Nitrogen-containing triterpenoids as well as preparation method and application thereof

Abstract

The invention relates to nitrogen-containing triterpenoids as well as a preparation method and application thereof. The nitrogen-containing triterpenoid compound has a structure as shown in a formula I which is described in the specification. The compound disclosed by the invention has anti-inflammatory activity and can be used for preparing related medicines for prevention or treatment or adjuvant treatment of immune-mediated inflammatory diseases. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to the field of biomedicine. Specifically, the present invention provides nitrogen-containing triterpenoid compounds of the following formula I, and pharmaceutically acceptable salts and solvates (such as hydrates) thereof, a preparation method and uses thereof. The nitrogen-containing triterpenoid compounds and pharmaceutically acceptable salts and solvates (such as hydrates) thereof have anti-inflammatory and immunosuppressive activities and can be used for preventing, treating or adjuvantly treating immune-mediated inflammatory diseases. Background Art

[0002] Immune-mediated inflammatory diseases (IMIDs) are a group of common and clinically diverse diseases, including rheumatoid arthritis (RA), cutaneous inflammatory diseases (including psoriasis and atopic dermatitis), inflammatory bowel disease (IBD), systemic lupus erythematosus (SLE), asthma, spondyloarthritis (SpA), connective tissue diseases and autoimmune neurological diseases (such as multiple sclerosis). Therefore, they pose a major systemic medical challenge. In addition, these diseases are usually accompanied by various comorbidities, including cardiovascular diseases, metabolic and skeletal diseases, and cognitive deficits, which will further affect the quality of life and increase the mortality rate.

[0003] Glucocorticoids, as the main therapeutic drugs for various IMIDs, although having multiple uses and good efficacy, studies have shown that the therapeutic benefit decreases over time and they have significant toxicity in terms of bone, cardiovascular system and metabolic functions. Other main therapeutic drugs include methotrexate, azathioprine, sodium aurothiomalate (gold salts), sulfasalazine, hydroxychloroquine, D-penicillamine, mycophenolate and other drugs. However, due to significant adverse events, the sequential use or combined use of these drugs requires great caution. Subsequent studies have confirmed the clinical relevance of several immune targets, such as IL-1, IL-6, IL-17, IL-23 and IFN-α receptor, etc. However, not all IMIDs patients respond to targeted therapies and many patients relapse after treatment. In addition, many treatment methods can cause side effects, such as atrophy, organ toxicity, immunosuppression, infection and carcinogenesis, which leads to limitations in long-term use. Therefore, it is necessary to develop alternative treatment methods for IMIDs to achieve good efficacy and few side effects.

[0004] Traditional Chinese medicine has received extensive attention in the search for new therapies. Traditional Chinese medicine is a rich source of potentially bioactive compounds. Many plants in the Rubiaceae family have the effect of clearing heat and detoxifying. The plant Mussaenda pubescens W.T.Aiton in this family is sweet and slightly bitter in nature, mainly for relieving exterior syndrome, dissipating summer heat, promoting diuresis, detoxifying, and activating blood circulation, and is used to treat colds, heatstroke, fever, cough, sore throat, summer damp diarrhea, dysentery, abscesses, bruises, snake bites, etc. Previous chemical and biological studies found that plants in the genus Mussaenda mainly contain triterpenoid saponins and iridoid glycosides, and have pharmacological activities such as antibacterial, anti-inflammatory, anti-tumor, antiviral, and insecticidal. Heinsiagenin A is a nitrogen-containing triterpenoid compound, and it was first reported to be obtained by hydrolysis of the glycoside compound contained in the Rubiaceae plant Heinsiacrinata (Tetrahedron, 1989, 45:5907-5916.). The immunosuppressive and anti-inflammatory activities of Heinsiagenin A, its glycosides and esters, and 3-oxo-heinsiagenin A have not been reported. Summary of the Invention

[0005] The inventors of the present invention have found through research a class of nitrogen-containing triterpenoid compounds that can be used for preventing, treating, or adjuvantly treating immune-mediated inflammatory diseases.

[0006]

[0007] According to one aspect of the present invention, there is provided a nitrogen-containing triterpenoid compound represented by the following formula I, its pharmaceutically acceptable salts or solvates (such as hydrates),

[0008] wherein, R1 is selected from hydroxy, C1-C6 alkanoyloxy, or a sugar chain group composed of one or more sugar groups selected from glucose, rhamnose, and xylose, and R2 is selected from hydrogen; or R1 and R2 form a carbonyl group.

[0009] Preferably, R1 is selected from hydroxy, C1-C4 alkanoyloxy (such as C2 alkanoyloxy, i.e., acetoxy), or a sugar chain group composed of one or more sugar groups selected from glucose, rhamnose, and xylose, and R2 is selected from hydrogen; or R1 and R2 form a carbonyl group.

[0010] In one embodiment, R1 is hydroxy, acetoxy, or a sugar chain group composed of sugar groups selected from the following group: 1, 2, 3, or 4 glucose groups, 1 or 2 rhamnose groups, 1 or 2 xylose groups, and R2 is selected from hydrogen.

[0011] In another embodiment, R1 and R2 form a carbonyl group.

[0012] In particular, the compound is selected from the following structures, where Xyl represents xylosyl, and G, G', G", R, and R' are conventional representations for differentiating the same types of glucosyl and rhamnosyl groups:

[0013]

[0014] According to another aspect of the present invention, a method for preparing the compound represented by Formula I of the present invention is provided.

[0015] In the case where the specific structure of the compound is disclosed in the present invention, those skilled in the art can extract it from specific plants of the Rubiaceae family or obtain it through chemical synthesis. For example, it can be synthesized from small molecule compounds by known chemical synthesis methods through structural design, or obtained by modifying the structure of known compounds.

[0016] In some embodiments, the method includes the following steps:

[0017] (1) After pulverizing the above-ground part of Mussaenda densiflora with a pulverizer, extract it with an ethanol aqueous solution, concentrate the extract to obtain a total extract. Preferably, extract it 3 - 5 times with a 90% - 99%, preferably 95% ethanol aqueous solution for a total of 5 - 8 days, combine the extracts, and concentrate them under reduced pressure to obtain the total extract. More preferably, extract it 3 - 5 times with a 95% ethanol aqueous solution for a total of 7 days, with the mass-volume ratio of Mussaenda densiflora to the ethanol aqueous solution being 1.5 kg:3 - 7 L, preferably 1.5 kg:5 L, combine the extracts, and concentrate them under reduced pressure to obtain the total extract.

[0018] (2) Suspend the total extract in water and extract it successively with petroleum ether, ethyl acetate, and n-butanol to obtain a petroleum ether fraction, an ethyl acetate fraction, and an n-butanol fraction, respectively.

[0019] (3) Separate and purify the n-butanol fraction sample through a macroporous resin column, using methanol / water as the elution solvent, and elute it successively with 0%, 20%, 40%, 60%, and 80% methanol, and combine the 80% methanol fraction. Separate and purify the 80% methanol fraction through semi-preparative HPLC, elute it with a 50% - 90% acetonitrile / water gradient for 45 min, and collect it at a wavelength of 265 nm to obtain Compounds 3 - 6.

