Pulsatilla saponin b4 derivative, preparation method therefor, and use thereof

Abstract

Disclosed are a Pulsatilla saponin B4 derivative, a preparation method therefor, and a use thereof. A compound Pulsatilla saponin B4 (referred to as B4 or AB4) is used as a raw material, a nucleophilic substitution reaction, an electrophilic addition reaction, an esterification reaction, or an amidation reaction is separately performed on a C-19 exocyclic double bond or a C-28 carboxyl group to prepare a derivative, and the derivative is used as an active ingredient to prepare a drug having anti-inflammatory and immunomodulatory effects. In the present invention, it is disclosed for the first time that the B4 derivative has the effect of treating inflammatory diseases such as inflammatory bowel disease, atopic dermatitis, eczema, and psoriasis, and the effect is superior to that of B4 or a clinically common drug. In addition, it is also disclosed for the first time that most B4 derivatives do not show obvious cytotoxicity to macrophages, and can reduce the level of P-IκBa protein in LPS and ATP-induced macrophage NF-κB signaling pathways, inhibit the activation of NLRP3 signaling pathways and obviously reduce the level of Pro-IL-1β (p<0.05), i.e., inhibiting the activation of an inflammasome pathway, and the effect is superior to that of B4. These results show that the B4 derivative of the present invention has better anti-inflammatory activity.

Description

A pulsatilla saponin B4 derivative and its preparation method and application Technical Field

[0001] The present invention belongs to the field of biomedicine technology and relates to a pulsatilla saponin B4 derivative and a preparation method thereof, as well as the application of such derivative in anti-inflammatory, such as in treating or alleviating inflammatory diseases such as skin inflammation and intestinal inflammation and autoimmune diseases. Background Art

[0002] Generally speaking, inflammation is the body's defensive response to irritation, with common symptoms including redness, heat, swelling, and pain. Inflammation is broadly categorized based on its causes, including acute and chronic inflammation, local and systemic inflammation, and infectious and non-infectious inflammation. Inflammation is closely linked to maintaining homeostasis. During inflammation, damaging factors directly or indirectly damage tissues and cells. Inflammatory congestion and exudation dilute, kill, and surround the damaging factors. Simultaneously, 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 various tissues. However, when inflammatory responses become uncontrollable within the body, they can lead to a range of diseases, such as arthritis, pneumonia, gastritis, nephritis, enteritis, and skin inflammation. To maintain health and restore normal physiological functions, anti-inflammatory medications are essential.

[0003] As we all know, the classic anti-inflammatory drugs are non-steroidal anti-inflammatory drugs and steroids, but both types of drugs have a series of adverse reactions or toxic side effects, such as gastrointestinal dysfunction, 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 committed to finding and developing anti-inflammatory drugs with good efficacy, low toxic side effects and easy acceptance by patients.

[0004] SUMMARY OF THE INVENTION Technical issues

[0005] As we all know, the classic anti-inflammatory drugs are non-steroidal anti-inflammatory drugs and steroids, but both types of drugs have a series of adverse reactions or toxic side effects, such as gastrointestinal dysfunction, 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 committed to finding and developing anti-inflammatory drugs with good efficacy, low toxic side effects and easy acceptance by patients. Technical Solutions

[0006] Existing anti-inflammatory drugs have obvious toxic side effects, and some drugs have the defect of a short half-life. To understand and solve this problem, the present invention discloses a pulsatilla saponin B4 derivative and a preparation method, as well as the use of such derivatives in the preparation of anti-inflammatory drugs (including those for skin inflammation, intestinal inflammation and other diseases). The derivatives are compounds or drugs with few side effects, high safety and good efficacy.

[0007] Pulsatilla chinensis (Bunge) Regel is a perennial herbaceous plant of the genus Pulsatilla in the family Ranunculaceae. Its dried root is used medicinally, with the effects of clearing heat and detoxifying, cooling blood and stopping dysentery. Extensive research has been conducted on the various components of Pulsatilla chinensis, with Pulsatilla saponin B4 (abbreviated as AB4) being the main active substance. The applicant previously used Pulsatilla saponin B4 to treat psoriasis, atopic dermatitis, and inflammatory bowel disease, but found that its lipid solubility was low and its efficacy needed further improvement. The present invention uses Pulsatilla saponin B4 as the lead compound and undergoes a series of structural modifications and alterations, unexpectedly improving the drug's activity and lipid solubility. The resulting series of derivatives overcomes the aforementioned issue of AB4's poor drugability and exhibits superior activity to B4.

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

[0009] A pulsatilla saponin B4 derivative having the following general chemical structure:

[0010] In the formula, R1 is 3-O-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosyl; R2 is hydroxyl or acetoxy; R3 is 2-allyl, 2-propyl, 3-hydroxypropenyl, 3-bromopropenyl, 2- Oxiranylmethyl; R4 is 1-oxybenzotriazolyl, methoxy, hydroxy, 28-O-α-L-rhamnose-(1→4)-β-D-glucopyranose-(1→6)-β-D-glucopyranose, 28-O-α-L-[2,3,4-triacetoxy-rhamnose]-(1→4)-β-D-[2,3,6-triacetoxy-glucopyranose]-(1→6)-β-D-[2,3,4-triacetoxy-glucopyranose]-(1→6)-β-D-[2,3,4-triacetoxy-glucopyranose]-yl, 2-methoxyethylamino, 4-aminobutyric acid methyl ester, cyclopentylamino, 3-chloropropylamino, 2-fluoroethylamino, 1-(3-aminopropyl)benzotriazolyl, cyclohexylamino, 1-cyclopropylethylamino, cyclobutylmethylamino, 1-(3-aminopropyl)imidazolyl, N-(2-aminoethyl)pyrrolidinyl, 4-aminofuranyl, 3-aminocyclopentanecarboxylic acid methyl ester, 4-aminocyclohexylcarboxylic acid methyl ester, allylamino, 1-(2-aminoethyl)piperidinyl, 2-thiazolylethylamino, tetrahydrofuranmethylamino, N-aminoethylmorpholinyl, 1-methyl-4-piperidinylmethylamino, 1-methylpyrrolidin-3-methylamino, 4-aminocyclohexanol, β-phenylethylamino, 2-thiopheneethylamino, p-hydroxyphenylethylamino, 4-oxazolemethylamino, glycine, N-(2-aminoethyl)acetamido, 4-aminobutyric acid, 5-aminopentanoic acid, 6-aminohexanoic acid methyl ester, 3-L-aminocyclopentanol, 3-D-aminocyclopentanol. Preferably, R3 is one of 2-allyl, 2-propyl, and 3-hydroxypropenyl; R4 is one of 1-oxybenzotriazolyl, 1-(3-aminopropyl)benzotriazolyl, N-aminoethylmorpholinyl, and p-hydroxyphenylethylamino; further preferably, the derivative of pulsatilla saponin B4 is compound B4-19, B4-33, and B4-39, etc.

[0011] The invention discloses a drug system, which uses the pulsatilla saponin B4 derivative as an active ingredient.

[0012] The present invention discloses the application of the pulsatilla saponin B4 derivative or drug system in the preparation of anti-inflammatory drugs.

[0013] The present invention also discloses the use of the pulsatilla saponin B4 derivative or drug system in the preparation of immune-regulating drugs.

[0014] The present invention also discloses the use of the above-mentioned pulsatilla saponin B4 derivative or drug system in the preparation of drugs for improving psoriasis and atopic dermatitis.

[0015] The present invention also discloses the use of the above-mentioned pulsatilla saponin B4 derivative or drug system in the preparation of drugs for improving inflammatory bowel disease.

[0016] In the present invention, the pulsatilla saponin B4 derivative is formulated into a drug for treating or alleviating inflammation; or the pulsatilla saponin B4 derivative is used to treat or alleviating inflammation.

[0017] The present invention discloses a method for treating or alleviating inflammation, comprising administering a drug; the drug comprises the pulsatilla saponin B4 derivative or the drug system. Furthermore, the drug is administered to a patient in need thereof to treat or alleviate inflammation, wherein the patient in need thereof is a patient in need of treatment or alleviation of inflammation.

[0018] In the present invention, the inflammation includes inflammation on the body surface and inflammation in the body. Further preferably, the inflammation includes inflammatory diseases such as skin inflammation and intestinal inflammation (e.g., inflammatory bowel disease). As an example, the present invention discloses the use of the above-mentioned pulsatilla saponin B4 derivative or drug system in the preparation of drugs for treating atopic dermatitis, psoriasis, skin erythema, scales, thickening of skin lesions, ichthyosis, melasma, keratosis, keratoderma, dry and rough skin, colitis (or inflammatory bowel disease), etc.

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

[0020] In the present invention, the drugs (such as anti-inflammatory drugs or immunomodulatory drugs) include drugs for external use, oral administration, injection, rectal administration or parenteral administration. In the above-mentioned method for treating or alleviating inflammation, administration includes external use, injection, bolus, oral administration or inhalation.

[0021] In the present invention, the drug (such as an anti-inflammatory drug or an immunomodulatory drug) is prepared into a pharmaceutically acceptable dosage form, such as a pill, tablet, powder, capsule, granule (powder), ointment, solution, gel or suppository, and a solution includes a pellet, drop, spray, injection, and suspension.

