Pharmaceutical use of guaiane sesquiterpene derivative

Abstract

Provided is use of a guaiane sesquiterpene derivative or a pharmaceutically acceptable salt thereof in preparing a medicament for treating an inflammatory disease or tumor or soothing the skin. Using parthenolide as a raw material, a guaiane sesquiterpene lactone derivative, a prodrug, or a pharmaceutically acceptable salt thereof, and a composition are prepared, which in studies have exhibited good therapeutic effects on acute lung injury, hepatitis, lupus nephritis, atopic dermatitis, inflammatory diseases such as sepsis caused by bacterial and / or viral infections, and tumors.

Description

Medical uses of guaiacyl sesquiterpenoid derivatives Technical Field

[0001] The present invention relates to medical use of a guaiacyl sesquiterpene derivative, and in particular to use of a guaiacyl sesquiterpene derivative and a pharmaceutically acceptable salt thereof in treating inflammatory diseases or tumors. Background Art

[0002] Inflammatory diseases, a general term for a class of conditions characterized by excessive or persistent inflammatory responses leading to tissue damage, have become a significant threat to global health, encompassing sepsis, rheumatoid arthritis, acute lung injury, non-alcoholic steatohepatitis, and drug-induced hepatitis. These diseases are spreading globally and have become a major public health issue, seriously endangering human health and sustainable socioeconomic development, and are receiving increasing attention from governments and the public. In my country, these diseases have an incidence rate of approximately 10%, affecting over 100 million people. Their protracted course, high rates of complications, and disability pose a serious threat to human health and well-being. While the etiology of inflammatory diseases remains unclear, immune dysregulation is currently believed to be the primary culprit behind this uncontrolled inflammatory response. Clinically, the main treatments for inflammatory diseases include nonsteroidal anti-inflammatory drugs (NSAIDs), glucocorticoids, immunosuppressants, and biologics. Long-term use of these drugs can lead to various complications (such as osteoporosis and adrenal insufficiency), induce infections (such as intestinal infections), and increase the risk of cancer (such as lymphoma and melanoma). Furthermore, the high cost of biologics imposes a significant financial burden on patients. Therefore, there is an urgent need to explore new drugs or more effective treatment options.

[0003] Malignant tumors have long posed a serious threat to human health. According to statistics, at least 15-20% of cancer deaths worldwide are related to persistent infection and chronic inflammation. Chronic inflammation is one of the ten hallmarks of tumors, and the presence of numerous inflammatory cells in the tumor microenvironment plays a crucial role in tumor development and progression.

[0004] Natural products are an important source of drugs for the treatment of inflammatory diseases and malignant tumors. Among them, guaiacyl sesquiterpene lactones, as an important natural product, have good anti-inflammatory and anti-tumor activities. The guaiacyl sesquiterpene lactone Arglabin (Arg) is derived from the plant Artemisia absinthium (Artemisia absinthium) in Kazakhstan and is reported to have good anti-inflammatory and anti-tumor activities. In a rat arthritis model, Arg can significantly inhibit rat joint swelling, reduce the arthritis index, reduce ankle joint tissue inflammatory lesions and cartilage and bone damage, reduce the levels of pro-inflammatory factors TNF-α, IL-1β and IL-6 in serum, and ultimately significantly relieve rat arthritis. In clinical trials in patients with heart failure, Arglabin can inhibit cardiac inflammation in the middle stage of myocardial hypertrophy, protect cardiac function, and reduce fibrosis [Circulation, 2020, 141: 1704-1719]. At the same time, some studies have reported that Arg can achieve anti-tumor effects by inhibiting farnesyltransferase (FTase) to prevent the farnesylation of H-Ras protein; however, in vitro experiments showed that Arg inhibited the IC of H-Ras protein. 50 The concentration of arginine is only at the micromolar level, and this mechanism of action cannot fully support its anti-tumor effect in vivo. Its application in tumor indications requires further optimization. Furthermore, the natural content of arginine is low, approximately 0.27% [J Nat Prop, 1999, 62:1068-1071]; the compound's solubility in water is only 7.9 μg / mL; its chemical stability in gastric fluid is poor, with a degradation rate of 50% within 8 hours and an oral bioavailability of only 5%. These drawbacks, such as its low natural content and poor drugability, also limit the clinical application of arginine, and its suitable anti-inflammatory indications have yet to be confirmed.

[0005] Another guaiacyl-type sesquiterpenoid lactone, michelinolactone (MCL), is found in the root bark of Michelia serrata and Michelia taiwanensis, both of the Magnoliaceae family. It can also be synthesized from parthenolide. Modern pharmacological studies have shown that MCL has significant anti-inflammatory effects. In addition, in vitro cell models have reported that MCL inhibits tumor cell growth by exerting cytotoxic effects, particularly on tumor stem cells. Its IC 50 At the micromolar level. MCL can promote DCs maturation and present DAMPs signals to immune cells such as T and B cells to mediate immunogenic cell death. However, the level of T cell activation promoted by DCs maturation is not high, resulting in weak anti-HCC activity of MCL. In an in vivo pharmacodynamic evaluation study on liver cancer, a single dose of MCL (30 mg / kg) was administered intraperitoneally for 14 consecutive days, and the tumor suppression rate of the anti-Hepa1-6 transplant tumor model was only 50%. In the human HepG2 and Huh7 liver cancer cell transplant tumor model inoculated with normal immune mice, the effective dose of intraperitoneal injection was as high as 25 mg / kg.

[0006] Therefore, in the fields of inflammatory diseases and malignant tumors, new derivatives with optimized structures, stronger activity and better drugability are needed.

[0007] Summary of the Invention

[0008] The first aspect of the present invention provides the use of a guaiacyl sesquiterpene derivative or a pharmaceutically acceptable salt, solvate, or stereoisomer thereof in the preparation of a drug for treating tumors. Preferably, the tumor is hepatocellular carcinoma, colorectal cancer, or lymphoma.

[0009] The second aspect of the present invention further provides the use of a guaiacyl sesquiterpene derivative or a pharmaceutically acceptable salt, solvate, or stereoisomer thereof in the preparation of a drug for treating inflammatory diseases or soothing skin. Preferably, the inflammatory disease is selected from (1) acute lung injury; (2) hepatitis; (3) sepsis caused by bacterial and / or viral infection; (4) acute and chronic kidney disease; (5) renal fibrosis; (6) lupus nephritis; (7) diabetic nephropathy; and (8) atopic dermatitis.

[0010] Preferably, the soothing of the skin is by increasing skin hyaluronic acid levels and / or inhibiting mast cell degranulation.

[0011] In the first and second aspects of the present invention, the structure of the guaiacyl sesquiterpene derivative is shown in Formula I:

[0012] Wherein, R1 and R2 together form a double bond; or R1 is hydrogen or deuterium, and R2 is wherein R3 and R4 are alkyl groups, or R3, R4 and the nitrogen atom to which they are connected form a 3-10 membered ring structure;

[0013] R5 is selected from hydroxy, C1-C6 alkoxy, C1-C6 alkyl ester or halogen, and -OCONR6R7, wherein R6 is selected from hydrogen or C1-C6 alkyl; R7 is C1-C6 alkyl, fluorine-substituted C1-C6 alkyl, C1-C6 cycloalkyl, hydroxy-substituted C1-C6 alkyl, C1-C6 alkoxy-substituted C1-C6 alkyl, OH-(CH2) n -O-substituted C1~C6 alkyl, amine-substituted C1~C6 alkyl, 5~6-membered nitrogen-containing heterocyclic C1~C6 alkyl, benzene ring-substituted C1~C6 alkyl; the 5~6-membered nitrogen-containing heterocyclic or benzene ring is optionally substituted by one or more C1~C6 alkyl; or R6, R7 and the nitrogen atom to which they are connected form a 3~10-membered cyclic structure; or the carbon atom at position 3 is connected to the carbon atom at position 4 to form a double bond; or the carbon atom at position 4 is connected to the carbon atom at position 5 to form a double bond;

[0014] n is selected from 1, 2, 3, 4, 5 or 6;

[0015] R8 is hydrogen or hydroxy;

[0016] R9 is C1-C6 alkyl, R 10 With R 11 connected to form cyclopropane; or R9 is a C1-C6 alkyl group, and the carbon atom at position 1 is connected to the carbon atom at position 10 to form a double bond.

[0017] In some preferred embodiments, when R9 is a C1-C6 alkyl group, and the carbon atom at position 1 is connected to the carbon atom at position 10 to form a double bond, R5 is not a hydroxyl group.

[0018] In some preferred embodiments, R1 and R2 together form a double bond;

[0019] In some preferred embodiments, R1 is hydrogen;

[0020] In some preferred embodiments, R2 is R3 and R4 are independently selected from C1, C2, C3, C4, C5, C6, C7, C8, C9 or C 10 alkyl;

[0021] In some preferred embodiments, R2 is R3 and R4 are independently selected from C1 to C6 alkyl groups;

[0022] In some preferred embodiments, R2 is R3 and R4 are independently selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl; more preferably, R3 and R4 are both methyl;

[0023] In some preferred embodiments, R2 is R3, R4 and the N atom to which they are attached form a 3-, 4-, 5-, 6-, 7-, 8-, 9- or 10-membered ring structure;

[0024] In some preferred embodiments, R2 is R3, R4 and the N atom to which they are connected form a 5- to 8-membered ring structure;

[0025] In some preferred embodiments, R2 is R3, R4 and the N atom to which they are connected form a 5-6 membered ring structure;

[0026] In some preferred embodiments, R2 is R3, R4 and the N atom to which they are connected form a 4-6 membered ring structure;

[0027] In the above preferred embodiments, R3, R4 and the N atom to which they are attached form a monocyclic, spirocyclic or fused ring structure;

[0028] In some preferred embodiments, R2 is The cyclic structure formed by R3, R4 and the N atom to which they are connected is selected from azetidine, tetrahydropyrrole, piperidine, piperazine, morpholine, azocyclooctane, 6-azaspiro[2,5]octane, 8-azaspiro[4,5]decane and octahydrocyclopenta[c]pyrrole.

[0029] In the above preferred embodiment, the cyclic structure formed by R3, R4 and the N atom to which they are connected is substituted by one or more substituents; preferably, the substituents are selected from C1~C6 alkyl, 5-6 membered nitrogen-containing heterocyclic group, C1~C6 alkyloxycarbonyl (such as ethoxycarbonyl); further preferably, the 5-6 membered nitrogen-containing heterocyclic group is selected from tetrahydropyrrolyl, piperidinyl, piperazine or morpholinyl.

[0030] In some preferred embodiments, R5 is hydroxy;

[0031] In some preferred embodiments, R5 is selected from C1-C6 alkoxy, C1-C6 alkyl ester;

[0032] In some preferred embodiments, R5 is selected from fluoro, chloro, bromo or iodo.

[0033] In some preferred embodiments, R5 is -OCONR6R7, R6 is hydrogen, and R7 is C1-C6 alkyl, fluorine-substituted C1-C6 alkyl, C1-C6 cycloalkyl, hydroxyl-substituted C1-C6 alkyl, C1-C6 alkoxy-substituted C1-C6 alkyl, amino-substituted C1-C6 alkyl, 5- to 6-membered nitrogen-containing heterocyclic ring-substituted C1-C6 alkyl, or benzene ring-substituted C1-C6 alkyl; the 5- to 6-membered nitrogen-containing heterocyclic ring or benzene ring is optionally substituted by one or more C1-C6 alkyl groups.

[0034] In some preferred embodiments, R5 is -OCONR6R7, R6 is C1-C6 alkyl, and R7 is C1-C6 alkyl;

[0035] In some preferred embodiments, R5 is -OCONR6R7, and R6, R7 and the N atom to which they are attached form a 3-, 4-, 5-, 6-, 7-, 8-, 9- or 10-membered ring structure;

[0036] In some preferred embodiments, R5 is -OCONR6R7, and R6, R7 and the N atom to which they are attached form a 5-6 membered ring structure;

[0037] In some preferred embodiments, R5 is -OCONR6R7, and R6, R7 and the N atom to which they are connected form a 5- to 6-membered cyclic structure selected from tetrahydropyrrole, piperidine, piperazine, and morpholine.

[0038] In the above preferred embodiment, the cyclic structure formed by R6, R7 and the N atom to which they are connected is substituted by one or more substituents; preferably, the substituents are selected from C1 to C6 alkyl groups, 5-6 membered nitrogen-containing heterocyclic groups; further preferably, the 5-6 membered nitrogen-containing heterocyclic groups are selected from tetrahydropyrrolyl, piperidinyl, piperazinyl or morpholinyl.