[0020] (4) Take one or more selected from Compounds 3 - 6, add 10% hydrochloric acid, and hydrolyze it by heating in a water bath. The hydrolyzate is extracted, concentrated, and purified to obtain Compound 1. In particular, heat it in a water bath at 80 °C for 3 hours, extract it with dichloromethane, concentrate the dichloromethane layer, and purify it through a C18 medium-pressure column chromatography (elute it with an 80% - 100% methanol / water gradient for 45 min) to obtain Compound 1, namely heinsiagenin A.

[0021] (5) Dissolve heinsiagenin A in anhydrous dichloromethane, add 2 equivalents of acetic anhydride, catalyze with pyridine, stir at room temperature. After detecting that the reaction is complete by LC-MS, separate and purify by C18 medium-pressure column chromatography (gradient elution with 80%-100% methanol / water for 40 min) to obtain acetylated heinsiagenin A, namely compound 2.

[0022] (6) Dissolve 10 mg of heinsiagenin A in anhydrous dichloromethane, add 1 equivalent of Dess-Martin periodinane, stir at room temperature. After detecting that the reaction is complete by LC-MS, separate and purify by C18 medium-pressure column chromatography (gradient elution with 80%-100% methanol / water for 40 min) to obtain heinsiagenin A with the 3-position oxidized to a carbonyl group, 3-oxo-heinsiagenin A, namely compound 7.

[0023] According to another aspect of the present invention, there is provided a pharmaceutical composition, which comprises a therapeutically effective amount of the nitrogen-containing triterpenoid compound represented by formula I, its pharmaceutically acceptable salt or solvate (such as hydrate), and optionally pharmaceutically acceptable excipients.

[0024] According to the present invention, the nitrogen-containing triterpenoid compound represented by formula I, its pharmaceutically acceptable salt or solvate (such as hydrate) or the pharmaceutical composition can be used for preparing an immunosuppressant.

[0025] According to another aspect of the present invention, there is provided the use of the nitrogen-containing triterpenoid compound represented by formula I, its pharmaceutically acceptable salt or solvate (such as hydrate), or the pharmaceutical composition in the preparation of a drug for preventing, treating or adjuvantly treating immune-mediated inflammatory diseases.

[0026] According to the present invention, preferably, the immune-mediated inflammatory diseases are such as psoriasis, inflammatory bowel disease, systemic lupus erythematosus, lupus nephritis, rheumatoid arthritis, asthma, psoriasis vulgaris, idiopathic pulmonary fibrosis, etc.

[0027] According to another aspect of the present invention, there is provided the use of the nitrogen-containing triterpenoid compound represented by formula I, its pharmaceutically acceptable salt or solvate (such as hydrate), or the pharmaceutical composition in the preparation of an immunosuppressant.

[0028] According to the present invention, the nitrogen-containing triterpenoid compounds include heinsiagenin A and its esters, glycoside compounds and 3-oxo-heinsiagenin A.

[0029] Experiments have proven that the nitrogen-containing triterpenoid compounds according to the present invention have excellent immunosuppressive activity against mouse T and B lymphocytes, can inhibit the infiltration of inflammatory cells in the skin tissue of experimental mice, down-regulate the levels of related pro-inflammatory cytokines, and significantly improve the proteinuria level of MRL / lpr mice. Pharmacodynamic and pharmacological studies have confirmed that this type of compound has good anti-inflammatory and immunosuppressive activity and high safety, and has good application prospects in the clinical treatment and adjuvant treatment of psoriasis, inflammatory bowel disease, systemic lupus erythematosus, lupus nephritis, etc. Brief Description of the Drawings

[0030] Figure 1 Shows the experimental process of evaluating the anti-psoriasis activity of heinsiagenin A (Compound 1) against IMQ-induced psoriasis in mice and the results of its effect on IMQ-induced psoriasis. Among them, (A) Experimental outline of treating IMQ-induced psoriasis in mice with heinsiagenin A; (B) Body weight changes of mice during 7 days of treatment; (C) Representative photos of the back skin morphology of mice in each group after 7 days of treatment; (D-G) Erythema, scale, skin thickening scores and total scores (sum of scores for erythema, desquamation and thickening) of the back skin; (H) Representative photos of H&E staining of the back skin of mice in each group; (I-L) Levels of pro-inflammatory cytokines such as IL-1β, IL-6, TNF-α, IL-17 in the back skin tissue of mice in each group; *, p < 0.05; **, p < 0.01; ***, p < 0.001; n = 5, compared with the model group.

[0031] Figure 2 Shows the experimental process of evaluating the anti-inflammatory bowel disease activity of heinsiagenin A against DSS-induced inflammatory bowel disease in mice and the results of its effect on DSS-induced colitis: (A) Experimental outline of treating DSS-induced colitis mice with heinsiagenin A; (B) Body weight changes of mice during 7 days of treatment; (C-E) Fecal morphology, occult blood and disease activity index (DAI) scores; (F) Representative photos of H&E staining of the colon tissue of mice in each group; (G-I) Levels of pro-inflammatory cytokines IL-1β, IL-6, TNF-α and IL-17 in the colon tissue detected by ELISA; *p < 0.05, **p < 0.01, ***p < 0.001; n = 5, compared with the model group.

[0032] Figure 3 Shows the results of the effect of heinsiagenin A on spontaneous systemic lupus erythematosus in MRL / lpr mice, *p < 0.05, ***p < 0.001; n = 5, compared with the model group. Detailed Description of the Invention

[0033] Compound Preparation and Isolation Experiment

[0034] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods.

[0035] Unless otherwise specified, the materials, reagents, etc. used in the following examples are all commercially available.

[0036] Experimental equipment and reagents

[0037] NMR spectrum detection: Bruker Avance III 400 and Bruker Avance III 800 (Bruker, Germany); LC-MS analysis: Shimadzu LC-MS-2020 spectrometer (Shimadzu Corporation, Japan); semi-preparative high performance liquid chromatography: Unimicro Technologies EsaySep TM-1020 dual pump system and ultraviolet detector (Tongwei Analytical Technology Co., Ltd., Shanghai); semi-preparative chromatographic column: YMC-Pack ODS-A chromatographic column (250×20 mm, 5 μm); Athena HILIC chromatographic column (250 mm×21.2 mm, 10 μm) (Shanghai Anpu Experimental Technology Co., Ltd.).

[0038] Elution solvents: Analytical grade methanol, ethanol, dichloromethane, petroleum ether, ethyl acetate and other solvents are from Sinopharm Chemical Reagent Co., Ltd.; acetonitrile is from J&K Scientific or Suyuan Chemical Reagent Co., Ltd.; column chromatography packing materials: macroporous resin HZ-801 (Shanghai Huazhen Technology Co., Ltd.); reversed-phase silica gel LiChroprep RP-18 (40-63 μm) (Fuji Silysia Chemical Ltd., Japan); gel HW 40F (Tosoh Corporation, Japan).

[0039] Preparation Example 1: Preparation and structural identification of Heinsiagenin A and its glycoside compounds

[0040] (1) After pulverizing 1.5 kg of the dried aerial parts of Mussaenda densiflora collected from Luodian County, Qiannan Prefecture, Guizhou Province using a pulverizer, it was soaked and extracted with 95% ethanol-water for 7 days, three times in total (5 L×3). The extraction solutions were combined and concentrated under reduced pressure to obtain the total extract.

