen sulfide derivative, in Example 1 of this invention.

[0061] like Figure 2 The image shows a stigmasterol hydrogen sulfide derivative. 1 H NMR spectrum; 1H NMR (500MHz, CDCl3) δ7.69(d,J=8.5Hz,2H),7.41(s,1H),7.27(d,J=8.4Hz,2H),5.39(d,J=5.0Hz,1H),5.18(dd,J=15.2,8.6Hz,1H),5.04(dd, J=15.1,8.6Hz,1H),4.72-4.63(m,1H),2.95-2.88(m,2H),2.75(t,J=6.7Hz,2H),2.35(d,J=8.1Hz,2H),2.13-1.93(m,3H),1.89(d,J=10.4Hz,1 H),1.72(ddd,J=14.7,9.4,5.6Hz,1H),1.63(ddd,J=14.5,8.9,3.3Hz,1H),1.59(s,1H),1.56(d,J=5.2Hz,2H),1.51-1.41(m,1H),1.33-1.22( m,1H),1.22-1.14(m,3H),1.05(s,2H),1.04(d,J=2.6Hz,7H),0.98(td, J=11.4,4.9Hz,1H),0.91-0.84(m,4H),0.83-0.79(m,6H),0.72(s,3H).

[0062] Figure 3 The stigmasterol hydrogen sulfide donor derivative prepared in Example 1 13 C NMR spectrum; 13 C NMR (126MHz, CDCl3) δ215.55,171.67,171.38,170.55,153.63,139.51,138. 32,136.08,129.38,129.29,128.25,122.91,122.89,74.77,56.84,56.01,51 .29,50.11,42.27,40.51,39.68,38.14,37.01,36.66,31.93,29.48,29.41,28.94,27.82,25.44,24.40,21.27,21.12,21.08,19.36,19.04,12.29,12.10.

[0063] Example 2

[0064] 1. Synthesis of β-sitosterol succinate monoester

[0065] Succinic anhydride (4.8 g, 0.048 mol) was placed in a 100 mL reaction flask and dissolved in 40 mL of dichloromethane by stirring. The reaction flask was placed on an ice bath, and triethylamine (15 mL, 0.108 mol) was slowly added dropwise to the reaction solution. After the addition was complete, commercially available β-sitosterol (5.00 g, 0.012 mol) was added to the reaction solution in small amounts several times. After all the solution was added, the mixture was stirred for another ten minutes, then the ice bath was removed, and the reaction was allowed to proceed at room temperature for 24 h. After the reaction was complete, water and ethyl acetate were added to the reaction solution for extraction, and the organic layer was collected. The aqueous layer was extracted again with ethyl acetate, and the organic layers were combined. Anhydrous sodium sulfate was added, and the mixture was allowed to stand for 1 h. The mixture was filtered, and the organic solvent was recovered by rotary evaporation to obtain crude β-sitosterol succinic acid monoester. The crude product was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate) to obtain a white solid with a yield of 90% and a melting point of 146-149 °C. The theoretical value of ESI-MS m / z is C. 33 H 54 O4[MH] - 513.39, measured value 513.40.

[0066] 2. Synthesis of β-sitosterol hydrogen sulfide derivative (BSD)

[0067] β-sitosterol succinate monoester (1.54 g, 0.003 mol) was placed in a 100 mL reaction flask and dissolved in 30 mL of dichloromethane. EDCI (1.15 g, 0.006 mol), DMAP (0.037 g, 0.0003 mol), and ADT-OH (0.68 g, 0.003 mol) prepared in Example 1 were added sequentially. The inner wall of the reaction flask was then rinsed with 10 mL of dichloromethane. The reaction was carried out at room temperature, and the reaction progress was monitored by TLC. After the reaction was completed for 4 hours, water extraction was performed. Glacial acetic acid was added to the reaction solution to adjust the pH to approximately 2, and the dichloromethane layer was collected. The aqueous layer was extracted again with dichloromethane. The dichloromethane solutions were combined, anhydrous sodium sulfate was added, and the mixture was allowed to stand for 1 hour. The mixture was filtered, and the organic solvent was recovered by rotary evaporation to obtain the crude β-sitosterol hydrogen sulfide derivative. This crude product was purified by silica gel column chromatography (eluent: petroleum ether / chloroform) to obtain a brownish-red solid powder with a yield of 75% and a melting point of 176-178℃. The theoretical value of APCI-MS m / z is C0. 42 H 58 O4S3[M+H] + 723.3497, measured value 723.36. 1HNMR(500MHz,CDCl3)δ7.69(d,J=8.7Hz,2H),7.41(s,1H),7.27(d,J=8.7Hz,2H),5.39(d,J=3.8Hz,1H),4.68(dtd,J=11.6,9.0,4.1Hz,1H),2 .91(t,J=5.7Hz,2H),2.75(t,J=5.8Hz,2H),2.35(d,J=7.6Hz,2H),2.01(ddt,J=22.3,17.2,4.3Hz,3H),1.88(dp,J=9.4,3.5Hz,3H),1.68(dq ,J=9.8,3.8Hz,1H),1.66-1.54(m,3H),1.55-1.44(m,3H),1.42-1.32(m,2H),1.33-1.24(m,2H),1.17(dd,J=11.5,4.2Hz,2H),1.15-1.10(m, 3H),1.09-0.99(m,6H),0.94(d,J=6.5Hz,5H),0.88(d,J=1.8Hz,1H),0.86(d,J=2.1Hz,3H),0.85(d,J=3.5Hz,3H),0.83(s,2H),0.70(s,3H).