[0022] The invention discloses a preparation method of the pulsatilla saponin B4 derivative. The pulsatilla saponin B4 derivative is prepared by using B4 as a raw material through reactions such as nucleophilic substitution, electrophilic addition, esterification or amidation.

[0023] The B4 derivative of pulsatilla saponin, disclosed for the first time in this invention, showed no significant cytotoxicity against THP-1 macrophages, but was able to reduce the level of P-IκBa protein in the NF-κB signaling pathway, inhibit the activation of the NLRP3 signaling pathway, and significantly reduce Pro-IL-1β levels (p < 0.05), with an effect superior to that of B4. These results indicate that the B4 derivative of the present invention has superior anti-inflammatory and immunomodulatory activity. In mice with DNCB-induced atopic dermatitis (eczema), the B4 derivative had a significant therapeutic effect on atopic dermatitis, significantly reducing ear swelling and effectively improving ulceration and edema on the back and ear skin of the mice, with an effect superior to that of the positive drugs dexamethasone and B4.

[0024] The present invention also employed a mouse colitis model, administering B4-39, B4, and the control drug mesalazine via oral gavage. Studies of mouse colon length, DAI scores, biochemical markers, and pathological observations demonstrated that the B4-39 derivative was superior to B4 and mesalazine in treating colitis. Similarly, B4-39 was superior to the control drugs B4 and mesalazine in inhibiting colon epithelial cell apoptosis, while exhibiting lower toxic side effects. Furthermore, the B4 derivative exhibited a certain immunomodulatory effect while treating the aforementioned diseases, while the present derivative lacked the immunosuppressive side effects of dexamethasone. Beneficial effects

[0025] The present invention uses a derivative of Pulsatilla saponin B4 as the sole active ingredient to be applied externally to the psoriasis site to achieve effective treatment of psoriasis. Furthermore, the drug does not contain other Pulsatilla extracts. Existing Chinese medicines all use decoctions to treat or alleviate psoriasis, which are less effective than Western medicines. In addition, the decoctions are inconvenient to prepare and have side effects. Long-term use of drugs, even Chinese medicines, can cause adverse stimulation to the gastrointestinal tract. The externally applied medicament of the present invention is convenient to use and avoids side effects while exerting a better therapeutic effect than existing Western medicines. Animal experiments showed that after modeling with imiquimod, the weight of the animals showed a downward trend on the second day, and the model group subsequently showed a downward trend every day. The weight of the halometasone-administered group dropped sharply, and the weight of the B4-33-administered group and the B4-39-administered group was significantly better than that of the halometasone group from the 4th day, with statistically significant differences (p < 0.05); in the PASI scores of the drug-administered groups, B4-33 and B4-39 both had an improvement effect on the skin of psoriasis mice, and the effect was better than that of the B4 group, and was equivalent to that of the halometasone group. After modeling, the spleen of the mice was significantly enlarged, with significant differences compared to the normal group. The B4-39 administration group also showed a certain improvement in the spleen index, and the difference was significant. In short, the present invention uses the pulsatilla saponin B4 derivative for the treatment of psoriasis, which not only solves the problem of toxic side effects of existing drugs with good clinical therapeutic effects, but also unexpectedly achieves better therapeutic effects than B4, which is creative. Compared with other modes of administration, the pulsatilla saponin B4 derivative disclosed in the present invention has the advantages of convenient and safe medication as a topical drug for the treatment of psoriasis. In particular, the present invention avoids the problems of obvious side effects and large dosage of oral medication. The therapeutic effect of topical administration is significantly better than that of existing clinical medication, achieving unexpected technical effects. For example, the drug for the treatment of psoriasis uses the pulsatilla saponin B4 derivative as the active ingredient and does not contain other pulsatilla extracts. A small dose of administration can achieve a better technical effect than the existing halometasone cream that is considered to be effective. Animal experimental results show that the pulsatilla saponin B4 derivative has a protective effect on changes in psoriasis lesions, and the effect is better than that of glucocorticoid drugs. In particular, the pulsatilla saponin B4 derivative of the present invention unexpectedly achieves a significant technical improvement over B4. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] FIG1 is a reaction diagram of the preparation scheme (1) of the pulsatilla saponin B4 derivative of the present invention.

[0027] FIG2 is a reaction diagram of the preparation scheme (2) of the pulsatilla saponin B4 derivative of the present invention.

[0028] FIG3 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] FIG5 shows the nitrite production levels of non-cytotoxic AB4 derivatives (10 μM) under LPS stimulation.

[0031] FIG6 is a western blotting diagram of the anti-inflammatory activity of AB4 derivatives.

[0032] FIG7 shows the statistical results of western blotting for the anti-inflammatory activity of AB4 derivatives.

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

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

[0035] FIG10 is a schematic diagram of the weight changes and back scores of mice.

[0036] FIG11 is a schematic diagram showing the difference in ear thickness and ear weight in mice.

[0037] FIG12 is a schematic diagram of mouse spleen index.

[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 administration group vs. halometasone group *p<0.05, **p<0.01, **p<0.001; B4-39 administration group vs. halometasone group #p<0.05, ##p<0.01; A3-9 administration group vs. halometasone group $p<0.05, $$p<0.01.

[0039] FIG14 shows the effects of pulsatilla saponin B4 derivatives on the external morphology of psoriatic skin in psoriasis mice.

[0040] Figure 15 shows the effect of B4-33, a derivative of pulsatilla saponin B4, on the psoriasis severity index (PASI) of mice, normal group vs. model group **p<0.01, **p<0.001, **p<0.0001; B4-33-administered group vs. model group #p<0.05, ##p<0.01, ###p<0.001, ####p<0.0001; halometasone-administered group vs. model group $p<0.05, $$p<0.01, $$$$p<0.0001; B4-administered group vs. model group &&p<0.01, &&&p<0.001.

[0041] Figure 16 shows the effect of B4-39, a derivative of pulsatilla saponin B4, on the psoriasis severity index (PASI) of mice, normal group vs. model group **p<0.01, **p<0.001, **p<0.0001; B4-39-administered group vs. model group #p<0.05, ##p<0.01, ###p<0.001, ####p<0.0001; halometasone-administered group vs. model group $p<0.05, $$p<0.01, $$$$p<0.0001; B4-administered group vs. model group &&p<0.01, &&&p<0.001.

[0042] Figure 17 shows the effects of A3-9, a derivative of Pulsatilla saponin B4, on the psoriasis severity index (PASI) of mice. Normal group vs. model group **p<0.01, **p<0.001, **p<0.0001; A3-9-administered group vs. model group #p<0.05, ##p<0.01, ###p<0.001, ####p<0.0001; halometasone-administered group vs. model group $p<0.05, $$p<0.01, $$$$p<0.0001; B4-administered 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 psoriasis 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] FIG20 shows the effects of B4-39 on colon length (A&B), DAI score (C), and body weight (D) of UC mice.

[0046] FIG21 shows the effect of B4-39 on pathological changes in colon tissue of UC mice.

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

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

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

[0050] Figure 25 shows the effects of B4-39 on the release of inflammatory factors in the colon tissue of UC mice: (A) IL-6 levels; (B) IL-1β levels; (C) TNF-α levels. Modes for Carrying Out the Invention

[0051] Atopic dermatitis (AD) is a common, immune-mediated inflammatory skin disease characterized by recurrent, pruritic, localized eczema, often with seasonal fluctuations. Atopic dermatitis is also known as atopic eczema, neurodermatitis, atopic dermatitis, and, most commonly, eczema. Current treatments for eczema include various topical corticosteroids (TCSs), the topical calcineurin inhibitors tacrolimus and pimecrolimus, and the phosphodiesterase 4 (PDE4) inhibitor crisaborole. For more severe AD, in addition to ultraviolet light, current treatment guidelines recommend cyclosporine A, methotrexate, azathioprine, and mycophenolate mofetil, but these treatments have significant side effects and are expensive. Pulsatilla saponin B4 (B4 or AB4) has therapeutic effects on atopic dermatitis, but AB4 currently suffers from issues such as high molecular weight and water solubility, resulting in low bioavailability.

[0052] Psoriasis is a chronic inflammatory skin disease whose pathogenesis is believed to be multifactorial, involving genetic, environmental, and immune factors. Psoriasis is a complex disease with a multifactorial inheritance pattern, involving the interaction of multiple genes. Current research has identified psoriasis susceptibility loci, including PSO RS1 on 6p21.3, PSORS2 on 17q, PSORS3 on 4q, PSORS4 on 1cen-q21, PSORS5 on 3q21, and PSORS6 on 19p13. The various treatments for psoriasis provide short-term relief rather than a complete cure. Commonly used clinical treatments are primarily categorized as chemical agents and biologics. Chemical agents are further divided into topical and oral agents. Topical agents include emollients, moisturizers, vitamin D3 derivatives, retinoids, glucocorticoids, 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 obvious, and there are many adverse reactions. For example, topical preparations often cause skin atrophy, swelling, stinging, itching, burning sensation and other reactions. In addition, long-term use of glucocorticoids will also produce a large number of adverse reactions. Currently, biological agents for the treatment of psoriasis mainly include four categories, namely TNF-α inhibitors, IL-12 / 23 inhibitors, IL-17 inhibitors and IL-23 inhibitors. Although emerging biological agents targeting psoriasis have good clinical application prospects, they are expensive and are prone to drug resistance after long-term use. In addition to skin reactions, biological agents may also cause adverse reactions such as gastrointestinal reactions, infections and autoimmune diseases. Therefore, more drugs are still needed to treat psoriasis.