[0041] (2) The total extract was suspended in 1 L of water and successively extracted three times with 1 L of petroleum ether, ethyl acetate, and n-butanol respectively to obtain the petroleum ether fraction, ethyl acetate fraction, and n-butanol fraction (30 g).

[0042] (3) The n-butanol fraction sample (30 g) was separated and purified by a macroporous resin column with a length of 25 cm and a diameter of 6 cm. Using methanol / water as the elution solvent, it was eluted successively with 0%, 20%, 40%, 60%, and 80% methanol, and the 80% fraction was combined; the 80% methanol fraction was separated and purified by semi-preparative HPLC, eluted with a 50%-90% acetonitrile / water gradient for 45 min, and collected at a wavelength of 265 nm to obtain Compound 3 (170 mg), Compound 4 (250 mg), Compound 5 (260 mg), and Compound 6 (150 mg);

[0043] (4) Portions of Compounds 3 - 6 were each added with 10% hydrochloric acid and heated in a water bath at 80 °C for 3 hours. The hydrolysis solution was extracted with dichloromethane. After the dichloromethane layer was concentrated, it was separated and purified by C18 medium-pressure column chromatography (gradient elution with 80%-100% methanol / water for 45 min) to obtain Compound 1, namely heinsiagenin A;

[0044] (5) 10 mg of heinsiagenin A was dissolved in anhydrous dichloromethane, 2 equivalents of acetic anhydride were added, catalyzed by pyridine, and stirred at room temperature. After the reaction was detected to be complete by LC-MS, it was separated and purified by C18 medium-pressure column chromatography (gradient elution with 80%-100% methanol / water for 40 min) to obtain acetylated heinsiagenin A, namely Compound 2;

[0045] (6) 10 mg of heinsiagenin A was dissolved in anhydrous dichloromethane, 1 equivalent of Dess-Martin periodinane was added, and stirred at room temperature. After the reaction was detected to be complete by LC-MS, it was separated and purified by C18 medium-pressure column chromatography (gradient elution with 80%-100% methanol / water for 40 min) to obtain heinsiagenin A with the 3-position oxidized to a carbonyl group, 3-oxo-heinsiagenin A, namely Compound 7.

[0046] Compound 1 (heinsiagenin A), molecular formula: C 36 H 55 NO4. 11H NMR (400 MHz, C5D5N): δ 9.09 (1H, d, J = 7.6 Hz, NH), 1.20 (3H, d, J = 6.5 Hz, 4'-Me), 0.89 (3H, d, J = 7.3 Hz, 3'-Me), 5.69 (1H, dd, J = 7.6, 7.2 Hz, H-2'), 0.96 (3H, s, H-30), 1.27 (3H, s, H-29), 1.15 (3H, s, H-28), 2.23 (3H, s, H-26), 7.30 (1H, d, J = 11.1 Hz, H-24), 6.46 (1H, dd, J = 15.0, 11.0 Hz, H-23), 5.69 (1H, dd, J = 14.4, 8.4 Hz, H-22), 1.04 (3H, d, J = 5.2 Hz, H-21), 0.35 / 0.60 (1H each, br s, H-19), 1.04 (3H, s, H-18). 13 13C NMR (125 MHz, C5D5N): δ 31.8 (C-1, t), 29.3 (C-2, t), 78.4 (C-3, d), 41.8 (C-4, s), 47.9 (C-5, d), 20.9 (C-6, t), 26.7 (C-7, t), 48.6 (C-8, d), 20.4 (C-9, s), 26.8 (C-10, s), 27.1 (C-11, t), 32.9 (C-12, t), 46.2 (C-13, s), 49.7 (C-14, s), 36.3 (C-15, t), 29.3 (C-16, t), 52.5 (C-17, d), 20.0 (C-18, q), 30.5 (C-19, t), 41.6 (C-20, d), 20.5 (C-21, q), 148.3 (C-22, d), 124.2 (C-23, d), 135.3 (C-24, d), 129.6 (C-25, s), 14.0 (C-26, q), 171.2 (C-27, s), 20.4 (C-28, q), 26.7 (C-29, q), 16.0 (C-30, q), 176.2 (C-1', s), 55.9 (C-2', d), 39.1 (C-3', d), 77.5 (C-4', d), 8.6 (C-3'-Me, q), 15.4 (C-4'-Me, q).

[0047] Compound 2, molecular formula: C 38 H 47 NO5. 11H NMR (400 MHz, C5D5N): δ 9.11 (1H, d, J = 7.6 Hz, NH), 1.20 (3H, d, J = 6.6 Hz, 4'-Me), 0.89 (3H, d, J = 7.2 Hz, 3'-Me), 5.69 (1H, dd, J = 7.6, 7.2 Hz, H-2'), 0.96 (3H, s, H-30), 1.27 (3H, s, H-29), 1.15 (3H, s, H-28), 2.23 (3H, s, H-26), 2.10 (3H, s, H-OAc), 7.30 (1H, d, J = 11.1 Hz, H-24), 6.46 (1H, dd, J = 15.0, 11.0 Hz, H-23), 5.69 (1H, dd, J = 14.4, 8.4 Hz, H-22), 1.04 (3H, d, J = 5.2 Hz, H-21), 0.31 / 0.53 (1H each, br s, H-19), 1.04 (3H, s, H-18). 13 13C NMR (125 MHz, C5D5N): δ 32.2 (C-1, t), 29.2 (C-2, t), 80.9 (C-3, d), 41.8 (C-4, s), 47.6 (C-5, d), 20.9 (C-6, t), 26.1 (C-7, t), 48.3 (C-8, d), 20.3 (C-9, s), 26.8 (C-10, s), 29.1 (C-11, t), 33.4 (C-12, t), 46.1 (C-13, s), 49.6 (C-14, s), 36.2 (C-15, t), 29.3 (C-16, t), 52.4 (C-17, d), 19.9 (C-18, q), 30.1 (C-19, t), 41.8 (C-20, d), 20.6 (C-21, q), 148.3 (C-22, d), 124.2 (C-23, d), 135.2 (C-24, d), 129.7 (C-25, s), 14.0 (C-26, q), 171.2 (C-27, s), 171.1, 26.0 (C-OAc), 20.4 (C-28, q), 26.7 (C-29, q), 16.0 (C-30, q), 176.2 (C-1', s), 55.9 (C-2', d), 39.1 (C-3', d), 77.5 (C-4', d), 8.6 (C-3'-Me, q), 15.9 (C-4'-Me, q).

