An anemoside b4 derivative, and a preparation method and application thereof

Abstract

The application discloses a paeonoside B4 derivative, a preparation method and application, and relates to a paeonoside B4 (AB4) compound as raw material, a nucleophilic substitution, an electrophilic addition, an esterification reaction or an amidation reaction for C-19 ring outside double bond or C-28 carboxyl to prepare a derivative, and an active ingredient of the AB4 derivative to prepare a medicine with anti-inflammatory and immune regulation effects. Most of the AB4 derivatives disclosed for the first time in the application do not show obvious cytotoxicity to macrophages, can reduce the P-IκBa protein level in the NF-κB signal path, inhibit the NLRP3 signal path activation, and obviously reduce the Pro-IL-1β level. p< 0.05), and the effect is better than that of AB4; the results show that the AB4 derivative has better anti-inflammatory activity. In addition, the AB4 derivative has remarkable treatment effect on atopic dermatitis, reduces the ear swelling level, obviously improves the skin ulceration of the back and ears, has better treatment effect than positive drugs dexamethasone and AB4, and has obviously smaller toxic side effects than dexamethasone.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to a Pulsatilla saponin B4 derivative and its preparation method, as well as the application of this derivative in the preparation of anti-inflammatory drugs, such as those for atopic dermatitis. Background Technology

[0002] Generally, inflammation is a defensive response of the body to stimuli, with common symptoms including redness, heat, swelling, and pain. Inflammation is broadly classified according to its causes, such as acute and chronic inflammation, local and systemic inflammation, infectious and non-infectious inflammation, etc. Inflammation is closely related to maintaining homeostasis in the body. During inflammation, on the one hand, damaging factors directly or indirectly cause damage to tissues and cells; on the other hand, through inflammatory hyperemia and exudation, damaging factors are diluted, killed, and surrounded. Simultaneously, the regeneration of parenchymal and interstitial cells allows damaged tissues to be repaired and healed. Therefore, inflammation plays an important role in the repair, remodeling, and renewal of different tissues. However, when the inflammatory response becomes uncontrollable in the body, it can trigger a series of diseases, such as arthritis, pneumonia, gastritis, etc. To ensure the body's health and restore normal physiological function, anti-inflammatory drugs are essential.

[0003] As is well known, classic anti-inflammatory drugs are nonsteroidal anti-inflammatory drugs (NSAIDs) and steroids, both of which have certain therapeutic effects on different types of inflammatory diseases. However, both classes of drugs have a series of adverse side effects, such as gastrointestinal disorders, cardiac changes, nephrotoxicity, hypertension, type 2 diabetes, visceral obesity, and atherosclerosis. With the development of the times and people's higher demands for health, more and more people are committed to finding and developing anti-inflammatory drugs with advantages such as good efficacy, low toxicity, and high acceptance. Natural products have the characteristics of fewer side effects, high safety, and good efficacy.

[0004] Atopic dermatitis (AD) is a common, immune-mediated inflammatory skin disease characterized by recurrent, itchy, localized eczema, often with seasonal fluctuations. It is also known as atopic eczema, neurodermatitis, atopic skin inflammation, and most commonly, eczema. Current treatments for eczema include various topical corticosteroids (TCS), topical calcineurin inhibitors such as tacrolimus and pimecrolimus, and phosphodiesterase 4 (PDE4) inhibitors such as criborole. For more severe AD, in addition to ultraviolet light therapy, current treatment guidelines recommend cyclosporine A, methotrexate, azathioprine, and mycophenolate mofetil, but these have drawbacks such as significant side effects or high cost. Pulsatilla saponin B4 (AB4) has therapeutic effects on atopic dermatitis, but its molecular weight and water solubility are too large, resulting in low bioavailability. Summary of the Invention

[0005] Existing anti-inflammatory drugs have significant toxic side effects, and some drugs have the drawback of short half-life. In order to solve this problem, this invention discloses a Pulsatilla saponin B4 derivative and its preparation method, as well as the application of this derivative in the preparation of anti-inflammatory (including atopic dermatitis) drugs.

[0006] Pulsatilla saponin B4 (AB4) has a therapeutic effect on atopic dermatitis, but it currently suffers from problems such as its large molecular weight and water solubility, and low bioavailability. To address this, this invention employs chemical methods to modify and transform the structure of AB4, obtaining a series of derivatives to solve the aforementioned problem of poor drug-likeness of AB4 and to obtain compounds with superior activity compared to AB4.

[0007] The present invention adopts the following technical solution: A derivative of Pulsatilla saponin B4 has the following general chemical structural formula:

[0008] In the formula, R1 is 3-O- α -L-pyrano-rhamnosyl-(1→2)- α -L-Pyranoarabinose; R2 is hydroxyl or acetoxy; R3 is 2-allyl, 2-propyl, 3-hydroxypropenyl, 3-bromopropenyl, 2-ethylene oxide methyl; R4 is 1-oxobenzotriazolyl, methoxy, hydroxyl, 28-O- α -L-pyranoside-(1→4)- β -D-glucopyranose-(1→6)- β -D-glucopyranosyl, 28-O- α -L-[2,3,4-triacetoxy-rhamnopyranose]-(1→4)- β -D-[2,3,6-triacetoxy-glucose pyranoside]-(1→6)- β-D-[2,3,4-triacetoxy-glucose pyranoyl]yl, 2-methoxyethylamino, 4-aminobutyric acid methyl ester, cyclopentanamino, 3-chloropropylamino, 2-fluoroethylamino, 1-(3-aminopropyl)benzotriazolyl, cyclohexylamino, 1-cyclopropylethylamino, cyclobutylmethylamino, 1-(3-aminopropyl)imidazolyl, N-(2-aminoethyl)pyrrolidinyl, 4-aminofuranyl, 3-aminocyclopentanecarboxylic acid methyl ester, 4-aminocyclohexylcarboxylic acid methyl ester, allylamino, 1-(2-aminoethyl)piperidinyl, 2 -Thiazolethylamino, tetrahydrofuranmethylamino, N-aminoethylmorpholino, 1-methyl-4-piperidinemethylamino, 1-methylpyrrolidine-3-methylamino, 4-aminocyclohexanol, β-phenylethylamino, 2-thiopheneethylamino, p-hydroxyphenylethylamino, 4-oxazolmethylamino, glycine, N-(2-aminoethyl)acetamiprido, 4-aminobutyric acid, 5-aminovaleric acid, 6-aminohexanoic acid methyl ester, 3-L-aminocyclopentanol, 3-D-aminocyclopentanol. Preferably, R3 is one of 2-allyl, 2-propyl, and 3-hydroxypropenyl; R4 is one of 1-oxobenzotriazolyl, 1-(3-aminopropyl)benzotriazolyl, N-aminoethylmorpholinyl, and p-hydroxyphenethylamine; more preferably, the derivative of Pulsatilla saponin B4 is compound B4-19, B4-33, and B4-39, etc.

[0009] This invention discloses a pharmaceutical composition using the above-mentioned Pulsatilla saponin B4 derivative as the active ingredient.

[0010] This invention discloses the use of the above-mentioned Pulsatilla chinensis saponin B4 derivative or pharmaceutical composition in the preparation of anti-inflammatory drugs. Preferably, the inflammation includes superficial inflammation and internal inflammation. More preferably, the inflammation includes skin inflammation. As an example, this invention discloses the use of the above-mentioned Pulsatilla chinensis saponin B4 derivative or pharmaceutical composition in the preparation of a drug for treating atopic dermatitis.

[0011] The present invention also discloses the application of the above-mentioned Pulsatilla saponin B4 derivative or pharmaceutical composition in the preparation of immunomodulatory drugs.

[0012] In this invention, the drug includes topical, oral, rectal, or parenteral medications. The drug is formulated into a pharmaceutically permissible dosage form, such as pills, tablets, powders, capsules, granules (powders), ointments, solutions, gels, or suppositories. Solutions include pellets, drops, sprays, injections, and suspensions.

[0013] This invention discloses a method for preparing the Pulsatilla saponin B4 derivative, which uses compound AB4 as a raw material and prepares the Pulsatilla saponin B4 derivative by nucleophilic substitution, electrophilic addition, esterification or amidation reaction.

[0014] The purpureus saponin B4 derivative disclosed in this invention did not show significant cytotoxicity against THP-1 macrophages, but it could reduce the level of P-IκBa protein in the NF-κB signaling pathway, inhibit the activation of the NLRP3 signaling pathway, and significantly reduce the level of Pro-IL-1β. p< The AB4 derivative showed better anti-inflammatory activity than AB4 (0.05%). Furthermore, in DNCB-induced atopic dermatitis mice, the AB4 derivative demonstrated a good therapeutic effect, significantly reducing ear swelling and effectively improving skin ulceration and edema on the back and ears of mice, outperforming the positive control drugs dexamethasone and AB4. Simultaneously, the AB4 derivative exhibited certain immunomodulatory effects; notably, the derivative of this invention did not have the immunosuppressive side effects of dexamethasone. Attached Figure Description

[0015] Figure 1 This is a schematic diagram of the reaction in the preparation scheme (1) of the Pulsatilla saponin B4 derivative of the present invention.

[0016] Figure 2 This is a schematic diagram of the reaction in the preparation scheme (2) of the Pulsatilla saponin B4 derivative of the present invention.

[0017] Figure 3 This is a schematic diagram of the reaction in the preparation scheme (3) of the Pulsatilla saponin B4 derivative of the present invention.

[0018] Figure 4 The cytotoxicity of AB4 derivatives against HIEC and THP-1 cells at 50 μM was measured.

[0019] Figure 5 The content of nitrite produced by a non-cytotoxic AB4 derivative (10 μM) under LPS stimulation.

[0020] Figure 6 WB diagram showing the anti-inflammatory activity of AB4 derivatives.

[0021] Figure 7 The results of Western blot analysis show the anti-inflammatory activity of AB4 derivatives.

[0022] Figure 8 The condition of the skin on the back of mice with DNCB-induced atopic dermatitis.

[0023] Figure 9 The condition of the ear in mice with DNCB-induced atopic dermatitis.

[0024] Figure 10 This diagram illustrates the changes in mouse body weight and back score.

[0025] Figure 11 This is a schematic diagram of the difference in ear thickness and ear weight in mice.

[0026] Figure 12 This is a schematic diagram of the spleen index in mice. Detailed Implementation

[0027] This invention discloses a method for preparing the Pulsatilla saponin B4 derivative, which uses compound AB4 as a raw material and prepares the Pulsatilla saponin B4 derivative by nucleophilic substitution, electrophilic addition, esterification or amidation reaction.

[0028] The following scheme was adopted to modify and transform the structure of AB4: (1) Using AB4 as a raw material, intermediate A3 was obtained by hydrolysis in sodium hydroxide aqueous solution at 105℃. The glycosyl moiety was then protected with acetyl groups. The double bond moiety was modified by oxidation, nucleophilic substitution, reduction, and hydrolysis to obtain a partial derivative of Pulsatilla saponin B4. See the reaction diagram. Figure 1 Reaction conditions: (a) NaOH / H2O, 105℃, 10h; (b) AC2O, Py, DMAP, rt; (c) i m-CPBA, NaHCO3, DCM; ii m-CPBA, CHCl3, reflux, 2 days; iii NBS, CCl4, 3 days; (d) NaOH, THF / CH3OH / H2O, room temperature, overnight.

[0029] (2) After obtaining intermediate A3 according to scheme (1), the C28 carboxyl group was used as the modified group, and amide condensation was carried out under the action of TBTU (O-benzotriazole-N,N,N',N'-tetramethylurea tetrafluoroborate), DIEA (N,N-diisopropylethylamine), and DMF (N,N-dimethylformamide) to obtain a series of amide derivatives, which are some of the derivatives of Pulsatilla chinensis saponin B4. See the reaction diagram. Figure 2 Reaction conditions: (a) NaOH / H2O, 105℃, 10h; (b) TBTU, DIEA, DMF, room temperature, overnight; (c) DIEA, Amine, room temperature, overnight; (d) NaOH, THF / CH3OH / H2O, room temperature, overnight.

[0030] (3) Using AB4 as a raw material, the double bond portion was modified by oxidation, reduction, nucleophilic substitution, etc., while protecting the entire sugar group, to obtain some Pulsatilla saponin B4 derivatives. See the reaction diagram. Figure 3 Reaction conditions: (a) AC2O, Py, DMAP, rt; (b) i NBS, CCl4, 3 days; ii H2, Pd / C; (c) i NaOH, THF / CH3OH / H2O or CHCl3-CH3OH, K2CO3, room temperature, overnight.

[0031] Those skilled in the art can obtain the product of the present invention (lupinane-type pentacyclic triterpenoid saponin compound) using conventional techniques based on the raw materials and reaction conditions of the present invention, or other methods that can obtain the product of the present invention can be used.

[0032] This invention discloses the application of the above-mentioned Pulsatilla chinensis saponin B4 derivative in the preparation of anti-inflammatory drugs. In particular, this invention discloses the application of the above-mentioned Pulsatilla chinensis saponin B4 derivative in the preparation of drugs for treating atopic dermatitis.