[0039] In some preferred embodiments, the carbon atom at position 3 is connected to the carbon atom at position 4 to form a double bond; or the carbon atom at position 4 is connected to the carbon atom at position 5 to form a double bond;

[0040] In some preferred embodiments, R8 is hydrogen;

[0041] In some preferred embodiments, R9 is methyl and R 10 With R 11 Connected to form cyclopropane;

[0042] In some preferred embodiments, R9 is methyl, and the carbon atom at position 1 is connected to the carbon atom at position 10 to form a double bond.

[0043] In the first and second aspects of the present invention, the structure of the guaiacyl sesquiterpene derivative is shown in Formula II:

[0044] R1 and R2 together form a double bond;

[0045] Or R1 is hydrogen or deuterium, R2 is wherein R3 and R4 are alkyl groups, or R3, R4 and N atoms form a 3-10 membered ring structure,

[0046] R5 is hydroxy, C1-C6 alkoxy, C1-C6 alkyl ester or halogen; or the carbon atom at position 3 is connected to the carbon atom at position 4 to form a double bond; or the carbon atom at position 4 is connected to the carbon atom at position 5 to form a double bond;

[0047] R8 is hydrogen or hydroxy.

[0048] In some preferred embodiments, R1 and R2 together form a double bond;

[0049] In some preferred embodiments, R2 is R3 and R4 are independently selected from C1, C2, C3, C4, C5, C6, C7, C8, C9 or C 10 alkyl;

[0050] In some preferred embodiments, R2 is R3 and R4 are independently selected from C1 to C6 alkyl groups;

[0051] In some preferred embodiments, R2 is R3 and R4 are independently selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl; more preferably, R3 and R4 are both methyl;

[0052] In some preferred embodiments, R2 is R3, R4 and the nitrogen atom to which they are attached form a 3-, 4-, 5-, 6-, 7-, 8-, 9- or 10-membered ring structure;

[0053] In some preferred embodiments, R2 is R3, R4 and the N atom to which they are connected form a 5- to 8-membered ring structure;

[0054] In some preferred embodiments, R2 is R3, R4 and the N atom to which they are connected form a 4-6 membered ring structure;

[0055] In some preferred embodiments, R2 is R3, R4 and the N atom to which they are connected form a 5-6 membered ring structure;

[0056] In the above preferred embodiment, the cyclic structure formed by R3, R4 and the N atom to which they are connected is a monocyclic, spirocyclic or fused ring structure;

[0057] In the above preferred embodiment, the cyclic structure formed by R3, R4 and the N atom to which they are connected is selected from azetidine, tetrahydropyrrole, piperidine, piperazine, morpholine, azocyclooctane, 6-azaspiro[2,5]octane, 8-azaspiro[4,5]decane and octahydrocyclopenta[c]pyrrole.

[0058] In the above preferred embodiment, the cyclic structure formed by R3, R4 and the N atom to which they are connected is substituted by one or more substituents; preferably, the substituents are selected from C1~C6 alkyl, 5-6 membered nitrogen-containing heterocyclic group, C1~C6 alkyloxycarbonyl; further preferably, the 5-6 membered nitrogen-containing heterocyclic group is selected from tetrahydropyrrolyl, piperidinyl, piperazinyl or morpholinyl.

[0059] In some preferred embodiments, R5 is hydroxy;

[0060] In some preferred embodiments, R8 is hydrogen;

[0061] In the first and second aspects of the present invention, the guaiacyl sesquiterpene derivative is represented by the general formula III:

[0062] R1 and R2 together form a double bond;

[0063] Or R1 is hydrogen or deuterium, R2 is wherein R3 and R4 are alkyl groups, or R3, R4 and the N atom form a 3-10 membered ring structure;

[0064] R5 is selected from hydroxy, C1-C6 alkoxy, C1-C6 alkyl ester or halogen, and -OCONR6R7, wherein R6 is hydrogen, C1-C6 alkyl; R7 is C1-C6 alkyl, fluorine-substituted C1-C6 alkyl, C1-C6 cycloalkyl, hydroxy-substituted C1-C6 alkyl, C1-C6 alkoxy-substituted C1-C6 alkyl, OH-(CH2) n -O-substituted C1~C6 alkyl, amine-substituted C1~C6 alkyl, 5~6-membered nitrogen-containing heterocyclic C1~C6 alkyl, benzene ring-substituted C1~C6 alkyl; the 5~6-membered nitrogen-containing heterocyclic or benzene ring is optionally substituted with one or more C1~C6 alkyl groups or R6, R7 and the nitrogen atom to which they are connected form a 3~10-membered ring structure;

[0065] n is selected from 1, 2, 3, 4, 5 or 6.

[0066] In some preferred embodiments, R1 and R2 together form a double bond;

[0067] In some preferred embodiments, R1 is hydrogen;

[0068] In some preferred embodiments, R2 is R3 and R4 are independently selected from C1, C2, C3, C4, C5, C6, C7, C8, C9 or C 10 alkyl;

[0069] In some preferred embodiments, R2 is R3 and R4 are independently selected from C1 to C6 alkyl groups;

[0070] In some preferred embodiments, R2 is R3 and R4 are independently selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl; more preferably, R3 and R4 are both methyl;

[0071] In some preferred embodiments, R2 is R3, R4 and the N atom to which they are attached form a 3-, 4-, 5-, 6-, 7-, 8-, 9- or 10-membered ring structure;

[0072] In some preferred embodiments, R2 is R3, R4 and the N atom to which they are connected form a 5- to 8-membered ring structure;

[0073] In some preferred embodiments, R2 is R3, R4 and the N atom to which they are connected form a 5-6 membered ring structure;

[0074] In some preferred embodiments, R2 is R3, R4 and the N atom to which they are connected form a 4-6 membered ring structure;

[0075] In the above preferred embodiments, R3, R4 and the N atom to which they are attached form a monocyclic, spirocyclic or fused ring structure;

[0076] In some preferred embodiments, R2 is The cyclic structure formed by R3, R4 and the N atom to which they are connected is selected from azetidine, tetrahydropyrrole, piperidine, piperazine, morpholine, azocyclooctane, 6-azaspiro[2,5]octane, 8-azaspiro[4,5]decane and octahydrocyclopenta[c]pyrrole.

[0077] In the above preferred embodiment, the cyclic structure formed by R3, R4 and the N atom to which they are connected is substituted by one or more substituents; preferably, the substituents are selected from C1 to C6 alkyl, 5-6 membered nitrogen-containing heterocyclic group, C1 to C6 alkoxycarbonyl (e.g., ethoxycarbonyl); further preferably, the 5-6 membered nitrogen-containing heterocyclic group is selected from tetrahydropyrrolyl, piperidinyl, piperazinyl or morpholinyl.

[0078] In some preferred embodiments, R5 is hydroxy;

[0079] In some preferred embodiments, R5 is selected from C1-C6 alkoxy, C1-C6 alkyl ester;

[0080] In some preferred embodiments, R5 is selected from fluoro, chloro, bromo or iodo.

[0081] In some preferred embodiments, R5 is -OCONR6R7, R6 is hydrogen, and R7 is C1-C6 alkyl, fluorine-substituted C1-C6 alkyl, C1-C6 cycloalkyl, hydroxyl-substituted C1-C6 alkyl, C1-C6 alkoxy-substituted C1-C6 alkyl, amino-substituted C1-C6 alkyl, 5-6 membered nitrogen-containing heterocyclic ring-substituted C1-C6 alkyl, or benzene ring-substituted C1-C6 alkyl; the 5-6 membered nitrogen-containing heterocyclic ring or benzene ring is optionally substituted by one or more C1-C6 alkyl groups.

[0082] In some preferred embodiments, R5 is -OCONR6R7, R6 is C1-C6 alkyl, and R7 is C1-C6 alkyl;

[0083] In some preferred embodiments, R5 is -OCONR6R7, and R6, R7 and the N atom to which they are attached form a 3-, 4-, 5-, 6-, 7-, 8-, 9- or 10-membered ring structure;

[0084] In some preferred embodiments, R5 is -OCONR6R7, and R6, R7 and the N atom to which they are attached form a 5-6 membered ring structure;

[0085] In some preferred embodiments, R5 is -OCONR6R7, and R6, R7 and the nitrogen atom to which they are connected form a 5- to 6-membered cyclic structure selected from tetrahydropyrrole, piperidine, piperazine or morpholine.

[0086] In the above preferred embodiment, the cyclic structure formed by R6, R7 and the N atom to which they are connected is substituted by one or more substituents; preferably, the substituents are selected from C1 to C6 alkyl groups, 5-6 membered nitrogen-containing heterocyclic groups; further preferably, the 5-6 membered nitrogen-containing heterocyclic groups are selected from tetrahydropyrrolyl, piperidinyl, piperazinyl or morpholinyl.

[0087] In the first and second aspects of the present invention, the guaiacyl sesquiterpene derivative is represented by the general formula IV:

[0088] Among them, R1 and R2 together form a double bond;

[0089] Or R1 is hydrogen or deuterium, R2 is Wherein R3 and R4 are alkyl groups respectively, or R3, R4 and the nitrogen atom to which they are connected form a 3-10 membered ring structure.

[0090] In some preferred embodiments, R2 is R3 and R4 are independently selected from C1, C2, C3, C4, C5, C6, C7, C8, C9 or C 10 alkyl;

[0091] In some preferred embodiments, R2 is R3 and R4 are independently selected from C1 to C6 alkyl groups;

[0092] In some preferred embodiments, R2 is R3 and R4 are independently selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl; more preferably, R3 and R4 are both methyl;

[0093] In some preferred embodiments, R2 is R3, R4 and the N atom to which they are attached form a 3-, 4-, 5-, 6-, 7-, 8-, 9- or 10-membered ring structure;

[0094] In some preferred embodiments, R2 is R3, R4 and the N atom to which they are connected form a 5- to 8-membered ring structure;

[0095] In some preferred embodiments, R2 is R3, R4 and the N atom to which they are connected form a 5-6 membered ring structure;

[0096] In some preferred embodiments, R2 is R3, R4 and the N atom to which they are connected form a 4-6 membered ring structure;

[0097] In the above preferred embodiments, R3, R4 and the N atom to which they are attached form a monocyclic, spirocyclic or fused ring structure;

[0098] In some preferred embodiments, R2 is The cyclic structure formed by R3, R4 and the N atom to which they are connected is selected from azetidine, tetrahydropyrrole, piperidine, piperazine, morpholine, azocyclooctane, 6-azaspiro[2,5]octane, 8-azaspiro[4,5]decane and octahydrocyclopenta[c]pyrrole.

[0099] In the above preferred embodiment, the cyclic structure formed by R3, R4 and the N atom to which they are connected is substituted by one or more substituents; preferably, the substituents are selected from C1 to C6 alkyl, 5-6 membered nitrogen-containing heterocyclic group, C1 to C6 alkoxycarbonyl (e.g., ethoxycarbonyl); further preferably, the 5-6 membered nitrogen-containing heterocyclic group is selected from tetrahydropyrrolyl, piperidinyl, piperazinyl or morpholinyl.

[0100] In the first and second aspects of the present invention, the pharmaceutically acceptable salt of the guaiacyl sesquiterpene derivative is selected from hydrochloride, sulfate, phosphate, maleate, fumarate or citrate.

[0101] The third aspect of the present invention provides a use of a guaiacyl sesquiterpene derivative and a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating tumors. Preferably, the tumor is hepatocellular carcinoma, colorectal cancer, or lymphoma.

[0102] In the third aspect of the present invention, the guaiacyl sesquiterpene derivative is represented by the general formula I:

[0103] Wherein, R1 and R2 together form a double bond; or R1 is hydrogen or deuterium, and R2 is wherein R3 and R4 are C1-C3 alkyl groups, or R3, R4 and the N atom form a 5-6 membered ring structure, and the 5-6 membered ring structure is preferably selected from: pyrrole, proline, piperidine, piperazine, and morpholine;

[0104] R5 is hydroxy, methoxy, methyl ester, halogen, and -OCONR6R7, wherein R6 is hydrogen, C1-C6 alkyl; R7 is C1-C6 alkyl, fluorine-substituted C1-C3 alkyl, cyclopropyl, hydroxy-substituted C1-C6 alkyl, methoxy-substituted C1-C6 alkyl, dimethylaminoethyl, morpholine-substituted C1-C6 alkyl, piperazine-substituted C1-C6 alkyl, or benzene ring-substituted methyl; or R6, R7 and the N atom form a substituted 5-6 membered cyclic structure; or the carbon atom at position 3 is connected to the carbon atom at position 4 to form a double bond; or the carbon atom at position 4 is connected to the carbon atom at position 5 to form a double bond;

[0105] R8 is hydrogen or hydroxy;

[0106] R9 is methyl, R 10 With R 11 Connected to form cyclopropane; or the carbon atom at position 1 is connected to the carbon atom at position 10 to form a double bond.