[0048] Compound 3, ESIMS m / z 1205 [M+Na] + , molecular formula: C 60 H 95 NO 22 .1 1H NMR (400 MHz, C5D5N): δ 9.10 (1H, d, J = 7.7 Hz, NH), 1.20 (3H, d, J = 6.4 Hz, 4'-Me), 0.89 (3H, d, J = 7.2 Hz, 3'-Me), 5.70 (1H, dd, J = 7.5, 7.4 Hz, H-2'), 0.92 (3H, s, H-30), 1.42 (3H, s, H-29), 1.17 (3H, s, H-28), 2.22 (3H, s, H-26), 7.29 (1H, d, J = 10.8 Hz, H-24), 6.45 (1H, dd, J = 14.8, 11.2 Hz, H-23), 5.71 (1H, dd, J = 14.4, 8.4 Hz, H-22), 1.03 (3H, d, J = 5.2 Hz, H-21), 0.22 / 0.51 (1H each, br s, H-19), 1.02 (3H, s, H-18), 4.93 (1H, d, J = 7.2 Hz, H- G1 ), 5.82 (1H, d, J = 7.6 Hz, H- G'1 ), 6.48 (1H, br s, H- R'1 ), 5.83 (1H, br s, H- R1 ). 13 13C NMR (125 MHz, C5D5N): δ 31.8 (C-1, t), 29.4 (C-2, t), 89.4 (C-3, d), 41.1 (C-4, s), 47.3 (C-5, d), 20.9 (C-6, t), 26.1 (C-7, t), 47.7 (C-8, d), 19.7 (C-9, s), 26.0 (C-10, s), 26.3 (C-11, t), 32.8 (C-12, t), 45.4 (C-13, s), 48.9 (C-14, s), 35.5 (C-15, t), 28.5 (C-16, t), 51.7 (C-17, d), 18.2 (C-18, q), 29.4 (C-19, t), 41.1 (C-20, d), 19.6 (C-21, q), 147.7 (C-22, d), 123.3 (C-23, d), 134.6 (C-24, d), 128.8 (C-25, s), 13.2 (C-26, q), 170.5 (C-27, s), 19.2 (C-28, q), 25.7 (C-29, q), 15.1 (C-30, q), 175.5 (C-1', s), 55.2 (C-2', d), 38.4 (C-3', d), 76.8 (C-4', d), 7.9 (C-3'-Me, q), 15.3 (C-4′-Me, q), 104.4 (C-G1 , d), 78.8 (C- G2 , d), 77.3 (C- G3 , d), 79.2 (C- G4 , d), 76.2 (C- G5 , d), 61.3 (C- G6 , t), 101.8 (C- G'1 , d), 78.1 (C- G'2 , d), 79.1 (C- G'3 , d), 72.5 (C- G'4 , d), 77.5 (C- G'5 , d), 63.2 (C- G'6 , t), 102.7 (C- R1 , d), 72.3 (C- R2 , d), 72.4 (C- R3 , d), 73.8 (C- R4 , d), 70.7 (C- R5 , d), 18.6 (C- R6 , q), 102.4 (C- R'1 , d), 72.2 (C- R'2 , d), 72.3 (C- R'3 , d), 73.7 (C- R'4 , d), 70.3 (C- R'5 , d), 18.4 (C- R'6 , q). [J. Nat. Prod. 1994, 57: 1613 - 1618.]

[0049] Compound 4, ESIMS m / z 1367 [M + Na] + , Molecular formula: C 66 H 105 NO 27 . 11H NMR (400 MHz, C5D5N): δ 9.15 (1H, d, J = 7.7 Hz, NH), 1.20 (3H, d, J = 6.4 Hz, 4'-Me), 0.89 (3H, d, J = 7.2 Hz, 3'-Me), 5.70 (1H, dd, J = 7.5, 7.4 Hz, H-22), 0.91 (3H, s, H-30), 1.42 (3H, s, H-29), 1.24 (3H, s, H-28), 2.24 (3H, s, H-26), 7.39 (1H, d, J = 10.8 Hz, H-24), 6.46 (1H, dd, J = 14.8, 11.2 Hz, H-23), 5.68 (1H, dd, J = 14.4, 8.4 Hz, H-22), 1.02 (3H, d, J = 5.2 Hz, H-21), 0.22 / 0.46 (1H each, br s, H-19), 1.02 (3H, s, H-18), 4.93 (1H, d, J = 7.2 Hz, H- G1 ), 5.76 (1H, d, J = 7.6 Hz, H- G'1 ), 6.43 (1H, br s, H- R'1 ), 5.76 (1H, br s, H- R1 ). 13 13C NMR (125 MHz, C5D5N): δ 32.0 (C-1, t), 29.7 (C-2, t), 89.9 (C-3, d), 41.2 (C-4, s), 47.5 (C-5, d), 21.1 (C-6, t), 26.2 (C-7, t), 47.9 (C-8, d), 19.7 (C-9, s), 26.2 (C-10, s), 26.4 (C-11, t), 33.0 (C-12, t), 45.5 (C-13, s), 49.0 (C-14, s), 35.6 (C-15, t), 28.6 (C-16, t), 51.8 (C-17, d), 18.3 (C-18, q), 29.7 (C-19, t), 41.3 (C-20, d), 19.7 (C-21, q), 147.8 (C-22, d), 123.5 (C-23, d), 134.8 (C-24, d), 128.9 (C-25, s), 13.3 (C-26, q), 170.7 (C-27, s), 15.4 (C-28, q), 26.0 (C-29, q), 19.3 (C-30, q), 175.7 (C-1', s), 55.3 (C-2', d), 38.5 (C-3', d), 76.9 (C-4', d), 8.0 (C-3'-Me, q), 15.4 (C-4'-Me, q), 104.7 (C- G1, d), 79.2 (C- G2 , d), 77.6 (C- G3 , d), 79.2 (C- G4 , d), 76.1 (C- G5 , d), 61.4 (C- G6 , t), 101.9 (C- G'1 , d), 78.1 (C- G'2 , d), 78.1 (C- G'3 , d), 72.5 (C- G'4 , d), 76.7 (C- G'5 , d), 70.5 (C- G'6 , t), 105.1 (C- G”1 , d), 75.2 (C- G”2 , d), 78.7 (C- G”3 , d), 71.5 (C- G”4 , d), 78.0 (C- G”5 , d), 62.7 (C- G”6 , t), 102.6 (C- R'1 , d), 72.4 (C- R'2 , d), 72.4 (C- R'3 , d), 73.8 (C- R'4 , d), 70.5 (C- R'5 , d), 18.5 (C- R'6 , q). [Phytochemistry, 1996, 42: 1131 - 1134.]