[0033] The pharmaceutical composition disclosed in this invention uses the aforementioned Pulsatilla chinensis saponin B4 derivative as the active ingredient and further includes a pharmaceutically acceptable carrier. The active ingredient and pharmaceutical composition are used to prepare a medicament for atopic dermatitis. For example, the medicament contains a therapeutically effective amount of the Pulsatilla chinensis saponin B4 derivative or its hydrochloride, perchlorate, mesylate, phosphate, citrate, or sulfate, and a pharmaceutically acceptable carrier.

[0034] In this invention, a pharmaceutically acceptable carrier refers to one or more compatible solid or liquid fillers or gel substances that are pharmaceutically usable, have sufficient purity and low toxicity, and can be mixed with other components in the pharmaceutical composition and with the active ingredient of this invention without reducing the efficacy of the active ingredient. Pharmaceutically acceptable carriers include diluents, solubilizers, cosolvents, disintegrants, dispersants, lubricants, flavoring agents, antioxidants, binders, absorbents, humectants, buffers, and crosslinking agents. Pharmaceutically acceptable examples of carrier components include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerin, mannitol, sorbitol, etc.), cyclodextrins (such as hydroxypropyl β-cyclodextrin), emulsifiers (such as Tween), wetting agents (such as sodium dodecyl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0035] In this invention, the drug includes topical, oral, rectal, or parenteral medications. The drug is formulated into a pharmaceutically permissible dosage form, such as pills, tablets, powders, capsules, granules (powders), ointments, liquids, gels, or suppositories. Liquids include pellets, drops, sprays, injections, and suspensions.

[0036] This invention discloses the application of a Pulsatilla saponin B4 derivative in the preparation of anti-inflammatory drugs. The derivative of this invention can be administered alone or in combination with other therapeutic agents. There are no particular limitations on the administration method of the active ingredient or pharmaceutical composition of this invention; representative administration methods include external, oral, rectal, parenteral (e.g., intravenous, intramuscular, or subcutaneous), etc. Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules; liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, or tinctures. In addition to the active ingredient, liquid dosage forms may contain diluents conventionally used in the art, such as water or other solvents, solubilizers, and emulsifiers, such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butanediol, dimethylformamide, and oils, particularly cottonseed oil, peanut oil, corn germ oil, olive oil, sesame oil, and sesame oil, or mixtures thereof. In addition to these aqueous diluents, the composition may also contain adjuvants such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents, and fragrances. Besides the active ingredient, the suspension may contain suspending agents such as ethoxylated isooctadecyl alcohol, polyoxyethylene sorbitol and dehydrated sorbitan esters, microcrystalline cellulose, aluminum methacrylate, and agar, or mixtures thereof. Compositions for parenteral injection may contain physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions, or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents, or excipients include water, ethanol, polyols, and suitable mixtures thereof.

[0037] Existing technologies disclose the anti-inflammatory applications of scutellaria baicalensis saponin B4 (AB4), but its high water solubility, short half-life, and low oral bioavailability limit its clinical application. This invention modifies the structure of AB4 to obtain compounds with better anti-inflammatory activity and very low toxicity. The synthetic route for the scutellaria baicalensis saponin B4 derivative of this invention is described below. Figures 1 to 3 In vitro and in vivo activity results are shown in Figure 4-12 .

[0038] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise stated, percentages and parts refer to weight percentages and weight parts. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as are familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein can be applied to the methods of the present invention. In the following preparation embodiments, the reagents are existing products, mainly provided by Shanghai Chemical Reagent Company; the TLC thin-layer chromatography silica gel plates are from Shandong Yantai Jiangyou Silica Gel Development Company, model HSGF 254; and the normal-phase column chromatography silica gel used for compound purification is produced by Beijing Innocare Technology Co., Ltd., 200-300 mesh. NMR was recorded using a Varian Mercury 400M NMR spectrometer, and chemical shifts are expressed as δ (ppm). The abbreviations used in this article correspond to the following Chinese terms: DMF: N,N-dimethylformamide; DCM: dichloromethane; THF: tetrahydrofuran; TBTU: O-benzotriazole-N,N,N',N'-tetramethylurea tetrafluoroborate; DIPEA: N,N-diisopropylethylamine; PE: petroleum ether; EA: ethyl acetate; K2CO3: potassium carbonate; amine: amine.

[0039] Statistical analysis of all data was performed using GraphPad Prism 8 software. Differences between statistically significant means were compared using GraphPad Prism 8. All data were analyzed using one-way or two-way ANOVA, and the p-value was typically used to represent the difference between the two groups. If p ≤ 0.05, it indicates a statistically significant difference, usually indicated by "#" or "*", where "#" indicates the difference between the normal group and the model group, and "*" indicates the difference between the treated group and the model group (in cytotoxicity experiments, "*" indicates the difference between the treated group and the normal group). If p ≤ 0.01, it indicates a significant statistical difference, indicated by "##" or "**". If p ≤ 0.001, it indicates a highly significant statistical difference, indicated by "###" or "***"; if p ≤ 0.0001, it indicates an extremely significant statistical difference, indicated by "####" or "****".

[0040] The specific structures of the various Pulsatilla chinensis saponin B4 derivatives in the following examples are as follows:

[0041]

[0042]

[0043]

[0044]

[0045]

[0046] Example 1 B4-1: Pulsatilla saponin B4 (1 g, 0.819 mmol) was dissolved in 20 ml of pyridine, followed by the addition of 2.5 ml of acetic anhydride and DMAP (14 mg, 0.082 mmol). The mixture was stirred at room temperature for 28 hours. Then, 50 ml of ethyl acetate was added to the reaction solution, followed by 100 ml of water for extraction to obtain the organic phase. The organic phase was washed twice with water and dried, then evaporated to dryness to obtain fully acetylated B4. Fully acetylated B4 (300 mg, 0.2456 mmol) was dissolved in 15 ml of carbon tetrachloride, followed by the addition of NBS (48 mg, 0.2702 mmol). The mixture was stirred at room temperature for 24 hours to obtain intermediate 3-O- α -L-[3,4-diacetoxy-rhamnopyranosyl]-(1→2)- α -L-[2,3,4-triacetoxy-arabinopyranosyl]-3 β ,23-Dihydroxylussane-Δ 20(29) 30-Bromo-28-O- α -L-[2,3,4-triacetoxy-rhamnopyranose]-(1→4)- β -D-[2,3,6-triacetoxy-glucose pyranoside]-(1→6)- β -D-[2,3,4-triacetoxy-pyranose]. The above intermediate (100 mg, 0.052 mmol) was dissolved in 4 mL of a dichloromethane:methanol (2:1) mixture, and potassium carbonate (161 mg, 1.165 mmol) was added. The mixture was stirred at room temperature for 16 hours. After the reaction was completed, the solution was concentrated under reduced pressure and purified by preparative liquid chromatography (60% methanol-water) to obtain 12 mg of white solid, with a yield of 22%. 1 H NMR (400 MHz, Methanol- d 4) δ 5.51 (1H,d, J = 8.1 Hz, 1-H of glc), 5.20 (1H, brs, 1-H of rha ), 5.00 (1H, brs, 1´-H ofrha ), 4.98 (1H, brs, H1-29 ), 4.60 (1H, d, J = 4.7 Hz, 1-H of ara ), 4.42 (1H,d, J = 7.8 Hz, 1´-H of glc), 4.16 (1H, d, J= 11.9 Hz, H1-23), 3.95 (2H, s, H-30), 1.30 (3H, d, J = 6.3 Hz, 6-H3 of rha), 1.28 ( 3H, d, J = 6.2 Hz, 6´-H3 ofrha ), 1.07(3H, s, H-27), 1.00 (3H, s, H-26 ), 0.93 (3H, s, H-25 ), 0.72 (3H,s, H-24). 13 C NMR (101 MHz, MeOD) δ 176.33, 152.63, 110.16, 104.60, 104.28,102.91, 101.86, 95.28, 82.30, 79.56, 78.26, 77.98, 76.88, 76.71, 76.63,76.12, 75.28, 73.94, 73.74, 73.66, 72.43, 72.21, 72.14, 72.02, 70.98, 70.67,70.16, 69.62, 69.11, 64.73, 64.59, 61.93, 58.55, 57.98, 51.92, 51.10, 49.50, 44.44, 44.04, 43.61, 41.99, 39.91, 39.37, 37.82, 37.42, 34.93, 33.00, 32.77, 30.87, 27.99, 26.69, 22.20, 18.78, 17.96, 17.84, 17.28, 16.79, 15.11, 13.54.

[0047] Example 2 B4-4: Pulsatilla saponin B4 (1 g, 0.82 mmol) was dissolved in 15 ml of water. Sodium hydroxide (65.6 mg, 1.64 mmol) was added, and the mixture was stirred at 105 °C for 12 hours. The reaction solution was filtered, and the precipitate was washed twice with water to obtain pulsatilla saponin A3. Pulsatilla saponin A3 (1 g, 1.33 mmol) was then dissolved in 20 ml of pyridine. DMAP (20 mg, 0.164 mmol) was added, and the mixture was stirred at room temperature for 18 hours. 50 ml of ethyl acetate was added to the reaction solution, followed by 100 ml of water for extraction to obtain the organic phase. The organic phase was washed twice with water, dried, and then evaporated to dryness to obtain intermediate 3-O- α -L-[3,4-diacetoxy-rhamnopyranosyl]-(1→2)- α-L-[2,3,4-triacetoxy-arabinopyranosyl]-3 β ,23-Dihydroxylussane-Δ 20(29) Alkene-28-acid. The above intermediate (1 g, 0.998 mmol) was dissolved in a mixed solution of DCM and methanol (2:1), and Pd / C (50 mg) was added. The reaction was carried out at room temperature under hydrogen atmosphere for 18 hours. The reaction was monitored by HPLC. After the reaction was completed, the mixture was filtered, concentrated under reduced pressure to remove the solvent, and separated by C18 preparation (75% methanol and water) to give 523 mg of white solid, with a yield of 52.3%. 1 H NMR (400 MHz, Chloroform- d ) δ 5.04 (1H, brs, 1-Hof rha), 4.42 (1H, d, J = 6.4 Hz, 1-H of ara ),4.11 (1H, d, J = 11.5 Hz, H1-23),3.58 (1H, d, J = 11.5 Hz, H2-23 ), 2.13 (3H, s, H3 of -OAc ), 2.10 (3H, s, H3of -OAc), 2.09 (3H, s, H3 of -OAc), 2.05 (3H, s, H3 of -OAc), 2.03 (3H, s, H3of -OAc), 1.96 (3H, s, H3 of -OAc), 1.21 (3H, d, J = 6.2 Hz, 6-H3 of rha ),0.93 (3H, s, H-27), 0.91 (3H, s, H-26 ), 0.86 (3H, s, H-25 ), 0.85 (3H, d, J =7.2 Hz, H-29 ), 0.77 (3H, s, H-24), 0.75 (3H, d, J = 6.7 Hz, H-30). 13C NMR (101MHz, CDCl3) δ 181.66, 170.58, 170.52, 170.44, 170.29, 170.19, 169.78, 103.69,98.31, 82.11, 77.36, 74.47, 72.01, 71.15, 69.69, 68.73, 67.98, 67.27, 65.25,62.86, 56.89, 50.63, 48.85, 48.13, 44.25, 42.64, 42.11, 40.83, 38.74, 38.36,37.53, 36.87, 34.24, 32.13, 29.86, 29.71, 27.04, 25.84, 23.10, 22.85, 21.17, 21.11, 21.07, 20.95, 20.91, 20.80, 18.10, 17.47, 16.71, 16.13, 14.80, 14.58, 12.62.

[0048] B4-5: Dissolve B4-4 (200 mg, 0.20 mmol) in 4 mL of a methanol / tetrahydrofuran / water (2:1:1) mixture, add sodium hydroxide (72 mg, 1.8 mmol), stir at room temperature for 12 h, and when the reaction is complete, remove the solvent under reduced pressure, wash with 50 mL of water to remove salt, and dry to give 120 mg of white solid, yield 80.1%. 1 H NMR (400 MHz, DMSO- d 6) δ 4.96 (1H,brs, 1-H of rha),4.22 (1H, d, J = 4.6 Hz, 1-H of ara) , 4.04 (1H, d, J = 11.1 Hz,H1-23), 1.06 (3H, d, J = 5.8 Hz, 6-H3 of rha ), 0.89 (3H, s, H-27 ), 0.86 (3H,s, H-26), 0.82 (3H, s, H-25), 0.80 (3H, d, J = 6.1 Hz, H-29), 0.72 (3H, d, J =6.2 Hz, H-30), 0.69 (3H, s, H-24). 13C NMR (101 MHz, DMSO) δ 170.16, 103.36,100.28, 80.72, 74.67, 72.23, 72.03, 70.63, 70.42, 68.60, 67.53, 64.70, 64.04,56.11, 50.13, 48.38, 47.81, 43.95, 42.26, 41.69, 40.48, 38.38, 37.55, 36.51,33.91, 29.62, 29.36, 29.24, 26.83, 25.42, 23.23, 22.73, 20.90, 20.75, 17.98, 16.53, 16.04, 14.81, 14.27, 12.55.