[0107] In the third aspect of the present invention, the guaiacyl sesquiterpene derivative is represented by the general formula II:

[0108] R1 and R2 together form a double bond; or R1 is hydrogen or deuterium, and R2 is wherein R3 and R4 are C1-C3 alkyl groups, or R3, R4 and the N atom form a 5-6 membered cyclic structure, wherein the cyclic structure is selected from pyrrole, proline, piperidine, piperazine, and morpholine;

[0109] R5 is hydroxy, methoxy, methyl ester, or halogen; or the carbon atom at position 3 is connected to the carbon atom at position 4 to form a double bond; or the carbon atom at position 4 is connected to the carbon atom at position 5 to form a double bond;

[0110] R8 is hydrogen or hydroxy.

[0111] In the third aspect of the present invention, the guaiacyl sesquiterpene derivative is represented by the general formula III:

[0112] R1 and R2 together form a double bond; or R1 is hydrogen or deuterium, and R2 is wherein R3 and R4 are each a C1-C3 alkyl group, or R3, R4 and the N atom form a pyrrole;

[0113] R5 is -OCONR6R7, wherein R6 is hydrogen or C1-C6 alkyl; R7 is C1-C6 alkyl, fluorine-substituted C1-C3 alkyl, cyclopropyl, hydroxyl-substituted C1-C6 alkyl, methoxy-substituted C1-C6 alkyl, dimethylaminoethyl, morpholine-substituted C1-C6 alkyl, piperazine-substituted C1-C6 alkyl, or benzene ring-substituted methyl; or R6, R7 and the N atom form a substituted 5-6 membered cyclic structure.

[0114] In the third aspect of the present invention, the pharmaceutically acceptable salt of the guaiacyl sesquiterpene derivative is selected from hydrochloride, sulfate, phosphate, maleate, fumarate or citrate.

[0115] A fourth aspect of the present invention provides a use of a guaiacyl sesquiterpene derivative and a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating inflammatory diseases or soothing skin. Preferably, the inflammatory disease is selected from (1) acute lung injury; (2) hepatitis; (3) sepsis caused by bacterial and / or viral infection; (4) acute and chronic kidney disease; (5) renal fibrosis; (6) lupus nephritis; (7) diabetic nephropathy; and (8) atopic dermatitis.

[0116] In the fourth aspect of the present invention, the guaiacyl sesquiterpene derivative is represented by the general formula IV:

[0117] Wherein, R1 and R2 together form a double bond; or R1 is hydrogen or deuterium, and R2 is wherein R3 and R4 are respectively C1-C4 alkyl and isopropyl; or R3, R4 and the N atom form a 4-6 membered monocyclic structure, the 4-6 membered monocyclic structure is selected from azetidine, pyrrole, morpholine, piperidine and piperazine, and the substituents on the ring are selected from ethyl, ethoxy, morpholine and piperidine; or R3, R4 and the N atom form an 8-membered cyclic structure, the 8-membered cyclic structure is selected from azacyclooctane; or R3, R4 and the N atom form an 8-membered spirocyclic structure, the 8-membered spirocyclic structure is selected from 6-azaspiro[2,5]octane; or R3, R4 and the N atom form a 10-membered spirocyclic structure, the 10-membered spirocyclic structure is selected from 8-azaspiro[4,5]decane; or R3, R4 and the N atom form an 8-membered fused ring structure, the 8-membered spirocyclic structure is selected from octahydrocyclopenta[c]pyrrole.

[0118] In the fourth aspect of the present invention, the pharmaceutically acceptable salt of the guaiacyl sesquiterpene derivative is selected from hydrochloride, sulfate, phosphate, maleate, fumarate or citrate.

[0119] In all aspects of the present invention, the guaiacyl sesquiterpene derivative can be selected from the following compounds:

[0120] Preferably, the compounds of the present invention are selected from the following compounds:

[0121] The fifth aspect of the present invention provides the use of guaiacyl sesquiterpene derivatives or pharmaceutically acceptable salts, prodrugs, solvates, and stereoisomers thereof in the preparation of drugs for treating tumors; preferably, the tumor is: hepatocellular carcinoma, colorectal cancer, or lymphoma.

[0122] Also provided is the use of a guaiacyl sesquiterpene derivative or a pharmaceutically acceptable salt, prodrug, solvate, or stereoisomer thereof in the preparation of a drug for treating inflammatory diseases or soothing skin; preferably, the inflammatory disease is selected from (1) acute lung injury; (2) hepatitis; (3) sepsis caused by bacterial and / or viral infection; (4) acute and chronic kidney disease; (5) renal fibrosis; (6) lupus nephritis; (7) diabetic nephropathy; and (8) atopic dermatitis.

[0123] Preferably, the soothing of the skin is by increasing skin hyaluronic acid levels and / or inhibiting mast cell degranulation.

[0124] The guaiacyl sesquiterpene derivative is compound 1

[0125] Preferably, the guaiacyl sesquiterpene derivative prodrug can be selected from compounds 2-44:

[0126] Preferably, the guaiacyl sesquiterpene derivative prodrug can be selected from the following compounds:

[0127] The sixth aspect of the present invention provides a Michelia lactone prodrug and a pharmaceutically acceptable salt thereof. The structure of the Michelia lactone prodrug is shown in Formula III:

[0128] R1 and R2 together form a double bond;

[0129] Or R1 is hydrogen or deuterium, R2 is wherein R3 and R4 are alkyl groups, or R3, R4 and the N atom form a 3-10 membered ring structure;

[0130] R5 is selected from hydroxy, C1-C6 alkoxy, C1-C6 alkyl ester or halogen, and -OCONR6R7, wherein R6 is hydrogen, C1-C6 alkyl; R7 is C1-C6 alkyl, fluorine-substituted C1-C6 alkyl, C1-C6 cycloalkyl, hydroxy-substituted C1-C6 alkyl, C1-C6 alkoxy-substituted C1-C6 alkyl, OH-(CH2) n-O-substituted C1~C6 alkyl, amine-substituted C1~C6 alkyl, 5~6-membered nitrogen-containing heterocyclic C1~C6 alkyl, benzene ring-substituted C1~C6 alkyl; the 5~6-membered nitrogen-containing heterocyclic or benzene ring is optionally substituted with one or more C1~C6 alkyl groups or R6, R7 and the nitrogen atom to which they are connected form a 3~10-membered ring structure;

[0131] n is selected from 1, 2, 3, 4, 5 or 6.

[0132] In some preferred embodiments, R1 and R2 together form a double bond;

[0133] In some preferred embodiments, R1 is hydrogen;

[0134] In some preferred embodiments, R2 is R3 and R4 are independently selected from C1, C2, C3, C4, C5, C6, C7, C8, C9 or C 10 alkyl;

[0135] In some preferred embodiments, R2 is R3 and R4 are independently selected from C1 to C6 alkyl groups;

[0136] In some preferred embodiments, R2 is R3 and R4 are independently selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl; more preferably, R3 and R4 are both methyl;

[0137] In some preferred embodiments, R2 is R3, R4 and the N atom to which they are attached form a 3-, 4-, 5-, 6-, 7-, 8-, 9- or 10-membered ring structure;

[0138] In some preferred embodiments, R2 is R3, R4 and the N atom to which they are connected form a 5- to 8-membered ring structure;

[0139] In some preferred embodiments, R2 is R3, R4 and the N atom to which they are connected form a 5-6 membered ring structure;

[0140] In some preferred embodiments, R2 is R3, R4 and the N atom to which they are connected form a 4-6 membered ring structure;

[0141] In the above preferred embodiments, R3, R4 and the N atom to which they are attached form a monocyclic, spirocyclic or fused ring structure;

[0142] In some preferred embodiments, R2 is The cyclic structure formed by R3, R4 and the N atom to which they are connected is selected from azetidine, tetrahydropyrrole, piperidine, piperazine, morpholine, azocyclooctane, 6-azaspiro[2,5]octane, 8-azaspiro[4,5]decane and octahydrocyclopenta[c]pyrrole.

[0143] In the above preferred embodiment, the cyclic structure formed by R3, R4 and the N atom to which they are connected is substituted by one or more substituents; preferably, the substituents are selected from C1~C6 alkyl, 5-6 membered nitrogen-containing heterocyclic group, C1~C6 alkoxycarbonyl; further preferably, the 5-6 membered nitrogen-containing heterocyclic group is selected from tetrahydropyrrolyl, piperidinyl, piperazinyl or morpholinyl.

[0144] In some preferred embodiments, R5 is hydroxy;

[0145] In some preferred embodiments, R5 is selected from C1-C6 alkoxy, C1-C6 alkyl ester;

[0146] In some preferred embodiments, R5 is selected from fluoro, chloro, bromo or iodo.

[0147] In some preferred embodiments, R5 is -OCONR6R7, R6 is hydrogen, and R7 is C1-C6 alkyl, fluorine-substituted C1-C6 alkyl, C1-C6 cycloalkyl, hydroxyl-substituted C1-C6 alkyl, C1-C6 alkoxy-substituted C1-C6 alkyl, amino-substituted C1-C6 alkyl, 5- to 6-membered nitrogen-containing heterocyclic ring-substituted C1-C6 alkyl, or benzene ring-substituted C1-C6 alkyl; the 5- to 6-membered nitrogen-containing heterocyclic ring or benzene ring is optionally substituted by one or more C1-C6 alkyl groups.

[0148] In some preferred embodiments, R5 is -OCONR6R7, R6 is C1-C6 alkyl, and R7 is C1-C6 alkyl;

[0149] In some preferred embodiments, R5 is -OCONR6R7, and R6, R7 and the N atom to which they are attached form a 3-, 4-, 5-, 6-, 7-, 8-, 9- or 10-membered ring structure;

[0150] In some preferred embodiments, R5 is -OCONR6R7, and R6, R7 and the N atom to which they are attached form a 5-6 membered ring structure;

[0151] In some preferred embodiments, R5 is -OCONR6R7, and R6, R7 and the N atom to which they are connected form a 5- to 6-membered cyclic structure selected from tetrahydropyrrole, piperidine, piperazine, and morpholine.

[0152] In the above preferred embodiment, the cyclic structure formed by R6, R7 and the N atom to which they are connected is substituted by one or more substituents; preferably, the substituents are selected from C1 to C6 alkyl groups, 5-6 membered nitrogen-containing heterocyclic groups; further preferably, the 5-6 membered nitrogen-containing heterocyclic groups are selected from tetrahydropyrrolyl, piperidinyl, piperazinyl or morpholinyl.

[0153] The seventh aspect of the present invention further discloses a pharmaceutical composition comprising the guaiacyl terpenoid derivative of the present invention or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0154] The present invention also discloses a pharmaceutical composition comprising the guaiacyl-type sesquiterpene derivative of the present invention or a pharmaceutically acceptable salt thereof, a pharmaceutically acceptable carrier, and an immune checkpoint inhibitor.

[0155] Preferably, the immune checkpoint inhibitor is an anti-PD-L1 / PD-1 monoclonal antibody or an anti-CTLA-4 monoclonal antibody.

[0156] The present invention also discloses the use of the above-mentioned pharmaceutical composition in preparing medicines for resisting tumors or treating inflammatory diseases or soothing skin.

[0157] The tumor is selected from the group consisting of hepatocellular carcinoma, colorectal cancer, and lymphoma;

[0158] The inflammatory disease is selected from (1) acute lung injury; (2) hepatitis; (3) sepsis caused by bacterial and / or viral infection; (4) acute and chronic kidney disease; (5) renal fibrosis; (6) lupus nephritis; (7) diabetic nephropathy; and (8) atopic dermatitis.

[0159] Preferably, the soothing of the skin is by increasing skin hyaluronic acid levels and / or inhibiting mast cell degranulation.

[0160] In the seventh aspect of the present invention, the pharmaceutically acceptable salt of the guaiacyl sesquiterpene derivative is selected from hydrochloride, sulfate, phosphate, maleate, fumarate or citrate.

[0161] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: (1) strong anti-inflammatory activity, significantly improving the damage to the kidney, liver, lung and other tissues of septic mice; (2) significantly improving LPS-induced acute lung injury in mice; (3) alleviating CCl4-induced liver damage reaction; (4) obtaining a salt-type compound with significantly improved water solubility, and the salt-type compound can be converted into the prototype compound in HEPES buffer solution and mouse plasma, thereby exerting anti-inflammatory activity.

