Pulsatilla saponin B4 derivative as well as preparation method and application thereof

Abstract

The invention discloses an anemoside B4 derivative as well as a preparation method and application thereof, a compound anemoside B4 (abbreviated as B4 or AB4) is taken as a raw material, and the derivative is prepared by respectively carrying out nucleophilic substitution, electrophilic addition, esterification reaction or amidation and other reactions on C-19 exocyclic double bonds or C-28 carboxyl. The compound is used as an active component to prepare medicines with anti-inflammatory and immunoregulation effects. The invention discloses the effect of the B4 derivative on treating inflammatory diseases such as inflammatory bowel disease, atopic dermatitis, eczema and psoriasis for the first time, and the drug effect of the B4 derivative is superior to that of B4 or clinically common drugs. In addition, the invention also discloses that most B4 derivatives do not show obvious cytotoxicity to macrophages for the first time, can reduce the P-I kappa Ba protein level in LPS and ATP induced macrophage NF-kappa B signaling pathways, inhibit the activation of NLRP3 signaling pathways and obviously reduce the Pro-IL-1beta level (p is less than 0.05), namely inhibit the activation of inflammasome pathways, and the effect is superior to that of B4; the results show that the B4 derivative disclosed by the invention has better anti-inflammatory activity.

Description

A derivative of Pulsatilla saponin B4, its preparation method and application

[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 type of derivative in anti-inflammatory treatment, such as in treating or alleviating inflammatory diseases such as skin inflammation and intestinal inflammation, and in autoimmune diseases.

[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, and infectious and non-infectious inflammation. Inflammation is closely related to maintaining homeostasis in the body. During inflammation, damaging factors directly or indirectly cause damage to tissues and cells; on the other hand, inflammatory hyperemia and exudation dilute, kill, and surround the damaging factors; simultaneously, the regeneration of parenchymal and interstitial cells allows damaged tissues to repair and heal. Therefore, inflammation plays a crucial 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, nephritis, enteritis, and skin inflammation. Anti-inflammatory drugs are essential to ensure the body's health and restore normal physiological function.

[0003] As we all know, classic anti-inflammatory drugs are nonsteroidal anti-inflammatory drugs (NSAIDs) and steroids, but both of these drugs have a series of adverse reactions or toxic side effects, such as causing gastrointestinal dysfunction, cardiotoxicity, nephrotoxicity, hypertension, type 2 diabetes, visceral obesity, and atherosclerosis. With the development of the times and people's higher demand for health, more and more people are committed to finding and developing anti-inflammatory drugs with good efficacy, low toxicity and side effects, and easy acceptance by patients.

[0004] Technical issues

[0005] As is well known, classic anti-inflammatory drugs are nonsteroidal anti-inflammatory drugs (NSAIDs) and steroids, but both classes of drugs have a range of adverse reactions or toxic side effects, such as gastrointestinal disorders, cardiotoxicity, 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 dedicated to finding and developing anti-inflammatory drugs with good efficacy, low toxicity, and easy patient acceptance. Technical Solutions

[0006] Existing anti-inflammatory drugs have significant toxic side effects, and some drugs have the drawback of short half-life. In order to understand and 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 drugs (including drugs for skin inflammation, intestinal inflammation, etc.). It is a compound or drug with few side effects, high safety and good efficacy.

[0007] *Pulsatilla chinensis* (Bunge) Regel is a perennial herb belonging to the genus *Pulsatilla* in the family Ranunculaceae. Its dried root is used medicinally for its heat-clearing, detoxifying, blood-cooling, and dysentery-relieving effects. Extensive research has been conducted on the components of *Pulsatilla chinensis*, with sclerosing saponin B4 (AB4) being the main active substance. Previously, the applicant used sclerosing saponin B4 to treat psoriasis, atopic dermatitis, and inflammatory bowel disease, but found its low lipid solubility and efficacy required further improvement. This invention uses sclerosing saponin B4 as a lead compound, and through a series of structural modifications and alterations, unexpectedly improved the drug's activity and lipid solubility. The resulting series of derivatives solved the aforementioned problem of poor drug-likeness of AB4 and exhibited superior activity compared to B4.

[0008] The present invention adopts the following technical solution:

[0009] A derivative of Pulsatilla saponin B4 has the following general chemical structural formula:

[0010] In the formula, R1 is 3-O-α-L-pyranorhamnosyl-(1→2)-α-L-pyranarabinose; R2 is hydroxyl or acetoxy; R3 is 2-allyl, 2-propyl, 3-hydroxypropenyl, 3-bromopropenyl, 2- Ethylene oxide; R4 is 1-oxobenzotriazolyl, methoxy, hydroxy, 28-O-α-L-rhamnopyranose-(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-glucopyranose]-(1→6)-β-D-[2,3,4-triacetoxy-glucopyranosyl]yl, 2-methoxyethylamino, methyl 4-aminobutyrate, cyclopentanamino, 3-chloropropylamino, 2-fluoroethylamino, 1-(3-aminopropyl)benzotriazolyl, cyclohexylamino, 1-cyclopropylethylamino, cyclobutylmethylamino. 1-(3-aminopropyl)imidazolyl, N-(2-aminoethyl)pyrrolidinyl, 4-aminofuranyl, methyl 3-aminocyclopentanecarboxylate, methyl 4-aminocyclohexylcarboxylate, allylamino, 1-(2-aminoethyl)piperidinyl, 2-thiazolylethylamino, tetrahydrofuranmethylamino, N-aminoethylmorpholinyl, 1-methyl-4-piperidinylmethylamino, 1-methylpyrrolidine-3-methylamino, 4-aminocyclohexanol, β-phenylethylamino, 2-thiopheneethylamino, p-hydroxyphenylethylamino, 4-oxazolylmethylamino, glycine, N-(2-aminoethyl)acetamyl, 4-aminobutyric acid, 5-aminovaleric acid, methyl 6-aminohexanoate, 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.

[0011] This invention discloses a pharmaceutical system using the above-mentioned Pulsatilla saponin B4 derivative as the active ingredient.

[0012] This invention discloses the application of the above-mentioned Pulsatilla saponin B4 derivative or drug system in the preparation of anti-inflammatory drugs.

[0013] This invention also discloses the application of the above-mentioned Pulsatilla saponin B4 derivative or drug system in the preparation of immunomodulatory drugs.

[0014] This invention also discloses the application of the above-mentioned Pulsatilla saponin B4 derivative or drug system in the preparation of drugs for improving psoriasis and atopic dermatitis.

[0015] This invention also discloses the application of the above-mentioned Pulsatilla saponin B4 derivative or drug system in the preparation of drugs to improve inflammatory bowel disease.

[0016] In this invention, the Pulsatilla saponin B4 derivative is formulated into a drug for treating or relieving inflammation; or the Pulsatilla saponin B4 derivative is used to treat or relieve inflammation.

[0017] This invention discloses a method for treating or alleviating inflammation, including the step of administering a drug; the drug includes the Pulsatilla chinensis saponin B4 derivative or the drug system. Further, the drug is administered to a patient in need to achieve treatment or relief of inflammation, wherein a patient in need refers to a patient requiring treatment or relief of inflammation.

[0018] In this invention, the inflammation includes superficial inflammation and internal inflammation. More preferably, the inflammation includes inflammatory diseases such as skin inflammation and intestinal inflammation (e.g., inflammatory bowel disease). As an example, this invention discloses the application of the above-mentioned Pulsatilla chinensis saponin B4 derivative or pharmaceutical system in the preparation of drugs for treating atopic dermatitis, psoriasis, erythema, scaling, thickened skin lesions, ichthyosis, melasma, keratosis, keratoderma, dry and rough skin, colitis (or inflammatory bowel disease), etc.

[0019] In this invention, the active ingredient of the drug (such as an anti-inflammatory drug or an immunomodulatory drug) is a derivative of Pulsatilla saponin B4; it may also include conventional pharmaceutical excipients, including one or more of diluents, dispersants, binders, lubricants, and penetration enhancers.

[0020] In this invention, the drug (such as an anti-inflammatory drug or an immunomodulatory drug) includes drugs administered via topical, oral, injection, rectal, or parenteral routes. In the above-mentioned methods of treating or relieving inflammation, administration includes topical application, injection, suppository, oral administration, or inhalation.

[0021] In this invention, the drug (such as an anti-inflammatory drug or an immunomodulatory 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.

[0022] This invention discloses a method for preparing the Pulsatilla saponin B4 derivative, which uses B4 as a raw material and employs reactions such as nucleophilic substitution, electrophilic addition, esterification, or amidation to prepare the Pulsatilla saponin B4 derivative.

[0023] The Pulsatilla saponin B4 derivative disclosed in this invention did not show significant cytotoxicity against THP-1 macrophages, but it could reduce the level of P-IκB 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<0.05), with better effects than B4. These results indicate that the B4 derivative of this invention has better anti-inflammatory and immunomodulatory activities. In DNCB-induced atopic dermatitis (eczema) mice, the B4 derivative showed significant therapeutic effects on atopic dermatitis, significantly reducing ear swelling and effectively improving the skin ulceration and edema on the back and ears of mice, with better effects than the positive control drugs dexamethasone and B4.

[0024] This invention also uses a mouse colitis model, administering B4-39, B4, and the control drug mesalazine via gavage. Studies on mouse colon length, DAI score, biochemical indicators, and pathological observations showed that derivative B4-39 was more effective than B4 and mesalazine in treating colitis. Similarly, B4-39 was superior to B4 and mesalazine in inhibiting colonic epithelial cell apoptosis, with lower toxicity. Simultaneously, the B4 derivative exhibited certain immunomodulatory effects while treating the aforementioned diseases; however, the derivative of this invention did not have the immunosuppressive side effects of dexamethasone. Beneficial Effects

[0025] This invention uses Pulsatilla saponin B4 derivative as the sole active ingredient, applied topically to psoriasis lesions to achieve effective treatment. Furthermore, the medication does not contain other Pulsatilla extracts. Existing traditional Chinese medicine treatments for or relief of psoriasis are typically done in decoction form, which is less effective than Western medicine. Furthermore, decoction preparation is inconvenient and can cause side effects. Long-term use of even traditional Chinese medicine can irritate the gastrointestinal tract. The topical medication of this invention is convenient to use and achieves better therapeutic effects than existing Western medicines while avoiding side effects. Animal experiments show that after imiquimod modeling, the animals' body weight began to decrease on the second day, and subsequently decreased daily in the model groups. The halometasone group experienced a sharp decrease in body weight, while the B4-33 and B4-39 groups showed significantly better body weight from the fourth day onwards (p < 0.05). In the PASI scores of the treatment groups, B4-33 and B4-39 both improved the skin of psoriatic mice, with better effects than the B4 group and comparable to the halometasone group. After modeling, the spleens of mice were significantly enlarged, showing a significant difference from the normal group. The B4-39 administration group also showed a certain improvement in spleen index, with a significant difference. In summary, this invention uses Pulsatilla chinensis saponin B4 derivatives to treat psoriasis, not only solving the toxicity and side effects problems of existing clinically effective drugs, but also unexpectedly achieving better therapeutic effects than B4, demonstrating inventiveness. Compared with other administration methods, the Pulsatilla chinensis saponin B4 derivatives disclosed in this invention, as a topical drug for treating psoriasis, have the advantages of convenient and safe administration. In particular, this invention avoids the problems of significant side effects and high dosage associated with oral medications. The topical administration effect is significantly better than existing clinical drugs, achieving unexpected technical effects. For example, the psoriasis treatment drug uses Pulsatilla chinensis saponin B4 derivatives as the active ingredient, without other Pulsatilla chinensis extracts, and small doses can achieve better technical effects than the currently considered effective halometasone cream. Animal experimental results show that Pulsatilla chinensis saponin B4 derivatives have a protective effect on psoriatic skin lesions, and the effect is better than that of glucocorticoid drugs. In particular, the Pulsatilla saponin B4 derivative of this invention has unexpectedly achieved significantly better technical progress than B4.