[0049] Example 3 B4-6: The intermediate 3-O- α -L-[3,4-diacetoxy-rhamnopyranosyl]-(1→2)- α -L-[2,3,4-triacetoxy-arabinopyranosyl]-3 β ,23-Dihydroxylussane-Δ 20(29) 1 g of 28-ene acid (0.998 mmol) was dissolved in 10 mL of chloroform. At low temperature, 207 mg of m-chloroperoxybenzoic acid (1.20 mmol) was added. After complete dissolution, the mixture was heated to 65 °C and refluxed for 16 hours. After the reaction was completed, the solvent was removed by concentration under reduced pressure. Silica gel column chromatography (dichloromethane:methanol = 80:1 → 70:1) yielded 305 mg of white solid, with a yield of 30%. 1 H NMR (400 MHz, Chloroform- d ) δ 5.22 (1H,brs, 1-H of rha), 5.04 (1H, brs, H1-29), 4.97 (1H, s, H1 of -OH on C-30), 4.92 (1H, brs, H2-29), 4.41 (1H, d, J= 6.4 Hz, 1-H of ara ), 4.12 (2H, s, H2of H-30 ), 2.13 (3H, s, H3 of -OAc), 2.10 (3H, s, H3 of -OAc), 2.10 (3H, s,H3 of -OAc), 2.05 (3H, s, H3 of -OAc) -OAc), 2.03 (3H, s, H3 of -OAc), 1.96 (3H, s,H3 of -OAc), 1.21 (3H, d, J = 6.2 Hz, 6-H3 of rha ), 0.96 (3H, s, H-27), 0.91 (3H, s, H-26), 0.85 (3H, s, H-25), 0.77 (3H, s, H-24). 13 C NMR (101 MHz, CDCl3)δ 181.05, 170.90, 170.84, 170.76, 170.61, 170.51, 170.10, 155.14, 107.35,104.01, 98.63, 82.36, 77.68, 74.80, 72.34, 71.48, 70.01, 69.05, 68.30, 67.58,65.71, 65.57, 56.72, 51.14, 50.40, 48.47, 43.00, 42.76, 42.42, 41.15, 39.03,38.79, 37.21, 34.51, 32.81, 32.40, 30.16, 27.21, 21.49, 21.43, 21.39, 21.27, 21.23, 21.12, 17.79, 17.08, 16.44, 14.98, 12.93.

[0050] B4-7: Dissolve B4-6 (100 mg, 0.098 mmol) in 4 mL of a methanol / tetrahydrofuran / water (2:1:1) mixture, add sodium hydroxide (35.3 mg, 0.882 mmol), stir at room temperature for 12 h, and after the reaction is complete, concentrate under reduced pressure to remove the solvent, and perform silica gel column chromatography (dichloromethane:methanol = 10:1) to give 15 mg of white solid, yield 20%. 1 H NMR (400 MHz, DMSO- d6) δ12.03 (1H, s, H1 of -COOH ),4.95 (1H, brs, 1-H of rha ), 4.85 (1H, s, H1 of -OH on C-30), 4.76 (1H, brs, H1-29), 4.66 (1H, brs, H2-29),4.21 (1H, d, J = 4.7Hz, 1-H of ara), 4.03 (1H, d, J = 11.3 Hz, H1-23), 3.69 (1H, d, J = 11.8 Hz, H2-23), 3.87 (2H, s, H2of H-30 ), 1.05 (3H, d, J = 6.1 Hz, 6-H3 of rha ), 0.90(3H, s, H-27), 0.84 (3H, s, H-26), 0.79 (3H, s, H-25), 0.67 (3H, s, H-24 ). 13 CNMR (101 MHz, DMSO) δ 170.49, 155.94, 106.07, 103.68, 100.58, 81.06, 74.97,72.54, 72.33, 70.95, 70.72, 68.92, 67.85, 63.40, 55.94, 50.66, 49.42, 48.14,42.85, 42.71, 42.38, 42.01, 40.78, 38.63, 38.07, 36.85, 34.12, 32.44, 29.56,29.11, 27.09, 25.75, 22.63, 21.22, 18.30, 18.01, 16.88, 16.28, 14.70, 14.49, 12.86.

[0051] Example 4 B4-8: The intermediate 3-O- α -L-[3,4-diacetoxy-rhamnopyranosyl]-(1→2)- α -L-[2,3,4-triacetoxy-arabinopyranosyl]-3 β ,23-Dihydroxylussane-Δ 20(29)500 mg of 28-ene acid (0.499 mmol) was dissolved in 5 mL of dichloromethane and sodium bicarbonate (46.1 mg, 0.55 mmol) was added. The mixture was stirred at room temperature for 8 hours. After the reaction was completed, the mixture was filtered, concentrated under reduced pressure, and purified by silica gel column chromatography (dichloromethane:methanol = 70:1) to give 211 mg of white solid, with a yield of 41.5%. 1 H NMR (400 MHz, DMSO- d 6) δ 12.09 (1H, s, H1 of -COOH), 5.02 (1H, brs,1-H of rha), 4.50 (1H, d, J = 6.9 Hz, 1-H of ara), 3.99 (1H, d, J = 11.4 Hz, H1-23), 2.56 (2H, dd, J = 3.4 Hz, H-29), 2.10 (3H, s, H3 of -OAc), 2.07 (3H, s, H3of -OAc), 2.06 (3H, s, H3 of -OAc), 2.02 (3H, s, H3 of -OAc), 1.95 (3H, s, H3of -OAc), 1.93 (3H, s, H3 of -OAc), 1.16 (3H, s, H-30), 1.10 (3H, d, J = 6.2Hz, 6-H3 of rha), 0.92 (3H, s, H-27), 0.86 (3H, s, H-26), 0.83 (3H, s, H-25), 0.73 (3H, s, H-24). 13C NMR (101 MHz, DMSO) δ 177.14, 170.10, 169.90, 169.88,169.64, 169.61, 169.48, 102.35, 97.34, 80.44, 73.92, 71.88, 70.03, 68.83,68.03, 67.83, 66.38, 64.42, 62.63, 59.57, 58.48, 55.77, 55.52, 49.89, 49.08,47.57, 45.10, 41.91, 41.30, 40.22, 38.12, 36.86, 36.28, 36.09, 33.55, 31.56, 28.94, 26.96, 26.22, 25.43, 20.74, 20.71, 20.66, 20.52, 20.45, 20.40, 18.05, 17.44, 17.10, 16.30, 15.68, 14.05, 12.20.

[0052] Example 5 B4-9: The intermediate 3-O- α -L-[3,4-diacetoxy-rhamnopyranosyl]-(1→2)- α -L-[2,3,4-triacetoxy-arabinopyranosyl]-3 β ,23-Dihydroxylussane-Δ 20(29) 500 mg of olefinic acid (0.499 mmol) was dissolved in 10 mL of carbon tetrachloride, and NBS (90 mg, 0.499 mmol) was added. The mixture was stirred at room temperature for 20 hours. After the reaction was completed, the mixture was filtered, concentrated under reduced pressure, and subjected to silica gel column chromatography (dichloromethane:methanol = 80:1) to give 324 mg of an off-white solid, with a yield of 60%. 1 HNMR (400 MHz, Chloroform- d ) δ 5.21 (1H, brs, 1-H of rha), 5.14 (1H, brs, H1-29), 5.04 (1H, brs, H2-29), 4.41 (1H, d, J = 6.3 Hz, 1-H of ara), 4.11 (1H, d, J = 11.4 Hz, H1-23 ), 3.99 (2H, s, H-30 ), 3.88 (1H, d, J= 10.9 Hz, H2-23), 2.13(3H, s, H3 of -OAc), 2.10 (3H, s, H3 of -OAc), 2.09 (3H, s, H3 of -OAc), 2.05(3H, s, H3 of -OAc), 2.03 (3H, s, H3 of -OAc), 1.96 (3H, s, H3 of -OAc), 1.21(3H, d, J = 6.1 Hz, 6-H3 of rha), 0.97 (3H, s, H-27 ), 0.92 (3H, s,H-26 ), 0.86 (3H, s, H-25 ), 0.77 (3H, s, H-24). 13 C NMR (101 MHz, CDCl3) δ 170.56, 170.50,170.42, 170.28, 170.17, 169.77, 151.40, 113.62, 103.65, 98.32, 82.02, 77.36,74.50, 71.98, 71.19, 69.71, 68.75, 67.97, 67.28, 65.27, 62.82, 56.52, 50.86,48.18, 43.19, 42.50, 42.12, 40.87, 38.76, 38.54, 36.92, 34.23, 33.18, 29.84, 29.46, 27.35, 26.96, 25.84, 22.83, 21.15, 21.10, 21.06, 20.94, 20.91, 20.79, 18.10, 17.47, 16.77, 16.17, 14.69, 14.25, 12.62.

[0053] B4-10: Dissolve B4-9 (200 mg, 0.185 mmol) in 4 mL of a methanol / tetrahydrofuran / water (2:1:1) mixture, add sodium hydroxide (66.6 mg, 1.67 mmol), stir at room temperature for 12 h, and after the reaction is complete, concentrate under reduced pressure to remove the solvent, and perform silica gel column chromatography (dichloromethane:methanol = 10:1) to give 100 mg of white solid, yield 66.7%. 1 H NMR (400 MHz, Methanol-) d4) δ 5.19 (1H, brs, 1-H of rha), 5.09 (1H, brs, H1-29), 4.96 (1H,brs, H2-29), 4.59 (1H, d, J = 4.7 Hz, 1-H of ara), 4.09 (1H, s, H1 of -OH on C-23), 3.93 (2H, s, H-30), 1.27 (3H, d, J = 6.3 Hz, 6-H3 of rha), 1.07 (3H, s, H-27), 1.00 (3H, s, H-26), 0.92 (3H, s, H-25 ), 0.71 (3H, s, H-24 ). 13 C NMR (101MHz, MeOD) δ 153.40, 113.70, 104.28, 101.88, 82.26, 76.66, 76.19, 73.93,73.64, 72.14, 72.02, 70.16, 69.11, 66.65, 64.72, 64.57, 58.54, 57.53, 51.91,51.03, 44.39, 44.04, 43.63, 41.89, 39.91, 39.68, 37.81, 35.03, 34.27, 33.21,30.85, 28.23, 26.69, 22.23, 18.77, 17.95, 17.20, 16.69, 15.14, 13.51.

[0054] Example 6 B4-11: The intermediate 3-O- α -L-[3,4-diacetoxy-rhamnopyranosyl]-(1→2)- α -L-[2,3,4-triacetoxy-arabinopyranosyl]-3 β ,23-Dihydroxylussane-Δ 20(29) Alkene-28-acid (500 mg, 0.499 mmol) was dissolved in 12 mL of DMF, and TBTU (240.3 mg, 0.7485 mmol) and DIEA (726 mg, 2.495 mmol) were added. The mixture was stirred at room temperature for 10 hours. After the reaction was completed, 60 mL of water was added to the reaction solution, and a white solid precipitated. The solid was filtered and purified by silica gel column chromatography (petroleum ether: ethyl acetate = 2:1) to give 452 mg of white solid, with a yield of 81%. 1 H NMR (400 MHz, Chloroform-d ) δ8.08 (1H, d, J = 8.4 Hz, H1 of benzene), 7.52–7.56 (1H, m, H1 of benzene ),7.40–7.44(1H, m, H1 of benzene), 7.36 (1H, d, J = 8.3 Hz, H1 of benzene ), 5.04(1H, brs, 1-H of rha), 4.73 (1H, brs, H1-29), 4.64 (1H, brs, H2-29), 4.42 (1H,d, J = 6.4 Hz,1-H of ara), 4.12 ( 1H, d, J = 12.1 Hz, H1-23), 3.57 (1H, d, J = 11.2Hz, H2-23), 2.13 (3H, s, H3 of -OAc), 2.11 (3H, s, H3 of -OAc), 2.10 (3H, s,H3 of -OAc), 2.05 (3H, s, H3 of -OAc), 2.03 (3H, s, H3 of -OAc), 1.97 (3H, s,H3 of -OAc), 1.71 (3H, s, H-30), 1.21 (3H, d, J = 6.2 Hz, 6-H3 of rha), 1.03(3H, s, H-27 ), 0.98 (3H, s, H-26), 0.85 (3H, s, H-25), 0.78 (3H, s, H-24). 13CNMR (101 MHz, CDCl3) δ 171.97, 170.54, 170.50, 170.42, 170.28, 170.17,169.77, 149.36, 143.76, 129.01, 128.79, 124.84, 120.78, 110.53, 108.04,103.67, 98.24, 81.97, 77.36, 74.34, 72.05, 71.16, 69.70, 68.72, 67.99, 67.24,65.28, 62.88, 57.13, 50.89, 50.08, 48.21, 46.68, 42.54, 42.11, 40.93, 38.76, 38.61, 36.92, 34.21, 30.44, 30.17, 29.83, 25.84, 25.55, 21.18, 21.11, 21.07, 20.95, 20.91, 20.80, 19.55, 18.08, 17.46, 16.78, 16.25, 14.80, 12.64.