[0053] Inflammatory bowel disease, primarily encompassing ulcerative colitis (UC) and Crohn's disease, is a chronic, relapsing inflammatory disease. UC primarily affects the colonic mucosa and proximal rectum. Its pathogenesis is characterized by diffuse disease, recurrent and progressively worsening symptoms, prolonged disease resolution, and a high incidence of malignant transformation. Typical clinical symptoms of UC include chronic diarrhea, bloody and mucous stools, abdominal pain, weight loss, fatigue, and a feeling of tenesmus. The persistence of these symptoms not only impacts patients' quality of life but can also lead to psychological distress and social dysfunction. The pathogenesis of UC is complex, and its exact cause remains unclear. However, it is generally believed to be associated with multiple factors, including genetic susceptibility genes, environmental factors, immune system abnormalities, and an imbalance in the intestinal microbiome. Pathophysiologically, UC involves disruption of the intestinal mucosal barrier, abnormal activation of immune cells, and an imbalance between pro- and anti-inflammatory factors. Currently, drug therapy is the core treatment strategy for UC, encompassing a wide range of medications to address the treatment needs of patients at different stages and severity levels. Topically acting medications, such as sulfapyridine, a 5-aminosalicylic acid (5-ASA) drug, act directly on the colonic mucosa to reduce inflammation and are suitable for patients with mild to moderate UC. Glucocorticoids, such as prednisone, are often used to rapidly induce clinical remission due to their rapid and potent anti-inflammatory properties and are particularly 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 who require long-term maintenance of remission. Biologics, such as ustekinumab and adalimumab, are targeted agents that target inflammatory mediators such as tumor necrosis factor (TNF)-α, providing precise anti-inflammatory effects and are particularly suitable for patients who have not responded well to conventional treatments. For patients with UC who do not respond to medical treatment or develop severe complications, surgical treatment may be necessary. Common surgeries include colectomy, which involves partial or complete removal of the colon and rectum to eliminate diseased tissue. Although the drugs currently used to treat ulcerative colitis (UC) play a key role in controlling symptoms, they generally have certain side effects, such as possible nephrotoxicity, gastrointestinal discomfort, or bone marrow suppression. In addition, for some patients with severe or refractory UC, surgical treatment and the use of expensive biological agents may impose a heavy economic burden. Therefore, the development of drugs that are both highly effective, less toxic, and low-cost is of great significance for improving the treatment effect and quality of life of UC patients. This will not only provide patients with more treatment options, but also reduce the economic and psychological burden, thereby improving the overall level of disease management.

[0054] The invention discloses a preparation method of the pulsatilla saponin B4 derivative. The pulsatilla saponin B4 derivative is prepared by using compound AB4 as a raw material through reactions such as nucleophilic substitution, electrophilic addition, esterification or amidation.

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

[0056] (1) AB4 was used as the raw material and hydrolyzed in a sodium hydroxide aqueous solution at 105°C to obtain the intermediate A3, and then the glycosyl portion was acetyl protected; the double bond portion was modified by oxidation, nucleophilic substitution, reduction, hydrolysis and other reactions to obtain some pulsatilla saponin B4 derivatives. The reaction schematic is shown in Figure 1. The reaction conditions are: (a) NaOH / H2O, 105°C, 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 was used as the modified group, and amide condensation was carried out under the action of TBTU (O-benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroborate), DIEA (N,N-diisopropylethylamine), and DMF (N,N-dimethylformamide) to obtain a series of amide derivatives, which are some derivatives of pulsatilla saponin B4. The reaction schematic is shown in Figure 2. The reaction conditions are: (a) NaOH / H2O, 105°C, 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 the raw material, while protecting the entire sugar group, the double bond part was modified by oxidation, reduction, nucleophilic substitution, etc. to obtain some pulsatilla saponin B4 derivatives. The reaction schematic is shown in Figure 3. The reaction conditions are: (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 (lupane-type pentacyclic triterpenoid saponin compound) according to the raw materials and reaction conditions of the present invention according to conventional techniques, or adopt other methods to obtain the product of the present invention.

[0060] The pharmaceutical system disclosed herein comprises the aforementioned pulsatilla saponin B4 derivative as an active ingredient and also includes a pharmaceutically acceptable carrier. The active ingredient and pharmaceutical system are used to prepare a therapeutic drug for inflammatory diseases or immune diseases. For example, the drug comprises a therapeutically effective amount of a pulsatilla saponin B4 derivative or its hydrochloride, perchlorate, methanesulfonate, phosphate, citrate, or sulfate, and a pharmaceutically acceptable carrier.

[0061] In the present invention, a pharmaceutically acceptable carrier refers to one or more compatible solid or liquid fillers or gels that are pharmaceutically acceptable, have sufficient purity, and low toxicity, and are compatible with each other and with the active ingredient of the present invention without reducing the efficacy of the active ingredient. Pharmaceutically acceptable carriers include diluents, solubilizers, cosolvents, disintegrants, dispersants, lubricants, flavoring agents, antioxidants, binders, absorbents, wetting agents, buffers, and crosslinking agents. Some examples of pharmaceutically acceptable carriers 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, glycerol, mannitol, sorbitol, etc.), cyclodextrins (such as hydroxypropyl β-cyclodextrin), emulsifiers (such as Tween), wetting agents (such as sodium lauryl sulfate), colorants, flavorings, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0062] In the present invention, the drug includes topical, oral, rectal or parenteral drugs. The drug is prepared into pharmaceutically acceptable dosage forms, such as pills, tablets, powders, capsules, granules (powders), ointments, liquids, gels or suppositories, and liquids include pills, drops, sprays, injections and suspensions.

[0063] The present invention discloses the use of a pulsatilla saponin B4 derivative in the preparation of an anti-inflammatory drug. The derivative of the present invention can be administered alone or in combination with other therapeutic agents. The administration method of the active ingredient or pharmaceutical system of the present invention is not particularly limited; representative administration methods include topical, oral, rectal, and parenteral (e.g., intravenous, intramuscular, or subcutaneous). 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, the liquid dosage form may contain diluents commonly used in the art, such as water or other solvents, solubilizers, and emulsifiers, such as ethanol, isopropyl alcohol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butylene glycol, dimethylformamide, and oils, particularly cottonseed oil, peanut oil, corn germ oil, elm oil, sesame oil, and sesame oil, or mixtures thereof. In addition to these inert diluents, the composition may also contain adjuvants such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents, and fragrances. In addition to the active ingredient, the suspension may contain a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide, and agar, or mixtures thereof. Compositions for parenteral injection may include 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] Prior art discloses that scutellaria saponin B4 (abbreviated as AB4 or B4) has applications in treating inflammation. However, AB4's strong water solubility, short half-life, and low oral availability limit its clinical application. The present invention structurally modifies AB4 to produce compounds with enhanced anti-inflammatory activity and very low toxicity. The synthetic routes of the scutellaria saponin B4 derivatives of the present invention are shown in Figures 1 to 3, and the in vitro and in vivo activity results are shown in Figures 4-25.

[0065] The present invention will be further described below with reference to specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. Unless otherwise stated, percentages and parts are percentages by weight and parts by weight. Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein can be applied to the methods of the present invention, and the animal experiments involved meet the relevant requirements of Soochow University. In the following preparation examples, reagents are existing products, mainly provided by Shanghai Chemical Reagent Company; 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 Inokai Technology Co., Ltd., 200-300 mesh. NMR measurements were recorded on a Varian Mercury 400M NMR spectrometer, and chemical shifts are expressed in δ (ppm); DMF: N,N-dimethylformamide; DCM: dichloromethane; THF: tetrahydrofuran; TBTU: O-benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroborate; DIPEA: N,N-diisopropylethylamine; PE: petroleum ether; EA: ethyl acetate.

[0066] Statistical analysis of all data was performed using GraphPad Prism 8 software. Statistically significant differences between mean values ​​were compared using GraphPad Prism 8. All data were analyzed using one-way or two-way ANOVA. P values ​​were used to represent differences between the two groups. If P ≤ 0.05, a statistically significant difference was observed between the two groups, indicated by “#” or “*.” “#” indicates the difference between the normal group and the model group, and “*” indicates the difference between the drug-treated group and the model group (in cytotoxicity experiments, “*” indicates the difference between the drug-treated group and the normal group). If P ≤ 0.01, a significant statistical difference was observed between the two groups, indicated by “##” or “**.” If P ≤ 0.001, a highly significant statistical difference was observed between the two groups, indicated by “###” or “***.” If P ≤ 0.0001, an extremely significant statistical difference was observed between the two groups, indicated by “####” or “***.”