[0162] Compared with the existing technology, the present invention has the following significant advantages: (1) strong anti-inflammatory activity, significantly reducing the renal inflammatory response induced by folic acid or unilateral ureteral ligation, and simultaneously reducing folic acid-induced renal fibrosis; (2) reducing the renal inflammatory response in mice with lupus nephritis; (3) reducing the renal inflammatory response and renal pathological damage in mice with diabetic nephropathy; (4) alleviating ear and back damage and related inflammatory factor levels in mice with atopic dermatitis (AD); (5) obtaining a salt-type compound with significantly improved water solubility, and the salt-type compound can be converted into the prototype compound in both HEPES buffer solution and mouse plasma, thereby exerting anti-inflammatory activity. At the same time, the preparation process of the related compounds is simple and the yield is high.

[0163] Compared with the prior art, the present invention has the following significant advantages: (1) It shows a better therapeutic effect on hot tumors such as hepatocellular carcinoma, colorectal cancer or lymphoma; oral administration of compound 20 (40 μmol / kg / d) can achieve a tumor inhibition rate of up to 96.78% in the Hepa1-6 mouse transplant tumor model; at a concentration of 10 μM, the inhibition rate of compound 1 on the chemotaxis of myeloid-derived suppressor cells MDSCs can reach up to 83.3%; (2) Combination with immune checkpoint inhibitors can synergistically enhance the effect and significantly improve the anti-tumor effect; intraperitoneal injection of compound 20 (1 mg / kg / d) combined with intraperitoneal injection of anti-PD-L1 monoclonal antibody (5 mg / kg / d) can achieve a tumor inhibition rate of up to 96.99% in the Hepa1-6 mouse transplant tumor model; (3) The derivatives of the present invention show extremely low toxicity when used in anti-tumor treatment.

[0164] Compared with the prior art, the present invention has the following significant advantages: (1) it can soothe the skin to a certain extent by down-regulating hyaluronidase in human keratinocytes and up-regulating hyaluronic acid levels; (2) it can soothe the skin to a certain extent by inhibiting mast cell degranulation. DETAILED DESCRIPTION

[0165] The technical solution of the present invention is further described below with reference to the examples. The test materials used in the examples can all be purchased through conventional channels.

[0166] Example 1

[0167] Preparation of compounds:

[0168] Preparation of Parthenolide: 5 kg of dried root bark of Magnolia dahurica was ground into a coarse powder, soaked in 10-fold volume of 95% ethanol for 12 hours, and extracted twice under reflux for 2 hours each time. The extract was filtered, the filtrates were combined, concentrated under reduced pressure, and dried to obtain a crude extract of Magnolia dahurica. The extract was then purified by silica gel column chromatography using a petroleum ether-ethyl acetate gradient elution. Fractions rich in parthenolide and costunolide were collected, combined, concentrated, and recrystallized to obtain parthenolide with a yield of 4.0% and a purity of 96.3%.1 H NMR (500MHz, CDCl3): δ6.31(d,J=2.9Hz,1H),5.62(d,J=3.4Hz,1H),5.20(d,J=11.8Hz,2H),3.85(t,J=8.6Hz,1H),2.78 (d,J=8.9Hz,1H),2.45-2.32(m,2H),2.22-2.10(m,4H),1.70(s,3H),1.69-1.66(m,1H),1.29(s,3H),1.27-1.18(m,1H). ESI-MS(m / z):[M+H] + =249.1(calcd:249.1).

[0169] Preparation of compound 1

[0170] To a 150 mL round-bottom flask, dichloromethane (50 mL), p-toluenesulfonic acid (125 mg, 0.73 mmol), and parthenolide (5 g, 20.16 mmol) were added sequentially and stirred at room temperature until the reaction was complete as determined by TLC. The reaction solution was washed sequentially with water (10 mL x 3) and saturated brine (10 mL x 3), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. Separation by silica gel column chromatography afforded the intermediate, parthenolide (MCL), in a 90% yield. 1 H NMR (500MHz, CDCl3): δ6.21(d,J=3.5Hz,1H),5.51(d,J=3.0Hz,1H),3.81(t,J=10.5Hz,1H),2.73(d,J=10.5Hz,1H),2.68-2.64( m,2H),2.42-2.37(m,1H),2.26-2.16(m,3H),2.11-2.08(m,1H),1.83-1.75(m,2H),1.69(s,3H),1.31(s,3H),1.27-1.25(m,1H). ESI-MS(m / z):[M+Na] + =271.1(calcd:271.1).

[0171] In an ice bath under nitrogen, ethylene glycol dimethyl ether (1.67 mL, 21.26 mmol) was added to anhydrous dichloromethane (67 mL). After stirring, 13.3 mL of diethylzinc solution (1 M in n-hexane) was added, followed by the slow dropwise addition of diiodomethane (2.67 mL, 3.11 mmol) and stirring for 10 min to prepare the cyclopropanation reagent. In a separate round-bottom flask, 300 mg of Michelia lactone (MCL) and 5 mL of anhydrous dichloromethane were added. After stirring to dissolve, nitrogen was applied and the mixture was placed in an ice bath. The cyclopropanation reagent was added dropwise to the substrate solution. After the addition was complete, the reaction was continued for 1 h, then brought to room temperature and allowed to react overnight. The reaction solution was quenched with saturated ammonium chloride, filtered, washed sequentially with water (10 mL x 3) and saturated brine (10 mL x 3), dried over anhydrous sodium sulfate, concentrated under reduced pressure, and separated by silica gel column chromatography to obtain compound 1 in a yield of 75%. 1 HNMR (500MHz, CDCl3): δ6.14(d,J=3.5Hz,1H),5.45(d,J=3.0Hz,1H),3.82 (t,J=10.5Hz,1H),2.52-2.48(m,1H),2.26-2.21(m,1H),2.09-1.99(m,2H) ,1.93-1.89(m,1H),1.88-1.86(m,1H,H5),1.85-1.83(m,1H),1.55(s,3H) ,1.52-1.44(m,2H),1.26-1.14(m,2H),1.11(s,3H),0.81(d,J=4.0Hz,1H,H 16a ),0.54(d,J=4.0Hz,1H,H 16b ). ROESY spectrum shows that H5 and H 16a There is signal correlation, confirming that cyclopropane is α-configuration. ESI-MS (m / z): [M+Na] + =285.2(calcd:285.2).

[0172] Preparation of compound 2

[0173] Compound 1 (262 mg, 1.00 mmol), dimethylamine (68 mg, 1.50 mmol), DBU (380 mg, 2.5 mmol), and dimethyl sulfoxide (3 mL) were added to a round-bottom flask and stirred for 8 h. The reaction solution was diluted with water (50 mL) and then extracted with ethyl acetate (20 mL × 3). The combined organic phases were washed sequentially with water (10 mL × 3) and saturated brine (10 mL × 3), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. Compound 2 was isolated by silica gel column chromatography (petroleum ether:ethyl acetate:triethylamine = 1:1:0.02) to obtain compound 2 in an 85% yield.1 H NMR (500MHz, CDCl3): δ3.92 (t, J = 10.5Hz, 1H), 3.55-3.47 (m, 1H), 3.33-3.29 (m, 1H), 2.38-2.34(m,1H),2.28(s,6H),2.21-2.16(m,2H),1.98(d,J=15.0Hz,2H),1.88(t,J =5.0Hz,2H),1.72(d,J=10.0Hz,1H),1.60-1.52(m,2H),1.50(s,3H),1.20(t,J=5.0H z, 1H), 1.12 (s, 3H), 1.08-1.04 (m, 1H), 0.76 (d, J = 5.0Hz, 1H), 0.53 (d, J = 5.0Hz, 1H). ESI-MS(m / z):[M+Na] + =330.2(calcd:330.2).

[0174] Preparation of compound 3

[0175] The preparation method is the same as compound 2, and the yield of compound 3 is 86%. 1 H NMR (500MHz, CDCl3): δ3.76 (t, J = 10.2Hz, 1H), 2.86-2.74 (m, 2H), 2.53-2.43 (m, 4H), 2.35-2.28(m,1H),2.14(dd,J=14.3,6.6Hz,1H),1.93-1.83(m,3H),1.76(s,1H),1.7 5-1.68(m,5H),1.51(s,3H),1.48-1.37(m,2H),1.29-1.23(m,1H),1.09(s,3H),1.03 (d,J=13.1Hz,1H),0.75(d,J=3.4Hz,1H),0.49(d,J=3.4Hz,1H).ESI-MS(m / z):[M+H] + =334.2(calcd:334.2).

[0176] Preparation of compound 4

[0177] The preparation method is the same as compound 2, and the yield of compound 4 is 83%. 1H NMR (500MHz, CDCl3): δ4.14 (m, J=7.1Hz, 2H), 3.78 (t, J=10.3Hz, 1H), 3.05 (dd, J=13.4, 5.0Hz, 1H), 3. 00-2.87(m,2H),2.46(q,J=7.1,6.7Hz,1H),2.36(s,1H),2.29(d,J=11.4Hz,1H),2.16(dd,J=14.6,6.6 Hz,1H),2.11-2.01(m,3H),1.93-1.84(m,4H),1.78(d,J=10.4Hz,2H),1.52(s,3H),1.49-1.34(m,2H) ,1.26(t,J=7.4Hz,4H),1.10(s,4H),0.78(d,J=3.6Hz,1H),0.50(d,J=3.6Hz,1H).ESI-MS(m / z):[M+H] + =406.3(calcd:406.3).

[0178] Preparation of compound 5

[0179] The preparation method is the same as compound 2, and the yield of compound 5 is 88%. 1 H NMR (500MHz, CDCl3): δ3.79 (t, J = 10.5Hz, 1H), 2.79-2.75 (m, 1H), 2.56-2.53 (m ,1H),2.46-2.40(m,3H),2.39-2.32(m,3H),2.19-2.08(m,4H),1.94-1.85(m,3 H),1.77(d,J=10.5Hz,1H),1.54(s,6H),1.45-1.39(m,4H),1.12(s,3H),1.09- 1.04(m,1H),0.79(d,J=4.0Hz,1H),0.52(d,J=4.0Hz,1H).ESI-MS(m / z):[M+H] + =348.3(calcd:348.3).

[0180] Preparation of compound 6

[0181] The preparation method is the same as compound 2, and the yield of compound 6 is 86%. 1H NMR (500MHz, CDCl3): δ3.81 (t, J = 10.5Hz, 1H), 3.74-3.67 (m, 3H), 2.82-2.79 (m, 1H), 2.65-2. 61(m,1H),2.53-2.44(m,5H),2.40-2.35(m,1H),2.21-2.17(m,1H),2.13-2.07(m,2H),1.95- 1.86(m,3H),1.78(d,J=10.5Hz,1H),1.55(s,3H),1.52-1.39(m,2H),1.31-1.27(m,1H),1.12 (s,3H),1.09-1.04(m,1H),0.80(d,J=4.0Hz,1H),0.54(d,J=4.0Hz,1H).ESI-MS(m / z):[M+H] + =350.2(calcd:350.2).

[0182] Preparation of compound 7

[0183] The preparation method is the same as compound 2, and the yield of compound 7 is 82%. 1 H NMR (500MHz, CDCl3): δ3.79 (t, J = 10.5Hz, 1H), 2.82-2.79 (m, 1H), 2.64-2.60 (m, 1H), 2.55- 2.42(m,5H),2.38-2.34(m,2H),2.29(s,3H),2.20-2.16(m,1H),2.15-2.05(m,2H),1.93-1. 83(m,3H),1.76(d,J=10.5Hz,1H),1.54(s,3H),1.49-1.38(m,2H),1.29-1.25(m,1H),1.12( s,3H),1.09-1.03(m,1H),0.78(d,J=4.0Hz,1H),0.52(d,J=4.0Hz,1H).ESI-MS(m / z):[M+H] + =349.2(calcd:349.2).

[0184] Preparation of compound 8

[0185] The preparation method is the same as compound 2, and the yield of compound 8 is 76%. 1H NMR (500MHz, CDCl3): δ3.75 (t, J=10.2Hz, 1H), 2.77 (dd, J=13.2, 4.2Hz, 1H), 2.58 (dd, J=13. 2,6.8Hz,2H),2.47(d,J=30.9Hz,4H),2.41-2.35(m,4H),2.32(dd,J=12.1,6.5Hz,1H),2.26– 2.17(m,4H),1.92-1.77(m,3H),1.72(d,J=10.6Hz,1H),1.50(s,3H),1.47-1.20(m,3H),1.08 (s,3H),1.06-0.95(m,3H),0.74(d,J=3.7Hz,1H),0.48(d,J=3.5Hz,1H).ESI-MS(m / z):[M+H] + =377.3(calcd:377.3).