[0050] Compound 5, ESIMS m / z 1498.7169 [M + Na] + , Molecular formula: C 71 H 113 NO 31 . 11H NMR (800 MHz, C5D5N): δ 9.11 (1H, d, J = 7.7 Hz, NH), 1.20 (3H, d, J = 6.5 Hz, 4'-Me), 0.89 (3H, d, J = 7.2 Hz, 3'-Me), 5.70 (1H, d, J = 7.4 Hz, H-2'), 0.91 (3H, s, H-30), 1.47 (3H, s, H-29), 1.28 (3H, s, H-28), 2.23 (3H, s, H-26), 7.30 (1H, d, J = 7.0 Hz, H-24), 6.44 (1H, dd, J = 14.9, 11.0 Hz, H-23), 5.66 (1H, dd, J = 14.9, 9.0 Hz, H-22), 1.02 (3H, d, J = 7.0 Hz, H-21), 0.30 / 0.54 (1H each, brs, H-19), 1.00 (3H, s, H-18), 4.90 (1H, d, J = 7.8 Hz, H- G1 ), 5.77 (1H, d, J = 7.7 Hz, H- G'1 ), 5.43 (1H, d, J = 7.8 Hz, H- G”1 ), 5.84 (1H, br s, H- R1 ), 6.44 (1H, br s, H- R'1 ), 5.33 (1H, d, J = 7.3 Hz, H- Xyl1 ). 1313C NMR (125 MHz, C5D5N): δ 32.7 (C-1, t), 30.3 (C-2, t), 91.0 (C-3, d), 42.0 (C-4, s), 48.2 (C-5, d), 21.7 (C-6, t), 26.8 (C-7, t), 48.6 (C-8, d), 20.4 (C-9, s), 26.9 (C-10, s), 27.0 (C-11, t), 33.5 (C-12, t), 46.1 (C-13, s), 49.7 (C-14, s), 36.7 (C-15, t), 29.3 (C-16, t), 52.5 (C-17, d), 19.6 (C-18, q), 30.3 (C-19, t), 41.8 (C-20, d), 20.3 (C-21, q), 148.4 (C-22, d), 124.3 (C-23, d), 135.3 (C-24, d), 129.6 (C-25, s), 14.0 (C-26, q), 171.3 (C-27, s), 16.1 (C-28, q), 26.8 (C-29, q), 20.0 (C-30, q), 176.3 (C-1, s), 55.9 (C-2', d), 39.2 (C-3', d), 77.5 (C-4', d), 8.6 (C-3'-Me, q), 16.0 (C-4'-Me, q), 105.7 (C- G1 , d), 78.8 (C- G2 , d), 77.4 (C- G3 , d), 80.1 (C- G4 , d), 76.8 (C- G5 , d), 62.9 (C- G6 , t), 102.6 (C- G'1 , d), 78.9 (C- G'2 , d), 78.4 (C- G'3 , d), 76.3 (C- G'4 , d), 78.5 (C- G'5 , d), 70.7 (C- G'6 , t), 102.6 (C- G”1 , d), 84.1 (C- G”2 , d), 78.2 (C- G”3 , d), 71.8 (C- G”4 , d), 79.8 (C- G”5 , d), 62.1 (C- G”6 , t), 103.3 (C- R1 , d), 73.0 (C- R2 , d), 72.9 (C- R3,d), 74.4 (C- R4 ,d), 71.3 (C- R5 ,d), 19.0 (C- R6 ,q), 102.4 (C- R'1 ,d), 72.9 (C- R'2 ,d), 73.2 (C- R'3 ,d), 74.7 (C- R'4 ,d), 70.0 (C- R'5 ,d), 19.2 (C- R'6 ,q), 107.2 (C- Xyl1 ,d), 77.5 (C- Xyl2 ,d), 77.5 (C- Xyl3 ,d), 71.2 (C- Xyl4 ,d), 67.9 (C- Xyl5 ,t).

[0051] Compound 6, ESIMS m / z 1529 [M+Na] + , molecular formula: C 72 H 115 NO 32 . 1 H NMR (400 MHz, C5D5N): δ 9.11 (1H, d, J = 7.7 Hz, NH), 1.19 (3H, d, J = 6.5 Hz, 4'-Me), 0.88 (3H, d, J = 7.2 Hz, 3'-Me), 5.69 (1H, dd, J = 7.4, 7.4 Hz, H-2'), 0.90 (3H, s, H-30), 1.47 (3H, s, H-29), 1.27 (3H, s, H-28), 2.21 (3H, s, H-26), 7.29 (1H, d, J = 11.1 Hz, H-24), 6.44 (1H, dd, J = 14.8, 11.1 Hz, H-23), 5.66 (1H, dd, J = 14.8, 8.9 Hz, H-22), 1.01 (3H, d, J = 6.5 Hz, H-21), 0.29 / 0.55 (1H each, br s, H-19), 0.99 (3H, s, H-18), 4.89 (1H, d, J = 7.6 Hz, H- G1 ), 5.76 (1H, d, J = 7.4 Hz, H- G'1 ), 5.37 (1H, d, J = 7.7 Hz, H- G”1 ), 5.34 (1H, d, J = 6.8 Hz, H- G”'1 ), 5.80 (1H, br s, H- R1 ), 6.39 (1H, br s, H-R'1 )。 13 CNMR (125 MHz, C5D5N): δ 32.2 (C-1, t), 29.8 (C-2, t), 90.5 (C-3, d), 41.4 (C-4, s), 47.8 (C-5, d), 21.2 (C-6, t), 26.4 (C-7, t), 48.1 (C-8, d), 19.8 (C-9, s), 26.3 (C-10, s), 26.6 (C-11, t), 33.0 (C-12, t), 45.6 (C-13, s), 49.2 (C-14, s), 35.8 (C-15, t), 28.8 (C-16, t), 52.0 (C-17, d), 18.5 (C-18, q), 29.9 (C-19, t), 41.3 (C-20, d), 19.9 (C-21, q), 147.9 (C-22, d), 123.8 (C-23, d), 134.8 (C-24, d), 129.1 (C-25, s), 13.5 (C-26, q), 170.8 (C-27, s), 15.6 (C-28, q), 26.2 (C-29, q), 19.5 (C-30, q), 175.8 (C-1', s), 55.4 (C-2', d), 38.6 (C-3', d), 77.0 (C-4', d), 8.1 (C-3'-Me, q), 15.5 (C-4'-Me, q), 105.1 (C- G1 , d), 78.5 (C- G2 , d), 78.0 (C- G3 , d), 79.4 (C- G4 , d), 76.3 (C- G5 , d), 61.6 (C- G6 , t), 101.9 (C- G'1 , d), 79.3 (C- G'2 , d), 78.3 (C- G'3 , d), 72.3 (C- G'4 , d), 76.0 (C- G'5 , d), 70.3 (C- G'6 , t), 102.8 (C- G”1 , d), 84.5 (C- G”2 , d), 77.8 (C- G”3 , d), 71.2 (C- G”4 , d), 77.8 (C- G”5 , d), 62.5 (C- G”6 , t), 102.8 (C- R1 , d), 72.5 (C- R2, d), 72.7 (C- R3 , d), 73.9 (C- R4 , d), 70.7 (C- R5 , d), 18.7 (C- R6 , q), 102.1 (C- R'1 , d), 72.4 (C- R'2 , d), 72.7 (C- R'3 , d), 74.2 (C- R'4 , d), 69.5 (C- R'5 , d), 19.1 (C- R'6 , q), 106.5 (C- G”'1 , d), 76.5 (C- G”'2 , d), 78.1 (C- G”'3 , d), 71.1 (C- G”'4 , d), 78.8 (C- G”'5 , d), 62.3 ( G”'6 , t). [Data same as Phytochemistry, 1997, 45: 1073 - 1078.]