[0067] The specific structures of the 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, 2.5 ml of acetic anhydride and DMAP (14 mg, 0.082 mmol) were added, and the mixture was stirred at room temperature for 28 hours. 50 ml of ethyl acetate was added to the reaction solution, and then 100 ml of water was added to extract the organic phase. The organic phase was washed twice with water, dried, and spin-dried to obtain fully acetylated B4. Fully acetylated B4 (300 mg, 0.2456 mmol) was dissolved in 15 ml of carbon tetrachloride, NBS (48 mg, 0.2702 mmol) was added, and the mixture was stirred at room temperature for 24 hours to obtain the intermediate 3-O-α-L-[3,4-diacetoxy-rhamnopyranosyl]-(1→2)-α-L-[2,3,4-triacetoxy-arabinopyranosyl]-3β,23-dihydroxylupane-Δ 20(29) Olefin-30-bromo-28-O-α-L-[2,3,4-triacetoxy-rhamnose]-(1→4)-β-D-[2,3,6-triacetoxy-glucopyranose]-(1→6)-β-D-[2,3,4-triacetoxy-glucopyranose]. The above intermediate (100 mg, 0.052 mmol) was dissolved in 4 ml of a dichloromethane:methanol (2:1) mixture, and potassium carbonate (161 mg, 1.165 mmol) was added. The mixture was stirred at room temperature for 16 hours. After the reaction, the mixture was concentrated under reduced pressure and purified by preparative liquid chromatography (60% methanol-water) to obtain 12 mg of a white solid with a yield of 22%. 1 H NMR(400MHz,Methanol-d4)δ5.51(1H,d,J=8.1Hz,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=4.7Hz,1-H of ara),4.42(1H,d,J=7.8Hz,1′-H of glc),4.16(1H,d,J=11.9Hz,H1-23),3.95(2H,s,H-30),1.30(3H,d,J=6.3Hz,6-H3 of rha),1.28(3H,d,J=6.2Hz,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). 13C NMR(101 MHz, MeOD) δ176.33,152.63,110.16,104.60,104.28,102.91,101.86,95.28,82.30,79.56,78.26,77.98,76. 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 mixture was filtered and the precipitate was washed twice with water to obtain ulsatilla 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 mixture, followed by 100 ml of water. The organic phase was extracted and washed twice with water. The organic phase was dried and spin-dried to obtain the intermediate 3-O-α-L-[3,4-diacetoxy-rhamnopyranosyl]-(1→2)-α-L-[2,3,4-triacetoxy-arabinopyranosyl]-3β,23-dihydroxylupane-Δ 20(29) En-28-acid. The above intermediate (1 g, 0.998 mmol) was dissolved in a mixture of DCM and methanol (2:1), and Pd / C (50 mg) was added. The mixture was reacted at room temperature under hydrogen for 18 hours. The reaction was monitored by HPLC. After completion of the reaction, the mixture was filtered, concentrated under reduced pressure to remove the solvent, and purified by C18 preparative separation (75% methanol in water) to obtain 523 mg of a white solid with a yield of 52.3%. 1H 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). 13 C 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: B4-4 (200 mg, 0.20 mmol) was dissolved in 4 mL of a mixed solution of methanol / tetrahydrofuran / water (2:1:1). Sodium hydroxide (72 mg, 1.8 mmol) was added and stirred at room temperature for 12 h. After the reaction was completed, the solvent was removed under reduced pressure. The product was washed with 50 mL of water to remove salts and dried to obtain 120 mg of a white solid with a yield of 80.1%. 1H 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). 13 C 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-rhamnopyranosyl]-(1→2)-α-L-[2,3,4-triacetoxy-arabinopyranosyl]-3β,23-dihydroxylupane-Δ 20(29) En-28-acid (1 g, 0.998 mmol) was dissolved in 10 ml of chloroform, and m-chloroperbenzoic acid (207 mg, 1.20 mmol) was added at low temperature. After complete dissolution, the temperature was raised to 65°C and refluxed for 16 hours. After the reaction was completed, the solvent was concentrated under reduced pressure and purified by silica gel column chromatography (dichloromethane:methanol = 80:1 → 70:1) to obtain 305 mg of a white solid with a yield of 30%. 1H 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). 13 C 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: B4-6 (100 mg, 0.098 mmol) was dissolved in 4 mL of a mixed solution of methanol / tetrahydrofuran / water (2:1:1), and sodium hydroxide (35.3 mg, 0.882 mmol) was added. The mixture was stirred at room temperature for 12 h. After the reaction was completed, the solvent was removed by concentration under reduced pressure. Silica gel column chromatography (dichloromethane:methanol = 10:1) gave 15 mg of a white solid, with a yield of 20%. 1H 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). 13 C 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-rhamnopyranosyl]-(1→2)-α-L-[2,3,4-triacetoxy-arabinopyranosyl]-3β,23-dihydroxylupane-Δ 20(29) En-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, it was filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane:methanol=70:1) to obtain 211 mg of a white solid with a yield of 41.5%. 1H 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). 13 C 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-rhamnopyranosyl]-(1→2)-α-L-[2,3,4-triacetoxy-arabinopyranosyl]-3β,23-dihydroxylupane-Δ 20(29)En-28-acid (500 mg, 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, it was filtered, concentrated under reduced pressure, and purified by silica gel column chromatography (dichloromethane:methanol=80:1) to obtain 324 mg of an off-white solid with a yield of 60%. 1 H 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: B4-9 (200 mg, 0.185 mmol) was dissolved in 4 mL of a mixed solution of methanol / tetrahydrofuran / water (2:1:1), and sodium hydroxide (66.6 mg, 1.67 mmol) was added. The mixture was stirred at room temperature for 12 h. After the reaction was completed, the solvent was removed by concentration under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane:methanol = 10:1) to obtain 100 mg of a white solid with a yield of 66.7%. 1 H 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-rhamnopyranosyl]-(1→2)-α-L-[2,3,4-triacetoxy-arabinopyranosyl]-3β,23-dihydroxylupane-Δ 20(29) En-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, 60 ml of water was added to the reaction solution to precipitate a white solid. The solid was filtered and purified by silica gel column chromatography (petroleum ether:ethyl acetate = 2:1) to obtain 452 mg of a white solid, with a yield of 81%.1 1H 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, 20.9, 20.8, 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-rhamnopyranosyl]-(1→2)-α-L-[2,3,4-triacetoxy-arabinopyranosyl]-3β,23-dihydroxylupane-Δ 20(29) En-28-acid (100 mg, 0.0998 mmol) was dissolved in 5 ml of DMF, and potassium carbonate (13.8 mg, 0.0998 mmol) and iodomethane (14.2 mg, 0.0998 mmol) were added. The mixture was stirred at room temperature for 8 hours. After the reaction was completed, the mixture was filtered and concentrated under reduced pressure to remove the solvent. 89.4 mg of white solid was obtained by silica gel column chromatography (petroleum ether:ethyl acetate = 2:1). The yield was 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] Embodiment 9

[0087] B4-14: Compound A3 (3-O-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosyl-3β,23-dihydroxylupane-Δ 20(29) En-28-acid) (100 mg, 0.1333 mmol) was dissolved in 4 ml of DMF, and DIEA (70 ul, 0.3999 mmol) was added. The mixture was stirred at room temperature for 8 hours. After the reaction was completed and the intermediate was completely generated, 2-methoxyethylamine (15 mg, 0.2 mmol) and DIEA (116 ul, 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 to precipitate a solid, which was filtered and purified by silica gel column chromatography (dichloromethane:methanol=10:1→8:1) to obtain 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 preparation method of the following compounds is similar to that of B4-14. By replacing 2-methoxyethylamine, the corresponding products can be obtained.

[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 on the hydromethyl ester is newly added (3.57 ppm). 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 B4-14, with a yield of 54%; in addition to the amide group, the newly added marker hydrogen is the monohydrogen 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 B4-14, with a yield of 43%; in addition to the amide group, the newly added marker hydrogen is a methylene group connected to the chlorine atom (3.60 ppm). 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 B4-14, with a yield of 38%; in addition to the amide group, a 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 (377 MHz, DMSO) δ 19.44.