[0186] Preparation of compound 9

[0187] The preparation method is the same as compound 2, and the yield of compound 9 is 78%. 1 H NMR (500MHz, CDCl3): δ3.77(t,J=10.3Hz,1H),2.83(dd,J=13.6,4.6Hz,1H),2 .63(dd,J=13.7,6.6Hz,1H),2.58-2.50(m,2H),2.45m,J=6.8Hz,2H),2.38(s, 1H),2.29(dt,J=11.7,5.6Hz,1H),2.19-2.11(m,2H),2.11-2.04(m,1H),1.91 -1.78(m,3H),1.75(d,J=10.5Hz,1H),1.52(s,3H),1.50-1.36(m,2H),1.09(s, 3H),1.05(d,J=13.1Hz,1H),0.98(t,J=7.1Hz,6H),0.76(d,J=4.0Hz,1H),0.49(d,J=4.0Hz,1H).ESI-MS(m / z):[M+H] + =336.2(calcd:336.2).

[0188] Preparation of compound 10

[0189] The preparation method is the same as compound 2, and the yield of compound 10 is 78%. 1H NMR (500MHz, CDCl3): δ3.78(t,J=10.2Hz,1H),2.87-2.80(m,1H),2.66(dd,J=13.5,6.6Hz,1H ),2.38(d,J=12.2Hz,3H),2.34-2.25(m,3H),2.15(d,J=11.7Hz,2H),2.11-2.04(m,1H),1.93 -1.82(m,3H),1.75(d,J=10.6Hz,1H),1.52(s,3H),1.45-1.38(m,5H),1.10(s,3H),1.04(t,J=12. 2Hz,1H),0.85(t,J=7.3Hz,6H),0.76(d,J=3.9Hz,1H),0.50(d,J=3.9Hz,1H).ESI-MS(m / z):[M+H] + =364.3(calcd:364.3).

[0190] Preparation of compound 11

[0191] The preparation method is the same as compound 2, and the yield of compound 11 is 78%. 1 H NMR (500MHz, CDCl3): δ3.78 (t, J=10.2Hz, 1H), 2.82 (dd, J=13.6, 4.2Hz, 1H), 2.64 (dd, J=13.5 ,6.6Hz,1H),2.42(m,J=14.3,7.7Hz,2H),2.38-2.24(m,4H),2.19-2.04(m,3H),1.93-1.82(m ,3H),1.75(d,J=10.6Hz,1H),1.52(s,3H),1.43-1.32(m,5H),1.28(m,J=7.1Hz,5H),1.10(s, 3H),0.90(t,J=7.3Hz,6H),0.77(d,J=3.8Hz,1H),0.50(d,J=3.8Hz,1H).ESI-MS(m / z):[M+H] + =392.3(calcd:392.3).

[0192] Preparation of compound 12

[0193] The preparation method is the same as compound 2, and the yield of compound 12 is 75%. 1H NMR (500MHz, CDCl3): δ3.78(t,J=10.3Hz,1H), 2.83(d,J=8.7Hz,1H), 2.65(dd,J=13.2,6.2Hz,1H), 2.38(m,J=12.7,9.1Hz,3H),2.14(d,J=12.2Hz,2H),2.11-2.01(m,1H),1.88(m,J=10.0,7.8Hz,3H), 1.74(d,J=10.5Hz,1H),1.51(s,3H),1.49-1.35(m,6H),1.09(s,3H),1.03(t,J=12.2Hz,1H),0.85( q,J=9.0,7.3Hz,6H),0.75(d,J=3.6Hz,1H),0.49(d,J=3.5Hz,1H),0.06(s,2H).ESI-MS(m / z):[M+H] + =364.3(calcd:364.3).

[0194] Preparation of compound 13

[0195] The preparation method is the same as compound 2, and the yield of compound 13 is 80%. 1 H NMR (500MHz, CDCl3): δ3.76 (t, J = 10.3Hz, 1H), 2.76-2.66 (m, 2H), 2.62-2.47 (m, 4H), 2.38(s,1H),2.31-2.25(m,1H),2.20-2.03(m,5H),1.94-1.82(m,3H),1.74(d,J=10. 6Hz,1H),1.67-1.55(m,3H),1.51(s,3H),1.49-1.37(m,3H),1.33(dd,J=11.0,6.7Hz ,2H),1.09(s,3H),0.76(d,J=3.6Hz,1H),0.49(d,J=3.6Hz,1H).ESI-MS(m / z):[M+H] + =374.3(calcd:374.3).

[0196] Preparation of compound 14

[0197] The preparation method is the same as compound 2, and the yield of compound 14 is 88%. 1H NMR (500MHz, CDCl3): δ3.72(t,J=10.3Hz,1H),3.22-3.14(m,4H),2.75-2.68(m,2H),2.37(s,1H),2.17-2.08(m,2H),2.04-1.95(m,3H),1.91-1. 81(m,3H),1.74(d,J=10.6Hz,1H),1.50(s,3H),1.46-1.24(m,3H),1.07( s,3H),0.74(d,J=3.7Hz,1H),0.48(d,J=3.7Hz,1H).ESI-MS(m / z):[M+H] + =320.2(calcd:320.2).

[0198] Preparation of compound 15

[0199] The preparation method is the same as compound 2, and the yield of compound 15 is 88%. 1 H NMR (500MHz, CDCl3): δ3.76(t,J=10.3Hz,1H),2.74(dd,J=13.1,4.4Hz,1H),2.53(dd,J=13.1,6.8Hz,1H),2.40(s,3H),2.34-2.28 (m,2H),2.17-2.02(m,3H),1.87(dd,J=10.0,3.1Hz,2H),1.85-1.79(m,1H),1.73(d,J=10.6Hz,1H),1.56(s,3H),1.51(s,3H),1.44 -1.41(m,8H),1.36(d,J=6.7Hz,4H),1.08(s,3H),1.03(t,J=12.5Hz,1H),0.75(d,J=3.8Hz,1H),0.48(d,J=3.7Hz,1H).ESI-MS(m / z):[M+H] + =402.3(calcd:402.3).

[0200] Preparation of compound 16

[0201] The preparation method is the same as compound 2, and the yield of compound 16 is 82%. 1H NMR (500MHz, CDCl3): δ3.72(t,J=10.2Hz,1H),2.81(dd,J=13.3,4.4Hz,2H),2.69(dd,J=13.2,4.2Hz,1H ),2.50(dd,J=13.3,7.0Hz,1H),2.44(m,4H),2.32-2.25(m,1H),2.18-2.07(m,2H),2.05-1.97(m,2H),1. 91-1.80(m,3H),1.79-1.67(m,4H),1.57-1.50(m,4H),1.47(s,3H),1.45-1.37(m,4H),1.36-1.20(m,2H ),1.04(s,3H),0.98(t,J=12.7Hz,1H),0.71(d,J=3.6Hz,1H),0.45(d,J=3.5Hz,1H).ESI-MS(m / z):[M+H] + =431.3(calcd:431.3).

[0202] Preparation of compound 17

[0203] The preparation method is the same as compound 2, and the yield of compound 17 is 82%. 1 H NMR (500MHz, CDCl3): δ3.75(t,J=10.2Hz,1H),3.69(s,4H),2.87(d,J=11.1Hz,1H),2.80(d,J =11.2Hz,1H),2.72(dd,J=13.2,3.7Hz,1H),2.58-2.53(m,1H),2.51(s,4H),2.39(s,1H),2.35 -2.28(m,1H),2.14-2.04(m,4H),1.93(t,J=11.8Hz,1H),1.85(d,J=8.5Hz,2H),1.80(d,J=10.9Hz,3H),1.71(d,J=10.6Hz,1H),1.49(s,3H),1. 41(dd,3H),1.23(dd,J=13.5,5.7Hz,1H),1.07(s,3H),1.00(t,J=13.1Hz,1H),0.73(d,J=3.7Hz,1H),0.47(d,J=3.6Hz,1H).ESI-MS(m / z):[M+H] + =433.3(calcd:433.3).

[0204] Preparation of compound 18

[0205] The preparation method is the same as compound 2, and the yield of compound 18 is 90%. 1 H NMR (500MHz, CDCl3): δ3.78(t,J=10.2Hz,1H),2.81(d,J=13.0Hz,1H),2.60(dd,J=12.6,6.2Hz,1H),2.49 (s,2H),2.15(d,J=12.0Hz,2H),2.07(t,J=11.6Hz,1H),1.86-1.80(m,3H),1.74(d,J=10.5Hz,1H),1.52(s ,3H),1.50-1.39(m,3H),1.33(d,J=20.0Hz,4H),1.26(d,J=13.6Hz,1H),1.09(s,3H),1.04(t,J=12.6Hz, 1H),0.86(d,J=7.1Hz,1H),0.76(d,J=3.8Hz,1H),0.49(d,J=3.7Hz,1H),0.24(s,4H).ESI-MS(m / z):[M+H] + =374.3(calcd:374.3).

[0206] Preparation of compound 19

[0207] The preparation method is the same as compound 2, and the yield of compound 19 is 83%. 1 H NMR(500MHz, CDCl3)δ3.78(t,J=10.2Hz,1H),2.89(dd,J=13.6,4.4Hz,1H),2.80(dd,J=13.6,4.8Hz,1H), 2.61-2.58(m,1H),2.58-2.51(m,4H),2.38(d,J=9.0Hz,1H),2.25-2.15(m,3H),2.12-2.05(m,1H),2.05- 1.97(m,3H),1.92-1.85(m,3H),1.75(d,J=10.7Hz,1H),1.68(d,J=10.9Hz,4H),1.56(s,3H),1.48-1.39( m,3H),1.11(s,3H),1.09-1.02(m,1H),0.78(d,J=3.9Hz,1H),0.50(d,J=3.7Hz,1H).ESI-MS(m / z):[M+H] + =376.3(calcd:376.3).

[0208] Preparation of the hydrochloride salt 20 of compound 2

[0209] Compound 2 (307 mg, 1 mmol) was dissolved in dichloromethane (2 mL) and stirred at room temperature for 2 h. Hydrochloric acid solution was then added dropwise to a pH of 4-5. The mixture was filtered and the resulting solid was washed with dichloromethane. The resulting white solid was the hydrochloride salt of compound 2 (compound 20) with a yield of 90%. 1 H NMR (500MHz, CD3OD): δ4.15-4.10(m,1H,H6),3.42-3.37(m,1H),3.31-3.26(m,1H),3.04-2.98(m,1H,H 11 ),2.91(s,6H),2.21-2.12(m,2H),2.02(d,J=15.0Hz,2H),1.88(t,J=5.0Hz,2H),1.78(d,J=10.0Hz,1H,H5), 1.64-1.54(m,2H),1.52(s,3H),1.26(t,J=5.0Hz,1H),1.14(s,3H),1.10-1.05(m,1H),0.76(d,J=5.0Hz,1H,H 16a ),0.53(d,J=5.0Hz,1H,H 16b ). ROESY spectrum shows H5 and H 16a of hydrogen, as well as H6 and H 11 The hydrogen signals of cyclopropane are correlated, confirming that cyclopropane is α-configuration and 11-H is β-configuration. ESI-MS (m / z): [M+H] + =308.2(calcd:308.2).

[0210] Preparation of fumarate salt 21 of compound 2

[0211] Fumaric acid was used instead of hydrochloric acid to prepare the fumarate compound 21 according to the preparation method of the hydrochloride of compound 2. The yield was 80%. 1 H NMR (500MHz, CD3OD): δ6.72(s,2H),4.13(t,J=10.0Hz,1H,H6),3.41-3.39(m,1H),3.30-3.27(m,1H),3.03-2.98(m,1H,H 11 ),2.91(s,6H),2.21-2.12(m,2H),1.94-1.91(m,1H),1.88(t,J=5.0Hz,2H),1.84-1.80(m,1H),1.78(d,J =15.0Hz,1H,H5),1.64-1.54(m,2H),1.52(s,3H),1.14(s,3H),1.10-1.04(m,1H),0.75(d,J=5.0Hz,1H,H16a ),0.52(d,J=5.0Hz,1H,H 16b ). ROESY spectrum shows H5 and H 16a , as well as H6 and H 11 There is signal correlation, confirming that cyclopropane is α-configuration and 11-H is β-configuration. ESI-MS (m / z): [M+H] + =308.2(calcd:308.2).