[0068] 13 C NMR (126MHz, CDCl3) δ215.64,171.77,171.47,170.64,153.72,136.17,128.33( x2),123.01(x2),122.98,74.87,56.83,56.20,50.17,46.00,42.46,39.86,38.2 3,37.09,36.73,36.29,34.09,32.05(x2),32.00,29.57,29.50,29.32,28.37,27 .91,26.26,24.43,23.23,21.18(x2),19.95,19.44,19.19,18.92,12.13,12.00.

[0069] Example 3

[0070] 1. Synthesis of campesterol succinate monoester

[0071] Succinic anhydride (4.8 g, 0.048 mol) was placed in a 100 mL reaction flask and dissolved in 40 mL of dichloromethane by stirring. The reaction flask was placed on an ice bath, and triethylamine (15 mL, 0.108 mol) was slowly added dropwise to the reaction solution. After the addition was complete, commercially available campesterol (4.80 g, 0.012 mol) was added to the reaction solution in small amounts several times. After all the solution was added, the mixture was stirred for another ten minutes, then the ice bath was removed, and the reaction was allowed to proceed at room temperature for 24 h. After the reaction was complete, water and ethyl acetate were added to the reaction solution for extraction, and the organic layer was collected. The aqueous layer was extracted again with ethyl acetate, and the organic layers were combined. Anhydrous sodium sulfate was added, and the mixture was allowed to stand for 1 h. The mixture was filtered, and the organic solvent was recovered by rotary evaporation to obtain crude campesterol succinic acid monoester. The crude product was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate) to obtain a white solid with a yield of 90% and a melting point of 144-145 °C. The theoretical value of ESI-MS m / z is C. 32 H 52 O4[MH] - 499.39, measured value 499.39.

[0072] 2. Synthesis of rapeseed oil sterol hydrogen sulfide derivative (CSD)

[0073] Take 1.54 g of campesterol succinate monoester (0.003 mol) and place it in a 100 mL reaction flask. Add 30 mL of dichloromethane to dissolve it. Then add EDCI (1.15 g, 0.006 mol), DMAP (0.037 g, 0.0003 mol), and ADT-OH (0.68 g, 0.003 mol) prepared in Example 1 in sequence. Add 10 mL of dichloromethane to rinse the inner wall of the reaction flask. React at room temperature and monitor the reaction progress by TLC. After the reaction was completed for 4 hours, water extraction was performed. Glacial acetic acid was added to the reaction solution to adjust the pH to approximately 2, and the dichloromethane layer was collected. The aqueous layer was extracted again with dichloromethane. The dichloromethane solutions were combined, anhydrous sodium sulfate was added, and the mixture was allowed to stand for 1 hour. The mixture was filtered, and the organic solvent was recovered by rotary evaporation to obtain a crude product of campesterol hydrogen sulfide derivative. This crude product was purified by silica gel column chromatography (eluent: petroleum ether / chloroform) to obtain a brownish-red solid powder with a yield of approximately 70% and a melting point of 172-174℃. The theoretical value of APCI-MS m / z is C0. 42 H 58 O4S3[M+H] + 709.34, measured value 709.34.