[0055] Example 7 B4-13: The intermediate 3-O- α -L-[3,4-diacetoxy-rhamnopyranosyl]-(1→2)- α -L-[2,3,4-triacetoxy-arabinopyranosyl]-3 β ,23-Dihydroxylussane-Δ 20(29) Alkene-28-acid (100 mg, 0.0998 mmol) was dissolved in 5 ml of DMF, and potassium carbonate (13.8 mg, 0.0998 mmol) and iodomethane (14.2 mg, 0.0998 mmol) were added. The mixture was stirred at room temperature for 8 hours. After the reaction was completed, the mixture was filtered, concentrated under reduced pressure to remove the solvent, and subjected to silica gel column chromatography (petroleum ether: ethyl acetate = 2:1) to give 89.4 mg of white solid, with a yield of 88%. 1 H NMR (400 MHz, Chloroform- d ) δ5.04(1H, brs, 1-H of rha )4.73 (1H, brs, H1-29), 4.60 (1H, brs, H2-29), 4.41(1H, d, J= 6.4 Hz,1-H of ara), 3.66 (3H, s, H3 of -COOCH3), 2.13 (3H, s, H3 of -OAc), 2.10 (3H, s, H3 of -OAc), 2.09 (3H, s, H3 of -OAc), 2.05 (3H, s, H3 of-OAc), 2.02 (3H, s, H3 of -OAc), 1.96 (3H, s, H3 of -OAc), 1.68 (3H, s, H-30), 1.21 (3H, d, J = 6.2 Hz, 6-H3 of rha), 0.94 (3H, s, H-27 ), 0.90 (3H, s,H-26), 0.85 (3H, s, H-25), 0.77 (3H, s, H-24). 13 C NMR (101 MHz, CDCl3) δ176.76, 170.55, 170.49, 170.41, 170.26, 170.18, 169.74, 150.72, 109.72,103.67, 98.27, 82.04, 74.42, 72.04, 71.18, 69.71, 68.74, 68.00, 67.25, 65.27,62.87, 56.68, 51.40, 50.89, 49.61, 48.18, 47.09, 42.45, 42.11, 40.81, 38.75,38.38, 37.07, 36.91, 34.17, 32.26, 30.75, 29.83, 29.74, 25.86, 25.64, 21.15, 21.09, 21.05, 20.93, 20.89, 20.78, 19.52, 18.09, 17.45, 16.73, 16.11, 14.66, 12.64.

[0056] Example 8 B4-14: Compound A3 (3-O- α -L-pyrano-rhamnosyl-(1→2)- α -L-arabinopyranosyl-3 β ,23-Dihydroxylussane-Δ 20(29)100 mg (0.1333 mmol) of olefinic acid (28-carboxylic acid) was dissolved in 4 ml of DMF. 70 μL (0.3999 mmol) of DIEA was added, and the mixture was stirred at room temperature for 8 hours. After the intermediate was completely formed, 15 mg (0.2 mmol) of 2-methoxyethylamine and 116 μL (0.6665 mmol) of DIEA were added, and the mixture was stirred at room temperature for 16 hours. After the reaction was completed, 50 ml of water was added to the reaction solution, and a solid precipitated. The solid was filtered and subjected to silica gel column chromatography (dichloromethane:methanol = 10:1 → 8:1) to give 72.6 mg of an off-white solid, with a yield of 66%. 1 H NMR (400 MHz, DMSO- d 6) δ 7.58 (1H, s, H1 of -CONH), 5.05 (1H, brs, 1-Hof rha), 4.65 (1H, brs, H1-29), 4.53 (1H, brs, H1-29), 4.33 (1H, d, J = 5.9 Hz,1-H of ara), 3.22 (3H, s, H3 of -OCH3), 1.62 (3H, s, H-30), 1.07 (3H, d, J =6.2 Hz, 6-H3 of rha), 0.90 (3H, s, H-27), 0.84 (3H, s, H-26), 0.78 (3H, s,H-25), 0.54 (3H, s, H-24). 13 C NMR (101 MHz, DMSO) δ 175.63, 150.93, 109.24,102.91, 99.90, 79.37, 74.19, 72.82, 72.03, 70.70, 70.43, 70.36, 68.13, 67.78,64.31, 62.44, 57.87, 54.89, 50.08, 49.69, 46.47, 46.23, 42.34, 41.93, 40.21,38.40, 38.07, 37.65, 36.69, 36.15, 33.53, 32.35, 30.35, 28.81, 25.49, 25.30, 20.57, 19.08, 17.78, 17.07, 16.40, 15.80, 14.27, 12.81.

[0057] The following compounds can be prepared in a similar manner to B4-14, except that 2-methoxyethylamine can be substituted to obtain the corresponding products.

[0058] B4-15: The preparation method is similar to B4-14, with a yield of 58%; in addition to the amide group, a methyl group (3.57 ppm) is added to mark the hydrogen methyl ester. 1 H NMR (400 MHz, DMSO- d 6) δ 7.60 (1H, s, H1 of -CONH), 5.05 (1H,brs, 1-H of rha), 4.64 (1H, brs, H1-29), 4.59 (1H, brs, H2-29), 4.53 (1H, s,H1 of -OH on C-23), 4.44 (1H, d, J = 5.9 Hz, 1-H of ara), 3.57 (3H, s, H3 of -COOCH3), 2.28 (2H, t, J = 7.5 Hz, H2 of -CH2-COOCH3), 1.62 (3H, s, H-30), 1.07(3H, d, J = 6.2 Hz, 6-H3 of rha), 0.90 (3H, s, H-27 ), 0.82 (3H, s, H-26), 0.78 (3H, s, H-25), 0.54 (3H, s, H-24). 13 C NMR (101 MHz, DMSO) δ 176.18, 173.79,151.55, 109.88, 103.54, 100.53, 80.00, 74.83, 73.44, 72.66, 71.06, 70.99,68.76, 68.42, 64.93, 63.07, 55.49, 51.87, 50.72, 50.30, 47.10, 46.80, 42.97,42.56, 40.83, 39.03, 38.35, 38.23, 37.27, 36.78, 34.16, 33.04, 31.33, 30.97, 29.47, 26.12, 25.93, 25.28, 21.22, 19.69, 18.41, 17.69, 17.04, 16.42, 14.88, 13.45.

[0059] B4-16: The preparation method is similar to that of B4-14, with a yield of 54%; in addition to the amide group, the additional marker hydrogen is the monobasic hydrogen on cyclopentane (3.97 ppm). 1 H NMR (400 MHz, DMSO-d 6) δ 7.28 (1H, s, H1 of -CONH), 5.06 (1H,brs, 1-H of rha), 4.65 (1H, brs, H1-29), 4.53 (1H, brs, H2-29), 4.43 (1H, d, J =5.7 Hz, 1-H of ara), 3.97 (1H, p, J = 6.7 Hz, H1 of -CH on cyclopentane), 1.63(3H, s, H-30), 1.07 (3H, d, J = 6.2 Hz, 6-H3 of rha), 0.91 (3H, s, H-27 ), 0.84 (3H, s, H-26), 0.79 (3H, s, H-25), 0.55 (3H, s, H-24). 13 C NMR (101 MHz, DMSO)δ 175.14, 150.99, 109.17, 102.89, 99.90, 79.37, 74.21, 72.79, 72.03, 70.43,70.36, 68.12, 67.76, 64.28, 62.44, 54.64, 50.23, 50.10, 49.79, 46.48, 46.17,42.34, 41.88, 40.22, 38.39, 37.66, 36.58, 36.15, 33.51, 32.43, 32.32, 31.46, 30.41, 28.77, 25.49, 25.30, 23.57, 23.53, 20.59, 19.07, 17.77, 17.05, 16.39, 15.81, 14.24, 12.82.

[0060] B4-17: The preparation method is similar to that of B4-14, with a yield of 43%; in addition to the amide group, a new marker hydrogen is added, which is a methylene group (3.60 ppm) linked to a chlorine atom. 1 H NMR (400 MHz, DMSO- d 6) δ 7.66 (1H, s, H1 of -CONH), 5.06 (1H, brs, 1-H of rha), 4.65 (1H, brs, H1-29), 4.54 (1H, brs, H2-29), 4.33 (1H,d, J= 5.8 Hz, 1-H of ara), 3.60 (2H, t, J = 6.7 Hz, H2 of -CH2Cl), 1.63 (3H, s,H-30), 1.07 (3H, d, J = 6.2 Hz, 6-H3 of rha), 0.91 (3H, s, H-27), 0.83 (3H, s,H-26), 0.78 (3H, s, H-25), 0.54 (3H, s, H-24). 13 C NMR (101 MHz, DMSO) δ175.91, 151.11, 109.47, 103.12, 100.10, 79.57, 74.39, 73.05, 72.24, 70.65,70.57, 68.33, 68.01, 64.53, 62.64, 55.09, 50.30, 49.86, 48.81, 46.68, 46.38,43.34, 42.55, 42.14, 40.43, 38.61, 37.89, 36.88, 36.36, 36.11, 33.73, 32.70, 30.56, 29.07, 25.70, 25.51, 20.80, 19.28, 17.99, 17.26, 16.63, 16.04, 14.46, 13.03.

[0061] B4-18: The preparation method is similar to that of B4-14, with a yield of 38%; in addition to the amide group, the new marker is a fluorine atom (19.44 ppm). 1 H NMR (400 MHz, DMSO- d 6) δ 7.76 (1H, s, H1 of -CONH), 5.04 (1H,brs, 1-H of rha ), 4.57 (1H, brs, H1-29), 4.56 ((1H, brs, H2-29)) ,4.53 (1H,s, H1 of -OH on C-23), 4.42 (1H,d, J = 5.9 Hz, 1-H of ara), 1.62 (3H, s, H-30), 1.06 (3H, d, J= 6.2 Hz, 6-H3 of rha), 0.90 (3H, s, H-27), 0.82 (3H, s, H-26), 0.77 (3H, s, H-25), 0.53 (3H, s, H-24). 13 C NMR (101 MHz, DMSO) δ 175.99,150.88, 109.27, 102.89, 99.91, 83.03, 81.39, 79.37, 74.23, 72.78, 72.04,70.45, 70.37, 68.14, 67.76, 64.27, 62.45, 54.96, 50.07, 49.69, 46.47, 46.23,42.35, 41.92, 40.21, 38.39, 37.57, 36.72, 36.15, 33.49, 32.28, 30.33, 29.84, 25.48, 25.29, 20.56, 19.07, 17.78, 17.05, 16.39, 15.72, 14.27, 12.82. 19 F NMR (377 MHz, DMSO) δ 19.44.

[0062]

[0063] B4-19: The preparation method is similar to B4-14, with a yield of 33%; in addition to the amide group, the additional marker hydrogen is the four hydrogens on the benzene ring (7-8 ppm). 1 H NMR (400 MHz, DMSO- d 6) δ 8.06 (1H, d, J = 8.4 Hz, H1 of benzene), 7.92 (1H, d, J = 8.4 Hz, H1 of benzene ), 7.73 (1H, s, H1 of -CONH), 7.65 –7.59 (1H, m, H1 of benzene), 7.50 – 7.44 (1H, m, H1 of benzene), 5.06 (1H,brs, 1-H of rha), 4.64 (1H, brs, H1-29), 4.57 (1H, brs, H2-29), 4.43 (1H, d, J=5.9 Hz, 1-H of ara), 1.62 (3H, s, H-30), 1.08 (3H, d, J = 6.2 Hz, 6-H3 of rha), 0.88 (3H, s, H-27), 0.71 (3H, s, H-26), 0.57 (3H, s, H-25), 0.54 (3H, s, H-24). 13 C NMR (101 MHz, DMSO) δ 175.79, 150.86, 142.75, 128.28, 126.97, 124.92,119.64, 109.41, 109.24, 102.88, 99.89, 79.35, 79.00, 74.18, 72.79, 72.02,70.43, 70.36, 68.13, 67.76, 64.27, 62.43, 54.88, 50.02, 49.61, 46.42, 46.16,42.32, 41.86, 40.10, 38.37, 37.68, 36.65, 36.09, 34.66, 33.39, 32.31, 30.31, 28.84, 28.40, 25.46, 25.26, 20.51, 19.05, 17.77, 17.01, 16.35, 15.54, 14.18, 12.79.

[0064] B4-20: The preparation method is similar to that of B4-14, with a yield of 47%; the additional marker besides the amide group is the carbon on the cyclohexane (20-40 ppm). 1 H NMR (400 MHz, DMSO- d 6) δ 7.17 (1H, s, H1 of -CONH), 5.05 (1H,brs, 1-H of rha), 4.58 (1H, brs, H1-29), 4.52 (1H, brs, H2-29), 4.32 (1H, d, J =5.9 Hz, 1-H of ara), 1.62 (3H, s, H-30), 1.06 (3H, d, J = 6.2 Hz, 6-H3 of rha), 0.90 (3H, s, H-27), 0.82 (3H, s, H-26), 0.77 (3H, s, H-25), 0.54 (3H, s, H-24). 13C NMR (101 MHz, DMSO) δ 174.51, 151.00, 109.18, 102.91, 99.90, 79.37,74.20, 72.83, 72.03, 70.43, 70.36, 68.12, 67.79, 64.32, 62.43, 54.68, 50.09,49.76, 48.60, 47.24, 46.48, 46.23, 42.34, 41.88, 40.23, 38.40, 37.79, 36.62,36.14, 33.50, 32.53, 32.36, 32.06, 30.40, 28.80, 25.49, 25.33, 24.97, 24.91, 20.59, 19.07, 17.78, 17.03, 16.39, 15.87, 14.25, 12.83.