[0052] Compound 7, ESIMS m / z 564 [M + H] + , molecular formula: C 36 H 53 NO4. 1 1H NMR (400 MHz, C5D5N) δ 9.09 (1H, d, J = 7.6 Hz, NH), 1.20 (3H, d, J = 6.5 Hz, 4'-Me), 0.89 (3H, d, J = 7.3 Hz, 3'-Me), 5.69 (1H, dd, J = 7.6, 7.2 Hz, H-2'), 0.91 (3H, s, H-30), 1.18 (3H, s, H-29), 1.09 (3H, s, H-28), 2.23 (3H, s, H-26), 7.31 (1H, d, J = 11.0 Hz, H-24), 6.47 (1H, dd, J = 14.9, 11.0 Hz, H-23), 5.69 (1H, dd, J = 14.4, 8.4 Hz, H-22), 1.03 (3H, d, J = 6.6 Hz, H-21), 1.05 (3H, s, H-18), 0.68 (1H, d, J = 4.2 Hz, H-19), 0.52 (1H, d, J = 4.2 Hz, H-19). 1313C NMR (151 MHz, C5D5N) δ 33.3 (C-1, t), 38.0 (C-2, t), 215.4 (C-3, s), 49.5 (C-4, s), 48.4 (C-5, d), 22.1 (C-6, t), 26.5 (C-7, t), 48.9 (C-8, d), 21.3 (C-9, s), 23.1 (C-10, s), 27.1 (C-11, t), 33.9 (C-12, t), 46.1 (C-13, s), 50.7 (C-14, s), 36.2 (C-15, t), 29.2 (C-16, t), 52.4 (C-17, d), 19.9 (C-18, q), 29.8 (C-19, t), 41.8 (C-20, d), 21.5 (C-21, q), 148.2 (C-22, d), 124.3 (C-23, d), 135.2 (C-24, d), 129.7 (C-25, s), 14.0 (C-26, q), 171.2 (C-27, s), 20.3 (C-28, q), 26.5 (C-29, q), 16.0 (C-30, q), 176.3 (C-1', s), 55.9 (C-2', d), 39.1 (C-3', d), 77.5 (C-4', d), 8.6 (C-3'-Me, q), 19.0 (C-4'-Me, q).

[0053] Biological activity test experiment

[0054] Experimental Example 1: Evaluation of the in vitro immunosuppressive activity of the compound

[0055] I. Experimental materials: BALB / c mice, female, 6 - 8 weeks old, purchased from Shanghai Slake Experimental Animal Co., Ltd. Concanavalin A (ConA), lipopolysaccharide (LPS), and MTT were all purchased from Sigma; fetal bovine serum (FBS) was purchased from Hyclone; 3H-thymidine (1 μCi / mL) was purchased from PerkinElmer; dimethyl sulfoxide (DMSO) was purchased from Sinopharm Chemical Reagent Co., Ltd.

[0056] II. Experimental methods

[0057] [I] Detection of the non-specific toxicity of the compound to mouse spleen lymphocytes by MTT method

[0058] Succinate dehydrogenase in the mitochondria of living cells can reduce 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to water-insoluble blue-violet crystalline formazan, while dead cells do not have this function. After dissolving formazan with DMSO and measuring its absorbance at a wavelength of 570 nm, the number of living cells can be indirectly reflected. Within a certain range of cell numbers, the amount of MTT crystal formation is proportional to the number of cells. It can be used for cytotoxicity analysis.

[0059] Single-cell suspensions were prepared from mouse spleens and adjusted to a concentration of 8×10 5 cells / well and plated in 96-well plates. Different concentrations of the positive control drug (cyclosporine A, CsA) and the compound (final concentrations of 200, 100, 50, 25, 12.5, 6.25, 3.13, and 1.56 μg / mL) were added at a volume of 200 μL / well. Corresponding blank controls (only adding cell culture medium without cells) and cell controls (only adding cells without the compound) were also set. The cells were cultured in a 37°C, 5% CO2 incubator for 48 hours. Four hours before the end of the culture, an MTT solution with a concentration of 5 mg / mL was added. At the end of the culture, the supernatant was aspirated, 150 μL of DMSO was added to each well to dissolve the purple crystals, and the OD value was measured at 570 nM using a microplate reader to calculate the compound CC 50 value.

[0060] [Two] 3 Determination of the effect of the compound on the proliferation function of mitogen or cluster of differentiation 3-induced primary mouse spleen lymphocytes by 3H-thymidine incorporation method

[0061] Single-cell suspensions were prepared from mouse spleens and adjusted to a concentration of 6×10 5 cells / well and inoculated in 96-well plates. 20 μg / mL lipopolysaccharide (LPS) or 1 μg / mL concanavalin A (ConA) was added, and different concentrations of the positive control drug (CsA) and the compound (final concentrations of = 100, 50, 25, 12.5, 6.25, 3.13, 1.56, and 0.78 μg / mL) were added and cultured for 48 hours. 25 μL of 3 3H-thymidine (10 μCi / mL) was added to each well 8 hours before the end of the culture. After the culture ended, the 96-well plates were frozen at -30°C overnight, and the cell proliferation was measured using a liquid scintillation counter the next day.

[0062] III. Experimental results:

[0063] The results showed (Table 1) that heinsiagenin A and its esters and glycosides had excellent immunosuppressive activities against T and B lymphocytes.

[0064] Table 1. In vitro immunosuppressive activity test of the compound

[0065]

[0066] As can be seen from the results in Table 1, this series of compounds have good activity and selectivity against ConA-induced T lymphocyte proliferation and LPS-induced B lymphocyte proliferation. Cyclosporin A (CsA) is a classical immunosuppressive compound and serves as the positive control drug in this experiment.

[0067] Experimental Example 2: Evaluation of the activity of Heinsiagenin A against imiquimod (IMQ)-induced psoriasis in mice

[0068] I. Experimental materials

[0069] BALB / c mice, female, 6 - 8 weeks old, were purchased from Shanghai Slake Experimental Animal Co., Ltd.

[0070] Imiquimod ointment was purchased from Sichuan Mingxin Pharmaceutical Co., Ltd.; azone, propylene glycol, absolute ethanol, and triethanolamine were all purchased from Sinopharm Chemical Reagent Co., Ltd.; mouse ELISA kits (TNF-α,

[0071] IL-1β, IL-6, IL-17) were purchased from BD Biosciences.

[0072] II. Experimental methods

[0073] 1. Reagent preparation

[0074] Preparation of Heinsiagenin A gel preparation (10 g)

[0075] a. 0.2 g of carbomer 940 was added to an appropriate amount of double-distilled water and allowed to stand. After sufficient swelling, it was stirred evenly.

[0076] b. 200 mg (2%), 50 mg (0.5%), and 10 mg (0.1%) of heinsiagenin A were dissolved in 2.8 g of 96% ethanol, and 0.1 g of azone and 0.2 g of propylene glycol were added and mixed evenly.

[0077] c. a and b were mixed together and stirred thoroughly until it became gel-like, and an appropriate amount of triethanolamine was added to adjust the pH value to neutral. d. The volume was made up to 10 g with double-distilled water and stored at 4°C.

[0078] e. The gel matrix was the preparation part after removing heinsiagenin A in steps a - d above.

[0079] 2. Establishment of an IMQ-induced psoriasis mouse model and pharmacodynamic evaluation

[0080] a. Female BALB / c mice were anesthetized by intraperitoneal injection of 50 mg / kg sodium pentobarbital injection. After removing the hair on the back about 3 cm × 2 cm, the modeling started the next day. The mice were randomly divided into 5 groups (n = 5), including a normal group (Normal), a model group (Vehicle; IMQ + gel matrix), a 2% heinsiagenin A gel treatment group (IMQ + 2% heinsiagenin A), a 0.5% heinsiagenin A gel treatment group (IMQ + 0.5%

[0081] heinsiagenin A), and a 0.1% heinsiagenin A gel treatment group (IMQ + 0.1% heinsiagenin A). Except for the Normal group, mice in other groups were topically applied with 62.5 mg of IMQ on the back every day for 7 consecutive days.

[0082] b. From the day of mouse induction of modeling to the end of the experiment, 62.5 mg of heinsiagenin A gel preparation at each concentration was applied. The model group was applied with 62.5 mg of blank control gel matrix. The application time of the drug and the modeling agent was at least 8 hours apart. The back skin of the mice was scored every day. At the end of the experiment, the spleens, peripheral blood, and skin tissues of the mice were collected.