[0074] Pharmacological tests and results

[0075] Experimental Example 1: Xylene-induced ear swelling test in mice

[0076] Kunming mice, purchased from the Shanghai Laboratory Animal Center, Chinese Academy of Sciences, weighing 18-22g, male. They were kept at a temperature of 20-24℃ and humidity of 65-70%, fed standard feed, with bedding and food changed daily, and the cages kept clean.

[0077] All test compounds were prepared as suspensions using 0.5% CMC-Na solution. Mice were fasted for 12 hours before administration but had free access to water. Mice were administered the compound by gavage (90.4 mg / kg) at a volume of 0.2 mL / 10 g body weight. One hour after administration, 20 μL of xylene was evenly applied to both sides of the right ear using a microsyringe to induce inflammation; the left ear served as a control. One hour after inflammation induction, the mice were euthanized by cervical dislocation, and both ears were removed along the auricular baseline. One earpiece was taken from the same location using a 7 mm diameter punch and weighed using an electronic balance. The weight of the inflamed earpiece minus the weight of the control earpiece represented the degree of swelling. The swelling inhibition rate was calculated using the following formula, and the swelling degrees of the control and administered groups were statistically analyzed:

[0078] Swelling inhibition rate (%) = (average swelling degree of model group - average swelling degree of drug treatment group) / average swelling degree of model group × 100%.

[0079] Effects of stigmasterol hydrogen sulfide derivatives on xylene-induced mouse ear swelling: The anti-inflammatory activity of stigmasterol hydrogen sulfide derivatives was evaluated using a xylene-induced mouse ear swelling model. Results are shown below. Figure 4 Wherein, SS: stigmasterol, DI: diclofenac, SSD: stigmasterol hydrogen sulfide derivative, the same below; from Figure 4 The results show that the tested compound exhibits a fairly good inhibitory effect on ear swelling, with an inhibition rate close to that of the positive control drug diclofenac, but significantly different from that of stigmasterol. This indicates that the derivative of stigmasterol combined with the hydrogen sulfide donor significantly enhances its anti-inflammatory activity.

[0080] Experiment Example 2: Gastrointestinal Injury Experiment

[0081] 1. Effects of a single dose of the test compound on the gastrointestinal tract of rats.

[0082] Healthy male SD rats, purchased from Shanghai Slater Experimental Animal Center, with an average weight of 100g, were randomly assigned to groups. They were fasted for 24 hours before the experiment, but water was allowed. They were fed standard feed, with bedding and food changed daily to maintain cleanliness in the cages. During the experiment, four groups were established: a solvent control group (CTR), a diclofenac group (DI), a stigmasterol group (SS), and a stigmasterol hydrogen sulfide derivative group (SSD). In the solvent control group, each animal received the solvent via gavage; in the diclofenac control group, each animal received diclofenac sodium solution at a dose of 20 mg / kg via gavage; and in the test drug groups (SS, SSD), each animal received the test drug at a dose of 45.2 mg / kg via gavage. Animals in each group were sacrificed 6 hours after administration. The effects of the drugs on the rat gastrointestinal tract were observed and compared using the following methods, and relevant indices were calculated. The stomach was opened and flattened along the greater curvature. The gastric ulcer index (UI) was calculated according to the Guth criteria: 1 point for ulcer length less than 1 mm, 2 points for 1–2 mm, 3 points for 2–3 mm, 4 points for 3–4 mm, and ulcers longer than 4 mm were divided into segments, each scored using the same method. For ulcer widths > 1 mm, the score was multiplied by 2. Point bleeding was scored as 0.5 points each. The cumulative scores for each rat were the total gastric ulcer index. A t-test was used to analyze whether there were significant differences in gastric ulcer indices among the groups. Experimental results are shown below. Figure 5 .

[0083] Figure 5 The results showed that, compared with the diclofenac group, the gastric ulcer index of the SS and SSD groups was almost zero, with a highly significant difference (P<0.01). Furthermore, necropsy revealed varying degrees of color changes in the stomachs of rats in the diclofenac group, mainly yellowish-white or pale white, while no such changes were observed in the stomachs of rats in the SS and SSD groups, and no other abnormal changes were noticeable.