[0065] B4-21: The preparation method is similar to B4-14, with a yield of 59%; in addition to the amide group, the additional marker hydrogen is the methyl group on cyclopropyl ethane (1.08 ppm). 1 H NMR (400 MHz, DMSO- d 6) δ 7.31 (1H, s, H1 of -CONH), 5.06 (1H, brs, 1-H of rha), 4.66 (1H, brs, H1-29), 4.54 (1H, brs, H2-29), 4.33 (1H,d, J = 5.9 Hz, 1-H of ara), 1.63 (3H, s, H-30), 1.07 (3H, d, J= 6.2 Hz, 6-H3 ofrha) 1.08 (3H, s, H3 of -CH3on 1-cyclopropyletan), 0.91 (3H, s, H-27), 0.86(1H, s, H1 of -CH to CONH ), 0.83 (3H, s, H-26 ), 0.78 (3H, s, H-25), 0.55(3H, s, H-24), 0.42–0.35 (1H, m, H1 of cyclopropyl), 0.31–0.25 (1H, m, H1 of cyclopropyl), 0.22 – 0.15 (1H, m, H1 of cyclopropyl), 0.14 – 0.06 (1H, m, H1 of cyclopropyl). 13 C NMR (101 MHz, DMSO) δ 174.75, 151.20, 109.38, 103.12,100.11, 79.58, 74.41, 73.03, 72.24, 70.64, 70.57, 68.33, 68.00, 64.52, 62.64,55.00, 50.30, 49.96, 47.90, 46.69, 46.38, 42.55, 42.09, 40.41, 38.60, 38.10,36.78, 36.36, 33.72, 32.50, 30.63, 28.99, 25.70, 25.51, 20.74, 19.33, 17.99, 17.48, 17.24, 17.06, 16.60, 16.00, 14.46, 13.04, 3.14, 2.72.

[0066] B4-22: The preparation method is similar to that of B4-14, with a yield of 51%; in addition to the amide group, the additional marker hydrogen is the monohydric hydrogen on the cyclobutyl group (2.34-2.45 ppm). 1 H NMR (400 MHz, DMSO- d 6) δ 7.53 (1H, s, H1 of -CONH), 5.05 (1H, brs, 1-H of rha), 4.64 (1H, brs, H1-29), 4.52 (1H, brs, H2-29), 4.32(1H, d, J= 5.9 Hz, 1-H of ara ), 2.45 – 2.34 (1H, m, H1 of -CH on cyclobutyl), 1.62 (3H, s, H-30), 1.06 (3H, d, J = 6.2 Hz, 6-H3 of rha ), 0.90 (3H, s, H-27), 0.83 (3H, s, H-26), 0.78 (3H, s, H-25), 0.53 (3H, s, H-24). 13 C NMR (101MHz, DMSO) δ 175.23, 150.75, 108.98, 102.68, 99.67, 79.15, 73.96, 72.61,71.82, 70.22, 70.15, 67.90, 67.56, 64.09, 62.22, 54.69, 49.90, 49.48, 46.26,45.93, 43.14, 42.12, 41.72, 40.00, 38.19, 37.63, 36.41, 35.93, 34.82, 33.34,32.30, 30.14, 28.61, 25.27, 25.11, 24.95, 24.87, 20.39, 18.86, 17.56, 17.52, 16.83, 16.20, 15.64, 14.04, 12.60.

[0067] B4-23: The preparation method is similar to that of B4-14, with a yield of 55%; in addition to the amide group, the additional marker hydrogens are hydrogen (6.5-8 ppm) and carbon (115-140 ppm) on the imidazole group. 1 H NMR (400 MHz, DMSO- d 6) δ 7.65 (1H, s, H1 of -CONH), 7.61 (1H, s, H1 of imidazole ), 7.16 (1H, s, H1 of imidazole ), 6.88 (1H, s, H1 of imidazole ), 5.05 (1H, brs, 1-H of rha), 4.59 (1H, brs, H1-29),4.54 (1H, brs, H2-29), 4.33 (1H, d, J = 5.8 Hz, 6-H3 of rha ), 3.92 (2H, t, J=7.5 Hz, H2 of -CH2to imidazole ), 1.63 ( 3H, s, H-30), 1.07 (3H, d, J = 6.2 Hz,6-H3 of rha ), 0.91 (3H, s, H-27), 0.82 (3H, s, H-26), 0.77 (3H, s, H-25), 0.54 (3H, s, H-24). 13 C NMR (101 MHz, DMSO) δ 176.25, 151.36, 137.74, 128.85,119.82, 109.75, 103.37, 100.37, 79.84, 74.68, 73.27, 72.51, 70.91, 70.84,68.60, 68.24, 64.75, 62.92, 55.37, 50.57, 50.13, 49.07, 46.95, 46.62, 44.15,42.82, 42.42, 40.71, 38.87, 38.21, 37.13, 36.62, 36.09, 33.99, 32.90, 31.52, 30.84, 29.38, 25.96, 25.77, 21.06, 19.53, 18.25, 17.53, 16.89, 16.35, 14.72, 13.28.

[0068] B4-24: The preparation method is similar to B4-14, with a yield of 44%; in addition to the amide group, the additional marker hydrogen is the carbon on the tetrahydropyrrolidine (20-50 ppm). 1 H NMR (400 MHz, DMSO- d 6) δ 7.51 (1H, s, H1 of -CONH), 5.06 (1H, brs, 1-H of rha), 4.65 (1H, brs, H1-29), 4.54 (1H, brs, H2-29), 4.33 (1H,d, J = 5.8 Hz, 1-H of ara ), 1.63 (3H, s, H-30), 1.07 (3H, d, J = 6.2 Hz, 6-H3 of rha), 0.91 (3H, s, H-27), 0.84 (3H, s, H-26), 0.78 (3H, s, H-25), 0.54 (3H,s, H-24). 13C NMR (101 MHz, DMSO) δ 175.43, 150.90, 109.23, 102.88, 99.90,79.36, 74.21, 72.78, 72.03, 70.43, 70.35, 68.13, 67.76, 64.27, 62.44, 54.87,54.83, 53.58, 50.07, 49.64, 46.46, 46.23, 42.34, 41.94, 40.20, 38.38, 37.67,37.52, 36.69, 36.14, 33.52, 32.41, 30.33, 28.82, 25.47, 25.29, 23.11, 20.57, 19.05, 17.77, 17.07, 16.39, 15.82, 14.25, 12.79.

[0069] B4-25: The preparation method is similar to B4-14, with a yield of 50%; in addition to the amide group, the additional marker hydrogen is the carbon on the tetrahydropyran ring (20-70 ppm). 1 H NMR (400 MHz, DMSO- d 6) δ 7.33 (1H, s, H1 of -CONH), 5.05 (1H, brs, 1-H of rha), 4.64 (1H, brs, H1-29), 4.52 (1H, brs, H1-29), 4.32 (1H,d, J = 5.9 Hz, 1-H of ara), 1.61 (3H, s, H-30), 1.05 (3H, d, J = 6.2 Hz, 6-H3 of rha), 0.90 (3H, s, H-27), 0.81 (3H, s, H-26), 0.76 (3H, s, H-25), 0.53 (3H,s, H-24). 13C NMR (101 MHz, DMSO) δ 174.85, 150.95, 109.23, 102.92, 99.91,79.38, 74.21, 72.84, 72.03, 70.44, 70.37, 68.13, 67.80, 66.24, 66.15, 64.33,62.44, 54.73, 50.09, 49.73, 46.48, 46.20, 44.71, 42.35, 41.89, 40.22, 38.40,37.72, 36.61, 36.15, 33.50, 32.56, 32.32, 32.09, 30.38, 28.80, 25.49, 25.30, 20.59, 19.07, 17.78, 17.04, 16.40, 15.86, 14.25, 12.83.

[0070] B4-26: The preparation method is similar to B4-14, with a yield of 53%; in addition to the amide group, a new marker hydrogen is added, which is the methyl group on the methyl ester (3.60 ppm). 1 H NMR (400 MHz, DMSO- d 6) δ 7.38 (1H, s, H1 of -CONH), 5.06 (1H,brs, 1-H of rha), 4.66 (1H, brs, H1-29), 4.54 (1H, brs, H2-29), 4.34 (1H, d, J =5.9 Hz, 1-H of ara), 3.60 (3H, s, H3 of -COOCH3), 1.63 (3H, s, H-30), 1.08(3H, d, J = 6.2 Hz, 6-H3 of rha), 0.91 (3H, s, H-27), 0.83 (3H, s, H-26), 0.79 (3H, s, H-25), 0.55 (3H, s, H-24). 13C NMR (101 MHz, DMSO) δ 176.02, 175.24,150.94, 109.20, 102.89, 99.89, 79.36, 74.20, 72.80, 72.03, 70.43, 70.36,68.12, 67.76, 64.28, 62.44, 54.71, 51.50, 50.02, 49.83, 49.73, 46.47, 42.33,41.88, 41.26, 41.21, 38.38, 37.58, 36.63, 36.14, 35.03, 33.50, 31.88, 31.62, 30.37, 28.79, 27.59, 27.04, 25.48, 25.28, 20.58, 19.06, 17.77, 17.03, 16.39, 15.80, 14.24, 12.81.

[0071] B4-27: The preparation method is similar to B4-14, with a yield of 47%; in addition to the amide group, the additional marker hydrogen is the methyl group on the methyl ester (3.58 ppm). 1 H NMR (400 MHz, DMSO- d 6) δ 7.26 (1H, s, H1 of -CONH), 5.05 (1H,brs, 1-H of rha), 4.64 (1H, brs, H1-29), 4.52 (1H, brs, H2-29), 4.43 (1H, d, J =5.9 Hz, 1-H of ara), 3.58 (3H, s, H3 of -COOCH3), 1.62 (3H, s, H-30), 1.06(3H, d, J = 6.2 Hz, 6-H3 of rha), 0.90 (3H, s, H-27), 0.83 (3H, s, H-26), 0.77 (3H, s, H-25), 0.54 (3H, s, H-24). 13C NMR (101 MHz, DMSO) δ 175.21, 174.70,150.96, 109.18, 102.89, 99.90, 79.37, 74.21, 72.80, 72.03, 70.43, 70.35,68.12, 67.76, 64.27, 62.44, 54.70, 51.31, 50.08, 49.73, 46.72, 46.47, 46.21,42.34, 41.88, 41.69, 40.22, 38.38, 37.75, 36.63, 36.14, 33.49, 32.28, 31.25, 30.83, 30.38, 28.80, 27.77, 27.69, 25.48, 25.29, 20.58, 19.05, 17.77, 17.03, 16.38, 15.90, 14.24, 12.82.

[0072] B4-28: The preparation method is similar to B4-14, with a yield of 41%; in addition to the amide group, the additional marker hydrogen is the hydrogen on the propylene group (5-6 ppm). 1 H NMR (400 MHz, DMSO- d 6) δ 7.74 (1H, s, H1 of -CONH), 5.81– 5.72(1H, m, H1 of -CH on CH=CH2), 5.07 (1H, d, J = 13.5 Hz, H1of -CH2on CH=CH2),5.05 (1H, brs, 1-H of rha), 5.00 (1H, d, J = 10.2 Hz, H2of -CH2on CH=CH2), 4.65(1H, brs, H1-29), 4.53 (1H, brs, H2-29), 4.41 (1H, d, J = 5.9 Hz, 1-H of ara ),1.63 (3H, s, H-30 ), 1.07 (3H, d, J = 6.1 Hz, 6-H3 of rha ), 0.91 (3H, s, H-27), 0.82 (3H, s, H-26), 0.78 (3H, s, H-25), 0.54 (3H, s, H-24). 13C NMR (101MHz, DMSO) δ 175.27, 150.90, 136.21, 114.28, 109.22, 102.87, 99.88, 79.36,74.19, 72.78, 72.02, 70.42, 70.35, 68.12, 67.75, 64.26, 62.43, 54.90, 50.08,49.70, 46.46, 46.13, 42.33, 41.92, 40.60, 40.22, 38.38, 37.72, 36.61, 36.14,33.51, 32.34, 30.32, 28.86, 25.46, 25.28, 20.57, 19.06, 17.76, 17.04, 16.39, 15.85, 14.23, 12.80.

[0073] B4-30: The preparation method is similar to B4-14, with a yield of 41%; in addition to the amide group, the additional marker hydrogen is the carbon on the piperidine ring (20-70 ppm). 1 H NMR (400 MHz, DMSO- d 6) δ 7.39 (1H, s, H1 of -CONH), 5.05 (1H,brs, 1-H of rha), 4.62 (1H, brs, H1-29), 4.56 (1H, brs, H2-29), 4.33 (1H, d, J =6.2 Hz, 1-H of ara ), 1.62 (3H, s, H-30), 1.06 (3H, d, J = 6.2 Hz, 6-H3 of rha), 0.90 (3H, s, H-27), 0.83 (3H, s, H-26), 0.78 (3H, s, H-25), 0.54 (3H, s,H-24). 13C NMR (101 MHz, DMSO) δ 175.34, 150.88, 109.23, 102.86, 99.89, 79.36,74.21, 72.75, 72.02, 70.42, 70.34, 68.12, 67.73, 64.24, 62.44, 57.51, 54.88,53.92, 50.05, 49.58, 46.45, 46.24, 42.33, 41.95, 40.20,40.20, 38.37, 37.65,36.70, 36.13, 35.91, 33.51, 32.46, 30.32, 28.84, 25.47, 25.27, 23.91, 20.55, 19.03, 17.76, 17.05, 16.38, 15.85, 14.24, 12.77.