[0026] Figure 1 is a schematic diagram of the reaction in the preparation scheme (1) of the Pulsatilla saponin B4 derivative of the present invention.

[0027] Figure 2 is a reaction diagram of the preparation scheme (2) of the Pulsatilla saponin B4 derivative of the present invention.

[0028] Figure 3 is a reaction diagram of the preparation scheme (3) of the Pulsatilla saponin B4 derivative of the present invention.

[0029] Figure 4 shows the cytotoxicity of AB4 derivatives to HIEC and THP-1 cells at 50 μM.

[0030] Figure 5 shows the nitrite production of non-cytotoxic AB4 derivative (10 μM) under LPS stimulation.

[0031] Figure 6 shows the Western blotting diagram of the anti-inflammatory activity of the AB4 derivative.

[0032] Figure 7 shows the Western blotting statistical results of the anti-inflammatory activity of AB4 derivatives.

[0033] Figure 8 shows the skin condition on the back of mice with DNCB-induced atopic dermatitis.

[0034] Figure 9 shows the ear condition of mice with DNCB-induced atopic dermatitis.

[0035] Figure 10 is a schematic diagram of mouse weight changes and back score.

[0036] Figure 11 is a schematic diagram of the difference in ear thickness and ear weight in mice.

[0037] Figure 12 is a schematic diagram of the spleen index in mice.

[0038] Figure 13 shows the effects of Pulsatilla saponin B4 derivatives B4-33, B4-39, and A3-9 on the body weight of psoriatic mice; B4-33 group vs. halometasone group: *p<0.05, **p<0.01, ***p<0.001; B4-39 group vs. halometasone group: #p<0.05, ##p<0.01; A3-9 group vs. halometasone group: $p<0.05, $$p<0.01.

[0039] Figure 14 shows the effect of Pulsatilla saponin B4 derivative on the external morphology of psoriatic skin in mice.

[0040] Figure 15 shows the effect of B4-33, a derivative of Psoriatic saponin B4, on the Psoriasis Severity Index (PASI) in psoriasis mice. Normal group vs. model group: **p<0.01, ***p<0.001, ***p<0.0001; B4-33 treatment group vs. model group: #p<0.05, ##p<0.01, ###p<0.001, ####p<0.0001; Halometasone treatment group vs. model group: $p<0.05, $$p<0.01, $$$$p<0.0001; B4 treatment group vs. model group: &&p<0.01, &&&p<0.001.

[0041] Figure 16 shows the effect of Pulsatilla saponin B4 derivative B4-39 on the Psoriasis Severity Index (PASI) in psoriasis mice. Normal group vs. model group: **p<0.01, ***p<0.001, ***p<0.0001; B4-39 treatment group vs. model group: #p<0.05, ##p<0.01, ###p<0.001, ####p<0.0001; Halometasone treatment group vs. model group: $p<0.05, $$p<0.01, $$$$p<0.0001; B4 treatment group vs. model group: &&p<0.01, &&&p<0.001.

[0042] Figure 17 shows the effect of Pulsatilla saponin B4 derivative A3-9 on the Psoriasis Severity Index (PASI) in psoriasis mice. Normal group vs. model group: **p<0.01, ***p<0.001, ***p<0.0001; A3-9 treatment group vs. model group: #p<0.05, ##p<0.01, ###p<0.001, ####p<0.0001; Halometasone treatment group vs. model group: $p<0.05, $$p<0.01, $$$$p<0.0001; B4 treatment group vs. model group: &&p<0.01, &&&p<0.001.

[0043] Figure 18 shows the effects of Pulsatilla saponin B4 derivatives B4-33, B4-39, and A3-9 on the spleen of psoriatic mice. *p<0.05, ****p<0.0001.

[0044] Figure 19 shows the effects of Pulsatilla saponin B4 derivatives B4-33, B4-39, and A3-9 on the thymus of psoriatic mice. ***p<0.001, ***p<0.0001.

[0045] Figure 20 shows the effects of B4-39 on colon length (A & B), DAI score (C), and body weight (D) in UC mice.

[0046] Figure 21 shows the effect of B4-39 on pathological changes in the colon tissue of UC mice.

[0047] Figure 22 shows the effect of B4-39 on apoptosis-related proteins in the colon tissue of UC mice. (A) Western blotting bands, (B) ratio of cleaved-caspase 3 / caspase 3 protein, (C) ratio of Bax / Bcl-2 protein.

[0048] Figure 23 shows the effect of B4-39 on inflammation-related proteins in the colonic tissue of UC mice. (A) Western blotting protein bands; (B) IκBa level; (C) INOS level; (D) COX2 level; (E) P-P65 level.

[0049] Figure 24 shows the effect of B4-39 on epithelial intestinal barrier-related proteins in UC mice. (A) Western blotting protein bands; (B) Occludin level; (C) Claudin expression level; (D) ZO-1 level.

[0050] Figure 25 shows the effect of B4-39 on the release of inflammatory factors in the colonic tissue of UC mice. (A) Level of IL-6; (B) Level of IL-1β; (C) Level of TNF-α. Embodiments of the present invention

[0051] 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 (B4 or AB4) has therapeutic effects on atopic dermatitis, but its molecular weight and water solubility are too large, resulting in low bioavailability.

[0052] Psoriasis is a chronic inflammatory skin disease whose pathogenesis is considered multifactorial, involving genetic, environmental, and immune factors. It is a complex disease with a multifactorial inheritance pattern, involving interactions between multiple genes. Current research has identified several psoriasis susceptibility gene loci, including PSORS1 on 6p21.3, PSORS2 on 17q, PSORS3 on 4q, PSORS4 on 1cen-q21, PSORS5 on 3q21, and PSORS6 on 19p13. Most treatments for psoriasis provide short-term relief, not a complete cure. Clinically, treatments are mainly divided into chemical drugs and biological agents. Chemical drugs are further divided into topical and oral medications. Topical medications include emollients, moisturizers, vitamin D3 derivatives, retinoids, corticosteroids, calcineurin inhibitors, AhR agonists, anti-human IL-8 monoclonal antibodies, and tar preparations. Oral medications include retinoids, methotrexate, and cyclosporine. However, the efficacy of current chemical drugs is not significant, and they have many adverse reactions. For example, topical preparations often cause skin atrophy, swelling, stinging, itching, and burning sensations. Furthermore, long-term use of glucocorticoids also produces numerous adverse reactions. Currently, biologics for psoriasis treatment mainly fall into four categories: TNF-α inhibitors, IL-12 / 23 inhibitors, IL-17 inhibitors, and IL-23 inhibitors. While emerging biologics targeting psoriasis show promising clinical application prospects, they are costly, prone to drug resistance with long-term use, and, in addition to skin reactions, may also cause gastrointestinal reactions, infections, and autoimmune diseases. Therefore, more drugs are needed to treat psoriasis.

[0053] Inflammatory bowel disease (IBD), primarily including ulcerative colitis (UC) and Crohn's disease, is a chronic, relapsing inflammatory disease. UC mainly affects the colonic mucosa and proximal rectum, characterized by diffuse involvement, recurrent and progressively worsening symptoms, persistent nature, and a certain rate of malignant transformation. Typical clinical symptoms of UC include chronic diarrhea, bloody mucus stools, abdominal pain, weight loss, fatigue, and tenesmus. The persistence of these symptoms not only affects patients' quality of life but can also lead to psychological stress and social dysfunction. The pathogenesis of UC is complex, and the exact cause is not fully understood, but it is generally believed to be related to multiple factors, including genetic susceptibility genes, environmental factors, immune system abnormalities, and gut microbiota imbalance. Pathophysiologically, UC involves the disruption of the intestinal mucosal barrier, abnormal activation of immune cells, and an imbalance between pro-inflammatory and anti-inflammatory factors. Currently, drug therapy is the core of UC treatment strategies. Drug therapy strategies for UC encompass a variety of medications to meet the treatment needs of different stages and severity levels. Topical medications, such as 5-aminosalicylic acid (5-ASA) drugs like sulfapyridine, reduce inflammation by acting directly on the colonic mucosa and are suitable for patients with mild to moderate UC. Glucocorticoids, such as prednisone, with their rapid and potent anti-inflammatory properties, are often used to quickly induce clinical remission, especially suitable for patients with moderate to severe UC. Immunosuppressants, including azathioprine and methotrexate, help reduce chronic inflammation by inhibiting the proliferation and activity of immune cells and are suitable for patients requiring long-term maintenance remission. Biologics, such as ustekinumab and adalimumab, act as targeted therapies against inflammatory mediators such as tumor necrosis factor (TNF)-α, providing precise anti-inflammatory effects, particularly suitable for patients who do not respond well to conventional treatments. For UC patients who do not respond to drug therapy or have serious complications, surgical treatment may be necessary. Common surgeries include colectomy, which involves partial or total removal of the colon and rectum to eliminate diseased tissue. While current medications for ulcerative colitis (UC) play a crucial role in controlling symptoms, they generally have certain side effects, such as potential nephrotoxicity, gastrointestinal discomfort, or bone marrow suppression. Furthermore, for some patients with severe or refractory UC, surgical treatment and the use of expensive biologics can impose a heavy financial burden. Therefore, developing drugs that are both highly effective and low in toxicity, and at a lower cost, is of great significance for improving treatment outcomes and quality of life for UC patients. This would not only provide patients with more treatment options but also alleviate their economic and psychological burden, thereby improving overall disease management.

[0054] 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 reactions.

[0055] The following scheme was adopted to modify and transform the structure of AB4:

[0056] (1) Using AB4 as 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 some Pulsatilla saponin B4 derivatives. The reaction diagram is shown in Figure 1. The reaction conditions were: (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.

[0057] (2) After obtaining intermediate A3 according to scheme (1), the C28 carboxyl group is used as the modified group, and amide condensation is 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 saponin B4. The reaction schematic diagram is shown in Figure 2. The reaction conditions are: (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.

[0058] (3) Using AB4 as raw material, the double bond part was modified by oxidation, reduction and nucleophilic substitution while protecting the whole sugar group to obtain some Pulsatilla saponin B4 derivatives. See Figure 3 for the reaction diagram. 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.

[0059] 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.