[0084] 2. Effects of continuous administration of the test compound on the gastrointestinal tract of rats

[0085] Healthy male SD rats, purchased from Shanghai Slater Experimental Animal Center, with an average weight of 100g, were randomly assigned to groups. They were fasted for 24 hours before the experiment, but water was allowed. They were fed standard feed, with bedding and food changed daily to maintain cage cleanliness. During the experiment, separate groups were established: a solvent control group (CTR), a diclofenac group (DI), a stigmasterol group (SS), and a stigmasterol hydrogen sulfide derivative group (SSD). In the solvent control group, each animal received the solvent via gavage; in the diclofenac control group, each animal received diclofenac sodium solution at a dose of 40 μmol / kg via gavage; and in the test drug groups (SS, SSD), each animal received the drug at a dose of 40 μmol / kg via gavage, once daily for 5 consecutive days. Food and water were not controlled during the drug administration period. On day 5, animals in each group were sacrificed 6 hours after drug administration. The effects of the drugs on the rat gastrointestinal tract were observed and compared using the above method, and relevant indices were calculated. A t-test was used to analyze whether there were significant differences in gastric ulcer indices among the groups. Experimental results are as follows Figure 6 As shown.

[0086] Figure 6 The results showed that, compared with the diclofenac group, the gastric ulcer index of the SS and SSD groups was very small, with a highly significant difference (P<0.01). Meanwhile, during the experiment, it was observed that the abdomens of the rats in the diclofenac group were distended, and yellow or even brown ascites flowed out when the abdominal cavity was cut open. The mucosa covering the abdominal cavity hardened, the gastric mucosa had very little elasticity and showed erosion; some rats were unable to eat during the experiment.

[0087] The two experiments above show that no obvious linear gastrointestinal hemorrhagic injury was found in the test substance, whether it was a single dose or continuous dose, while the gastric damage caused by diclofenac during the experiment was very obvious, indicating that the gastrointestinal safety of the test substance is very good.

Claims

1. A phytosterol hydrogen sulfide donor derivative, characterized in that, The chemical structural formula of the derivative is as follows: ， Among them, R 1 Selected from one of the following A, B, or C groups: ； R 2 for: R 3 for: .

2. The method for preparing the phytosterol hydrogen sulfide donor derivative according to claim 1, characterized in that, The preparation method is shown in the following route: ， Among them, R 4 It is -CH2CH2-.

3. The preparation method according to claim 2, characterized in that, The preparation method of the phytosterol hydrogen sulfide donor derivative is as follows: Succinic anhydride is dissolved in a first solvent, and then an acid-binding agent and phytosterol are added sequentially for a substitution reaction. After the reaction, the first reaction solution is purified to obtain phytosterol succinic acid monoester. The phytosterol succinic acid monoester and 5-(p-hydroxyphenyl)-1,2-dithiocyclopentyl-4-ene-3-thione (ADT-OH) are dissolved in a second solvent, and a condensing agent is added for a condensation reaction. The second reaction solution is purified to obtain the phytosterol hydrogen sulfide donor derivative. The molar ratio of succinic anhydride, acid-binding agent, and phytosterol is (3~6):(6~12):

1. The first solvent is any one of dichloromethane, tetrahydrofuran, and dimethylformamide. The acid-binding agent is triethylamine or pyridine. The temperature of the substitution reaction is room temperature (25℃~60℃), and the reaction time is 6~24 hours. The molar ratio of the phytosterol succinate monoester to the ADT-OH reactant is (0.9~1.1):(0.9~1.1); the second solvent is dichloromethane or tetrahydrofuran; the condensing agent is a mixture of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 4-dimethylaminopyridine, or a mixture of dicyclohexylcarbodiimide and 4-dimethylaminopyridine; the condensation reaction temperature is room temperature (25℃~60℃), and the reaction time is 0.5~4 hours.

4. The preparation method according to claim 3, characterized in that, The molar ratio of succinic anhydride, acid binder, and phytosterol is 4:9:

1.

5. The preparation method according to claim 3, characterized in that, The molar ratio of the two reactants, phytosterol succinate monoester and ADT-OH, is 1:

1.

6. The preparation method according to claim 3, characterized in that, The substitution reaction was carried out at room temperature (25°C) for 24 hours.

7. The preparation method according to claim 3, characterized in that, The purification method for the first and second reaction solutions was silica gel column chromatography.

8. A pharmaceutical composition comprising, as an active ingredient, one or a mixture of two or three compounds of the phytosterol hydrogen sulfide donor derivatives of claim 1, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient.

9. Use of the phytosterol hydrogen sulfide donor derivative of claim 1 or the pharmaceutical composition of claim 8 in the preparation of an anti-inflammatory drug.

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