[0083] c. Scoring of the severity of skin inflammation

[0084] The severity of the back skin was scored successively for erythema, scaling, and the degree of skin infiltration and thickening (0 - 4 points) based on the psoriasis area and severity index. The scoring criteria are shown in Table 2. The cumulative score of the three was used to indicate the severity of skin inflammation (0 - 12 points).

[0085] Table 2. Scoring of the severity of skin inflammation

[0086]

[0087] 3. Detection of gross and microscopic pathology of skin tissue

[0088] At the end of the experiment, mouse skin tissue was taken, and pathological sections were made by H&E staining.

[0089] 4. Detection of the levels of pro-inflammatory cytokines in skin tissue

[0090] The skin tissues of mice in each group were homogenized. After centrifugation, a BCA kit was used to quantify the protein in the supernatant of the homogenate; the ELISA method was used to detect the content of pro-inflammatory cytokines in the supernatant, which was shown as the content of cytokines in the total protein per unit.

[0091] Figure 1Shows the evaluation process and results of the anti-IMQ-induced psoriasis activity of heinsiagenin A in mice. III. Experimental results:

[0092] The experimental results showed that the treatment with the heinsiagenin A gel preparation of the present invention could significantly improve the symptoms of IMQ-induced psoriasis-like diseases in mice and improve weight loss ( Figure 1 in B), and clinical symptoms such as skin erythema, scales and thickening ( Figure 1 in C-G). The pathogenesis of psoriasis is accompanied by a large number of immune cell infiltrations. These inflammatory cells and the cytokines they release play an important role in the occurrence and development of psoriasis. Through HE staining of skin tissue pathological sections, it was found that the skin epidermis of the mice in the psoriasis model group was thickened, the keratinized cells proliferated significantly, and there were a large number of inflammatory cell infiltrations, while the above changes in the treatment group mice were significantly alleviated ( Figure 1 in H). Figure 1 The results from I to J in showed that the treatment with the heinsiagenin A gel preparation could significantly reduce the release of TNF-α, IL-1β, IL-6, and IL-17 inflammatory factors induced by IMQ.

[0093] Therefore, the experimental research results of heinsiagenin A in the IMQ-induced psoriasis animal model in mice showed that topical administration could effectively improve weight loss in mice, and clinical symptoms such as skin erythema, scales and thickening; reduce the pathological damage of skin tissue, inhibit the infiltration of inflammatory cells in the skin tissue of experimental mice; down-regulate the levels of related pro-inflammatory cytokines. Pharmacodynamic and pharmacological studies confirmed that this type of compound had good anti-inflammatory and immunosuppressive activities and high safety, and had good application prospects in the clinical treatment and adjuvant treatment of psoriasis.

[0094] Experimental Example 3: Activity evaluation of heinsiagenin A against DSS-induced inflammatory bowel disease in mice

[0095] I. Experimental materials and methods

[0096] 1. BALB / c mice. The mice were randomly divided into a normal control group, a model group, and high, medium, and low dose (30 mg / kg, 10 mg / kg, 3 mg / kg) treatment groups of heinsiagenin A with anti-inflammatory and immunosuppressive activities, with 5 mice in each group. Except for the mice in the normal control group, 5%

[0097] DSS was added to the drinking water of other groups including the model group and each dose of heinsiagenin A treatment group of mice for 7 consecutive days to induce the establishment of an intestinal inflammation model. Dextran sulfate sodium (DSS, USA

[0098] MP Biomedicals (products of the biomedical company), mouse ELISA kits (TNF-α, IL-1β, IL-6, IL-17) were purchased from BD Biosciences (San Diego, CA, USA);

[0099] 2. From the day of inducing the mouse model to the end of the experiment, each treatment group was given heinsiagenin A (30 mg / kg, 10 mg / kg, and 3 mg / kg) by gavage, and the model group was given normal saline.

[0100] 3. Disease activity index evaluation

[0101] During the experiment, the weight of each mouse in each group was measured daily, and fecal occult blood was detected. The disease activity of each mouse was scored according to Table 3.

[0102] Table 3. Disease Activity Index (DAI) Scoring Table

[0103]

[0104] As shown in Table 3, weight loss was divided into 5 grades (0, no weight loss or gain; 1, 1 - 5% decrease; 2, 5 - 10% decrease; 3, 10 - 20% decrease; 4, more than 20% decrease); fecal hardness was divided into 3 grades (0, normal; 2, soft stools; 4, loose stools); fecal occult blood was divided into 5 grades (0, negative; 1, +; 2, ++; 3, +++; 4, perianal bleeding).

[0105] 4. Gross and microscopic pathological changes in mouse colon and skin tissues

[0106] At the end of the experiment, the colon of the mouse was taken, the length was measured, and the distal colon was intercepted and fixed in formalin, embedded in paraffin, sectioned, and stained with H&E to make pathological sections.

[0107] 5. Detection of pro-inflammatory cytokine levels in colon or skin tissues

[0108] The same segment of the colon of each group of mice was homogenized. After centrifugation, protein quantification was performed on the supernatant of the homogenate; the content of pro-inflammatory cytokines in the supernatant was detected by ELISA and shown as the content of cytokines in the total protein per unit.

[0109] Figure 2 The experimental process of evaluating the activity of heinsiagenin A against DSS-induced murine inflammatory bowel disease and the results of its effect on DSS-induced colitis are shown.

[0110] II. Experimental results

[0111] Figure 2The experimental results showed that intragastric administration of heinsiagenin A of the present invention could significantly improve the disease symptoms of DSS-induced inflammatory bowel disease in mice, and improve clinical symptoms such as weight loss, diarrhea, and bloody stools. Figure 2 Figure B shows that intragastric administration of heinsiagenin A can relieve weight loss in DSS-induced mice. Figure 2 Figures C to E show that intragastric administration of heinsiagenin A can improve clinical symptoms such as diarrhea and bloody stools in DSS-induced mice. Figure 2 Figure F shows that in the colitis group, the local glandular structure of the colon in mice was disordered, with inflammatory cell infiltration such as neutrophils, monocytes, and multinucleated cells in the mucosa and submucosa, local erosion of the mucosa, and partial crypt destruction. Intragastric administration of heinsiagenin A can significantly relieve the above changes. Pro-inflammatory cytokines (TNF-α, IL-1β, IL-6, IL-17, etc.) play a key role in regulating intestinal inflammatory immune responses and are involved in the occurrence and development of inflammatory bowel disease. The experimental results showed that intragastric administration of heinsiagenin A reduced the levels of pro-inflammatory cytokines such as TNF-α, IL-1β, IL-6, and IL-17 in colon tissues.

[0112] Therefore, the experimental research results of heinsiagenin A in the animal model of DSS-induced inflammatory bowel disease in mice showed that intragastric administration could effectively improve the clinical symptoms of colitis such as weight loss, diarrhea, and bloody stools in experimental animals, reduce the pathological damage of colon tissues, inhibit the infiltration of inflammatory cells in colon tissues of experimental mice, and down-regulate the levels of related pro-inflammatory cytokines. Pharmacodynamic and pharmacological studies confirmed that this type of compound has good anti-inflammatory and immunosuppressive activities and high safety, and has good application prospects in the clinical treatment and adjuvant treatment of inflammatory bowel disease.