[0060] The pharmaceutical system disclosed in this invention uses the aforementioned Pulsatilla saponin B4 derivative as the active ingredient and further includes a pharmaceutically acceptable carrier. The active ingredient and pharmaceutical system are used to prepare therapeutic drugs for inflammatory or immune diseases. For example, the drug contains a therapeutically effective amount of the Pulsatilla saponin B4 derivative or its hydrochloride, perchlorate, mesylate, phosphate, citrate, or sulfate, and a pharmaceutically acceptable carrier.

[0061] 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 system 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.

[0062] 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.

[0063] This invention discloses the application of a Pulsatilla saponin B4 derivative in the preparation of anti-inflammatory drugs. The derivative 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 system 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 sorbitol 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.

[0064] Existing technologies disclose the application of Pulsatilla saponin B4 (abbreviated as AB4 or B4) in the treatment of inflammation. However, AB4 is highly water-soluble, has a short half-life, and low oral bioavailability, thus limiting 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 of the Pulsatilla saponin B4 derivative of this invention is shown in Figures 1 to 3, and the in vivo and in vitro activity results are shown in Figures 4-25.

[0065] 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 are weight percentages and weight parts. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those familiar with 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, and the animal experiments involved comply with the relevant requirements of Suzhou University. 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); 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.

[0066] 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 p-values ​​were typically used to represent the differences 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 "***".

[0067] The specific structures of the various Pulsatilla saponin B4 derivatives in the following examples are as follows.

[0068] Example 1

[0069] B4-1: Pulsatilla saponin B4 (1 g, 0.819 mmol) was dissolved in 20 ml of pyridine, and 2.5 ml of acetic anhydride and DMAP (14 mg, 0.082 mmol) were added. After stirring at room temperature for 28 hours, 50 ml of ethyl acetate was added to the reaction solution, followed by 100 ml of water 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, and NBS (48 mg, 0.2702 mmol) was added. After stirring at room temperature for 24 hours, the intermediate 3-O-α-L-[3,4-diacetoxy-pyranorhamnosyl]-(1→2)-α-L-[2,3,4-triacetoxy-pyranaramonosyl]-3β,23-dihydroxylusane-Δ 20(29) Alkene-30-bromo-28-O-α-L-[2,3,4-triacetoxy-rhamnopyranose]-(1→4)-β-D-[2,3,6-triacetoxy-glucose pyranose]-(1→6)-β-D-[2,3,4-triacetoxy-glucose pyranose]. The above intermediate (100 mg, 0.052 mmol) was dissolved in 4 mL of a dichloromethane:methanol mixture (2:1), and potassium carbonate (161 mg, 1.165 mmol) was added. The mixture was stirred at room temperature for 16 hours. After the reaction was complete, the solution was concentrated under reduced pressure and purified by preparative liquid chromatography (60% methanol-water) to give 12 mg of a white solid, with a yield of 22%. 11H NMR (400 MHz, Methanol-d4) 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 of rha), 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 of rha), 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 13C 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.

[0070] Example 2

[0071] 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 extraction to obtain the organic phase. The organic phase was washed twice with water, dried, and then evaporated to dryness to obtain the intermediate 3-O-α-L-[3,4-diacetoxy-rhamnosylpyranosyl]-(1→2)-α-L-[2,3,4-triacetoxy-arabinosylpyranosyl]-3β,23-dihydroxylusane-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(400MHz,Chloroform-d)δ5.04(1H,brs,1-H of rha),4.42(1H,d,J=6.4Hz,1-H of ara),4.11(1H,d,J=11.5Hz,H1-23),3.58(1H,d,J=11.5Hz,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=6.2Hz,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=7.2Hz,H-29),0.77(3H,s,H-24),0.75(3H,d,J=6.7Hz,H-30). 13C NMR (101MHz, CDCl3) δ181.66,170.58,170.52,170.44,170.29,170.19,169.78,103.69,98.31, 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.

[0072] 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 (400MHz, DMSO-d6) δ4.96(1H,brs,1-H of rha),4.22(1H,d,J=4.6Hz,1-H of ara),4.04(1H,d,J=11.1Hz,H1-23),1.06(3H,d,J=5.8Hz,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=6.1Hz,H-29),0.72(3H,d,J=6.2Hz,H-30),0.69(3H,s,H-24). 13C NMR (101MHz, DMSO) δ170.16,103.36,100.28,80.72,74.67,72.23,72.03,70. 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.

[0073] Example 3

[0074] B4-6: The intermediate 3-O-α-L-[3,4-diacetoxy-rhamnosylpyranosyl]-(1→2)-α-L-[2,3,4-triacetoxy-arabinosylpyranosyl]-3β,23-dihydroxylusane-Δ 20(29) Alkene-28-acid (1 g, 0.998 mmol) was dissolved in 10 mL of chloroform. At low temperature, m-chloroperoxybenzoic acid (207 mg, 1.20 mmol) was added. After complete dissolution, the temperature was raised 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) gave 305 mg of white solid, yield 30%. 1 H NMR(400MHz,Chloroform-d)δ5.22(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=6.4Hz,1-H of ara),4.12(2H,s,H2 of 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),2.03(3H,s,H3 of-OAc),1.96(3H,s,H3 of-OAc),1.21(3H,d,J=6.2Hz,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). 13C NMR (101MHz, CDCl3) δ181.05,170.90,170.84,170.76,170.61,170.51,170.1 0,155.14,107.35,104.01,98.63,82.36,77.68,74.80,72.34,71.48,70.01,6 9.05, 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.

[0075] 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(400MHz,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=11.3Hz,H1-23),3.69(1H,d,J=11.8Hz,H2-23),3.87(2H,s,H2of H-30),1.05(3H,d,J=6.1Hz,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). 13C NMR (101MHz, DMSO) δ170.49,155.94,106.07,103.68,100.58,81.06,74.97,72. 54,72.33,70.95,70.72,68.92,67.85,63.40,55.94,50.66,49.42,48.14,42.8 5,42.71,42.38,42.01,40.78,38.63,38.07,36.85,34.12,32.44,29.56,29.11,27.09,25.75,22.63,21.22,18.30,18.01,16.88,16.28,14.70,14.49,12.86.

[0076] Example 4

[0077] B4-8: The intermediate 3-O-α-L-[3,4-diacetoxy-rhamnosylpyranosyl]-(1→2)-α-L-[2,3,4-triacetoxy-arabinosylpyranosyl]-3β,23-dihydroxylusane-Δ 20(29) Alkene-28-acid (500 mg, 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(400MHz,DMSO-d6)δ12.09(1H,s,H1 of-COOH),5.02(1H,brs,1-H of rha),4.50(1H,d,J=6.9Hz,1-H of ara),3.99(1H,d,J=11.4Hz,H1-23),2.56(2H,dd,J=3.4Hz,H-29),2.10(3H,s,H3 of-OAc),2.07(3H,s,H3 of-OAc),2.06(3H,s,H3 of-OAc),2.02(3H,s,H3 of-OAc),1.95(3H,s,H3 of-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(101MHz,DMSO)δ177.14,170.10,169.90,169.88,169.64,169.61,169.48,102.35 ,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.

[0078] Example 5

[0079] B4-9: The intermediate 3-O-α-L-[3,4-diacetoxy-rhamnosylpyranosyl]-(1→2)-α-L-[2,3,4-triacetoxy-arabinosylpyranosyl]-3β,23-dihydroxylusane-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%. 1H NMR(400MHz,Chloroform-d)δ5.21(1H,brs,1-H of rha),5.14(1H,brs,H1-29),5.04(1H,brs,H2-29),4.41(1H,d,J=6.3Hz,1-H of ara),4.11(1H,d,J=11.4Hz,H1-23),3.99(2H,s,H-30),3.88(1H,d,J=10.9Hz,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=6.1Hz,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 (101MHz, CDCl3) δ170.56,170.50,170.42,170.28,170.17,169.77,151.40,113.62,103.65,98. 32,82.02,77.36,74.50,71.98,71.19,69.71,68.75,67.97,67.28,65.27,62.82,56.52,50.86,48.1 8,43.19,42.50,42.12,40.87,38.76,38.54,36.92,34.23,33.18,29.84,29.46,27.35,26.96,25.84,22.83,21.15,21.10,21.06,20.94,20.91,20.79,18.10,17.47,16.77,16.17,14.69,14.25,12.62.

[0080] Example 6

[0081] 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 obtain 100 mg of white solid, yield 66.7%. 1H NMR(400MHz,Methanol-d4)δ5.19(1H,brs,1-H of rha),5.09(1H,brs,H1-29),4.96(1H,brs,H2-29),4.59(1H,d,J=4.7Hz,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=6.3Hz, 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)δ153.40,113.70,104.28,101.88,82.26,76.66,76.19,73 .93,73.64,72.14,72.02,70.16,69.11,66.65,64.72,64.57,58.54,57.53,5 1.91,51.03,44.39,44.04,43.63,41.89,39.91,39.68,37.81,35.03,34.27,33.21,30.85,28.23,26.69,22.23,18.77,17.95,17.20,16.69,15.14,13.51.

[0082] Example 7

[0083] B4-11: The intermediate 3-O-α-L-[3,4-diacetoxy-rhamnosylpyranosyl]-(1→2)-α-L-[2,3,4-triacetoxy-arabinosylpyranosyl]-3β,23-dihydroxylusane-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%. 11H NMR (400 MHz, Chloroform-d) δ 8.08 (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 = 11.2 Hz, 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). 13 13C NMR (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.

[0084] Example VIII

[0085] B4-13: The intermediate 3-O-α-L-[3,4-diacetoxy-rhamnosylpyranosyl]-(1→2)-α-L-[2,3,4-triacetoxy-arabinosylpyranosyl]-3β,23-dihydroxylusane-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 methyl iodide (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(400MHz,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=6.4Hz,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=6.2Hz,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 (101MHz, CDCl3) δ176.76,170.55,170.49,170.41,170.26,170.18,169.74,150.72,109.72,103.67, 98.27,82.04,74.42,72.04,71.18,69.71,68.74,68.00,67.25,65.27,62.87,56.68,51.40,50.89,49.6 1,48.18,47.09,42.45,42.11,40.81,38.75,38.38,37.07,36.91,34.17,32.26,30.75,29.83,29.74,25.86,25.64,21.15,21.09,21.05,20.93,20.89,20.78,19.52,18.09,17.45,16.73,16.11,14.66,12.64.

[0086] Example 9

[0087] B4-14: Compound A3(3-O-α-L-pyranorhamnosyl-(1→2)-α-L-pyranarabinosyl-3β,23-dihydroxylusulphoside-Δ) 20(29) 100 mg of olefinic acid (0.1333 mmol) was dissolved in 4 mL of DMF, and 70 μL of DIEA (0.3999 mmol) was added. The mixture was stirred at room temperature for 8 hours. After the intermediate was completely formed, 15 mg of methoxyethylamine (0.2 mmol) and 116 μL of DIEA (0.6665 mmol) were added. 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(400MHz,DMSO-d6)δ7.58(1H,s,H1 of-CONH),5.05(1H,brs,1-H of rha),4.65(1H,brs,H1-29),4.53(1H,brs,H1-29),4.33(1H,d,J=5.9Hz,1-H of ara),3.22(3H,s,H3 of-OCH3),1.62(3H,s,H-30),1.07(3H,d,J=6.2Hz,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(101MHz,DMSO)δ175.63,150.93,109.24,102.91, 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.