[0212] Preparation of the hydrochloride salt 22 of compound 3

[0213] Compound 3 (333 mg, 1 mmol) was dissolved in dichloromethane (2 mL) and stirred at room temperature for 2 h. Hydrochloric acid solution was then added dropwise to a pH of 4-5. The mixture was filtered and the resulting solid was washed with dichloromethane. The resulting white solid was the hydrochloride salt of compound 3 (compound 22) with a yield of 86%. 1 H NMR (500MHz, CD3OD): δ3.76 (t, J=10.2Hz, 1H, H6), 2.86-2.74 (m, 2H), 2.53-2.45 (m, 4H), 2.37-2.28 (m, 1H, H 11 ),2.14(dd,J=14.3,6.6Hz,1H),1.93-1.83(m,3H),1.76(s,1H),1.75-1.68(m,5H),1.51(s,3H),1. 48-1.37(m,2H),1.29-1.24(m,1H),1.09(s,3H),1.03(d,J=13.1Hz,1H,H5),0.75(d,J=3.4Hz,1H,H 16a ),0.49(d,J=3.4Hz,1H,H 16b ROESY spectrum shows H5 and H 16a The hydrogen of H6 and H11 have signal correlation, confirming that cyclopropane is α-configuration and 11-H is β-configuration. ESI-MS (m / z): [M+H] + =334.2(calcd:334.2).

[0214] Preparation of the hydrochloride salt 23 of compound 4

[0215] Compound 4 (404 mg, 1 mmol) was dissolved in dichloromethane (2 mL) and stirred at room temperature for 2 h. Hydrochloric acid solution was then added dropwise to a pH of 4-5. The mixture was filtered and the resulting solid was washed with dichloromethane. The resulting white solid was the hydrochloride salt of compound 4 (compound 23) with a yield of 83%. 1H NMR (500MHz, CD3OD): δ4.14 (d, J=7.0Hz, 2H), 3.78 (t, J=10.3Hz, 1H, H6), 3.15 (dd, J=13. 4,5.0Hz,1H),3.00-2.91(m,2H),2.46-2.42(m,1H),2.36(s,1H),2.29(d,J=11.4Hz,1H,H 11 ),2.16(dd,J=14.6,6.6Hz,1H),2.11-2.04(m,3H),1.93-1.84(m,4H),1.78(d,J=10.4Hz,1H,H 5),1.52(s,3H),1.49-1.37(m,3H),1.26(t,J=7.4Hz,4H),1.10(s,4H),0.78(d,J=3.6Hz,1H,H 16a ),0.50(d,J=3.6Hz,1H,H 16b ROESY spectrum shows H5 and H 16a of hydrogen, as well as H6 and H 11 The hydrogen signals of cyclopropane are correlated, confirming that cyclopropane is α-configuration and 11-H is β-configuration. ESI-MS (m / z): [M+H] + =406.2(calcd:406.2).

[0216] Preparation of the hydrochloride salt 24 of compound 5

[0217] Compound 5 (346 mg, 1 mmol) was dissolved in dichloromethane (2 mL) and stirred at room temperature for 2 h. Hydrochloric acid solution was then added dropwise to a pH of 4-5. The mixture was filtered and the resulting solid was washed with dichloromethane. The resulting white solid was the hydrochloride salt of compound 5 (compound 24) with a yield of 87%. 1 H NMR (500MHz, CD3OD): δ3.79(t,J=10.5Hz,1H,H6),2.79-2.75(m,1H),2.56-2.53(m,1H,H 11 ),2.46-2.40(m,3H),2.39-2.32(m,3H),2.19-2.08(m,4H),1.94-1.85(m,3H),1.77(d,J=10.4Hz ,1H,H5),1.54(s,6H),1.45-1.39(m,4H),1.12(s,3H),1.09-1.04(m,1H),0.79(d,J=4.0Hz,1H,H 16a ),0.52(d,J=4.0Hz,1H,H 16b ROESY spectrum shows H5 and H16a of hydrogen, as well as H6 and H 11 The hydrogen signals of cyclopropane are correlated, confirming that cyclopropane is α-configuration and 11-H is β-configuration. ESI-MS (m / z): [M+H] + =348.2(calcd:348.2).

[0218] Preparation of the hydrochloride salt 25 of compound 6

[0219] Compound 6 (348 mg, 1 mmol) was dissolved in dichloromethane (2 mL) and stirred at room temperature for 2 h. Hydrochloric acid solution was then added dropwise to a pH of 4-5. The mixture was filtered and the resulting solid was washed with dichloromethane to obtain a white solid, the hydrochloride salt of compound 6 (compound 25), with a yield of 88%. 1 H NMR (500MHz, CD3OD): δ3.81(t,J=10.5Hz,1H,H6),3.74-3.67(m,3H),2.82-2.79(m,1H,H 11 ),2.65-2.61(m,1H),2.53-2.44(m,5H),2.40-2.35(m,1H),2.21-2.17(m,1H),2.13-2.07(m,2H),1.95-1.86(m,3H),1.78(d, J=10.4Hz,1H,H5),1.55(s,3H),1.52-1.39(m,2H),1.31-1.27(m,1H),1.12(s,3H),1.09-1.04(m,1H),0.80(d,J=4.0Hz,1H,H 16a ),0.54(d,J=4.0Hz,1H,H 16b ROESY spectrum shows H5 and H 16a of hydrogen, as well as H6 and H 11 The hydrogen signals of cyclopropane are correlated, confirming that cyclopropane is α-configuration and 11-H is β-configuration. ESI-MS (m / z): [M+H] + =350.2(calcd:350.2).

[0220] Preparation of the hydrochloride salt 26 of compound 7

[0221] Compound 7 (347 mg, 1 mmol) was dissolved in dichloromethane (2 mL) and stirred at room temperature for 2 h. Hydrochloric acid solution was then added dropwise to a pH of 4-5. The mixture was filtered and the resulting solid was washed with dichloromethane. The resulting white solid was the hydrochloride salt of compound 7 (compound 26) with a yield of 86%. 1H NMR (500MHz, CD3OD): δ3.79(t,J=10.5Hz,1H,H6),2.82-2.79(m,1H),2.64-2.55(m,1H,H 11 ),2.55-2.42(m,5H),2.38-2.33(m,2H),2.29(s,3H),2.20-2.16(m,1H),2.15-2.05(m,2H),1.93-1.83(m,3H),1.76(d,J= 10.4Hz,1H,H5),1.54(s,3H),1.49-1.38(m,2H),1.29-1.25(m,1H),1.12(s,3H),1.09-1.03(m,1H),0.78(d,J=4.0Hz,1H,H 16a ),0.52(d,J=4.0Hz,1H,H 16b ROESY spectrum shows H5 and H 16a of hydrogen, as well as H6 and H 11 The hydrogen signals of cyclopropane are correlated, confirming that cyclopropane is α-configuration and 11-H is β-configuration. ESI-MS (m / z): [M+H] + =349.2(calcd:349.2).

[0222] Preparation of the hydrochloride salt 27 of compound 8

[0223] Compound 8 (375 mg, 1 mmol) was dissolved in dichloromethane (2 mL) and stirred at room temperature for 2 h. Hydrochloric acid solution was then added dropwise to a pH of 4-5. The mixture was filtered and the resulting solid was washed with dichloromethane to obtain a white solid, the hydrochloride salt of compound 8 (compound 27), with a yield of 88%. 1 H NMR (500MHz, CD3OD): δ3.75(t,J=10.2Hz,1H,H6),2.77(dd,J=13.2,4.2Hz,1H),2.58(dd,J =13.2,6.8Hz,2H),2.47(d,J=13.9Hz,4H),2.41-2.35(m,4H),2.32(dd,J=12.1,6.5Hz,1H,H 11 ),2.26-2.17(m,4H),1.92-1.87(m,3H),1.72(d,J=10.6Hz,1H,H5),1.50(s,3 H),1.47-1.20(m,3H),1.08(s,3H),1.06-0.95(m,3H),0.74(d,J=3.7Hz,1H,H 16a ),0.48(d,J=3.5Hz,1H,H16b ROESY spectrum shows H5 and H 16a of hydrogen, as well as H6 and H 11 The hydrogen signals of cyclopropane are correlated, confirming that cyclopropane is α-configuration and 11-H is β-configuration. ESI-MS (m / z): [M+H] + =378.3(calcd:378.3).

[0224] Preparation of the hydrochloride salt 28 of compound 9

[0225] Compound 9 (334 mg, 1 mmol) was dissolved in dichloromethane (2 mL) and stirred at room temperature for 2 h. Hydrochloric acid solution was then added dropwise to a pH of 4-5. The mixture was filtered and the resulting solid was washed with dichloromethane to obtain an oily liquid, the hydrochloride salt of compound 9 (compound 28), with a yield of 86%. 1 H NMR (500MHz, CD3OD): δ3.77(t,J=10.3Hz,1H,H6),2.89(dd,J=13.6,4.6Hz,1H),2.63(dd,J=13.7,6.6Hz,1H,H 11 ),2.58-2.50(m,2H),2.45(m,J=6.8Hz,2H),2.38(s,1H),2.29(m,J=11 .7,5.6Hz,1H),2.19-2.13(m,2H),2.11-2.04(m,1H),1.91-1.78(m,3H) ,1.75(d,J=10.5Hz,1H,H5),1.52(s,3H),1.50-1.36(m,2H),1.12(s,3H ),1.05(d,J=13.1Hz,1H),0.98(t,J=7.1Hz,6H),0.76(d,J=4.0Hz,1H,H 16a ),0.49(d,J=4.0Hz,1H,H 16b ROESY spectrum shows H5 and H 16a of hydrogen, as well as H6 and H 11 The hydrogen signals of cyclopropane are correlated, confirming that cyclopropane is α-configuration and 11-H is β-configuration. ESI-MS (m / z): [M+H] + =336.2(calcd:336.2).

[0226] Preparation of the hydrochloride salt 29 of compound 10

[0227] Compound 10 (362 mg, 1 mmol) was dissolved in dichloromethane (2 mL) and stirred at room temperature for 2 h. Hydrochloric acid solution was then added dropwise to a pH of 4-5. The mixture was filtered and the resulting solid was washed with dichloromethane to obtain an oily liquid, the hydrochloride salt of compound 10 (compound 29), with a yield of 85%. 1 H NMR (500MHz, CD3OD): δ3.78 (t, J=10.2Hz, 1H, H6), 2.87-2.80 (m, 1H), 2.66 (dd, J=13.5, 6.6Hz, 1H, H 11 ),2.38(d,J=12.2Hz,3H),2.34-2.25(m,3H),2.15(d,J=11.7Hz,2H),2.11-2.04(m,1H),1.93-1.82(m,3H),1.75(d,J=10.6H z,1H,H5),1.52(s,3H),1.45-1.38(m,5H),1.10(s,3H),1.04(t,J=12.2Hz,1H),0.85(t,J=7.3Hz,6H),0.76(d,J=3.9Hz,1H,H 16a ),0.50(d,J=3.9Hz,1H,H 16b ROESY spectrum shows H5 and H 16a of hydrogen, as well as H6 and H 11 The hydrogen signals of cyclopropane are correlated, confirming that cyclopropane is α-configuration and 11-H is β-configuration. ESI-MS (m / z): [M+H] + =364.3(calcd:364.3).

[0228] Preparation of the hydrochloride salt 30 of compound 11

[0229] Compound 11 (390 mg, 1 mmol) was dissolved in dichloromethane (2 mL) and stirred at room temperature for 2 h. Hydrochloric acid solution was then added dropwise to a pH of 4-5. The mixture was filtered and the resulting solid was washed with dichloromethane to obtain an oily liquid, the hydrochloride salt of compound 11 (compound 30), with a yield of 82%. 1 H NMR (500MHz, CD3OD): δ3.78(t,J=10.2Hz,1H,H6),2.82(dd,J=13.6,4.2Hz,1H),2.64(dd,J=13.5,6.6Hz,1H,H 11),2.46-2.42(m,2H),2.38-2.24(m,4H),2.19-2.04(m,3H),1.93-1.82(m,3H),1.75(d,J=10.6Hz,1H,H5),1.5 2(s,3H),1.43-1.32(m,5H),1.28(p,J=7.1Hz,5H),1.10(s,3H),0.90(t,J=7.3Hz,6H),0.77(d,J=3.8Hz,1H,H 16a ),0.50(d,J=3.8Hz,1H,H 16b ROESY spectrum shows H5 and H 16a of hydrogen, as well as H6 and H 11 The hydrogen signals of the cyclopropane and 11-H groups were correlated, confirming that the cyclopropane group was in the α configuration and the 11-H group was in the β configuration. ESI-MS (m / z): [M+H]+ = 392.3 (calcd: 392.3).