[0094] B4-19: The preparation method is similar to that of B4-14, with a yield of 33%; in addition to the amide group, the newly added marker hydrogens are 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%; in addition to the amide group, the new marker is the carbon on the 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 newly added marker hydrogen is the methyl group on cyclopropylethane (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 B4-14, with a yield of 51%; in addition to the amide group, the newly added marker hydrogen is the monohydrogen 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 B4-14, with a yield of 55%; in addition to the amide group, the newly added 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 newly added 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 newly added marker hydrogen is a 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 newly added 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 B4-14, with a yield of 47%; in addition to the amide group, the newly added 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 newly added 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 newly added marker hydrogen is a 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 newly added 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 newly added marker hydrogen is a 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 newly added marker hydrogen is a 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 B4-14, with a yield of 56%; in addition to the amide group, the newly added marker hydrogen is a 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 newly added 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 newly added marker hydrogen is the hydroxyl group on the 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 newly added 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 newly added 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 newly added 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 newly added 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 B4-14, with a yield of 34%; in addition to the amide group, a new marker is added, namely the 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 B4-14, with a yield of 54%; in addition to the amide group, the newly added marker hydrogen is a methyl group connected to the amide bond (1.79 ppm). 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: B4-15 (100 mg, 0.118 mmol) was dissolved in 4 ml of a mixed solution of tetrahydrofuran:methanol:water (2:1:1), and sodium hydroxide (42.4 mg, 1.06 mmol) was added. The mixture was stirred at room temperature for 12 hours. After the reaction, 5 ml of water was added, and the mixture was filtered. The mixture was purified by silica gel column chromatography (dichloromethane:methanol = 8:1 → 6:1) to obtain 30 mg of an off-white solid with a yield of 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 B4-14, with a yield of 42%; in addition to the amide group, the newly added marker hydrogens are the methylene group connected to the carboxyl group (2.19 ppm) and the 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 newly added 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, the newly added marker hydrogen 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, the newly added marker hydrogen 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,D 2O)δ178.18,152.27,110.02,104.30,101.88,82.27,76.65,73.93,73.66,73.31, 72.13,72.02,70.16,69.12,64.74,64.57,56.88,52.02,51.18,50.66,48.15,44.0 4,43.63,42.42,41.99,39.92,39.31,39.06,37.81,34.99,34.68,34.11,31.93,31.72,30.53,27.01,26.69,22.16,19.62,18.77,17.96,17.22,16.77,15.08,13.50.

[0122] The following experiments were conducted using pulsatilla saponin B4 or its derivatives as experimental drugs:

[0123] Example 11 Cytotoxicity Experiment 1

[0124] HIEC cells were seeded at 5000 cells / well in a 96-well plate, with 100 μL / well added for routine culture. A normal control group and a drug-treated group were set up. After the cells adhered, 1 μL of a B4 derivative other than the intermediate was added to each well except the normal control group, resulting in a final drug concentration of 50 μM. The cells were then placed in an incubator and incubated for 24 hours. After the incubation period, 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 a wavelength of 450 nm using a microplate reader, and the cell survival rate of each well was calculated to screen out compounds with no obvious cytotoxicity in the first round.

[0125] Example 12 Cytotoxicity Experiment 2

[0126] THP-1 cells were collected at 1×10 4 Each well was inoculated into a 96-well plate, 100 μL / well, and cultured conventionally.

[0127] After the cells reached 80% confluency, they were induced with 100 ng / mL phorbol-12-myristate-13-acetate (PMA) for 12 hours. The original culture medium was discarded and 100 μL of new complete culture medium was added. A zero-well culture without cells and a normal control group without drug were set up. 1 μL of the B4 derivative that showed no significant cytotoxicity in the first round was added to each well to a final drug concentration of 50 μM. The cells were then incubated in an incubator for 24 hours.

[0128] After incubation, 10 μL of CCK-8 was added to each well and incubated in the dark for 4 h. The absorbance of each well was measured at a wavelength of 450 nm using a microplate reader, and the cell viability of each well was calculated to screen out compounds with no obvious cytotoxicity in the second round.

[0129] Calculation formula: Cell survival rate (%) = [A(drug added) - A(blank)] / [A(0 drug added) - A(blank)] × 100

[0130] A(drug added): absorbance of the wells with cells, CCK-8 and drug solution

[0131] A (blank): absorbance of wells with culture medium and CCK-8 but no cells

[0132] A(0 drug addition): absorbance of a well containing cells and CCK-8 but no drug.

[0133] As shown in Figure 4, the cytotoxicity of B4 derivatives at 50 μM. The ones without the symbol "*" in the statistical graph indicate that there is no significant difference compared with the normal control, that is, the B4 derivatives do not have obvious cytotoxicity at a dose of 50 μM. It can be seen from the figure that there are many non-cytotoxic derivatives, including B4-1, B4-5, B4-10, etc.

[0134] Among them, these derivatives include B4-39, and the cytotoxicity of B4-39 was further evaluated. Three different cell lines, RAW264.7 (mouse monocytic cell line), THP-1 (human acute monocytic leukemia cell line) and HIEC (human intestinal epithelial cells) were used for testing. These cell lines represent different types of cells and can provide in vitro information about the toxicity of B4-39 to immune cells and intestinal epithelial cells. The test results showed that at a concentration of 50 μM and acting on the cells for 24 hours, B4-39 did not show obvious cytotoxicity to these cells, indicating that B4-39 still maintained good cell compatibility at relatively high concentrations, which is an important indicator of drug safety. In addition, these results further confirmed that B4-39 not only has anti-inflammatory activity, but also may have a lower risk of side effects, which makes it a potential drug candidate.

[0135] Example 13

[0136] Nitrite content detection: Raw 264.7 cells were cultured at 6×10 4 Cells were inoculated into 96-well plates at 100 μL / well for routine culture. A blank control group (N group), a modeling group (M group), a positive drug dexamethasone group (Y group), and a drug-treated group were set up. After the cells adhered to the wall, 1 μL of the corresponding drug was added to a final concentration of 10 μM. After 1 hour, 1 μL of LPS was added to a final concentration of 1 μg / ml. The cells were then placed in an incubator and incubated for 24 hours. 50 μL of culture medium was taken from each well of the seeded plate and transferred to a new 96-well plate. 50 μL of Griess reagent A solution was added to each well under dark conditions, followed by 50 μL of B solution. The absorbance of each well was measured at a wavelength of 540 nm using a microplate reader. As shown in Figure 5, the vertical axis is the nitrite content produced by the B4 derivative under LPS stimulation, and the numbers on the horizontal axis in the figure represent different B4 derivatives of pulsatilla saponin. 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 drug-treated group containing the B4 derivative and the model group, that is, the therapeutic effect is better and there is more considerable anti-inflammatory activity.

[0137] Example 14

[0138] Western blotting experiment: THP-1 cells were cultured at 2×10 6Cells were seeded into 6-well plates, with 2 ml per well, and cultured as usual. After reaching 80% cellularity, cells were induced with 100 ng / mL PMA for 12 hours, after which the original medium was discarded and fresh complete medium added. AB4 and its derivatives were added to the experimental groups, each at a final concentration of 10 μM; the control group served as a blank. One hour later, the blank group was removed, and 2 μL of 1 mg / mL LPS was added to the other groups, resulting in a final concentration of 1 μg / mL. Incubation continued for another 2 hours. The following procedures were then performed on ice: the supernatant was removed from the 6-well plates, and 4°C pre-chilled PBS was gently pipetted along the edges, washing twice. After adding 1 mL of PBS, cells were scraped off with a cell scraper, placed in a 1.5 mL centrifuge tube, and centrifuged at 2000 g at 4°C for 3 minutes. The supernatant was discarded, and the cell pellet was collected. 100 μL of RIPA lysis buffer (with protease and phosphatase inhibitors added immediately before use) was added to each tube, mixed by pipetting, and lysed on ice for 10 minutes. After further cell disruption using an ultrasonic disruptor, the cells were centrifuged at 12,000 g for 10 minutes at 4°C. The supernatant was carefully collected into a new EP tube and stored on ice. The total protein content was measured and calculated using a BCA protein quantification kit according to the manufacturer's instructions. The protein sample was diluted with PBS and added to 5× SDS-PAGE loading buffer (50 μL of β-mercaptoethanol per mL) to a final protein concentration of 2 μg / μL. The protein was denatured by boiling at 100°C for 10 minutes to prevent protein degradation. SDS-PAGE gels of varying concentrations were prepared according to the desired protein molecular weight, and protein samples were loaded onto the gels at a concentration of 20 mg / well for electrophoresis. The separated protein samples were transferred to polyvinylidene fluoride (PVDF) membranes and washed three times with Tris-HCl buffer (TBST buffer) for 10 minutes each. After blocking with protein blocking buffer at room temperature for 1 hour, the membranes were incubated with the specific primary antibody according to the manufacturer's instructions overnight at 4°C. The next day, the PVDF membrane was thoroughly washed with TBST buffer, bound to the corresponding secondary antibody at room temperature for 1 hour, and then thoroughly washed again with TBST buffer. Finally, the protein was exposed and analyzed by color development according to the instructions of the ultra-sensitive 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 abscissa represent different Pulsatilla saponin B4 derivatives. The first round of Western blotting revealed a significant increase in P-IκBα protein levels in the model group after 2 hours of LPS stimulation of THP-1 cells (P < 0.01).