[0088] Example 10

[0089] 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.

[0090] 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(400MHz,DMSO-d6)δ7.60(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=5.9Hz,1-H of ara),3.57(3H,s,H3 of-COOCH3),2.28(2H,t,J=7.5Hz,H2 of-CH2-COOCH3),1.62(3H,s,H-30),1.07(3H,d,J=6.2Hz,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 (101MHz, DMSO) δ176.18,173.79,151.55,109.88,103.54,100.53,80.00,74.83,73. 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.

[0091] 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). 1H NMR(400MHz,DMSO-d6)δ7.28(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=5.7Hz,1-H of ara),3.97(1H,p,J=6.7Hz,H1 of-CH on cyclopentane),1.63(3H,s,H-30),1.07(3H,d,J=6.2Hz,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(101MHz,DMSO)δ175.14,150.99,109.17,102.89,99.90,79.37,74.21,72.79,72.03 ,70.43,70.36,68.12,67.76,64.28,62.44,54.64,50.23,50.10,49.79,46.48,46.17,4 2.34,41.88,40.22,38.39,37.66,36.58,36.15,33.51,32.43,32.32,31.46,30.41,28.77,25.49,25.30,23.57,23.53,20.59,19.07,17.77,17.05,16.39,15.81,14.24,12.82.

[0092] 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(400MHz,DMSO-d6)δ7.66(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=5.8Hz,1-H of ara),3.60(2H,t,J=6.7Hz,H2of-CH2Cl),1.63(3H,s,H-30),1.07(3H,d,J=6.2Hz,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(101MHz,DMSO)δ175.91,151.11,109.47,103.12,100.10,79.57,74.39,73.05,7 2.24,70.65,70.57,68.33,68.01,64.53,62.64,55.09,50.30,49.86,48.81,46.68,4 6.38,43.34,42.55,42.14,40.43,38.61,37.89,36.88,36.36,36.11,33.73,32.70,30.56,29.07,25.70,25.51,20.80,19.28,17.99,17.26,16.63,16.04,14.46,13.03.

[0093] 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(400MHz,DMSO-d6)δ7.76(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=5.9Hz,1-H of ara),1.62(3H,s,H-30),1.06(3H,d,J=6.2Hz,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(101MHz,DMSO)δ175.99,150.88,109.27,102.89,99.91,83.03,81.39,79.37, 74.23,72.78,72.04,70.45,70.37,68.14,67.76,64.27,62.45,54.96,50.07,49.6 9,46.47,46.23,42.35,41.92,40.21,38.39,37.57,36.72,36.15,33.49,32.28,30.33,29.84,25.48,25.29,20.56,19.07,17.78,17.05,16.39,15.72,14.27,12.82. 19 F NMR (377MHz, DMSO) δ 19.44.

[0094] 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(400MHz, DMSO-d6)δ8.06(1H,d,J=8.4Hz,H1 of benzene),7.92(1H,d,J=8.4Hz,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=5.9Hz,1-H of ara),1.62(3H,s,H-30),1.08(3H,d,J=6.2Hz,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(101MHz,DMSO)δ175.79,150.86,142.75,128.28,126.97,124.92,119.64,109.41,109.24,1 02.88,99.89,79.35,79.00,74.18,72.79,72.02,70.43,70.36,68.13,67.76,64.27,62.43,54.8 8,50.02,49.61,46.42,46.16,42.32,41.86,40.10,38.37,37.68,36.65,36.09,34.66,33.39,32.31,30.31,28.84,28.40,25.46,25.26,20.51,19.05,17.77,17.01,16.35,15.54,14.18,12.79.

[0095] B4-20: The preparation method is similar to B4-14, with a yield of 47%; the additional marker besides the amide group is the carbon atom on cyclohexane (20-40 ppm). 1H NMR(400MHz,DMSO-d6)δ7.17(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=5.9Hz,1-H of ara),1.62(3H,s,H-30),1.06(3H,d,J=6.2Hz,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). 13 C NMR(101MHz,DMSO)δ174.51,151.00,109.18,102.91,99.90,79.37,74.20,72.83,72.03, 70.43,70.36,68.12,67.79,64.32,62.43,54.68,50.09,49.76,48.60,47.24,46.48,46.2 3,42.34,41.88,40.23,38.40,37.79,36.62,36.14,33.50,32.53,32.36,32.06,30.40,28.80,25.49,25.33,24.97,24.91,20.59,19.07,17.78,17.03,16.39,15.87,14.25,12.83.

[0096] 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). 1H NMR(400MHz,DMSO-d6)δ7.31(1H,s,H1of-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=5.9Hz,1-H of ara),1.63(3H,s,H-30),1.07(3H,d,J=6.2Hz,6-H3 of rha)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 ofcyclopropyl),0.31–0.25(1H,m,H1 ofcyclopropyl),0.22–0.15(1H,m,H1 ofcyclopropyl),0.14–0.06(1H,m,H1 ofcyclopropyl). 13 C NMR (101MHz, DMSO) δ174.75,151.20,109.38,103.12,100.11,79.58,74.41,73.03,72.2 4,70.64,70.57,68.33,68.00,64.52,62.64,55.00,50.30,49.96,47.90,46.69,46.38, 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.

[0097] 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). 1H NMR(400MHz,DMSO-d6)δ7.53(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=5.9Hz,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=6.2Hz,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)δ175.23,150.75,108.98,102.68,99.67,79.15,73.96,72.61,71.82 ,70.22,70.15,67.90,67.56,64.09,62.22,54.69,49.90,49.48,46.26,45.93,43.14,4 2.12,41.72,40.00,38.19,37.63,36.41,35.93,34.82,33.34,32.30,30.14,28.61,25.27,25.11,24.95,24.87,20.39,18.86,17.56,17.52,16.83,16.20,15.64,14.04,12.60.

[0098] 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. 1H NMR(400MHz,DMSO-d6)δ7.65(1H,s,H1 of-CONH),7.61(1H,s,H1 ofimidazole),7.16(1H,s,H1 ofimidazole),6.88(1H,s,H1 ofimidazole),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=5.8Hz,6-H3 of rha),3.92(2H,t,J=7.5Hz,H2 of-CH2to imidazole),1.63(3H,s,H-30),1.07(3H,d,J=6.2Hz,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(101MHz,DMSO)δ176.25,151.36,137.74,128.85,119.82,109.75,103.37,100.37,79.84 ,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.

[0099] 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). 1H NMR(400MHz,DMSO-d6)δ7.51(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=5.8Hz,1-H of ara),1.63(3H,s,H-30),1.07(3H,d,J=6.2Hz,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). 13 C NMR (101MHz, DMSO) δ175.43,150.90,109.23,102.88,99.90,79.36,74.21,72.78,72. 03,70.43,70.35,68.13,67.76,64.27,62.44,54.87,54.83,53.58,50.07,49.64,46.4 6,46.23,42.34,41.94,40.20,38.38,37.67,37.52,36.69,36.14,33.52,32.41,30.33,28.82,25.47,25.29,23.11,20.57,19.05,17.77,17.07,16.39,15.82,14.25,12.79.

[0100] 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(400MHz,DMSO-d6)δ7.33(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=5.9Hz,1-H of ara),1.61(3H,s,H-30),1.05(3H,d,J=6.2Hz,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(101MHz,DMSO)δ174.85,150.95,109.23,102.92,99.91,79.38,74.21,72.84,72.03 ,70.44,70.37,68.13,67.80,66.24,66.15,64.33,62.44,54.73,50.09,49.73,46.48,4 6.20, 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.

[0101] B4-26: 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 methyl group on the methyl ester (3.60 ppm). 1 H NMR(400MHz,DMSO-d6)δ7.38(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=5.9Hz,1-H of ara),3.60(3H,s,H3 of-COOCH3),1.63(3H,s,H-30),1.08(3H,d,J=6.2Hz,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). 13 C NMR(101MHz,DMSO)δ176.02,175.24,150.94,109.20,102.89,99.89,79.36,74.20,72.80,7 2.03,70.43,70.36,68.12,67.76,64.28,62.44,54.71,51.50,50.02,49.83,49.73,46.47,4 2.33,41.88,41.26,41.21,38.38,37.58,36.63,36.14,35.03,33.50,31.88,31.62,30.37,28.79,27.59,27.04,25.48,25.28,20.58,19.06,17.77,17.03,16.39,15.80,14.24,12.81.

[0102] B4-27: The preparation method is similar to that of 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(400MHz,DMSO-d6)δ7.26(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=5.9Hz,1-H of ara),3.58(3H,s,H3 of-COOCH3),1.62(3H,s,H-30),1.06(3H,d,J=6.2Hz,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). 13 C NMR (101MHz, DMSO) δ175.21,174.70,150.96,109.18,102.89,99.90,79.37,74.21,72.80,72. 03,70.43,70.35,68.12,67.76,64.27,62.44,54.70,51.31,50.08,49.73,46.72,46.47,46.2 1,42.34,41.88,41.69,40.22,38.38,37.75,36.63,36.14,33.49,32.28,31.25,30.83,30.38,28.80,27.77,27.69,25.48,25.29,20.58,19.05,17.77,17.03,16.38,15.90,14.24,12.82.

[0103] 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). 1H NMR (400MHz, DMSO-d6) δ7.74(1H,s,H1 of-CONH),5.81–5.72(1H,m,H1 of-CH on CH=CH2),5.07(1H,d,J=13.5Hz,H1of-CH2on CH=CH2),5.05(1H,brs,1-H of rha),5.00(1H,d,J=10.2Hz,H2of-CH2on CH=CH2),4.65(1H,brs,H1-29),4.53(1H,brs,H2-29),4.41(1H,d,J=5.9Hz,1-H of ara),1.63(3H,s,H-30),1.07(3H,d,J=6.1Hz,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). 13 C NMR(101MHz,DMSO)δ175.27,150.90,136.21,114.28,109.22,102.87,99.88,79.36, 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.

[0104] 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(400MHz,DMSO-d6)δ7.39(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=6.2Hz,1-H of ara),1.62(3H,s,H-30),1.06(3H,d,J=6.2Hz,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).13 C NMR (101MHz, DMSO) δ175.34,150.88,109.23,102.86,99.89,79.36,74.21,72.75,72.02,70.42,70.34,68.12,67.73,64.24,62.44,57.51,54.88, 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.

[0105] 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 (400MHz, DMSO-d6) δ7.72(1H,s,H1 of-CONH),7.70(1H,d,J=3.3Hz,H1ofthiazole),7.58(1H,d,J=3.3Hz,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=5.7Hz,1-H of ara),1.63(3H,s,H-30),1.07(3H,d,J=6.1Hz,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 (101MHz, DMSO) δ175.68,167.51,150.87,142.21,119.43,109.24,102.86,99.89,79. 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.