[0113] Experimental Example 4: Heinsiagenin A against spontaneous lupus in MRL / lpr mice

[0114] I. Experimental materials and methods

[0115] 1. MRL / lpr mice were purchased from Shanghai Slack Experimental Animal Co., Ltd. The mice were divided into a model group, a positive drug group (prednisone acetate, 2 mg / kg), and a treatment group of heinsiagenin A (30 mg / kg) with anti-inflammatory and immunosuppressive activities according to the initial proteinuria level, with 10 mice in each group. The proteinuria detection kit was purchased from Bio-Rad Company;

[0116] 2. During the experiment, proteinuria was detected in each group of mice weekly.

[0117] II. Experimental results

[0118] Figure 3 The results of the effects of heinsiagenin A on spontaneous systemic lupus erythematosus in MRL / lpr mice are shown. The experimental results show that intragastric administration of heinsiagenin A of the present invention can significantly improve the level of proteinuria in MRL / lpr mice.

[0119] Urine protein is one of the main manifestations of systemic lupus erythematosus and lupus nephritis, an important indicator affecting renal function, and also an important parameter for judging the curative effect and disease severity. Therefore, the study of heinsiagenin A in spontaneous systemic lupus erythematosus in MRL / lpr mice shows that heinsiagenin A has a good therapeutic effect on spontaneous systemic lupus erythematosus in MRL / lpr mice and has good application prospects in the clinical treatment and adjuvant treatment of systemic lupus erythematosus and lupus nephritis.

Claims

1. A nitrogen-containing triterpenoid compound represented by the following formula I, its pharmaceutically acceptable salt or solvate, Among them, R1 is selected from a hydroxyl group, a C1-C6 alkanoyloxy group, or a sugar chain group composed of one or more sugar groups selected from glucose, rhamnose, and xylose, and R2 is selected from hydrogen; or R1 and R2 form a carbonyl group.

2. The nitrogen-containing triterpenoid compound according to claim 1, its pharmaceutically acceptable salt or solvate, wherein, R1 is selected from a hydroxyl group, a C1-C4 alkanoyloxy group, or a sugar chain group composed of one or more sugar groups selected from glucose, rhamnose, and xylose, and R2 is selected from hydrogen; or R1 and R2 form a carbonyl group.

3. The nitrogen-containing triterpenoid compound according to claim 1, its pharmaceutically acceptable salt or solvate, wherein, R1 is a hydroxyl group, an acetoxy group, or a sugar chain group composed of sugar groups selected from the following group: 1, 2, 3, or 4 glucosyl groups, 1 or 2 rhamnosyl groups, 1 or 2 xylosyl groups, and R2 is selected from hydrogen; or R1 and R2 form a carbonyl group.

4. The nitrogen-containing triterpenoid compound according to claim 1, its pharmaceutically acceptable salt or solvate, wherein, The nitrogen-containing triterpenoid compound is selected from the following structures:

5. A method for preparing the nitrogen-containing triterpenoid compound represented by formula I according to any one of claims 1 to 4, the method comprising the following steps: (1) The above-ground part of Mussaenda densiflora is pulverized by a pulverizer and then extracted with an ethanol aqueous solution, and the extract is concentrated to obtain a total extract; (2) The total extract is suspended in water and successively extracted with petroleum ether, ethyl acetate, and n-butanol to obtain a petroleum ether fraction, an ethyl acetate fraction, and an n-butanol fraction, respectively; (3) The n-butanol fraction sample is separated and purified by a macroporous resin column, using methanol / water as the elution solvent, and successively eluted with 0%, 20%, 40%, 60%, and 80% methanol; the 80% methanol fraction is combined; the 80% methanol fraction is separated and purified by semi-preparative HPLC, eluted with a 50%-90% acetonitrile / water gradient for 45 min, and collected at a wavelength of 265 nm to obtain Compounds 3-6; (4) One or more selected from Compounds 3-6 are added with 10% hydrochloric acid and hydrolyzed by heating in a water bath. The hydrolyzate is extracted, concentrated, and purified to obtain Compound 1, namely heinsiagenin A; (5) Heinsiagenin A is dissolved in anhydrous dichloromethane, 2 equivalents of acetic anhydride are added, catalyzed by pyridine, and stirred at room temperature. After the reaction is detected to be complete by LC-MS, it is separated and purified by C18 medium-pressure column chromatography, eluted with an 80%-100% methanol / water gradient for 40 min to obtain acetylated heinsiagenin A, namely Compound 2; (6) 10 mg of heinsiagenin A is dissolved in anhydrous dichloromethane, 1 equivalent of Dess-Martin oxidant is added, and stirred at room temperature. After the reaction is detected to be complete by LC-MS, it is separated and purified by C18 medium-pressure column chromatography, eluted with an 80%-100% methanol / water gradient for 40 min to obtain heinsiagenin A with the 3-position oxidized to a carbonyl group, 3-oxo-heinsiagenin A, namely Compound 7.

6. According to the method of claim 5, wherein, Step (1): The above-ground part of *Mussaenda pubescens* Ait. f. is pulverized by a pulverizer and then extracted 3 - 5 times with an ethanol aqueous solution of 90% - 99%, preferably 95%, for a total of 5 - 8 days. The extraction solutions are combined and concentrated under reduced pressure to obtain a total extract. More preferably, it is extracted 3 - 5 times with an ethanol aqueous solution of 95% for a total of 7 days. The mass-to-volume ratio of *Mussaenda pubescens* Ait. f. to the ethanol aqueous solution is 1.5 kg: 3 - 7 L, preferably 1.5 kg: 5 L. The extraction solutions are combined and concentrated under reduced pressure to obtain a total extract; and / or In step (5), one or more selected from compounds 3 - 6 are added with 10% hydrochloric acid and heated in a water bath at 80 °C for 3 hours. After extraction with dichloromethane, the dichloromethane layer is concentrated and then purified by C18 medium-pressure column chromatography, eluted with a gradient of 80% - 100% methanol / water for 45 min to obtain compound 1.

7. A pharmaceutical composition, wherein, The pharmaceutical composition comprises a therapeutically effective amount of a nitrogen-containing triterpenoid compound represented by formula I according to any one of claims 1 to 4, a pharmaceutically acceptable salt or solvate thereof, and optionally a pharmaceutically acceptable excipient.

8. Use of a nitrogen-containing triterpenoid compound represented by formula I according to any one of claims 1 to 4, a pharmaceutically acceptable salt or solvate thereof, or the pharmaceutical composition according to claim 7 in the preparation of a drug for preventing, treating, or adjuvantly treating an immune-mediated inflammatory disease.

9. Use according to claim 8, wherein, The immune-mediated inflammatory diseases are selected from psoriasis, inflammatory bowel disease, systemic lupus erythematosus, lupus nephritis, rheumatoid arthritis, asthma, psoriasis vulgaris, idiopathic pulmonary fibrosis, multiple sclerosis, and vitiligo.

10. Use of a nitrogen-containing triterpenoid compound represented by formula I according to any one of claims 1 to 4, a pharmaceutically acceptable salt or solvate thereof, or the pharmaceutical composition according to claim 7 in the preparation of an immunosuppressant.

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