[0074] B4-31: The preparation method is similar to B4-14, with a yield of 52%; in addition to the amide group, the additional marker hydrogen is the hydrogen on the thiazole (7-8 ppm). 1 H NMR (400 MHz, DMSO- d 6) δ 7.72 (1H, s, H1 of -CONH), 7.70 (1H, d, J =3.3 Hz, H1 of thiazole ), 7.58 (1H, d, J = 3.3 Hz, H1´ of thiazole), 5.05 (1H,brs, 1-H of rha), 4.65 (1H, brs, H1-29), 4.54 (1H, brs, H2-29), 4.34 (1H, d, J =5.7 Hz, 1-H of ara ), 1.63 (3H, s, H-30), 1.07 (3H, d, J = 6.1 Hz, 6-H3 of rha), 0.90 (3H, s, H-27), 0.82 (3H, s, H-26), 0.78 (3H, s, H-25), 0.55 (3H,s, H-24). 13C NMR (101 MHz, DMSO) δ 175.68, 167.51, 150.87, 142.21, 119.43,109.24, 102.86, 99.89, 79.36, 74.21, 72.75, 72.02, 70.43, 70.34, 68.12,67.73, 64.24, 62.45, 54.86, 50.07, 49.61, 46.45, 46.14, 42.33, 41.90, 40.21,38.68, 38.37, 37.56, 36.60, 36.13, 33.47, 32.52, 32.34, 30.27, 28.89, 25.47, 25.27, 20.56, 19.03, 17.76, 17.07, 16.39, 15.85, 14.22, 12.79.

[0075] B4-32: The preparation method is similar to B4-14, with a yield of 53%; in addition to the amide group, the additional marker hydrogen is the carbon on the furan ring (20-70 ppm). 1 H NMR (400 MHz, DMSO- d 6) δ 7.58 (1H, s, H1 of -CONH), 5.05 (1H,brs, 1-H of rha), 4.64 (1H, brs, H1-29), 4.56 (1H, brs, H2-29), 4.53 (1H, s,H1 of -OH on C-23 ), 4.43 (1H, d, J = 5.8 Hz, 1-H of ara ), 1.62 (3H, s, H-30), 1.07 (3H, d, J = 6.2 Hz, 6-H3 of rha), 0.90 (3H, s, H-27), 0.83 (3H, s, H-26), 0.78 (3H, s, H-25), 0.54 (3H, s, H-24). 13C NMR (101 MHz, DMSO) δ 175.55,150.93, 109.22, 102.88, 99.89, 79.36, 77.37, 74.18, 72.79, 72.02, 70.43,70.35, 68.12, 67.76, 67.02, 64.27, 62.44, 54.88, 50.10, 49.71, 46.47, 46.17,42.46, 42.33, 41.92, 40.23, 38.39, 37.67, 36.66, 36.14, 33.53, 32.34, 30.33, 28.82, 28.57, 25.47, 25.30, 25.03, 20.58, 19.05, 17.76, 17.04, 16.40, 15.76, 14.24, 12.79.

[0076]

[0077] B4-33: The preparation method is similar to B4-14, with a yield of 66%; in addition to the amide group, the additional marker hydrogen is the carbon on the morpholine ring (40-75 ppm). 1 H NMR (400 MHz, DMSO- d 6) δ 7.44 (1H, s, H1 of -CONH), 5.05 (1H,brs, 1-H of rha), 4.63 (1H, brs, H1-29), 4.57 (1H, brs, H2-29), 4.41 (1H, d, J =5.9 Hz, 1-H of ara ), 1.63 (3H, s, H-30 ), 1.07 (3H, d, J = 6.2 Hz, 6-H3 of rha), 0.91 (3H, s, H-27), 0.83 (3H, s, H-26), 0.78 (3H, s, H-25), 0.54 (3H,s, H-24). 13C NMR (101 MHz, DMSO) δ 175.43, 150.91, 109.23, 102.88, 99.89,79.36, 74.21, 72.77, 72.02, 70.43, 70.35, 68.12, 67.74, 66.15, 64.25, 62.44,57.36, 54.87, 53.19, 53.15, 50.06, 49.61, 46.45, 46.21, 42.33, 41.94, 40.21,38.37, 37.67, 36.66, 36.14, 33.52, 32.44, 30.32, 28.85, 25.47, 25.26, 20.55, 19.03, 17.76, 17.06, 16.38, 15.89, 14.23, 12.77.

[0078] B4-34: The preparation method is similar to that of B4-14, with a yield of 56%; in addition to the amide group, the additional marker hydrogen is the methyl group on the piperidine ring (2.11 ppm). 1 H NMR (400 MHz, DMSO- d 6) δ 7.58 (1H, s, H1 of -CONH), 5.05 (1H,brs, 1-H of rha), 4.65 (1H, brs, H1-29), 4.57 (1H, brs, H2-29), 4.53 (1H, s,H1 of -OH on C-23), 4.35 (1H, d, J = 5.1 Hz, 1-H of ara ), 2.11 (3H, s, H3 of -CH3on piperidine ), 1.62 (3H, s, H-30), 1.07 (3H, d, J = 6.1 Hz, 6-H3 of rha ),0.90 (3H, s, H-27), 0.82 (3H, s, H-26), 0.78 (3H, s, H-25), 0.54 (3H, s, H-24). 13C NMR (101 MHz, DMSO) δ 175.41, 150.96, 109.20, 102.86, 99.90, 79.35,74.22, 72.75, 72.03, 70.44, 70.35, 68.13, 67.73, 64.24, 62.43, 55.16, 54.89,50.12, 49.70, 46.47, 46.20, 46.13, 44.06, 42.33, 41.94, 40.22, 38.39, 37.81,36.65, 36.14, 35.26, 33.59, 32.44, 30.37, 29.92, 29.85, 29.00, 28.86, 25.47, 25.33, 20.62, 19.07, 17.76, 17.07, 16.43, 15.78, 14.24, 12.77.

[0079] B4-35: The preparation method is similar to B4-14, with a yield of 47%; in addition to the amide group, the additional marker hydrogen is the methyl group on the tetrahydropyrrole ring (2.24 ppm). 1 H NMR (400 MHz, DMSO- d 6) δ 7.64 (1H, s, H1 of -CONH), 5.05 (1H, brs, 1-H of rha), 4.65 (1H, brs, H1-29), 4.53 (1H, brs, H2-29), 4.33 (1H,d, J = 5.6 Hz, 1-H of ara), 2.24 (3H, s, H3 of -CH3on pyrrolidine ), 1.63 (3H,s, H-30 ), 1.07 (3H, d, J = 6.1 Hz, 6-H3 of rha ), 0.91 (3H, s, H-27 ), 0.83 (3H, s, H-26 ), 0.78 (3H, s, H-25 ), 0.54 (3H, s, H-24 ). 13C NMR (101 MHz, DMSO) δ 175.49, 150.93, 109.22, 102.88, 99.89, 79.35, 74.20, 72.79, 72.03,70.44, 70.35, 68.13, 67.76, 64.27, 62.44, 59.79, 59.62, 55.50, 54.87, 50.10,49.63, 46.47, 46.13, 42.33, 41.94, 41.81, 40.22, 38.39, 37.72, 37.50, 36.65,36.14, 33.55, 32.44, 30.35, 28.82, 28.31, 25.47, 25.31, 20.60, 19.06, 17.76, 17.05, 16.41, 15.89, 14.24, 12.79.

[0080] B4-36: The preparation method is similar to B4-14, with a yield of 50%; in addition to the amide group, a new marker hydrogen is added, which is the hydroxyl group on cyclohexane (4.57 ppm). 1 H NMR (400 MHz, DMSO- d 6) δ 7.16 (1H, s, H1 of -CONH), 5.05 (1H,brs, 1-H of rha), 4.63 (1H, brs, H1-29), 4.57 (1H, s, H1 of -OH oncyclohexane ), 4.56 (1H, brs, H2-29), 4.53 (1H, s, H1 of -OH on C-23 ), 4.48(1H, d, J = 5.4 Hz, 1-H of ara ), 1.62 (3H, s, H-30 ), 1.07 (3H, d, J = 6.1 Hz,6-H3 of rha ), 0.90 (3H, s, H-27), 0.83 (3H, s, H-26), 0.78 (3H, s, H-25), 0.54 (3H, s, H-24). 13C NMR (101 MHz, DMSO) δ 174.75, 150.98, 109.17, 102.89,99.90, 79.37, 74.21, 72.79, 72.03, 70.43, 70.36, 68.40, 68.12, 67.75, 64.26,62.44, 54.70, 50.08, 49.74, 46.88, 46.47, 46.22, 42.34, 41.87, 40.22, 38.38,37.73, 36.63, 36.13, 34.28, 34.20, 33.50, 32.30, 30.35, 29.92, 28.79, 25.47, 25.28, 20.58, 19.05, 17.76, 17.02, 16.37, 15.88, 14.23, 12.81.

[0081] B4-37: The preparation method is similar to B4-14, with a yield of 67%; in addition to the amide group, the additional marker hydrogen is the hydrogen on the benzene ring (7-8 ppm). 1 H NMR (400 MHz, DMSO- d 6) δ 7.59 (1H, s, H1 of -CONH), 7.27 (1H, d, J =8.0 Hz, H1´ of benzene ), 7.26 (1H, d, J = 8.0 Hz, H1 of benzene ), 7.20 – 7.15(3H, m, each 1H of benzene), 5.05 (1H, brs, 1-H of rha), 4.64 (1H, brs, H1-29), 4.57 (1H, brs, H2-29), 4.42 (1H, d, J = 5.8 Hz, 1-H of ara ), 1.62 (3H, s,H-30 ), 1.07 (3H, d, J = 6.2 Hz, 6-H3 of rha ), 0.89 (3H, s, H-27 ), 0.79 (3H,s, H-26 ), 0.78 (3H, s, H-25 ), 0.54 (3H, s, H-24 ). 13C NMR (101 MHz, DMSO) δ175.40, 150.93, 139.67, 128.56, 128.23, 125.92, 109.21, 102.87, 99.91, 79.38,74.23, 72.75, 72.03, 70.43, 70.35, 68.13, 67.73, 64.23, 62.46, 54.81, 50.07,49.68, 46.46, 46.15, 42.33, 41.89, 40.19, 38.37, 37.63, 36.57, 36.13, 35.26, 33.46, 32.41, 30.27, 28.83, 25.47, 25.26, 20.55, 19.01, 17.76, 17.05, 16.37, 15.84, 14.22, 12.79.

[0082] B4-38: The preparation method is similar to B4-14, with a yield of 52%; in addition to the amide group, the additional marker hydrogen is the hydrogen on the thiophene ring (6.5-7.5 ppm). 1 H NMR (400 MHz, DMSO- d 6) δ 7.69 (1H, s, H1 of -CONH), 7.32(1H, d, J =5.1 Hz, H1of thiophene ), 6.93 (1H, dd, J = 5.1, 3.4 Hz, H2of thiophene), 6.86 (1H, d, J =8.0 Hz, H3of thiophene ), 5.05 (1H, brs, 1-H of rha ), 4.63(1H, brs, H1-29), 4.57 (1H, brs, H2-29), 4.42 (1H, d, J = 5.9 Hz, 1-H of ara ),1.62 (3H, s, H-30), 1.07 (3H, d, J = 6.2 Hz, 6-H3 of rha ), 0.90 (3H, s, H-27), 0.82 (3H, s, H-26), 0.78 (3H, s, H-25), 0.54 (3H, s, H-24). 13C NMR (101 MHz, DMSO) δ 175.60, 150.92, 141.81, 126.87, 124.98, 123.87, 109.24, 102.87,99.90, 79.37, 74.22, 72.76, 72.03, 70.44, 70.35, 68.13, 67.74, 64.24, 62.45,54.84, 50.09, 49.66, 46.46, 46.17, 42.34, 41.91, 40.35, 40.22, 37.61, 36.61,36.13, 33.48, 32.37, 30.30, 29.37, 29.00, 28.89, 25.47, 25.27, 20.57, 19.03, 17.76, 17.06, 16.39, 15.86, 14.23, 12.79.

[0083]

[0084] B4-39: The preparation method is similar to B4-14, with a yield of 70%; in addition to the amide group, the additional marker hydrogen is the hydrogen on the benzene ring (6-8 ppm). 1 H NMR (400 MHz, DMSO- d 6) δ 9.11 (1H, s, H1 of -OH on benzene), 7.53 (1H, s, H1 of -CONH), 6.96 (1H, d, J = 8.5 Hz, H1of benzene ), 6.96 (1H, d, J =8.5 Hz, H2of benzene ), 6.65 (1H, d, J = 8.4 Hz, H3of benzene), 6.65 (1H, d, J =8.4 Hz, H4of benzene), 5.05 (1H, brs, 1-H of rha), 4.63 (1H, brs, H1-29), 4.57(1H, brs, H2-29), 4.53 (1H, s, H1 of -OH on C-23 ), 4.42 (1H, d, J = 5.9 Hz, 1-H of ara ), 1.62 (3H, s, H-30 ), 1.07 (3H, d, J= 6.1 Hz, 6-H3 of rha), 0.89(3H, s, H-27), 0.80 (3H, s, H-26), 0.78 (3H, s, H-25), 0.54 (3H, s, H-24). 13 CNMR (101 MHz, DMSO) δ 175.33, 155.55, 150.95, 129.65, 129.36, 115.02, 109.21,102.87, 99.90, 79.38, 74.23, 72.76, 72.03, 70.44, 70.35, 68.13, 67.74, 64.24,62.46, 54.81, 50.07, 49.69, 46.46, 46.18, 42.34, 41.90, 40.36, 40.19, 38.38,37.65, 36.59, 36.13, 34.47, 33.44, 32.43, 30.30, 28.85, 25.47, 25.26, 20.55, 19.02, 17.77, 17.07, 16.38, 15.82, 14.22, 12.80.