[0106] 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(400MHz,DMSO-d6)δ7.58(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=5.8Hz,1-H of ara),1.62(3H,s,H-30),1.07(3H,d,J=6.2Hz,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). 13 C NMR(101MHz,DMSO)δ175.55,150.93,109.22,102.88,99.89,79.36,77.37,74.18,72.79 ,72.02,70.43,70.35,68.12,67.76,67.02,64.27,62.44,54.88,50.10,49.71,46.47,4 6.17,42.46,42.33,41.92,40.23,38.39,37.67,36.66,36.14,33.53,32.34,30.33,28.82,28.57,25.47,25.30,25.03,20.58,19.05,17.76,17.04,16.40,15.76,14.24,12.79.

[0107] 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(400MHz,DMSO-d6)δ7.44(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=5.9Hz,1-H of ara),1.63(3H,s,H-30),1.07(3H,d,J=6.2Hz, 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 (101MHz, DMSO) δ175.43,150.91,109.23,102.88,99.89,79.36,74.21,72.77,72. 02,70.43,70.35,68.12,67.74,66.15,64.25,62.44,57.36,54.87,53.19,53.15,50.0 6,49.61,46.45,46.21,42.33,41.94,40.21,38.37,37.67,36.66,36.14,33.52,32.44,30.32,28.85,25.47,25.26,20.55,19.03,17.76,17.06,16.38,15.89,14.23,12.77.

[0108] 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). 1H NMR(400MHz,DMSO-d6)δ7.58(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=5.1Hz,1-H of ara),2.11(3H,s,H3 of-CH3on piperidine),1.62(3H,s,H-30),1.07(3H,d,J=6.1Hz,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(101MHz,DMSO)δ175.41,150.96,109.20,102.86,99.90,79.35,74.22,72.75,72.03,70 .44,70.35,68.13,67.73,64.24,62.43,55.16,54.89,50.12,49.70,46.47,46.20,46.13,4 4.06, 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.

[0109] 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). 1H NMR(400MHz,DMSO-d6)δ7.64(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=5.6Hz,1-H of ara),2.24(3H,s,H3of-CH3on pyrrolidine),1.63(3H,s,H-30),1.07(3H,d,J=6.1Hz,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(101MHz,DMSO)δ175.49,150.93,109.22,102.88,99.89,79.35,74.20,72.79,72.03, 70.44,70.35,68.13,67.76,64.27,62.44,59.79,59.62,55.50,54.87,50.10,49.63,46.4 7,46.13,42.33,41.94,41.81,40.22,38.39,37.72,37.50,36.65,36.14,33.55,32.44,30.35,28.82,28.31,25.47,25.31,20.60,19.06,17.76,17.05,16.41,15.89,14.24,12.79.

[0110] B4-36: 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 hydroxyl group on cyclohexane (4.57 ppm). 1H NMR(400MHz,DMSO-d6)δ7.16(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 on cyclohexane),4.56(1H,brs,H2-29),4.53(1H,s,H1 of-OH on C-23),4.48(1H,d,J=5.4Hz,1-H of ara),1.62(3H,s,H-30),1.07(3H,d,J=6.1Hz,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). 13 C NMR (101MHz, DMSO) δ174.75,150.98,109.17,102.89,99.90,79.37,74.21,72.79,72.03 ,70.43,70.36,68.40,68.12,67.75,64.26,62.44,54.70,50.08,49.74,46.88,46.47,4 6.22,42.34,41.87,40.22,38.38,37.73,36.63,36.13,34.28,34.20,33.50,32.30,30.35,29.92,28.79,25.47,25.28,20.58,19.05,17.76,17.02,16.37,15.88,14.23,12.81.

[0111] 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). 1H NMR(400MHz,DMSO-d6)δ7.59(1H,s,H1 of-CONH),7.27(1H,d,J=8.0Hz,H1′of benzene),7.26(1H,d,J=8.0Hz,H1of 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=5.8Hz,1-H of ara),1.62(3H,s,H-30),1.07(3H,d,J=6.2Hz,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). 13 C NMR (101MHz, 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.

[0112] 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). 1H NMR(400MHz,DMSO-d6)δ7.69(1H,s,H1 of-CONH),7.32(1H,d,J=5.1Hz,H1ofthiophene),6.93(1H,dd,J=5.1,3.4Hz,H2ofthiophene),6.86(1H,d,J=8.0Hz,H3ofthiophene),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=5.9Hz,1-H of ara),1.62(3H,s,H-30),1.07(3H,d,J=6.2Hz,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 (101MHz, DMSO) δ175.60,150.92,141.81,126.87,124.98,123.87,109.24,102.87,99. 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.

[0113] 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). 1H NMR(400MHz,DMSO-d6)δ9.11(1H,s,H1 of-OH on benzene),7.53(1H,s,H1 of-CONH),6.96(1H,d,J=8.5Hz,H1of benzene),6.96(1H,d,J=8.5Hz,H2of benzene),6.65(1H,d,J=8.4Hz,H3of benzene),6.65(1H,d,J=8.4Hz,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=5.9Hz,1-H of ara),1.62(3H,s,H-30),1.07(3H,d,J=6.1Hz,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(101MHz,DMSO)δ175.33,155.55,150.95,129.65,129.36,115.02,109.21,102.87,99. 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.

[0114] 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). 1H NMR(400MHz,DMSO-d6)δ8.27(1H,s,H1of oxazole),8.02(1H,s,H1 of-CONH),7.76(1H,s,H2ofoxazole),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=5.9Hz,1-H of ara),1.62(3H,s,H-30),1.07(3H,d,J=6.2Hz,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). 13 C NMR(101MHz,DMSO)δ175.53,151.70,150.89,138.39,135.55,109.23,102.87,99.89, 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.

[0115] 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(400MHz,DMSO-d6)δ6.82(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=6.1Hz,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)δ174.25,171.56,150.86,109.28,102.88,99.88,79.37,74.22, 72.78,72.07,70.44,70.36,68.12,67.71,64.25,62.46,54.76,50.03,49.51,48.5 9,46.46,43.93,42.34,42.03,40.20,38.38,37.74,36.90,36.14,33.46,32.94,30.40,28.95,25.47,25.26,20.52,19.01,17.78,17.06,16.36,15.87,14.26,12.79.

[0116] 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(400MHz,DMSO-d6)δ7.83(1H,s,H1of-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=5.8Hz,1-H of ara),1.79(3H,s,H3 of-CH3to CONH),1.63(3H,s,H-30),1.07(3H,d,J=6.2Hz,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). 13 C NMR(101MHz,DMSO)δ175.54,169.14,150.68,109.02,102.68,99.68,79.15,74.00,7 2.57,71.82,70.22,70.14,67.91,67.54,64.06,62.23,54.66,49.84,49.43,46.25,4 5.99, 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.

[0117] 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(400MHz,DMSO-d6)δ7.78(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=5.7Hz,1-H of ara),2.04(2H,t,J=7.4Hz,H2 of-CH2to-COOH),1.62(3H,s,H-30),1.07(3H,d,J=6.1Hz,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). 13 C NMR(101MHz,DMSO)δ176.17,175.38,151.01,109.18,102.87,99.87,79.36,74.22,7 2.78,72.07,70.47,70.35,68.12,67.71,64.23,62.46,54.82,50.11,49.72,46.49,4 6.19, 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.

[0118] 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 hydrogens are methylene (2.19 ppm) attached to the carboxyl group and carboxyl carbon (175 ppm). 1H NMR(400MHz,DMSO-d6)δ7.58(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,H1of-OH on C-23),4.34(1H,d,J=5.7Hz,1-H of ara),2.19(2H,t,J=7.1Hz,H2 of-CH2to-COOH),1.63(3H,s,H-30),1.07(3H,d,J=6.2Hz,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 (101MHz, DMSO) δ175.33,174.66,150.97,109.19,102.86,99.88,79.34,74.24,72. 77,72.06,70.49,70.34,68.11,67.73,64.24,62.43,54.84,50.10,49.69,46.47,46.1 7,42.34,41.93,40.21,38.39,37.81,36.64,36.14,33.48,32.43,30.36,28.78,26.55,25.48,25.31,22.08,21.93,20.59,19.06,17.78,17.08,16.41,15.82,14.24,12.80.

[0119] 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). 1H NMR(400MHz,DMSO-d6)δ7.52(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=7.4Hz,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). 13 C NMR (101MHz, DMSO) δ175.31,173.25,150.96,109.15,70.30,64.43,54.80,51.13,50.10,49.66,48.58,46.69,46.15,41.92,40.20,38.13,37.9 4,37.71,36.61,36.42,33.57,33.26,32.44,30.33,28.91,28.83,26.75 ,25.82,25.24,24.14,20.59,19.03,17.44,16.29,15.82,14.30,12.39.

[0120] 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(400MHz,Methanol-d4)δ5.16(1H,brs,1-H of rha),4.72(1H,brs,H1-29),4.59((1H,brs,H2-29)),4.56(1H,d,J=4.8Hz,1-H of ara),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=6.2Hz,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). 13C NMR (101MHz, Methanol-d4) δ178.21,152.32,109.98,104.30,101.89,82.27,76.66,73. 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.

[0121] 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(400MHz,Methanol-d4)δ5.16(1H,brs,1-H of rha),4.72(1H,brs,H1-29),4.60(1H,brs,H2-29),4.57(1H,d,J=5.0Hz,1-H of ara),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=6.2Hz,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). 13 C NMR(101MHz,D2O)δ178.18,152.27,110.02,104.30,101.88,82.27,76.65,73.93,73. 66,73.31,72.13,72.02,70.16,69.12,64.74,64.57,56.88,52.02,51.18,50.66,48.1 5,44.04,43.63,42.42,41.99,39.92,39.31,39.06,37.81,34.99,34.68,34.11,31.93,31.72,30.53,27.01,26.69,22.16,19.62,18.77,17.96,17.22,16.77,15.08,13.50.

[0122] Using Pulsatilla saponin B4 or its derivatives as experimental drugs, the following experiments were conducted:

[0123] Example 11 Cytotoxicity Experiment 1

[0124] HIEC cells were seeded at 5000 cells / well in 96-well plates, with 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 bring the final drug concentration to 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.

[0125] Example 12 Cytotoxicity Experiment 2

[0126] THP-1 cells were used at a rate of 1×10 4 Seeds were inoculated into 96-well plates at 100 μL per well and cultured using standard methods.

[0127] Once the cells reached 80% confluence, they were induced with phorbol-12-myristate-13-acetate (PMA) at 100 ng / mL for 12 h. The original culture medium was then discarded, and 100 μL of fresh complete culture medium was added. Zero-concentration wells without cells and a normal control group without drugs were set up. 1 μL of a B4 derivative, which showed no significant cytotoxicity in the first round, was added to each well to bring the final drug concentration to 50 μM. The wells were then incubated for 24 h.

[0128] After incubation, 10 μL of CCK-8 was added to each well and incubated in the dark for 4 hours. 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 obvious cytotoxicity in the second round.

[0129] Calculation formula: Cell viability (%) = [A(drug-treated) - A(blank)] / [A(0-drug-treated) - A(blank)] × 100

[0130] A (Drug Addition): Absorbance of the pores containing cells, CCK-8, and drug solution.