[0140] Compared with the LPS model group, the compounds of the present invention and their AB4 can reduce the level of P-IκBa protein, and multiple derivatives, including B4-39, B4-36, etc., significantly reduce the level of P-IκBa protein compared with AB4 (p<0.05). Moreover, derivatives such as B4-19, B4-39, B4-36, B4-33, B4-28, and B4-40 can reduce the level of Pro-IL1β, 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 AB4 derivatives of dapoxetine on DNCB-induced atopic dermatitis in mice: Prior art has shown that AB4 has an improving effect on the DNCB (2,4-dinitrochlorobenzene)-induced mouse eczema model; and previous in vitro experiments have shown that there are AB4 derivatives with better activity than AB4, which are now further verified by in vivo experiments. Therefore, the following experiment was designed. 56 Balb / c mice were randomly divided into a normal control group, a model group, a dexamethasone-positive drug group (3 mg / kg), a dapoxetine B4 (AB4) control group (6.6 mg / kg), a B4-19-administered group (6.6 mg / kg), a B4-33-administered group (6.6 mg / kg), and a B4-39-administered group (6.6 mg / kg), with 8 mice in each group. One day before the experiment, the hair on the back of the mice was removed, and an area of ​​about 3 cm x 3 cm was selected for standby use. On day 1, mice in all groups except the normal control group were sensitized with 50 μL of 5% DNCB applied to the back of the mice. A booster application was performed on day 2, followed by a second sensitization. On day 3, mice were challenged with 50 μL of 1% DNCB applied to the inside and outside of the right auricle using a pipette. This challenge was repeated on days 4 and 5, with an equal amount of acetone matrix applied to the left auricle for three consecutive days. Success was determined by the development of varying degrees of redness, papules, blisters, erosions, exudates, crusting, and desquamation after repeated stimulation of the right ear and back with DNCB solution. For days 1-7, the dexamethasone group received a total of 0.08 g of dexamethasone cream applied to the back and inside and outside of the right auricle at 4:00 PM. The AB4, B4-19, B4-33, and B4-39 groups received a total of 200 μL of a 70% ethanol-water solution (0.66 mg / ml) applied to the back and inside and outside of the right auricle at 10:00 AM and once at 4:00 PM. In the model group, 200 μl of pure water was applied to the back and the inner and outer sides of the right auricle at 10:00 AM and again at 6:00 PM. This treatment continued for 7 days. On the eighth day, mice were sacrificed, and the spleen and ears were removed 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 trial, the dorsal skin of each group of mice was observed daily for eczema (visible redness, swelling, macules, erosions, and exudates) and photographed. The Eczema Area and Severity Index (EASI) was used to assess erythema, papules / pustules, scaling, and scabs, with scores ranging from 0 to 3: 0 = no symptoms; 1 = mild; 2 = moderate; 3 = severe. The sum of the scores for each indicator was used to obtain a total score. Two blinded observers performed scoring on intervention days 1, 3, 5, and 7, and the data were recorded using digital photography. Twenty-four hours after the last dose, the thickness of both ears was measured with a vernier caliper (three measurements were taken and the mean was calculated). The difference in ear thickness was calculated as: right ear thickness minus left ear thickness. Body weight was measured daily and recorded. The results are shown in Figures 8-12. Figure 8 shows the back skin condition of mice with DNCB-induced atopic dermatitis; Figure 9 shows the ear condition of mice with DNCB-induced atopic dermatitis; Figure 10 is a schematic diagram of mouse weight changes and back scores; Figure 11 is a schematic diagram of mouse ear thickness difference and ear weight difference; Figure 12 is a diagram of mouse spleen index.

[0144] When local tissues are stimulated by DNCB, cellular mediators such as histamine are released. These mediators, through H1 and H2 receptors, dilate capillaries in the ear skin and mucosa and increase capillary wall permeability, leading to edema. Experimental results showed that after the first application of DNCB on the third day, the auricles of the model group became slightly redder compared to the blank group. With continued drug action, the redness and swelling in the model group became increasingly severe starting on the fourth day, with desquamation. On the fifth day, exudation, ulceration, and scab formation began to form. On the seventh day, the difference in ear thickness between the control and model groups was statistically significant (P < 0.0001), indicating that the eczema mouse model was successfully established. Compared to the model group, the auricles of the AB4 group and its derivatives showed significantly less swelling and almost no ulceration during the same period. The dexamethasone-positive group showed slightly less effect, with some exudation and scab formation. The difference in ear thickness between the AB4, B4-19, B4-33, and B4-39 groups and the model group was statistically significant on day 7 (P < 0.0001). The difference in thickness between the positive drug group and the model group was also statistically significant on day 7 (P < 0.0001). After DNCB application to the dorsal skin of mice, the model group developed exudation, erosion, and scabs compared to the blank group, producing eczema-like lesions. With continued drug application, the lesions in the model group became increasingly severe from day 2 to 3, with prominent erythema, skin infiltration, and scabs. The EASI composite score between the control and model groups was statistically significant on days 3, 5, and 7 (P < 0.0001), indicating the successful establishment of an eczema mouse model. Skin lesions in the AB4 and AB4 derivative groups were significantly alleviated compared to the model group, with smoother skin, less exudation, and milder or earliest scabs to fall off. The positive drug group developed more severe scabs, which rarely fell off, and had noticeable scales and papules. On days 3 (P < 0.001), 5 (P < 0.0001), and 7 (P < 0.001) of treatment, the EASI composite scores of the AB4 derivative group and the model group were statistically significant. The difference in EASI composite scores between the positive drug group and the model group was statistically significant on day 3 (P < 0.001). The back scores of the AB4, B4-19, B4-33, and B4-39 groups were statistically significant compared with the model group (P < 0.0001), with AB4 being less effective than the derivatives. Furthermore, the experimental results showed that after 7 days of administration, the spleen index of the model group mice was significantly increased, while that of the AB4 derivative was significantly decreased. The spleen index of the dexamethasone group was significantly lower than that of the normal group compared with the model group, indicating that dexamethasone suppresses the mice's immunity, while the AB4 derivative can enhance their immunity. All these experimental results suggest that the AB4 derivative has a protective effect against DNCB-induced eczema lesions, surpassing the effects of AB4 and glucocorticoids.

[0145] Example 16

[0146] External use of pulsatilla saponin B4 can achieve some effects in the treatment of psoriasis. However, further research has shown that pulsatilla saponin B4 has some defects. After structural improvement, the present invention discloses a pulsatilla saponin B4 derivative with significant technical effects in treating psoriasis. The following experiments illustrate the technological progress of the present invention. The reagent used is an existing product, A3-9, recorded in CN2022112110168.

[0147] Weight 20

[0148] After one week of acclimatization, BALB / c mice weighing approximately 100 g were divided into seven groups according to body weight: a normal group, a model group, a B4-treated group (6.6 mg / kg), a B4-33-treated group (6.6 mg / kg), a B4-39-treated group (6.6 mg / kg), an A3-9-treated group (6.6 mg / kg), and a halometasone-treated group (0.1 g / mouse). Mice were initially numbered. Two days before modeling, a 3 cm × 3 cm area on the back of the mice was depilated. The day before modeling, the depilation was checked for completeness. If not, a second depilation was performed. On the day of modeling, 62.5 mg of imiquimod was applied to the backs of mice in all groups except the normal group for seven consecutive days. The drug-treated groups were administered once 4 hours before and 2 hours after modeling. At the same time, the model group was smeared with the solvent used for the test drug (70% ethanol and 2% glycerol in water, by volume). Mouse weights were recorded daily, and photos of the back skin condition were taken to record the condition. PASI scores were also calculated.

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

[0150] The experimental results show that, as shown in Figure 13, after the use of imiquimod to establish the model, the weight of the mice showed a downward trend on the second day, and the model group showed a downward trend every day thereafter. The AB4-administered group was slightly better than the model group, but the daily downward trend continued. The weight loss in the B4-33-administered group, the B4-39-administered group, and the A3-9-administered group was not obvious, and showed a trend of recovery on the 5th day. The weight of the halometasone-administered group dropped sharply. The weight of the B4-33-administered group and the B4-39-administered group was significantly better than that of the halometasone group starting from the 4th day, with statistically significant differences (p < 0.05). The weight of the A3-9-administered group was better than that of the halometasone group after the 5th day of modeling (p < 0.05). This shows that the AB4 derivative can improve the weight of psoriasis mice and has a higher safety than halometasone.

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

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

[0153] The Psoriasis Area and Severity Index (PASI) is an important indicator for assessing the severity of psoriasis. After modeling, the back skin of mice was photographed and scored daily. The scoring criteria are shown in Table 1. The back skin of mice is shown in Figure 14. The model group developed mild scaling one day after modeling. Three days after modeling, erythema and thickening appeared, and scaling increased. After five days, the scaling became flaky, the skin turned dark red, and the skin became significantly thickened. On the seventh day, the scaling became layered, and the skin was noticeably raised. The treatment groups all showed varying degrees of improvement.

[0154] As shown in Figures 15-17, the PASI scores of the drug-treated groups showed that B4-33, B4-39, and A3-9 all had a certain improvement effect on the skin of psoriasis mice, with the effect being superior to that of the B4 group and comparable to that of the halometasone group. As shown in Figure 15, the PASI scores of the model group and the control group showed significant differences from day 1, and the scores improved daily. The B4 group showed a slight improvement compared to the model group, but the score remained at a high level, while the B4-33 and halometasone groups significantly reduced the scores. As shown in Figures 16 and 17, the B4-39 and A3-9 groups also had comparable effects to the halometasone group. This indicates that the AB4 derivative has better efficacy than AB4 and is comparable to, or even superior to, the positive drug halometasone. (normal group vs model group **p<0.01, **p<0.001, **p<0.0001; B4-33, B4-39, A3-9 drug-treated groups vs model group #p<0.05, ##p<0.01, ###p<0.001, ####p<0.0001; halometasone drug-treated group vs model group $p<0.05, $$p<0.01, $$$$p<0.0001; B4 drug-treated group vs model group &&p<0.01, &&&p<0.001).