[0230] Preparation of the hydrochloride salt 31 of compound 12

[0231] Compound 12 (362 mg, 1 mmol) was dissolved in dichloromethane (2 mL) and stirred at room temperature for 2 h. Hydrochloric acid solution was then added dropwise to a pH of 4-5. The mixture was filtered and the resulting solid was washed with dichloromethane to obtain an oily liquid, the hydrochloride salt of compound 12 (compound 31), with a yield of 80%. 1 H NMR (500MHz, CD3OD): δ3.78(t,J=10.3Hz,1H,H6),2.83(d,J=8.7Hz,1H),2.65(dd,J=13.2,6.2Hz,1H,H 11 ),2.38(m,J=12.8,8.8Hz,3H),2.14(d,J=12.2Hz,2H),2.11-2.01(m,1H),1.88(q,J=10.0,7.8Hz,3H),1.74(d,J=10.5Hz,1H ,H5),1.51(s,3H),1.49-1.35(m,6H),1.09(s,3H),1.05(t,J=12.2Hz,1H),0.85(q,J=9.0,7.2Hz,6H),0.75(d,J=3.6Hz,1H,H 16a ),0.49(d,J=3.5Hz,1H,H 16b ),0.06(s,2H).ROESY spectrum shows H5 and H 16a of hydrogen, as well as H6 and H 11The hydrogen signals of cyclopropane are correlated, confirming that cyclopropane is α-configuration and 11-H is β-configuration. ESI-MS (m / z): [M+H] + =364.3(calcd:364.3).

[0232] Preparation of the hydrochloride salt 32 of compound 13

[0233] Compound 13 (375 mg, 1 mmol) was dissolved in dichloromethane (2 mL) and stirred at room temperature for 2 h. Hydrochloric acid solution was then added dropwise to a pH of 4-5. The mixture was filtered and the resulting solid was washed with dichloromethane to obtain a white solid, the hydrochloride salt of compound 13 (compound 32), with a yield of 81%. 1 H NMR (500MHz, CD3OD): δ3.76(t,J=10.3Hz,1H,H6),2.76-2.66(m,2H),2.62-2.47(m,4H),2.38(s,1H),2.31-2.25(m,1H,H 11 ),2.20-2.03(m,5H),1.94-1.83(m,3H),1.74(d,J=10.6Hz,1H,H5),1.67-1.55(m,3H),1.51 (s,3H),1.49-1.37(m,3H),1.33(dd,J=11.0,6.7Hz,2H),1.09(s,3H),0.76(d,J=3.6Hz,1H,H 16a ),0.49(d,J=3.6Hz,1H,H 16b ROESY spectrum shows H5 and H 16a of hydrogen, as well as H6 and H 11 The hydrogen signals of cyclopropane are correlated, confirming that cyclopropane is α-configuration and 11-H is β-configuration. ESI-MS (m / z): [M+H] + =374.3(calcd:374.3).

[0234] Preparation of the hydrochloride salt 33 of compound 14

[0235] Compound 14 (318 mg, 1 mmol) was dissolved in dichloromethane (2 mL) and stirred at room temperature for 2 h. Hydrochloric acid solution was then added dropwise to a pH of 4-5. The mixture was filtered and the resulting solid was washed with dichloromethane to obtain a white solid, the hydrochloride salt of compound 14 (compound 33), with a yield of 82%. 1 H NMR (500MHz, CD3OD): δ3.72(t,J=10.3Hz,1H,H6),3.22-3.14(m,4H),2.75-2.68(m,1H,H 11),2.37(s,1H),2.17-2.08(m,3H),2.04-1.95(m,3H),1.91-1.81(m,3H),1.74(d,J= 10.6Hz,1H,H5),1.50(s,3H),1.46-1.24(m,3H),1.07(s,3H),0.74(d,J=3.7Hz,1H,H 16a ),0.48(d,J=3.7Hz,1H,H 16b ROESY spectrum shows H5 and H 16a of hydrogen, as well as H6 and H 11 The hydrogen signals of cyclopropane are correlated, confirming that cyclopropane is α-configuration and 11-H is β-configuration. ESI-MS (m / z): [M+H] + =320.2(calcd:320.2).

[0236] Preparation of the hydrochloride salt 34 of compound 15

[0237] Compound 15 (400 mg, 1 mmol) was dissolved in dichloromethane (2 mL) and stirred at room temperature for 2 h. Hydrochloric acid solution was then added dropwise to a pH of 4-5. The mixture was filtered and the resulting solid was washed with dichloromethane to obtain a white solid, the hydrochloride salt of compound 15 (compound 34), with a yield of 81%. 1 H NMR (500MHz, CD3OD): δ3.76(t,J=10.3Hz,1H,H6),2.74(dd,J=13.1,4.4Hz,1H),2.53(dd,J=13.1,6.8Hz,1H,H 11 ),2.40(s,3H),2.34-2.28(m,2H),2.17-2.02(m,3H),1.87(dd,J=10.0,3.1Hz,2 H),1.85-1.79(m,1H),1.73(d,J=10.6Hz,1H,H5),1.56(s,3H),1.51(s,3H),1.44 -1.41(m,8H),1.36(d,J=6.7Hz,4H),1.08(s,3H),1.03(t,J=12.5Hz,1H),0.75(d,J=3.8Hz,1H,H 16a ),0.48(d,J=3.7Hz,1H,H 16b ROESY spectrum shows H5 and H 16a of hydrogen, as well as H6 and H 11 The hydrogen signals of cyclopropane are correlated, confirming that cyclopropane is α-configuration and 11-H is β-configuration. ESI-MS (m / z): [M+H] +=402.3(calcd:402.3).

[0238] Preparation of the hydrochloride salt 35 of compound 16

[0239] Compound 16 (429 mg, 1 mmol) was dissolved in dichloromethane (2 mL) and stirred at room temperature for 2 h. Hydrochloric acid solution was then added dropwise to a pH of 4-5. The mixture was filtered and the resulting solid was washed with dichloromethane to obtain a white solid, the hydrochloride salt of compound 16 (compound 35), with a yield of 83%. 1 H NMR (500MHz, CD3OD): δ3.72(t,J=10.2Hz,1H,H6),2.81(dd,J=13.3,4.4Hz,2H),2.69(dd,J=13.2,4.2Hz,1H,H 11 ),2.50(dd,J=13.3,7.0Hz,1H),2.44(m,4H),2.32-2.25(m,1H),2.18-2.0 7(m,2H),2.05-1.97(m,2H),1.91-1.80(m,3H),1.73(d,J=10.6Hz,1H,H5), 1.73-1.67(m,3H),1.57-1.50(m,4H),1.47(s,3H),1.45-1.37(m,4H),1.3 6-1.20(m,2H),1.04(s,3H),0.98(t,J=12.7Hz,1H),0.71(d,J=3.6Hz,1H,H 16a ),0.45(d,J=3.5Hz,1H,H 16b ROESY spectrum shows H5 and H 16a of hydrogen, as well as H6 and H 11 The hydrogen signals of cyclopropane are correlated, confirming that cyclopropane is α-configuration and 11-H is β-configuration. ESI-MS (m / z): [M+H] + =431.3(calcd:431.3).

[0240] Preparation of the hydrochloride salt 36 of compound 17

[0241] Compound 17 (431 mg, 1 mmol) was dissolved in dichloromethane (2 mL) and stirred at room temperature for 2 h. Hydrochloric acid solution was then added dropwise to a pH of 4-5. The mixture was filtered and the resulting solid was washed with dichloromethane to obtain a white solid, the hydrochloride salt of compound 17 (compound 36), with a yield of 84%. 1H NMR (500MHz, CD3OD): δ3.75(t,J=10.2Hz,1H,H6),3.69(s,4H),2.87(d,J=11.1H z,1H),2.80(d,J=11.2Hz,1H),2.72(dd,J=13.2,3.8Hz,1H),2.58-2.53(m,1H,H 11 ),2.51(s,4H),2.39(s,1H),2.35-2.28(m,1H),2.14-2.04(m,4H),1.93 (t,J=11.8Hz,1H),1.85(d,J=8.4Hz,2H),1.80(d,J=10.8Hz,3H),1.71( d,J=10.6Hz,1H,H5),1.49(s,3H),1.42-1.38(m,3H),1.23(dd,J=13.4,5.8Hz,1H),1.07(s,3H),1.00(t,J=13.1Hz,1H),0.73(d,J=3.7Hz,1H,H 16a ),0.47(d,J=3.6Hz,1H,H 16b ROESY spectrum shows H5 and H 16a of hydrogen, as well as H6 and H 11 The hydrogen signals of cyclopropane are correlated, confirming that cyclopropane is α-configuration and 11-H is β-configuration. ESI-MS (m / z): [M+H] + =433.3(calcd:433.3).

[0242] Preparation of the hydrochloride salt 37 of compound 18

[0243] Compound 18 (372 mg, 1 mmol) was dissolved in dichloromethane (2 mL) and stirred at room temperature for 2 h. Hydrochloric acid solution was then added dropwise to a pH of 4-5. The mixture was filtered and the resulting solid was washed with dichloromethane to obtain a white solid, the hydrochloride salt of compound 18 (compound 37), with a yield of 92%. 1 H NMR (500MHz, CD3OD): δ3.78 (t, J=10.2Hz, 1H, H6), 2.81 (d,J=13.0Hz,1H),2.60(dd,J=12.6,6.2Hz,1H),2.49(s,2H),2.15(d,J=12.0Hz,2H),2.07(t,J=11.6Hz,1H,H 11),1.86(m,3H),1.74(d,J=10.5Hz,1H,H5),1.52(s,3H),1.50-1.39(m,3H),1.33(d,J=20.0Hz,4H),1.2 6(d,J=13.6Hz,1H),1.09(s,3H),1.04(t,J=12.6Hz,1H),0.86(d,J=7.1Hz,1H),0.76(d,J=3.8Hz,1H,H 16a ),0.49(d,J=3.7Hz,1H,H 16b ),0.24(s,4H).ROESY spectrum shows H5 and H 16a of hydrogen, as well as H6 and H 11 The hydrogen signals of cyclopropane are correlated, confirming that cyclopropane is α-configuration and 11-H is β-configuration. ESI-MS (m / z): [M+H] + =374.3(calcd:374.3).

[0244] Preparation of the hydrochloride salt 38 of compound 19

[0245] Compound 19 (374 mg, 1 mmol) was dissolved in dichloromethane (2 mL) and stirred at room temperature for 2 h. Hydrochloric acid solution was then added dropwise to a pH of 4-5. The mixture was filtered and the resulting solid was washed with dichloromethane to obtain an oily liquid, the hydrochloride salt of compound 19 (compound 38), with a yield of 80%. 1 H NMR (500MHz, CD3OD): δ3.78(t,J=10.2Hz,1H,H6),2.97(dd,J=13.6,4.4Hz,1H),2.80(dd,J=13.6,4.8Hz,1H,H 11 ),2.61-2.58(m,1H),2.58-2.55(m,4H),2.38(d,J=9.0Hz,1H),2.25-2.15(m,3H),2.12-2.05(m,1H),2.05-1.97(m,3H),1.92-1.85(m,3H) ,1.75(d,J=10.7Hz,1H,H5),1.68(d,J=10.9Hz,4H),1.56(s,3H),1.48-1.39(m,3H),1.11(s,3H),1.09-1.02(m,1H),0.78(d,J=3.9Hz,1H,H 16a ),0.50(d,J=3.7Hz,1H,H 16b ROESY spectrum shows H5 and H 16a of hydrogen, as well as H6 and H 11The hydrogen signals of cyclopropane are correlated, confirming that cyclopropane is α-configuration and 11-H is β-configuration. ESI-MS (m / z): [M+H] + =376.3(calcd:376.3.

[0246] Preparation of Sulfate Salt 39 of Compound 2

[0247] The sulfate salt (Compound 39) was prepared by replacing hydrochloric acid with sulfuric acid according to the preparation method of the hydrochloride salt of Compound 2. The yield was 75%. 1 H NMR (500MHz, CD3OD): δ4.13-4.11(m,1H,H6),3.41-3.35(m,1H),3.31-3.27(m,1H),3.03-2.99(m,1H,H 11 ),2.92(s,6H),2.21-2.10(m,2H),2.00(d,J=15.0Hz,2H),1.85(t,J=5.0Hz,2H),1.77(d,J=10.0Hz,1H,H5), 1.62-1.53(m,2H),1.51(s,3H),1.25(t,J=5.0Hz,1H),1.13(s,3H),1.10-1.03(m,1H),0.75(d,J=5.0Hz,1H,H 16a ),0.52(d,J=5.0Hz,1H,H 16b ). ROESY spectrum shows H5 and H 16a of hydrogen, as well as H6 and H 11 The hydrogen signals of cyclopropane are correlated, confirming that cyclopropane is α-configuration and 11-H is β-configuration. ESI-MS (m / z): [M+H] + =308.2(calcd:308.2).