[0085] B4-40: The preparation method is similar to B4-14, with a yield of 33%; in addition to the amide group, the additional marker hydrogen is the hydrogen on the oxazole ring (7-8 ppm). 1 H NMR (400 MHz, DMSO- d 6) δ 8.27 (1H, s, H1 of oxazole), 8.02 (1H,s, H1 of -CONH), 7.76 (1H, s, H2of oxazole), 5.05 (1H, brs, 1-H of rha), 4.63(1H, brs, H1-29), 4.57 (1H, brs, H2-29), 4.41 (1H, d, J = 5.9 Hz, 1-H of ara ),1.62 (3H, s, H-30 ), 1.07 (3H, d, J = 6.2 Hz, 6-H3 of rha ), 0.89 (3H, s, H-27), 0.77 (3H, s, H-26), 0.73 (3H, s, H-25), 0.54 (3H, s, H-24). 13C NMR (101MHz, DMSO) δ 175.53, 151.70, 150.89, 138.39, 135.55, 109.23, 102.87, 99.89,79.36, 74.20, 72.77, 72.03, 70.43, 70.35, 68.13, 67.75, 64.26, 62.44, 54.86,50.08, 49.70, 48.58, 46.45, 46.16, 42.33, 41.90, 40.18, 38.39, 37.54, 36.65,36.13, 33.48, 32.22, 30.33, 28.79, 25.47, 25.29, 20.56, 19.06, 17.76, 17.05, 16.39, 15.64, 14.23, 12.78.

[0086] B4-41: The preparation method is similar to that of B4-14, with a yield of 34%; in addition to the amide group, the new marker is a carboxyl carbon (174 ppm). 1 H NMR (400 MHz, DMSO- d 6) δ 6.82 (1H, s, H1 of -CONH), 5.05 (1H, brs,1-H of rha), 4.65 (1H, brs, H1-29), 4.54 (1H, brs, H2-29), 4.33 (1H, d, J = 5.7Hz, 1-H of ara ), 1.63 (3H, s, H-30), 1.07 (3H, d, J = 6.1 Hz, 6-H3 of rha ),0.91 (3H, s, H-27), 0.82 (3H, s, H-26), 0.78 (3H, s, H-25), 0.54 (3H, s, H-24). 13C NMR (101 MHz, DMSO) δ 174.25, 171.56, 150.86, 109.28, 102.88, 99.88,79.37, 74.22, 72.78, 72.07, 70.44, 70.36, 68.12, 67.71, 64.25, 62.46, 54.76,50.03, 49.51, 48.59, 46.46, 43.93, 42.34, 42.03, 40.20, 38.38, 37.74, 36.90,36.14, 33.46, 32.94, 30.40, 28.95, 25.47, 25.26, 20.52, 19.01, 17.78, 17.06, 16.36, 15.87, 14.26, 12.79.

[0087] B4-42: The preparation method is similar to that of B4-14, with a yield of 54%; in addition to the amide group, a new marker hydrogen is added, which is a methyl group (1.79 ppm) attached to the amide bond. 1 H NMR (400 MHz, DMSO- d 6) δ 7.83 (1H, s, H1 of -CONH), 7.57(1H, s, H1´ of -CONH), 5.06 (1H, brs, 1-H of rha), 4.66 (1H, brs, H1-29), 4.54 (1H, brs, H2-29), 4.34 (1H, d, J = 5.8 Hz, 1-H of ara ), 1.79 (3H, s, H3of -CH3to CONH), 1.63 (3H, s, H-30), 1.07 (3H, d, J = 6.2 Hz, 6-H3 of rha ),0.91 (3H, s, H-27), 0.83 (3H, s, H-26), 0.78 (3H, s, H-25), 0.55 (3H, s, H-24). 13C NMR (101 MHz, DMSO) δ 175.54, 169.14, 150.68, 109.02, 102.68, 99.68,79.15, 74.00, 72.57, 71.82, 70.22, 70.14, 67.91, 67.54, 64.06, 62.23, 54.66,49.84, 49.43, 46.25, 45.99, 42.13, 41.71, 39.99, 38.34, 38.14, 37.41, 36.46,35.93, 33.26, 32.16, 30.12, 28.65, 25.26, 25.06, 22.41, 20.35, 18.83, 17.56, 16.84, 16.18, 15.63, 14.04, 12.59.

[0088] B4-43: Dissolve B4-15 (100 mg, 0.118 mmol) in 4 ml of a tetrahydrofuran:methanol:water (2:1:1) mixture, add sodium hydroxide (42.4 mg, 1.06 mmol), stir at room temperature for 12 hours, add 5 ml of water after the reaction is complete, filter, and precipitate by silica gel column chromatography (dichloromethane:methanol = 8:1 → 6:1) to give 30 mg of an off-white solid, yield 30.5%. 1 H NMR (400 MHz, DMSO- d 6) δ 7.78 (1H, s, H1 of -CONH), 5.05 (1H, brs, 1-H of rha), 4.65 (1H,brs, H1-29), 4.53 (1H, brs, H2-29), 4.33 (1H, d, J = 5.7 Hz, 1-H of ara ), 2.04(2H, t, J = 7.4 Hz, H2 of -CH2to -COOH ), 1.62 (3H, s, H-30), 1.07 (3H, d, J =6.1 Hz, 6-H3 of rha ), 0.90 (3H, s, H-27), 0.83 (3H, s, H-26), 0.78 (3H, s,H-25), 0.54 (3H, s, H-24). 13C NMR (101 MHz, DMSO) δ 176.17, 175.38, 151.01,109.18, 102.87, 99.87, 79.36, 74.22, 72.78, 72.07, 70.47, 70.35, 68.12,67.71, 64.23, 62.46, 54.82, 50.11, 49.72, 46.49, 46.19, 42.34, 41.92, 40.21,38.70, 38.40, 37.77, 36.65, 36.15, 33.80, 33.51, 32.40, 30.39, 28.87, 25.41, 25.32, 20.59, 19.07, 17.78, 17.06, 16.40, 15.87, 14.26, 12.80.

[0089] B4-44: The preparation method is similar to that of B4-14, with a yield of 42%; in addition to the amide group, the additional marker hydrogen is a methylene group (2.19 ppm) attached to the carboxyl group and a carboxyl carbon (175 ppm). 1 H NMR (400 MHz, DMSO- d 6) δ 7.58 (1H, s, H1 of -CONH), 5.06 (1H, brs, 1-H of rha), 4.68 (1H, brs, H1-29), 4.53 (1H, brs,H2-29), 4.39 (1H, s, H1 of -OH on C-23), 4.34 (1H, d, J = 5.7 Hz, 1-H of ara ),2.19 (2H, t, J = 7.1 Hz, H2 of -CH2to -COOH), 1.63 (3H, s, H-30 ), 1.07 (3H, d, J = 6.2 Hz, 6-H3 of rha), 0.91 (3H, s, H-27), 0.83 (3H, s, H-26), 0.78 (3H, s,H-25), 0.54 (3H, s, H-24). 13C NMR (101 MHz, DMSO) δ 175.33, 174.66, 150.97,109.19, 102.86, 99.88, 79.34, 74.24, 72.77, 72.06, 70.49, 70.34, 68.11,67.73, 64.24, 62.43, 54.84, 50.10, 49.69, 46.47, 46.17, 42.34, 41.93, 40.21,38.39, 37.81, 36.64, 36.14, 33.48, 32.43, 30.36, 28.78, 26.55, 25.48, 25.31, 22.08, 21.93, 20.59, 19.06, 17.78, 17.08, 16.41, 15.82, 14.24, 12.80.

[0090] B4-46: The preparation method is similar to B4-14, with a yield of 58%; in addition to the amide group, the additional marker hydrogen is the methyl group on the methyl ester (3.57 ppm). 1 H NMR (400 MHz, DMSO- d 6) δ 7.52 (1H, s, H1 of -CONH ), 4.64 (1H,brs, H1-29), 4.53 (1H, brs, H2-29), 4.35 (1H, s, H1 of -OH on -C3), 4.12 (1H,s, H1 of -OH on -C23), 3.57 (3H, s, H3 of -COOCH3), 3.41 (1H, m, H-19), 2.27(2H, t, J = 7.4 Hz, H2 of -CH2to -COOCH3), 1.62 (3H, s, H-30), 0.91 (3H, s, H-27), 0.83 (3H, s, H-26), 0.78 (3H, s, H-25), 0.51 (3H, s, H-24). 13C NMR (101MHz, DMSO) δ 175.31, 173.25, 150.96, 109.15, 70.30, 64.43, 54.80, 51.13,50.10, 49.66, 48.58, 46.69, 46.15, 41.92, 40.20, 38.13, 37.94, 37.71, 36.61,36.42, 33.57, 33.26, 32.44, 30.33, 28.91, 28.83, 26.75, 25.82, 25.24, 24.14,20.59, 19.03, 17.44, 16.29, 15.82, 14.30, 12.39.

[0091] B4-47: The preparation method is similar to B4-14, with a yield of 31%; in addition to the amide group, a new marker hydrogen is added, which is the hydroxyl group on the cyclopentane ring (4.30 ppm). 1 H NMR (400 MHz, Methanol- d 4 ) δ 5.16 (1H, brs, 1-H of rha), 4.72 (1H, brs, H1-29), 4.59 ((1H, brs, H2-29)), 4.56 (1H, d, J = 4.8 Hz, 1-H ofara ), 4.30 (1H, s, H1 of -OH on cyclopentane ), 4.20 (1H, s, H1 of -OH on C-23), 1.70 ( 3H, s, H-30), 1.25 (3H, d, J = 6.2 Hz, 6-H3 of rha ), 1.02 (3H, s,H-27), 0.98 (3H, s, H-26), 0.90 (3H, s, H-25), 0.69 (3H, s, H-24). 13 C NMR (101MHz, Methanol- d 4) δ 178.21, 152.32, 109.98, 104.30, 101.89, 82.27, 76.66,73.93, 73.65, 73.36, 72.13, 72.02, 70.17, 69.12, 64.74, 64.57, 56.83, 52.02,51.31, 50.77, 48.22, 44.04, 43.62, 42.05, 42.00, 39.93, 39.28, 39.09, 37.82,35.01, 34.69, 34.16, 32.10, 31.97, 30.50, 27.01, 26.69, 22.16, 19.63, 18.77, 17.95, 17.22, 16.78, 15.08, 13.51.

[0092] B4-48: The preparation method is similar to B4-14, with a yield of 32%; in addition to the amide group, a new marker hydrogen is added, which is the hydroxyl group on the cyclopentane ring (4.30 ppm). 1 H NMR (400 MHz, Methanol- d 4 ) δ 5.16 (1H, brs, 1-H of rha), 4.72 (1H, brs, H1-29), 4.60 (1H, brs, H2-29), 4.57 (1H, d, J = 5.0 Hz, 1-H ofara ), 4.30 (1H, s, H1 of -OH on cyclopentane), 4.21 (1H, s, H1 of -OH on C-23), 1.70 (3H, s, H-30), 1.25 (3H, d, J = 6.2 Hz, 6-H3 of rha), 1.02 (3H, s, H-27), 0.99 (3H, s, H-26), 0.90 (3H, s, H-25), 0.69 (3H, s, H-24). 13C NMR (101MHz, D2O) δ 178.18, 152.27, 110.02, 104.30, 101.88, 82.27, 76.65, 73.93,73.66, 73.31, 72.13, 72.02, 70.16, 69.12, 64.74, 64.57, 56.88, 52.02, 51.18,50.66, 48.15, 44.04, 43.63, 42.42, 41.99, 39.92, 39.31, 39.06, 37.81, 34.99,34.68, 34.11, 31.93, 31.72, 30.53, 27.01, 26.69, 22.16, 19.62, 18.77, 17.96, 17.22, 16.77, 15.08, 13.50.

[0093] Using Pulsatilla saponin B4 or its derivatives as the experimental group drugs, the following experiments were conducted.

[0094] Example 8 Cytotoxicity assay 1: HIEC cells were seeded at 5000 cells / well in 96-well plates, 100 μL / well, and cultured using standard methods. A normal control group and a drug-treated group were established. After cell attachment, except for the normal control group, 1 μL of a B4 derivative (excluding the intermediate) was added to each well to achieve a final drug concentration of 50 μM. The cells were incubated for 24 h. After incubation, 10 μL of CCK-8 was added to each well, and the cells were incubated in the dark for 4 h. The absorbance of each well was measured at 450 nm using a microplate reader, and the cell viability of each well was calculated to screen for compounds that showed no significant cytotoxicity in the first round.