[0131] A (Blank): Absorbance of wells containing culture medium and CCK-8 but without cells.

[0132] A(0 drug added): Absorbance of pores containing cells and CCK-8 without added drugs.

[0133] As shown in Figure 4, the cytotoxicity of B4 derivatives at 50 μM is statistically analyzed. The symbols without "*" indicate that there is no significant difference compared with the normal control, meaning that B4 derivatives do not have obvious cytotoxicity at a dose of 50 μM. The figure shows that there are many non-cytotoxic derivatives, including B4-1, B4-5, and B4-10.

[0134] Among these derivatives is B4-39. The cytotoxicity of B4-39 was further evaluated using three different cell lines: RAW264.7 (mouse mononuclear cell line), THP-1 (human acute monocytic leukemia cell line), and HIEC (human intestinal epithelial cells). These cell lines represent different cell types and can provide in vitro information on the cytotoxicity of B4-39 to immune cells and intestinal epithelial cells. The results showed that at a concentration of 50 μM and after 24 hours of treatment, B4-39 did not exhibit significant cytotoxicity to these cells, indicating that B4-39 maintains good cell compatibility even at relatively high concentrations, which is an important indicator of drug safety. Furthermore, these results further confirm that B4-39 not only possesses anti-inflammatory activity but also may have a low risk of side effects, making it a promising drug candidate.

[0135] Example 13

[0136] Nitrite content detection: Raw 264.7 cells were cultured at 6 × 10⁻⁶ cells / mL. 4 Cells were seeded at 100 μL / well in 96-well plates 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 attachment, 1 μL of the corresponding drug was added to a final concentration of 10 μM. One hour later, 1 μL of LPS was added to a final concentration of 1 μg / ml. The cells were then incubated for 24 hours. 50 μL of culture medium was transferred from each well of the seeded plate to a new 96-well plate. Under dark conditions, 50 μL of Griess reagent A and 50 μL of reagent B were added to each well. The absorbance of each well was measured at 540 nm using a microplate reader. As shown in Figure 5, 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.

[0137] Example 14

[0138] 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. 2 μL of AB4 and its derivatives were added to each experimental group to a final concentration of 10 μM, while the control group served as a blank. After 1 h, 2 μL of 1 mg / mL LPS was added to the other groups (excluding the blank group), bringing the final concentration to 1 μg / ml, and incubation continued 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 using a cell scraper and placed in a 1.5 mL centrifuge tube. The tubes were centrifuged at 2000 g for 3 min at 4°C, the supernatant was discarded, and the cell pellet was collected. 100 μL of RIPA lysis buffer (with protease and phosphatase inhibitors added before use) was added to each tube, and the mixture was pipetted and incubated on ice for 10 min. After re-disrupting the cells using an ultrasonic disruptor, centrifuge at 12000g, 4℃ for 10 min, and carefully collect the cell supernatant into a new EP tube, storing it on ice. Following the manufacturer's instructions, use the BCA protein quantification kit to measure and calculate the total protein content. 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℃ 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. Block the membrane with protein blocking buffer at room temperature for 1 hour, then incubate the PVDF membrane with a specific primary antibody overnight at 4℃ according to the manufacturer's instructions. The following 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 thoroughly washed again with TBST buffer. Finally, the protein was exposed and analyzed according to the instructions of the ultrasensitive ECL chemiluminescence kit.

[0139] As shown in Figures 6 and 7, AB4 and its derivatives can inhibit the activation of key proteins in the NF-κB and NLRP3 signaling pathways. The numbers on the horizontal axis in the figures represent different Pulsatilla chinensis saponin B4 derivatives. The first round of Western blotting showed that after 2 hours of LPS stimulation of THP-1 cells, the P-IκBa protein level in the model group significantly increased (P<0.01).

[0140] Compared with the LPS model group, the compounds of this invention and its AB4 can reduce P-IκBa protein levels. Moreover, several derivatives, including B4-39 and B4-36, significantly reduced P-IκBa protein levels compared with AB4 (p<0.05). Furthermore, derivatives B4-19, B4-39, B4-36, B4-33, B4-28, and B4-40 can reduce Pro-IL1β levels, and the effect is better than AB4. These results suggest that these derivatives have better anti-inflammatory activity.

[0141] Example 15

[0142] 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. This study further validates this effect through in vivo experiments, and therefore the following experiment was designed: 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 eight mice in each group. One day before the experiment, the fur on the back of the mice was removed, and a 3cm x 3cm area was selected for use. On day 1, except for the normal control group, other groups were sensitized by applying 5% DNCB 50μL 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 50μL was applied topically to the inside and outside of the right ear of 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 criterion 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 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 200μL 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. At 10:00 AM, 200 μl of pure water was applied to the back and the inside and outside of the right ear of the model group, and then again at 6:00 PM. This treatment was repeated for 7 days. On the 8th day, the mice were sacrificed, and the spleen and ear were harvested and weighed 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).

[0143] 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 evaluate the eczema based on four indicators: erythema, papules / pustules, scaling, and crusting, scored 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 ear thickness difference was calculated: Thickness difference = Right ear thickness - Left ear thickness. Body weight was measured and recorded daily. The experimental results are shown in Figure 8-12. Figure 8 shows the skin condition on the back of mice with DNCB-induced atopic dermatitis; Figure 9 shows the ear condition of mice with DNCB-induced atopic dermatitis; Figure 10 shows the mouse weight changes and back scores; Figure 11 shows the mouse ear thickness difference and ear weight difference; Figure 12 shows the mouse spleen index.

[0144] 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. On the fifth day, exudation, ulceration, and crusting began. On the seventh day, the difference in ear thickness between the control and model groups (P<0.0001) was statistically significant, 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). After applying DNCB to the skin on the back of mice, compared with the blank group, the model group showed exudation, erosion, and crusting, producing eczema-like skin lesions. With the continued action of the drug, the skin lesions in the model group became increasingly severe from day 2 to 3, with 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 that the eczema mouse model was successfully established. 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 crusting, and obvious scales and papules. On days 3 (P<0.001), 5 (P<0.0001), and 7 (P<0.001), the EASI scores of both the AB4 derivative group and the model group were statistically significant. The difference in EASI 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 back scores of the AB4, B4-19, B4-33, and B4-39 groups were all statistically significant (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 AB4 derivative group was significantly decreased. Compared with the model group, the spleen index of the dexamethasone group was significantly lower than that of the normal group, indicating that dexamethasone induced immunosuppression in mice, while the AB4 derivative could enhance the immunity of mice. All experimental results suggest that the AB4 derivative has a protective effect against DNCB-induced eczema lesions, and its effect is superior to that of AB4 and glucocorticoids.

[0145] Example 16

[0146] Topical application of Pulsatilla saponin B4 can achieve some effect in the treatment of psoriasis. However, further research has revealed some defects in Pulsatilla saponin B4. Through structural improvement, this invention discloses that the derivative of Pulsatilla saponin B4 has significant technical effects in the treatment of psoriasis. The following experiments illustrate the technical progress of this invention. The reagents used are existing products, and A3-9 are described in CN2022112110168.

[0147] Weight 20

[0148] After a week of acclimatization, BALB / c mice weighing approximately 100g were divided into seven groups according to their weight: a normal group, a model group, a B4-33 treatment group (6.6mg / kg), a B4-39 treatment group (6.6mg / kg), an A3-9 treatment group (6.6mg / kg), and a halometasone treatment group (0.1g / mouse). The mice were initially numbered. Two days before modeling, a 3cm × 3cm area on the back of each mouse was shaved. The day before modeling, the shaving was checked for completeness; if incomplete, a second shaving was performed. On the day of modeling, except for the normal group, 62.5mg of imiquimod was applied to the back of all other groups for seven consecutive days. The treatment groups received the drug once 4 hours before and 2 hours after modeling. Simultaneously, the model group was treated with the solvent used for the test drug (water containing 70% ethanol and 2% glycerol, volume percentage). Mouse weight was recorded daily, and the condition of the back skin was photographed and scored using the PASI (Patient Assessment and Evaluation) method.

[0149] ① Effects of AB4 derivatives B4-33, B4-39, and A3-9 on body weight in psoriatic mice

[0150] The experimental results, as shown in Figure 13, indicated that after imiquimod modeling, the body weight of mice began to decrease on the second day, and subsequently decreased daily in all model groups. The AB4 administration group showed slightly better results than the model group, but still maintained a downward trend daily. The B4-33, B4-39, and A3-9 administration groups showed no significant weight loss and exhibited a recovery trend on the fifth day. The halometasone administration groups experienced a sharp decrease in body weight. The B4-33 and B4-39 administration groups showed significantly better body weight than the halometasone group from the fourth day onwards (p < 0.05), while the A3-9 administration group showed better body weight than the halometasone group after the fifth day of modeling (p < 0.05). This demonstrates that the AB4 derivative can improve the body weight of psoriatic mice and has a higher safety profile compared to halometasone.

[0151] B4-33 treatment group vs. halometasone group: *p<0.05, **p<0.01, ***p<0.001; B4-39 treatment group vs. halometasone group: #p<0.05, ##p<0.01; A3-9 treatment group vs. halometasone group: $p<0.05, $$p<0.01.

[0152] ② Effects of AB4 derivatives B4-33, B4-39, and A3-9 on the area and severity index (PASI) of psoriasis lesions in mice.

[0153] The Psoriasis Skin Lesion Area and Severity Index (PASI) is an important indicator for evaluating the severity of psoriasis. After modeling, the skin on the backs of mice was photographed and scored daily, and the scoring criteria are shown in Table 1. Figure 14 shows the skin on the backs of the mice. In the model group, mild scaling appeared one day after modeling; erythema and thickening appeared three days later, with increased scaling; after five days, the scales became patchy, the skin turned dark red, and the skin thickened significantly; on the seventh day, the scales were layered, and the skin was noticeably raised. In contrast, the drug-treated groups showed varying degrees of improvement.

[0154] The PASI scores of the treatment groups are shown in Figures 15-17. B4-33, B4-39, and A3-9 all showed some improvement in the skin of psoriatic mice, with effects superior to the B4 group and comparable to the halometasone group. As shown in Figure 15, compared to the control group, the PASI scores of the model group showed a significant difference from day 1, and the scores increased daily. The B4 group showed slight improvement compared to the model group, but the scores remained at a high level, while the B4-33 and halometasone treatment groups significantly reduced the scores. As shown in Figures 16 and 17, the B4-39 and A3-9 treatment groups also showed effects comparable to the halometasone group. This indicates that the AB4 derivatives have better efficacy than AB4, and their efficacy is not weaker than, and may even be superior to, the positive control drug halometasone. (Normal group vs. model group: **p<0.01, ***p<0.001, ***p<0.0001; B4-33, B4-39, A3-9 treatment groups vs. model group: #p<0.05, ##p<0.01, ###p<0.001, ####p<0.0001; Halometasone treatment group vs. model group: $p<0.05, $$p<0.01, $$$$p<0.0001; B4 treatment group vs. model group: &&p<0.01, &&&p<0.001).