[0155] Table 1 Area and severity of psoriasis lesions

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

[0157] Psoriasis, as an autoimmune disease, is closely related to the immune system. The spleen and thymus, as the most important immune organs, will undergo obvious changes in psoriatic mice. At the end of the experiment on the 8th day, the mice were killed, and their spleens and thymuses were removed and weighed. The spleen index and thymus index were calculated, that is, (spleen or thymus weight / mouse weight) × 100.

[0158] The results are shown in Figure 18. After modeling, the spleens of the mice were significantly enlarged, which was significantly different from that of the normal group. The halometasone-treated group significantly suppressed the immune system, resulting in a significant decrease in the spleen index, which fell below the normal value. This is an obvious side effect. The B4-39-treated group suppressed the enlargement of the spleen in mice, but there was no statistical difference compared with the normal control group, indicating that there were no obvious side effects in terms of the spleen index.

[0159] As shown in Figure 19, the thymus index of mice decreased significantly after modeling, and all treatment groups showed varying degrees of recovery, with a significant difference observed in the B4-39 treatment group. This suggests that the scutellaria saponin B4 derivative can modulate the immune system of psoriasis mice and is safer than halometasone (*p<0.05, ***p<0.001, ***p<0.0001).

[0160] Example 17 Effect of B4-39 on DSS-induced colitis in mice

[0161] Establishment of 3% DSS-induced ulcerative colitis model in C57 mice: 56 C57 mice were randomly divided into normal control group, model group (3% DSS group), mesalazine group (400 mg / kg), 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 those in the normal control group, was replaced with a 3% DSS aqueous solution. The mice were housed for 8 days and treated on the 9th day. The day before modeling, the mice were weighed and given B4-39 (2.5, 5, or 10 mg / kg) orally once daily according to body weight. Positive controls, mesalazine and AB4, were also given orally once daily. The control group and model group mice were given the control solvent orally once daily for 8 consecutive days. The mice were then observed daily for survival, and body weight, stool characteristics, and bloody stools were recorded.

[0163] Colon length: As shown in Figures 20A and 20B, the model group mice had shortened colons and bleeding compared to the control group mice, with a highly significant difference (P < 0.0001). Administration of B4-39 alleviated the shortening of the colon in the mice, with the B4-39 (10 mg / kg) group showing a highly significant difference in colon length compared to the model group (P < 0.001). The B4 group also showed a statistically significant difference compared to the model group (P < 0.05). The mesalazine group also showed a significant difference in colon length compared to the model group (P < 0.01). These results preliminarily demonstrate that B4-39 has the efficacy of alleviating ulcerative colitis, and that B4-39 (10 mg / kg) is more effective than the AB4 (100 mg / kg) and mesalazine (400 mg / kg) groups.

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

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

[0166] Stool properties: Collect the mouse feces every day and observe the shape. Normal feces are formed and have a certain hardness, loose stools are loose stools, and unformed feces are watery stools.

[0167] Bloody stool: If bleeding is observed in the mouse feces every day, it is bloody stool. If it cannot be observed with the naked eye, place the mouse feces on a slide, first add 2% o-tolidine 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, there is occult blood in the feces. If there is no obvious change in color, it is normal feces.

[0168] Table 2 DAI scoring rules

[0169] The experimental results are shown in Figure 20C. Starting from the third day of modeling, all groups of mice showed varying degrees of fecal abnormalities, including loose stools and bloody stools or occult blood. The condition of the mice in the model group was significantly aggravated on the fourth day of modeling, with extremely significant statistical differences compared with the control group mice (P<0.0001). As the modeling time prolonged, the condition continued to worsen. The DAI scores of the mice in the B4-39 group (2.5, 5, 10 mg / kg) were always lower than those of the mice in the model group, and showed significant differences from the model group on the fourth day of modeling (P<0.01).

[0170] Furthermore, the B4-39 group (2.5 mg / kg) demonstrated comparable efficacy in reducing the DAI scores of DSS mice as the AB4 and mesalazine groups. In summary, B4-39 can reduce the DAI scores of DSS mice and improve the condition of ulcerative colitis mice, demonstrating superior efficacy compared to AB4 and mesalazine.

[0171] Body weight change: Body weight change is an important indicator for evaluating the IBD model. Mice were weighed at 10:00 am every day, and the body weight change rate was calculated using the following formula: body 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. The weight of mice in the DSS-induced IBD model group continued to decrease from the 5th day of modeling. Especially on the 6th day of modeling, the weight loss of mice in the model group showed an extremely significant difference compared with the normal control group (P<0.0001). The weight loss trend of mice in the B4-39 administration group was significantly alleviated, especially in the B4-39 (10 mg / kg) group. The weight loss rate of mice in this group slowed down significantly, and there was a highly significant difference in weight between the mice in the model group and the model group on the 6th day of modeling (P<0.001). In addition, the weight of mice in the positive control drug mesalazine treatment group began to rebound on the 7th day of modeling, and showed a significant difference compared with the model group (P<0.01).

[0173] Notably, B4-39 (10 mg / kg) was more effective than the mesalazine (5-ASA) group 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-induced mice and is superior to the positive controls AB4 and mesalazine.

[0174] Pathological changes in colon tissue: As shown in Figure 21, the pathological sections of colon tissue were carefully observed using an optical microscope (100x and 200x magnification). HE staining showed that the colon tissue of mice in the DSS-induced ulcerative colitis model group showed obvious pathological changes compared with the normal control group. Specifically, there was significant infiltration of inflammatory cells, destruction of colon structure, unevenness of the mucosal surface, a decrease in the number of crypts and structural incompleteness. However, after treatment with the drug B4-39, positive signs of recovery were observed in the colon tissue of mice. Specifically, the damage to the colon structure was repaired to a certain extent, the infiltration of inflammatory cells was alleviated, the number of crypts rebounded, and the structural integrity was improved. These observations further confirmed the potential efficacy of B4-39 in alleviating colon damage in mice with ulcerative colitis.

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

[0176] Apoptosis is a complex biological process that is finely regulated by multiple gene families, including key factors such as the Bcl-2 and caspase families. Bcl-2 and Bax are key factors in regulating apoptosis; the former has an anti-apoptotic effect, while the latter promotes apoptosis. Cleaved-caspase 3, 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, colon tissues were collected from three mice randomly selected from each group for protein extraction. Western blotting was used to detect the expression levels of the apoptosis-related proteins Bax / Bcl-2 and cleaved-caspase 3 / caspase 3.

[0177] As shown in Figure 22, the experimental results reveal the effects of DSS-induced inflammation on the apoptotic pathway in mouse colon epithelial cells. Compared with the normal control group, the expression of pro-apoptotic proteins Bax and cleaved-caspase 3 in the model group was significantly upregulated, while the expression of the anti-apoptotic protein Bcl-2 was significantly downregulated. These changes were statistically significant (P < 0.01), confirming that the DSS-induced inflammatory environment activates the apoptotic 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 was more effective 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 the apoptosis of colon epithelial cells by targeting the Bax / Bcl-2 / caspase 3 signaling pathway. The research results suggest that B4-39 has an anti-apoptotic effect.

[0178] Example 19 Effects of B4-39 on Inflammation-Related Proteins in Colonic 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 multiple genes related to immunity and inflammatory responses. During the pathogenesis of ulcerative 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 multiple proinflammatory 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 colon tissue of the model group mice were significantly increased, while the level of IκBα was significantly decreased, reflecting the activation of the NF-κB signaling pathway and the subsequent inflammatory response. After treatment with B4-39, the expression levels of these proteins were observed to return to normal, indicating that B4-39 has an alleviating effect on inflammation in colon tissue. It is worth noting that B4-39 was more effective than AB4 in regulating the expression of these proteins, which may indicate that B4-39 has higher potential and selectivity in anti-inflammatory effects.

[0181] Example 20 Effects of B4-39 on Epithelial Intestinal Barrier-Related Proteins in UC Mice

[0182] The integrity of intestinal epithelial cells is crucial for maintaining intestinal barrier function, and defects or damage to them 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 invasion by selectively regulating the absorption of nutrients and blocking pathogens and endotoxins. Tight junctions, adherens junctions, desmosomes, and gap junctions are the key forms of connection between intestinal epithelial cells. Tight junctions are particularly important and are composed of proteins such as occludin, claudin, and the Zonula Occlude (ZO) family of proteins. The integrity of tight junctions plays an important role in preventing the penetration of pathogens and antigens, inhibiting the activation of immune cells, and maintaining a stable internal environment.