[0095] Cytotoxicity experiment 2: THP-1 cells were used at 1×10⁻⁶ cells per cell line. 4 Cells were seeded per well in 96-well plates at 100 μL / well and cultured using standard methods. Once the cells reached 80% confluence, they were induced with 100 ng / mL phorbol-12-myristate-13-acetate (PMA) for 12 h. The original medium was then discarded, and 100 μL of fresh complete medium was added. Zero-concentration wells without cells and a normal control group without drugs were also included. 1 μL of a B4 derivative that showed no significant cytotoxicity in the first round was added to each well, bringing the final drug concentration to 50 μM. The wells were incubated for 24 h. After incubation, 10 μL of CCK-8 was added to each well, and the cells were incubated in the dark for 4 h. The absorbance of each well was measured at 450 nm using a microplate reader, and the cell viability was calculated to screen for compounds that showed no significant cytotoxicity in the second round.

[0096] Calculation formula: Cell viability (%) = [A(drug-treated) - A(blank)] / [A(0-drug-treated) - A(blank)] × 100 A (Drug Addition): Absorbance of pores containing cells, CCK-8, and drug solution. A (Blank): Absorbance of wells containing culture medium and CCK-8 but without cells. A(0 drug added): Absorbance of pores containing cells and CCK-8 without added drugs.

[0097] like Figure 4 As shown in the figure, the cytotoxicity of B4 derivatives at 50 μM is statistically analyzed. The absence of the asterisk (*) indicates that there is no significant difference compared to the normal control, meaning that B4 derivatives do not exhibit significant cytotoxicity at a dose of 50 μM. The figure also shows that there are many non-cytotoxic derivatives.

[0098] Example 9 Nitrite content detection: Raw 264.76 cells were used at 6 × 10⁻⁶ cells per cell line. 4 Cells were seeded per well in 96-well plates at 100 μL / well and cultured using standard methods. Four control groups (N group), a model group (M group), a positive control group (dexamethasone group) (Y group), and a treatment group were established. After cell adhesion, 1 μL of the corresponding drug was administered to achieve a final concentration of 10 μM. One hour later, 1 μL of lps was administered to achieve a final concentration of 1 μg / ml. After incubation for 24 h, 50 μL of culture medium was transferred from each well of the seeded plate to a new 96-well plate. Under light-protected conditions, 50 μL of Griess reagent A and 50 μL of solution B were added to each well. The absorbance of each well was measured at 540 nm using a microplate reader. Figure 5 As shown, the vertical axis represents the nitrite content produced by B4 derivatives under LPS stimulation, and the horizontal axis represents different Pulsatilla chinensis saponin B4 derivatives. The symbol "#" indicates that there is a significant difference between the model and the normal group, that is, the model is established; the symbol "*" indicates that there is a significant difference between the treatment group containing B4 derivatives and the model group, that is, the therapeutic effect is better and there is considerable anti-inflammatory activity.

[0099] Example 10 Western blotting experiment: THP-1 cells were blotting at 2×10⁻⁶ cells per cell line. 6Cells were seeded per well in 6-well plates at 2 ml per well and cultured using standard methods. Once cells reached 80% confluence, they were induced with 100 ng / mL PMA for 12 h, after which the original culture medium was discarded and fresh complete culture medium was added. AB4 and its derivatives were added to the experimental groups to a final concentration of 10 μM, while the control group served as a blank. After 1 h, the blank group was removed, and the other groups were incubated with 2 μL of 1 mg / mL LPS for 2 h. Then, the following steps were performed on ice: the supernatant in the 6-well plate was removed, and pre-chilled PBS (4°C) was gently added along the edge, followed by two washes. After adding 1 mL of PBS, cells were scraped off and placed in 1.5 mL centrifuge tubes. The cells were centrifuged at 2000 g, 4°C for 3 min, and the supernatant was discarded, collecting the cell pellet. 100 μL of LRIPA lysis buffer (with protease and phosphatase inhibitors added before use) was added to each tube, mixed thoroughly, and lysed on ice for 10 min. After re-disrupting the cells using an ultrasonic homogenizer, centrifuge at 12000 g, 4°C for 10 min. Carefully collect the cell supernatant into a new EP tube and store on ice. Refer to the manufacturer's instructions to measure and calculate the total protein content using the BCA protein quantification kit. Dilute the protein sample with PBS, then add 5×SDS-PAGE loading buffer (50 μL β-mercaptoethanol per mL) to achieve a final protein concentration of 2 μg / μL. Denature the protein by boiling at 100°C for 10 min to prevent degradation. Prepare SDS-PAGE gels of different concentrations according to the desired protein molecular weight, and load the protein sample at a concentration of 20 mg / well onto the gel for electrophoresis. Transfer the separated protein sample to a polyvinylidene fluoride (PVDF) membrane. Wash the PVDF membrane three times with Tris-HCl buffer (TBST buffer), 10 min each time. After blocking the PVDF membrane with protein blocking buffer at room temperature for 1 hour, it was washed repeatedly with TBST buffer until the blocking buffer was removed. The PVDF membrane was then incubated overnight with a specific primary antibody at 4°C according to the manufacturer's instructions. The next day, the PVDF membrane was thoroughly washed with TBST buffer and bound to the corresponding secondary antibody at room temperature for 1 hour. It was then washed thoroughly with TBST buffer again. Finally, the protein was exposed and analyzed for color development according to the instructions of the ultrasensitive ECL chemiluminescence kit.

[0100] AB4 and its derivatives can inhibit the activation of key proteins in the NF-κB / MAPK or NLRP3 signaling pathways, such as... Figure 6 as well as Figure 7 As shown in the figure, the numbers on the horizontal axis represent different Pseudobulbus saponin B4 derivatives. The first round of Western blotting was used to display the P-IκBa protein level in THP-1 macrophages. It was found that after LPS stimulation of THP-1 cells for 2 h, the P-IκBa protein level in the model group significantly increased. P<0.01); and compared with the LPS model group, the compounds of the present invention and their AB4 can reduce the level of P-IκBa protein, and several derivatives significantly reduce the level of P-IκBa protein compared with AB4 ( p< (0.05), these results suggest that these derivatives have better anti-inflammatory activity. The derivatives with better activity in the first round were used for the second round of anti-inflammatory activity screening. By detecting relevant indicators of the NLRP3 pathway, the results showed that five compounds, B4-19, B4-28, B4-33, B4-39, and B4-40, had better activity.

[0101] Example 11 The therapeutic effect of Pulsatilla saponin B4 derivative on DNCB-induced atopic dermatitis in mice: Existing technology has demonstrated that AB4 has an ameliorative effect on DNCB (2,4-dinitrochlorobenzene)-induced eczema in mice; and previous in vitro experiments have shown that AB4 derivatives have superior activity to AB4, which are now further verified through in vivo experiments. The experiment was designed as follows: Fifty-six Balb / c mice were randomly divided according to body weight into a normal control group, a model group, a dexamethasone positive control group (3 mg / kg), a Pulsatilla saponin B4 (AB4) control group (6.6 mg / kg), a B4-19 administration group (6.6 mg / kg), a B4-33 administration group (6.6 mg / kg), and a B4-39 administration group (6.6 mg / kg), with 8 mice in each group. One day before the experiment, the fur on the back of the mice was shaved with a disposable scalpel, and an area of ​​about 3 cm x 3 cm was selected for preparation. On day 1, except for the normal control group, other groups were sensitized by applying 5% DNCB 50uL topically to the back of mice. On day 2, the same method was used to reinforce the sensitization once more. On day 3, 1% DNCB 50uL was applied topically to the inside and outside of the right ear of the mice using a pipette to challenge them. Challenge was continued on days 4 and 5 for three consecutive days. An equal amount of acetone matrix was applied to the inside of the left ear. The standard for a successful model was the appearance of varying degrees of redness, papules, vesicles, erosion, exudation, crusting, and desquamation after repeated stimulation of the right ear and back skin of the mice with DNCB solution. On days 1-7, the dexamethasone group received 0.08g of dexamethasone cream applied to the back and inside and outside of the right ear at 4 pm. The AB4, B4-19, B4-33, and B4-39 groups received 200uL of 70% ethanol-water solution (0.66mg / ml) applied to the back and inside and outside of the right ear at 10 am and once at 4 pm. The model group was treated with 200 μL of pure water on the back and inside and outside of the right ear at 10:00 AM, and again at 6:00 PM. This treatment was repeated for 7 days. On the 8th day, the mice were sacrificed, and the spleen and ears were harvested to calculate the spleen index and ear weight difference (ear discs were obtained by punching the same area with a 6 mm diameter punch and weighing them).

[0102] During the experiment, the eczema condition (obvious redness, swelling, macules, erosion, and exudation) on the backs of mice in each group was observed and photographed daily. The Eczema Area and Severity Index (EASI) was used to assess the clinical symptoms of each group of mice, evaluating four indicators: erythema, papules / pustules, scaling, and crusting. Scores were assigned from 0 to 3: 0 = asymptomatic; 1 = mild; 2 = moderate; 3 = severe. The scores for each indicator were summed to obtain the total score. Two observers, in a blinded manner, scored the mice on days 1, 3, 5, and 7 of the intervention, and the results were recorded digitally. Twenty-four hours after the last administration, the thickness of both ears was measured using calipers (three measurements were taken and the average was used), and the thickness difference was calculated as: Thickness difference = Right ear thickness - Left ear thickness. Body weight was measured and recorded daily.

[0103] Figure 8 The condition of the skin on the back of mice with DNCB-induced atopic dermatitis; Figure 9 The condition of the ears in mice with DNCB-induced atopic dermatitis; Figure 10 A schematic diagram showing changes in mouse body weight and back score; Figure 11 This is a schematic diagram illustrating the differences in ear thickness and ear weight in mice. Figure 12 A graph showing the spleen index in mice.

[0104] When local tissues are stimulated by DNCB, cellular mediators such as histamine are released, causing edema by dilating capillaries in the ear skin and mucous membranes and increasing capillary permeability through H1 and H2 receptors. Experimental results showed that after the first application of DNCB on the third day, the auricle skin in the model group was slightly red compared to the control group. With continued drug action, the redness and swelling in the model group worsened from the fourth day, with desquamation appearing. Exudation, ulceration, and crusting began on the fifth day. The difference in ear thickness between the control and model groups was statistically significant on day 7 (P<0.0001), indicating the successful establishment of the eczema mouse model. Compared to the model group, the AB4 group and its derivatives showed significantly reduced ear swelling symptoms at the same time point, with almost no ear ulceration; the dexamethasone positive control group showed slightly worse effects, with some exudation and crusting. The differences in ear thickness between the AB4, B4-19, B4-33, and B4-39 groups and the model group were statistically significant on day 7 (P<0.0001); the differences in ear thickness between the positive control group and the model group were also statistically significant on day 7 (P<0.0001). Furthermore, the experimental results showed that after applying DNCB to the back skin, compared with the blank group, the model group exhibited exudation, erosion, and crusting, producing eczema-like skin lesions. With continued drug action, the skin lesions in the model group worsened from day 2 to 3, showing obvious erythema, skin infiltration, and significant crusting. The EASI comprehensive scores of the control group and the model group were statistically significant on days 3, 5, and 7 (P<0.0001), indicating the successful establishment of the eczema mouse model. Compared with the model group, the AB4 and AB4 derivative groups showed significantly reduced skin lesions at the same time point, smoother skin, less exudation, and milder or earliest crusting; the positive control group had more severe crusting, with almost no crust removal, and obvious scaling and papules. The differences in EASI comprehensive scores between the AB4 derivative group and the model group were statistically significant on days 3 (P<0.001), 5 (P<0.0001), and 7 (P<0.001). The difference in EASI comprehensive scores between the positive control group and the model group was statistically significant on day 3 (P<0.001). Compared with the model group, the AB4, B4-19, B4-33, and B4-39 groups all showed statistically significant differences in back scores (P<0.0001), with AB4 showing less efficacy than the derivative. Furthermore, the experimental results showed that after 7 days of administration, the spleen index of the model group mice was significantly increased, while the spleen index of the Pulsatilla chinensis saponin B4 derivative group was significantly decreased. The spleen index of the dexamethasone group was significantly lower than that of the model group compared to the normal group, indicating that dexamethasone induced immunosuppression in mice, while the Pulsatilla chinensis saponin B4 derivative group could enhance the immunity of mice. All experimental results suggest that Pulsatilla saponin B4 derivatives have a protective effect against eczema lesions caused by DNCB, and the effect is better than that of Pulsatilla saponin B4 and glucocorticoids.

Claims

1. A derivative of Pulsatilla saponin B4, having the following chemical structural formula: ； ； 。 2. The method for preparing the Pulsatilla saponin B4 derivative according to claim 1, characterized in that, The Pulsatilla saponin B4 derivative was prepared using compound AB4 as a raw material by nucleophilic substitution, electrophilic addition, esterification or amidation reaction. Compound AB4 has the following chemical structural formula: 。 3. A pharmaceutical composition comprising, as the active ingredient, the Pulsatilla saponin B4 derivative of claim 1.

4. The use of the Pulsatilla saponin B4 derivative of claim 1 or the pharmaceutical composition of claim 3 in the preparation of anti-inflammatory drugs.

5. The application according to claim 4, characterized in that, The inflammation in question is skin inflammation.

6. The use of the Pulsatilla saponin B4 derivative of claim 1 or the pharmaceutical composition of claim 3 in the preparation of immunomodulatory drugs.

7. The application according to any one of claims 4 to 6, characterized in that, The medications include topical, oral, rectal, or parenteral medications.

8. The application according to claim 6, characterized in that, The dosage forms of the drug include pills, tablets, powders, capsules, granules, ointments, solutions, injections, gels, or suppositories.

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