[0155] Table 1. Area and severity of psoriasis lesions

[0156] ③ Effects of Pulsatilla saponin B4 derivatives B4-33, B4-39, and A3-9 on organ coefficients in psoriatic mice

[0157] Psoriasis, as an autoimmune disease, is closely related to the immune system. The spleen and thymus, as the most important immune organs, undergo significant changes in psoriasis mice. At the end of the experiment on day 8, the mice were sacrificed, and their spleen and thymus were removed, weighed, and the spleen index and thymus index were calculated, i.e. (spleen or thymus weight / mouse weight) × 100.

[0158] As shown in Figure 18, after modeling, the spleen of the mice was significantly enlarged, showing a significant difference from the normal group. The halometasone administration group significantly suppressed the immune system, resulting in a sharp drop in the spleen index, which fell below the normal value. This is a clear side effect. The B4-39 administration group suppressed the splenomegaly of the mice, but there was no statistical difference compared with the normal control group, suggesting that there was no significant side effect in terms of spleen index.

[0159] As shown in Figure 19, the thymus index of mice decreased significantly after modeling, and all treatment groups showed a tendency to recover to varying degrees, with a significant difference observed in the B4-39 treatment group. This indicates that the Pulsatilla saponin B4 derivative can regulate the immune system of psoriatic mice and has a higher safety profile compared to halometasone (*p<0.05, ***p<0.001, ***p<0.0001).

[0160] Example XVII. Effects of B4-39 on DSS-induced colitis in mice

[0161] Establishment of a 3% DSS-induced ulcerative colitis model in C57 mice: 56 C57 mice were randomly divided into four groups according to body weight: normal control group, model group (3% DSS group), mesalazine group (400 mg / kg), Pulsatilla saponin B4 (AB4) control group (100 mg / kg), B4-39 administration group (2.5 mg / kg), B4-39 administration group (5 mg / kg), and B4-39 administration group (10 mg / kg), with 8 mice in each group.

[0162] On the day of modeling, the drinking water of all mice except the normal group was replaced with a 3% DSS aqueous solution. The mice were fed for 8 days, and treated on the 9th day. The day before modeling, the mice were weighed and administered B4-39 (2.5, 5, or 10 mg / kg) by gavage once a day, according to their body weight. The positive control drugs mesalazine and AB4 were administered by gavage once a day. The normal control group and model group mice were administered the control solution by gavage once a day for 8 consecutive days. Subsequently, the survival status of the mice was observed daily, and their body weight, fecal characteristics, and bloody stools were recorded.

[0163] Colon length: Experimental results, as shown in Figures 20A and 20B, indicated that compared to the control group, the model group mice exhibited significantly shorter colons accompanied by bleeding (P<0.0001). B4-39 administration alleviated the colon shortening in mice. Specifically, the colon length in the B4-39 (10 mg / kg) group was highly significantly different from the model group (P<0.001), and the B4 group also showed a statistically significant difference compared to the model group (P<0.05). The colon length in the mesalazine group was significantly different from that in the model group (P<0.01). These results preliminarily demonstrate that B4-39 has an efficacy in alleviating ulcerative colitis, and that the efficacy of B4-39 (10 mg / kg) is superior to that of the AB4 (100 mg / kg) group and the mesalazine (400 mg / kg) group.

[0164] The Disease Activity Index (DAI) score is a comprehensive indicator for assessing the severity of inflammatory bowel disease (IBD) in mice. It consists of three parts: weight loss rate score, fecal characteristics score, and bloody stool score. A higher DAI score indicates more severe IBD in the mouse. The scoring details are as follows:

[0165] Weight loss rate: Mice were weighed at 10:00 AM every day and the weight loss rate was calculated as follows: Weight loss rate = (body weight on day n - body weight on day 0) / body weight on day 0 × 100%.

[0166] Fecal characteristics: Collect mouse feces daily and observe the shape. Normal feces are formed and have a certain firmness, loose and soft feces are watery feces, and unformed feces are watery feces.

[0167] Bloody stool condition: If bleeding can be observed in mouse feces daily, it is considered bloody stool. If it cannot be observed with the naked eye, take mouse feces on a glass slide, first add 2% o-toluidine glacial acetic acid solution, then add 3% hydrogen peroxide solution, and observe the color change of the feces. If it turns blue-green within a certain period of time, the feces have occult blood. If there is no obvious color change, it is normal feces.

[0168] Table 2 DAI Scoring Details

[0169] The experimental results are shown in Figure 20C. From day 3 of modeling, mice in all groups showed varying degrees of fecal abnormalities, including loose stools, bloody stools, or occult blood. The condition of the model group mice significantly worsened on day 4 of modeling, showing a highly statistically significant difference compared to the control group (P<0.0001). With prolonged modeling time, the condition continued to deteriorate. The DAI scores of mice in the B4-39 group (2.5, 5, 10 mg / kg) were consistently lower than those in the model group, and showed a significant difference on day 4 of modeling (P<0.01).

[0170] Furthermore, the B4-39 group (2.5 mg / kg) showed comparable efficacy to the AB4 group and the mesalazine group in reducing the DAI score in DSS mice. In conclusion, B4-39 can reduce the DAI score in DSS mice and improve the condition of mice with ulcerative colitis, with superior efficacy compared to AB4 and mesalazine.

[0171] Weight change: Weight change is an important indicator for evaluating the IBD model. Mice were weighed at 10:00 a.m. every day and the weight change rate was calculated using the following formula: Weight change rate = (body weight on day n - body weight on day 0) / body weight on day 0 × 100%.

[0172] The experimental results are shown in Figure 20D. Mice in the DSS-induced IBD model group experienced a continuous decrease in body weight starting from day 5 of modeling. Particularly on day 6, the decrease in body weight in the model group was significantly different from the normal control group (P<0.0001). The weight loss trend in the B4-39 treatment group was significantly alleviated, especially in the B4-39 (10 mg / kg) group, where the rate of weight loss was significantly slower, showing a highly significant difference from the model group on day 6 (P<0.001). Furthermore, the body weight of mice in the positive control group treated with mesalazine began to recover on day 7 of modeling, showing a significant difference compared to the model group (P<0.01).

[0173] Notably, B4-39 (10 mg / kg) was more effective than mesalazine (5-ASA) and AB4 (B4 group) in alleviating DSS-induced weight loss in mice. Based on these results, it can be concluded that B4-39 can alleviate weight loss in DSS mice and is superior to the positive controls AB4 and mesalazine.

[0174] Pathological changes in colonic tissue: As shown in Figure 21, colonic tissue sections were meticulously observed using an optical microscope (100x and 200x magnification). HE staining revealed significant pathological changes in the colonic tissue of mice in the DSS-induced ulcerative colitis model group compared to the normal control group. Specifically, these included significant infiltration of inflammatory cells, disruption of colonic structure, uneven mucosal surface, and a reduction in the number and structural incompleteness of crypts. However, after treatment with B4-39, positive signs of recovery were observed in the mouse colonic tissue. Specifically, the damage to the colonic structure was repaired to some extent, inflammatory cell infiltration was reduced, the number of crypts increased, and structural integrity improved. These observations further confirm the potential efficacy of B4-39 in alleviating colonic damage in mice with ulcerative colitis.

[0175] Example 18: Effect of B4-39 on apoptosis-related proteins in colon tissue of DSS-induced colitis mice

[0176] Apoptosis is a complex biological process finely regulated by multiple gene families, including key factors such as the Bcl-2 family and the caspase family. Bcl-2 and Bax are key factors in the regulation of apoptosis; the former has anti-apoptotic effects, while the latter promotes apoptosis. Cleaved-caspase 3, as an activated form of the caspase family, is crucial for the execution of apoptosis. To investigate the effect of B4-39 on DSS-induced apoptosis in mouse colonic epithelial cells, colonic tissues from mice in the normal control group, model group (3% DSS group), Pulsatilla saponin B4 (AB4) group, and B4-39 treatment group (10 mg / kg) were collected. Colonic tissues from three mice in each group were randomly selected, and tissue proteins were extracted. The expression levels of apoptosis-related proteins Bax / Bcl-2 and cleaved-caspase 3 / caspase 3 were detected by Western blotting.

[0177] As shown in Figure 22, the experimental results revealed the effect of the DSS-induced inflammatory environment on the apoptosis pathway of mouse colonic epithelial cells. Compared with the normal control group, the expression of pro-apoptotic proteins Bax and cleaved-caspase 3 was significantly upregulated in the model group, while the expression of anti-apoptotic protein Bcl-2 was significantly downregulated. These changes showed statistically significant differences (P<0.01), thus confirming that the DSS-induced inflammatory environment activates the apoptosis pathway. After administration of B4-39, the levels of Bax and cleaved-caspase 3 in the model group were significantly reduced (Figure 22A&B), while the level of Bcl-2 increased accordingly (Figure 22A&C). This indicates that B4-39 has a significant regulatory effect on the expression of apoptosis-related proteins. In particular, B4-39 showed a more effective effect than AB4 in regulating the expression of these proteins. Although AB4 also showed some recovery effect, its effect was not as significant as that of B4-39. These results suggest that B4-39 may effectively inhibit apoptosis in colonic epithelial cells by targeting the Bax / Bcl-2 / caspase 3 signaling pathway, indicating that B4-39 has an anti-apoptotic effect.

[0178] Example 19 B4-39 Effects of inflammation-related proteins in colon tissue of colitis mice

[0179] NF-κB plays a central role in colitis, particularly inflammatory bowel disease (IBD). It is a transcription factor that controls the expression of various genes related to immune and inflammatory responses. In the pathogenesis of colitis (UC), abnormal activation of the NF-κB signaling pathway is associated with disease severity. Studies have shown that NF-κB activation can promote the production of various pro-inflammatory cytokines, such as tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, and IL-6, which further amplify the inflammatory response.

[0180] In this study, the experimental results are shown in Figure 23. Compared with the normal control group, the levels of phosphorylated p65 (p-p65), inducible nitric oxide synthase (iNOS), and cyclooxygenase 2 (COX2) in the colonic tissue of the model group mice were significantly increased, while the level of IκBα was significantly decreased. This reflects the activation of the NF-κB signaling pathway and the ensuing inflammatory response. After treatment with B4-39, the expression levels of these proteins were observed to return to normal, indicating that B4-39 has a relieving effect on colonic inflammation. Notably, B4-39 showed greater efficacy than AB4 in regulating the expression of these proteins, which may suggest that B4-39 has greater potential and selectivity in anti-inflammatory effects.

[0181] Example 20: Effects of B4-39 on intestinal barrier-related proteins in UC mice

[0182] The integrity of intestinal epithelial cells is crucial for maintaining intestinal barrier function, and their defects or damage play a key role in the pathogenesis of inflammatory bowel disease (IBD). Intestinal epithelial cells not only constitute the mechanical barrier of the intestine but also protect the body from harm by selectively regulating nutrient absorption and blocking pathogens and endotoxins. Tight junctions, adhesion junctions, desmosomes, and gap junctions are key forms of connection between intestinal epithelial cells, with tight junctions being particularly important. They are composed of proteins such as Occludin, Claudin, and the Zonula Occlude (ZO) protein family. The integrity of tight junctions plays a vital role in preventing the penetration of pathogens and antigens, inhibiting the activation of immune cells, and maintaining homeostasis.