[0183] In this study, as shown in Figure 24, the levels of ZO-1 (Figure 24A&D), Claudin-1 (Figure 24A&C) and Occludin (Figure 24A&B) proteins in the colon of mice in the model group were significantly reduced compared with the normal control group, indicating that the intestinal epithelial barrier function was impaired. However, after treatment with B4-39, the levels of these proteins were restored, indicating that B4-39 can alleviate the damage to the intestinal epithelial barrier. It is particularly noteworthy that B4-39 showed a more effective effect than AB4 in increasing the levels of ZO-1, Claudin-1 and Occludin proteins. This finding may indicate that B4-39 has better anti-inflammatory potential and therapeutic selectivity in regulating intestinal epithelial barrier function. In summary, B4-39 showed 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 Colonic Tissue of UC Mice

[0185] When the body is stimulated by external stimuli, it will stimulate an immune response, leading to the release of a large amount of inflammatory factors. Among the many inflammatory cytokines, tumor necrosis factor (TNF-α), IL-6 and IL-1β from the interleukin (IL) family are considered to be the main regulatory factors. In order to evaluate the expression of these inflammatory factors in the colon tissue of mice, enzyme-linked immunosorbent assay (ELISA) technology 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). The release of these three inflammatory factors in the colon tissue of mice treated with AB4 and B4-39 was effectively inhibited, and B4-39 was more effective in reducing inflammatory factors than AB4. These results indicate that B4-39 has potential anti-inflammatory effects and can significantly reduce 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] The present invention discloses a new application of B4-39, a derivative of pulsatilla saponin B4, in the treatment of ulcerative colitis. Existing drugs for the treatment of ulcerative colitis are often limited by their high cost and large side effects. As a new therapeutic candidate, B4-39 has shown significant therapeutic potential. In a UC mouse model induced by DSS (dextrose sodium sulfate), B4-39 showed a good therapeutic effect. Specifically, after administration, the weight change rate of the mice was improved, the disease activity index (DAI) score was reduced, the colon length was restored, and the infiltration of inflammatory cells was reduced (observed by HE staining). In addition, the levels of inflammatory factors IL-1β, TNF-α and IL-6 also decreased, and its efficacy was generally better than that of mesalazine and AB4. Western blotting technology detection showed that B4-39 was able to reduce the expression of pro-inflammatory proteins such as PP65, IKB-α, COX2 and iNOS, thereby showing a significant anti-inflammatory effect. At the same time, B4-39 effectively inhibited the apoptosis of intestinal epithelial cells by reducing the expression of pro-apoptotic proteins cleaved-caspase3 and Bax, and increasing the expression of anti-apoptotic protein Bcl-2. In addition, B4-39 can also increase the expression levels of tight junction proteins ZO-1, Claudin-1 and Occludin, thereby protecting the function of the intestinal barrier. In the cytotoxicity test on RAW264.7, THP-1, and HIEC cells, B4-39 did not show obvious cytotoxicity at a concentration of 50μM. These results show that B4-39 not only has good anti-inflammatory activity, but also has high safety. In summary, B4-39, as a derivative of scutellaria saponin B4, has shown great potential and advantages in the treatment of UC.

[0187] In the present invention, the drug for treating or preventing psoriasis can be specifically in the form of a solution, a gel for skin, an ointment for skin, a patch for skin, a microemulsion for skin, or a film spray for skin. Among them, the solution can be an aqueous solution of the drug or an aqueous solution of a scutellaria saponin B4 derivative with added excipients, which are conventional substances such as glycerol, sodium benzoate, and essence. In addition to the scutellaria saponin B4 derivative, the skin gel also includes conventional excipients such as carbomer, glycerol, propylene glycol, and water. In addition to the active drug, the skin ointment also includes conventional excipients such as stearic acid, glyceryl monostearate, liquid paraffin, white petrolatum, and water. In addition to the active drug, the skin patch also includes conventional excipients such as sodium polyacrylate, tranexamic acid, aluminum glycolate, EDTA (ethylenediaminetetraacetic acid), and water. In addition to the active drug, the skin microemulsion also includes conventional excipients such as oleic acid, Tween, co-emulsifiers, and water. In addition to the scutellaria saponin B4 derivative, the skin spray also includes conventional excipients such as polyacetylpyrrolidone, hydroxypropyl methylcellulose, and water. In each dosage form, the amount of excipients used is conventional and meets the requirements of general topical medications.

[0188] In summary, the present invention discloses a derivative of pulsatilla saponin B4, as well as its preparation method and application. Using the compound pulsatilla saponin B4 (AB4) as a raw material, the derivative is prepared by nucleophilic substitution, electrophilic addition, esterification, or amidation at the C-19 exocyclic double bond or the C-28 carboxyl group. The active ingredient of the AB4 derivative is then used to prepare a drug with anti-inflammatory and immunomodulatory effects. This invention discloses for the first time 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 that of AB4 or commonly used clinical drugs. Furthermore, it is disclosed for the first time that most AB4 derivatives exhibit no significant cytotoxicity against macrophages, reduce P-IκBα protein levels in the LPS- and ATP-induced NF-κB signaling pathway in macrophages, inhibit activation of the NLRP3 signaling pathway, and significantly reduce Pro-IL-1β levels (p < 0.05), indicating that they inhibit activation of the inflammasome pathway with superior efficacy to AB4. These results suggest that the AB4 derivatives of the present invention possess superior anti-inflammatory activity. Specifically, these AB4 derivatives have significant therapeutic effects on atopic dermatitis, reducing ear skin swelling and improving skin ulcers. Their therapeutic effects are superior to those of the positive drugs dexamethasone and AB4, and their toxic side effects are significantly less than those of dexamethasone. The present invention also uses an imiquimod-induced psoriasis mouse model. The weight of the mice showed a downward trend on the second day after modeling, and the model group showed a downward trend every day thereafter. The weight of the halometasone-treated group decreased sharply, and the weight of the B4-33 and B4-39-treated groups was significantly higher than that of the halometasone group starting on the fourth day, with statistically significant differences (p < 0.05). Based on the PASI score, B4-33 and B4-39 both showed an improvement effect on the skin of psoriatic mice, with effects superior to those of the AB4 group and comparable to those of the halometasone group. Furthermore, the present invention also uses a DSS-induced colitis mouse model, administering B4-39, AB4, and the control drug mesalazine by gavage. Judging from the three indicators of colitis severity evaluated by mouse colon length, DAI score and body weight, B4-39 is more effective than AB4 and mesalazine in treating colitis. Other biochemical experiments and pathological observations also show that B4-39 is superior to the control drugs AB4 and mesalazine in inhibiting colon epithelial cell apoptosis, inhibiting the release of inflammatory factors and alleviating colon tissue pathological changes, and has lower toxic side effects.

Claims

1. A pulsatilla saponin B4 derivative having the following general chemical structure: In the formula, R1 includes 3-O-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosyl; R2 includes hydroxyl or acetoxy; R3 includes 2-allyl, 2-propyl, 3-hydroxypropenyl, 3-bromopropenyl, 2-oxiranemethyl; R4 includes 1-oxybenzotriazolyl, methoxy, hydroxyl, 28-O-α-L-rhamnopyranosyl-(1→4)-β-D-glucopyranose-(1→6)-β-D-glucopyranose, 28-O-α-L-[2,3,4-triacetoxy-rhamnopyranosyl]-(1→4)-β-D-[2,3,6-triacetoxy-glucopyranose]-(1→6)-β-D-[2,3,4-triacetoxy-glucopyranose], 2-methoxyethylamine 4-aminobutyric acid methyl ester, cyclopentylamino, 3-chloropropylamino, 2-fluoroethylamino, 1-(3-aminopropyl)benzotriazolyl, cyclohexylamino, 1-cyclopropylethylamino, cyclobutylmethylamino, 1-(3-aminopropyl)imidazolyl, N-(2-aminoethyl)pyrrolidinyl, 4-aminofuranyl, 3-aminocyclopentanecarboxylic acid methyl ester, 4-aminocyclohexylcarboxylic acid methyl ester, allylamino, 1-(2-aminoethyl)piperidinyl, 2-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)ethyl Amide group, 4-aminobutyric acid group, 5-aminopentanoic acid group, 6-aminohexanoic acid methyl ester group, 3-L-aminocyclopentanol group, 3-D-aminocyclopentanol group.

2. The pulsatilla saponin B4 derivative according to claim 1, characterized in that: The scutellaria saponin B4 derivative is formulated into a drug for treating or alleviating inflammation; or the scutellaria saponin B4 derivative is used for treating or alleviating inflammation.

3. The method for preparing the pulsatilla saponin B4 derivative according to claim 1, characterized in that: The pulsatilla saponin B4 derivative is prepared by using pulsatilla saponin B4 as a raw material through nucleophilic substitution, electrophilic addition, esterification or amidation reactions.

4. A pharmaceutical system, comprising the pulsatilla saponin B4 derivative according to claim 1 as an active ingredient.

5. The pharmaceutical system according to claim 4, characterized in that The drug system includes topical, oral, rectal or parenteral drugs; the dosage form of the drug system includes pills, tablets, powders, capsules, granules, ointments, solutions, injections, gels or suppositories.

6. Use of the pulsatilla saponin B4 derivative according to claim 1 or the drug system according to claim 4 in the preparation of anti-inflammatory drugs or immunomodulatory drugs.

7. The use according to claim 6, characterized in that: The inflammation includes inflammation on the body surface and inflammation inside the body.

8. A method for treating or alleviating inflammation, characterized in that: The method comprises the step of administering a drug; the drug comprises the scutellaria saponin B4 derivative according to claim 1 or the drug system according to claim 4.

9. The method for treating or alleviating inflammation according to claim 8, characterized in that: The inflammation includes inflammation on the body surface and inflammation inside the body.

10. The method for treating or alleviating inflammation according to claim 8, characterized in that: The medicament is administered to a patient in need thereof.

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