[0183] In this study, as shown in Figure 24, compared with the normal control group, the levels of ZO-1 (Figure 24A&D), Claudin-1 (Figure 24A&C), and Occludin (Figure 24A&B) proteins in the colon of the model group mice were significantly reduced, indicating impaired intestinal epithelial barrier function. However, after treatment with B4-39, the levels of these proteins recovered, indicating that B4-39 can alleviate intestinal epithelial barrier damage. Notably, B4-39 showed a more effective effect than AB4 in upregulating the levels of ZO-1, Claudin-1, and Occludin proteins. This finding may suggest that B4-39 has superior anti-inflammatory potential and therapeutic selectivity in regulating intestinal epithelial barrier function. In conclusion, B4-39 demonstrates a protective effect on intestinal epithelial barrier function by upregulating the expression of ZO-1, Claudin-1, and Occludin proteins.

[0184] Example 21: Effects of B4-39 on inflammatory factors in the colonic tissue of UC mice

[0185] When the body is stimulated by external factors, it triggers an immune response, leading to the release of a large number of inflammatory cytokines. Among the many inflammatory cytokines, tumor necrosis factor (TNF-α), IL-6, and IL-1β from the interleukin (IL) family are considered to be major regulators. To assess the expression of these inflammatory cytokines in mouse colon tissue, enzyme-linked immunosorbent assay (ELISA) was used for detection. The experimental results are shown in Figure 25. In the DSS-induced colitis model, compared with the normal control group, the expression levels of TNF-α (Figure 25C), IL-1β (Figure 25B), and IL-6 (Figure 25A) in the colon tissue of the model group mice were significantly increased, and this difference was statistically significant (P<0.001). In mice treated with AB4 and B4-39, the release of these three inflammatory cytokines in their colon tissue was effectively inhibited, and B4-39 was more effective than AB4 in reducing inflammatory cytokines. These results indicate that B4-39 has potential anti-inflammatory effects, significantly reducing the expression levels of TNF-α, IL-6, and IL-1β in mouse colon tissue, which may have a positive impact on the treatment of colitis.

[0186] This invention discloses a novel application of B4-39, a derivative of Pulsatilla saponin B4, in the treatment of ulcerative colitis. Existing drugs for treating ulcerative colitis are often limited by high cost and significant side effects. B4-39, as a novel therapeutic candidate, shows significant therapeutic potential. In a DSS (glucosan sulfate)-induced ulcerative colitis mouse model, B4-39 demonstrated good therapeutic effects. Specifically, after administration, the rate of weight change in mice improved, the Disease Activity Index (DAI) score decreased, colon length recovered somewhat, and inflammatory cell infiltration was reduced (observed by HE staining). Furthermore, the levels of inflammatory factors IL-1β, TNF-α, and IL-6 were also decreased, and its efficacy was generally superior to mesalazine and AB4. Western blotting analysis showed that B4-39 could reduce the expression of pro-inflammatory proteins such as PP65, IKB-α, COX2, and iNOS, thus exhibiting a significant anti-inflammatory effect. Meanwhile, B4-39 effectively inhibited intestinal epithelial cell apoptosis by reducing the expression of pro-apoptotic proteins cleaved-caspase 3 and Bax, and increasing the expression of anti-apoptotic protein Bcl-2. Furthermore, B4-39 also enhanced the expression levels of tight junction proteins ZO-1, Claudin-1, and Occludin, thereby protecting the function of the intestinal barrier. In cytotoxicity tests on RAW264.7, THP-1, and HIEC cells, B4-39 at a concentration of 50 μM did not show significant cytotoxicity. These results indicate that B4-39 not only possesses good anti-inflammatory activity but also exhibits high safety. In conclusion, B4-39, as a derivative of Pulsatilla saponin B4, demonstrates great potential and advantages in the treatment of ulcerative colitis (UC).

[0187] In this invention, the drugs for treating or preventing psoriasis can specifically be solutions, skin gels, skin ointments, skin patches, skin microemulsions, or skin sprays. The dosage forms include: solutions (which can be aqueous solutions of the drug or aqueous solutions of Pulsatilla chinensis saponin B4 derivatives with added excipients such as glycerin, sodium benzoate, and fragrance); gels for skin use, in addition to Pulsatilla chinensis saponin B4 derivatives, also include conventional excipients such as carbomer, glycerin, propylene glycol, and water; ointments for skin use, in addition to the active drug, also include conventional excipients such as stearic acid, glyceryl monostearate, liquid paraffin, white petrolatum, and water; patches for skin use, in addition to the active drug, also include conventional excipients such as sodium polyacrylate, tranexamic acid, aluminum hydroxyl, EDTA (ethylenediaminetetraacetic acid), and water; microemulsions for skin use, in addition to the active drug, also include conventional excipients such as oleic acid, Tween, co-emulsifiers, and water; and spray films for skin use, in addition to Pulsatilla chinensis saponin B4 derivatives, also include conventional excipients such as polyacetylpyrrolidone, hydroxypropyl methylcellulose, and water. The amount of excipients used in each dosage form follows standard techniques and meets the requirements for general topical medications.

[0188] In summary, this invention discloses a Pulsatilla saponin B4 derivative, its preparation method, and its application. Using Pulsatilla saponin B4 (AB4) as a raw material, derivatives are prepared by targeting the C-19 exocyclic double bond or the C-28 carboxyl group through nucleophilic substitution, electrophilic addition, esterification, or amidation reactions. Drugs with anti-inflammatory and immunomodulatory effects are prepared from the active components of the AB4 derivatives. This invention is the first to disclose that AB4 derivatives have therapeutic effects on inflammatory diseases such as inflammatory bowel disease, atopic dermatitis, eczema, and psoriasis, and their efficacy is superior to AB4 or commonly used clinical drugs. Furthermore, this invention is the first to disclose that most AB4 derivatives did not show significant cytotoxicity against macrophages, and reduced the P-IκBa protein level in the LPS and ATP-induced macrophage NF-κB signaling pathway, inhibited NLRP3 signaling pathway activation, and significantly reduced Pro-IL-1β levels (p<0.05), i.e., inhibited the activation of the inflammasome pathway, with effects superior to AB4. These results suggest that the AB4 derivatives of this invention have better anti-inflammatory activity. Specifically, these AB4 derivatives showed significant therapeutic effects on atopic dermatitis, reducing ear swelling, improving skin ulceration, and exhibiting better therapeutic efficacy than the positive control drugs dexamethasone and AB4, with significantly fewer toxic side effects than dexamethasone. This invention also employed an imiquimod-induced psoriasis mouse model. On the second day after modeling, the mice showed a decreasing weight trend, with subsequent daily decreases in the model groups. The halometasone group experienced a sharp decrease in weight, while the B4-33 and B4-39 groups showed significantly higher weights from day 4 onwards (p < 0.05). Regarding the PASI score, B4-33 and B4-39 both improved the skin of psoriasis mice, with effects superior to the AB4 group and comparable to the halometasone group. Furthermore, this invention also used a DSS-induced mouse colitis model, administering B4-39, AB4, and the control drug mesalazine by gavage. Based on the three indicators for assessing the severity of colitis in mice—colon length, DAI score, and body weight—B4-39 showed better efficacy in treating colitis than AB4 and mesalazine. Similarly, using other biochemical experiments and pathological observations, B4-39 was superior to the control drugs AB4 and mesalazine in inhibiting colonic epithelial cell apoptosis, inhibiting the release of inflammatory factors, and alleviating pathological changes in colonic tissue, with lower toxicity and side effects.

Claims

A derivative of Pulsatilla saponin B4 has the following general chemical structural formula: In the formula, R1 includes 3-O-α-L-rhamnopyranose-(1→2)-α-L-arabinopyranose; R2 includes hydroxyl or acetoxy; R3 includes 2-allyl, 2-propyl, 3-hydroxypropenyl, 3-bromopropenyl, or 2-epoxyethylenemethyl; R4 includes 1-oxobenzotriazolyl, methoxy, hydroxyl, or 2-O-α-L-rhamnopyranose-(1→4)-β-D-glucopyranose. Sugar-(1→6)-β-D-glucopyranosyl, 28-O-α-L-[2,3,4-triacetoxy-rhamnopyranose]-(1→4)-β-D-[2,3,6-triacetoxy-glucopyranosyl]-(1→6)-β-D-[2,3,4-triacetoxy-glucopyranosyl]yl, 2-methoxyethylamino, methyl 4-aminobutyrate, cyclopentanamino, 3-chloropropylamino, 2- Fluoroethylamino, 1-(3-aminopropyl)benzotriazolyl, cyclohexylamino, 1-cyclopropylethylamino, cyclobutylmethylamino, 1-(3-aminopropyl)imidazolyl, N-(2-aminoethyl)pyrrolidinyl, 4-aminofuranyl, methyl 3-aminocyclopentanecarboxylate, methyl 4-aminocyclohexylcarboxylate, allylamino, 1-(2-aminoethyl)piperidinyl, 2-thiazolylethylamino, tetrahydrofuranmethylamino, N- Aminoethylmorpholino, 1-methyl-4-piperidinemethylamino, 1-methylpyrrolidine-3-methylamino, 4-aminocyclohexanol, β-phenylethylamino, 2-thiopheneethylamino, p-hydroxyphenylethylamino, 4-oxazolylmethylamino, glycine, N-(2-aminoethyl)acetamido, 4-aminobutyric acid, 5-aminovaleric acid, 6-aminohexanoic acid methyl ester, 3-L-aminocyclopentanol, 3-D-aminocyclopentanol. According to claim 1, the purslane saponin B4 derivative is characterized in that, The Pulsatilla saponin B4 derivative is formulated into a drug for treating or relieving inflammation; or the Pulsatilla saponin B4 derivative is used to treat or relieve inflammation. The method for preparing the Pulsatilla saponin B4 derivative according to claim 1 is characterized in that, The pulsatilla saponin B4 derivative was prepared by using nucleophilic substitution, electrophilic addition, esterification or amidation reactions as raw materials. A pharmaceutical system comprising the Pulsatilla saponin B4 derivative of claim 1 as an active ingredient. The drug system according to claim 4 is characterized in that, The drug system includes topical, oral, rectal, or parenteral medications; the dosage forms of the drug system include pills, tablets, powders, capsules, granules, ointments, solutions, injections, gels, or suppositories. The use of the Pulsatilla saponin B4 derivative of claim 1 or the pharmaceutical system of claim 4 in the preparation of anti-inflammatory drugs or immunomodulatory drugs. The application according to claim 6 is characterized in that, The inflammation includes inflammation on the body surface and inflammation inside the body. A method for treating or relieving inflammation, characterized in that, The procedure includes the step of administering a drug; the drug includes the Pulsatilla saponin B4 derivative of claim 1 or the drug system of claim 4. The method for treating or relieving inflammation according to claim 8 is characterized in that, The inflammation includes inflammation on the body surface and inflammation inside the body. The method for treating or relieving inflammation according to claim 8 is characterized in that, The drug is administered to patients who require it.

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