[0248] Preparation of the phosphate salt 40 of compound 2.

[0249] Phosphate (Compound 40) was prepared by replacing hydrochloric acid with phosphoric acid, following the preparation method of hydrochloride of Compound 2. The yield was 88%. 1 H NMR (500MHz, CD3OD): δ4.14-4.11(m,1H,H6), 3.43-3.36(m,1H), 3.31-3.28(m,1H), 3.04-2.99(m,1H,H 11),2.93(s,6H),2.21-2.11(m,2H),2.01(d,J=15.0Hz,2H),1.86(t,J=5.0Hz,2H),1.78(d,J=10.0Hz,1H,H5), 1.63-1.54(m,2H),1.52(s,3H),1.26(t,J=5.0Hz,1H),1.14(s,3H),1.10-1.04(m,1H),0.75(d,J=5.0Hz,1H,H 16a ),0.52(d,J=5.0Hz,1H,H 16b ). ROESY spectrum shows H5 and H 16a of hydrogen, as well as H6 and H 11 The hydrogen signals of cyclopropane are correlated, confirming that cyclopropane is α-configuration and 11-H is β-configuration. ESI-MS (m / z): [M+H] + =308.2(calcd:308.2).

[0250] Preparation of the maleate salt 41 of compound 2.

[0251] Maleic acid was used instead of hydrochloric acid, and the maleate salt (Compound 41) was prepared according to the preparation method of the hydrochloride salt of Compound 2. The yield was 83%. 1 H NMR (500MHz, CD3OD): δ6.27(s,2H),4.14-4.10(m,1H,H6),3.43-3.35(m,1H),3.30-3.27(m,1H),3.03-2.97(m,1H,H 11 ),2.91(s,6H),2.20-2.11(m,2H),2.00(d,J=15.0Hz,2H),1.85(t,J=5.0Hz,2H),1.76(d,J=10.0Hz,1H,H5), 1.62-1.52(m,2H),1.52(s,3H),1.25(t,J=5.0Hz,1H),1.13(s,3H),1.10-1.03(m,1H),0.74(d,J=5.0Hz,1H,H 16a ),0.51(d,J=5.0Hz,1H,H 16b ). ROESY spectrum shows H5 and H 16a of hydrogen, as well as H6 and H 11 The hydrogen signals of cyclopropane are correlated, confirming that cyclopropane is α-configuration and 11-H is β-configuration. ESI-MS (m / z): [M+H] + =308.2(calcd:308.2).

[0252] Preparation of Citrate Salt 42 of Compound 2

[0253] Citric acid was used instead of hydrochloric acid to prepare citrate (Compound 42) according to the preparation method of Compound 2 hydrochloride. The yield was 79%. 1 H NMR (500MHz, CD3OD): δ4.12(t,J=10.0Hz,1H,H6),3.40-3.39(m,1H),3.29-3.26(m,1H),3.02-2.97(m,1H,H 11 ),2.90(s,6H),2.73(d,J=5.2Hz,2H),2.55(d,J=5.2Hz,2H),2.20-2.12(m,2H),1.93-1.91(m,1H),1.87(t,J=5.0Hz,2H),1.83- 1.79(m,1H),1.78(d,J=15.0Hz,1H,H5),1.62-1.53(m,2H),1.51(s,3H),1.13(s,3H),1.09-1.02(m,1H),0.76(d,J=5.0Hz,1H,H 16a ),0.53(d,J=5.0Hz,1H,H 16b ). ROESY spectrum shows H5 and H 16a , as well as H6 and H 11 There is signal correlation, confirming that cyclopropane is α-configuration and 11-H is β-configuration. ESI-MS (m / z): [M+H] + =308.2(calcd:308.2).

[0254] Compounds 43-63 were prepared by referring to the preparation method described in CN 115403546 A.

[0255] Preparation of compound 64:

[0256] To a 5 mL reaction flask, MCL-containing smilage lactone (100 mg, 0.40 mmol), dichloromethane (1.0 mL), N,N-carbonylbis(1,2,4-triazole) (131 mg, 0.80 mmol), pyridine (64 mg, 0.8 mmol), and 4-dimethylaminopyridine (4 mg, 0.04 mmol) were added sequentially. The mixture was stirred at room temperature until the reaction was complete as determined by TLC, yielding a crude intermediate solution. N,N-diisopropylethylamine (52 mg, 0.4 mmol) and a methylamine aqueous solution (47 μL, 0.60 mmol, 40% aqueous solution) were added sequentially, and the mixture was stirred at room temperature until the reaction was complete as determined by TLC. The reaction solution was diluted with ethyl acetate (10 mL), washed successively with water (10 mL × 3), saturated citric acid solution (10 mL × 3, saturated copper sulfate solution was used if the subsequent product contained a basic group), saturated sodium bicarbonate solution (10 mL × 3), and saturated brine (10 mL × 3). After drying over anhydrous sodium sulfate, the mixture was concentrated under reduced pressure and separated by silica gel column chromatography to obtain compound 64 in a yield of 72%. 1 H NMR (500MHz, CDCl3): δ6.19 (d, J = 3.5Hz, 1H), 5.47 (d, J = 3.0Hz, 1H), 4.73 (s, 1H), 3.80 (t, J = 10.5Hz, 1H), 3.11 (d, J = 9.5Hz, 1H), 2.74 (d, J=4.5Hz,3H),2.70-2.65(m,1H),2.48-2.43(m,2H),2.28-2.20(m,3H),2.11-2.02(m,2H),1.72(s,3H),1.53(s,3H),1.40-1.32(m,1H). ESI-MS(m / z):[M+H] + =306.16(calcd:306.16).

[0257] Preparation of compound 65:

[0258] The preparation method of compound 65 was similar to that of compound 64, with a yield of 72%. 1H NMR (500MHz, CDCl3): δ6.20(d,J=3.5Hz,1H),5.48(d,J=3.0Hz,1H),4.76(s,1H),3.80(t,J=10.5Hz,1H),3.21-3.11(m,3H),2.76-2.6 6(m,1H),2.48-2.43(m,2H),2.28-2.20(m,3H),2.12-2.06(m,2H),1.72(s,3H),1.53(s,3H),1.40-1.32(m,1H),1.14(t,J=7.0Hz,3H). ESI-MS(m / z):[M+H] + =320.18(calcd:320.18).

[0259] Preparation of compound 66:

[0260] The preparation method of compound 66 was based on compound 64, with a yield of 76%. 1 H NMR (500MHz, CDCl3): δ6.20(d,J=3.5Hz,1H),5.47(d,J=3.0Hz,1H),4.80(s,1H),3.80(t,J=10.5Hz,1H),3.14-3.05(m,3H),2.70-2.66(m,1H), 2.48-2.41(m,2H),2.27-2.21(m,3H),2.11-2.05(m,2H),1.72(s,3H),1 .53(s,3H),1.55-1.48(m,2H),1.40-1.32(m,1H),0.92(t,J=7.5Hz,3H). ESI-MS(m / z):[M+H] + =334.19(calcd:334.19).

[0261] Preparation of compound 67:

[0262] The preparation method of compound 67 was based on compound 64, with a yield of 74%. 1H NMR (500MHz, CDCl3): δ6.17 (d, J = 3.5Hz, 1H), 5.46 (d, J = 3.0Hz, 1H), 4.78 (s, 1H), 3.79 (t, J = 10.5Hz, 1H), 3.17-3.10 (m, 3H), 2.69-2.64(m,1H),2.46-2.41(m,2H),2.26-2.18(m,3H),2.10-2.04(m,2H),1.71 (s,3H),1.51(s,3H),1.48-1.44(m,2H),1.37-1.31(m,3H),0.91(t,J=7.5Hz,3H). ESI-MS(m / z):[M+H] + =348.21(calcd:348.21).

[0263] Preparation of compound 68:

[0264] The preparation method of compound 68 was based on compound 64, with a yield of 41%. 1 H NMR (500MHz, CDCl3): δ6.20 (d, J = 3.5Hz, 1H), 5.48 (d, J = 3.0Hz, 1H), 4.66 (s, 1H), 3.83-3.74 (m, 2H), 3.13 (d, J = 10.0Hz, 1H), 2.71-2.6 6(m,1H),2.48-2.44(m,2H),2.29-2.20(m,3H),2.13-2.05(m,2H),1.73(s,3H),1.53(s,3H),1.41-1.33(m,1H),1.16(t,J=7.0Hz,6H). ESI-MS(m / z):[M+H] + =334.19(calcd:334.19).

[0265] Preparation of compound 69:

[0266] The preparation method of compound 69 was based on compound 64, with a yield of 33%. 1H NMR (500MHz, CDCl3): δ6.16(d,J=3.5Hz,1H),5.44(d,J=3.0Hz,1H),4.51(d,J =9.0Hz,1H),3.77(t,J=10.5Hz,1H),3.39-3.38(m,1H),3.19-3.17(m,1H),2.6 8-2.63(m,1H),2.45-2.34(m,2H),2.24-2.21(m,3H),2.17-2.06(m,2H),1.70 (s,3H),1.54-1.47(m,2H),1.49(s,3H),1.41-1.30(m,3H),0.92-0.86(m,6H). ESI-MS (m / z): [M+H] + =362.23(calcd:362.23).

[0267] Preparation of compound 70:

[0268] The preparation method of compound 70 was based on compound 64, with a yield of 69%. 1 H NMR (500MHz, CDCl3) δ6.19(d,J=3.3Hz,1H),5.47(d,J=3.0Hz,1H),5.01(s,1H),3.80(t,J=10.1Hz,1H),3.09(s,1H),2.71-2.62(m,1H),2.56(s ,1H),2.50-2.41(m,2H),2.32-2.16(m,3H),2.13-1.99(m,2H),1.72(s, 3H), 1.53 (s, 3H), 1.39-1.32 (m, 1H), 0.69 (d, J = 6.8Hz, 2H), 0.53 (s, 2H). ESI-MS(m / z):[M+H] + =332.18(calcd:332.18).

[0269] Preparation of compound 71:

[0270] The preparation method of compound 71 was based on compound 64, with a yield of 71%. 1H NMR (500MHz, CDCl3): δ6.13(d,J=3.5Hz,1H),5.42(d,J=3.0Hz,1H),5.30(s,1H ),4.66(s,1H),3.75(t,J=10.5Hz,1H),3.63-3.61(m,1H),3.24-3.20(m,1H),3 .13-3.11(m,1H),2.63-2.59(m,1H),2.54-2.47(m,2H),2.40-2.28(m,2H),2.2 0-2.15(m,3H),2.06-1.98(m,2H),1.64(s,3H),1.42(s,3H),1.33-1.26(m,1H). ESI-MS (m / z): [M+H] + =336.17(calcd:336.17).

[0271] Preparation of compound 72:

[0272] The preparation method of compound 72 was based on compound 64, with a yield of 62%. 1 H NMR (500MHz, CDCl3): δ6.21 (d, J=3.5Hz, 1H), 5.49 (d, J=3.0Hz, 1H), 3.83 (t, J= 10.5Hz,1H),3.68(t,J=5.5Hz,2H),3.37-3.26(m,2H),3.19(d,J=10.5Hz,1H),2 .71-2.67(m,1H),2.48-2.44(m,1H),2.40-2.37(m,1H),2.29-2.20(m,3H),2.1 6-2.10(m,2H),1.72(s,3H),1.71-1.68(m,2H),1.51(s,3H),1.40-1.35(m,1H). ESI-MS (m / z): [M+H] + =350.19(calcd:350.19).

[0273] Preparation of compound 73:

[0274] The preparation method of compound 73 was based on compound 64, with a yield of 59%. 1H NMR (500MHz, CDCl3): δ6.18(d,J=3.5Hz,1H),5.47(d,J=3.0Hz,1H),5.06(s,1H) ,4.49(s,1H),3.79(t,J=10.5Hz,1H),3.62(t,J=6.5Hz,2H),3.15-3.10(m,3H), 2.68-2.64(m,1H),2.45-2.38(m,2H),2.25-2.22(m,3H),2.10-2.04(m,2H),1.7 0(s,3H),1.60-1.55(m,2H),1.53-1.50(m,2H),1.49(s,3H),1.43-1.33(m,3H). ESI-MS (m / z): [M+H] + =377.24(calcd:377.24).

[0275] Preparation of compound 74:

[0276] The preparation method of compound 74 was similar to that of compound 64, with a yield of 75%. 1 H NMR (500MHz, CDCl3): δ6.20(d,J=3.5Hz,1H),5.47(d,J=3.0Hz,1H),5.51(s ,1H),3.80(t,J=10.5Hz,1H),3.48-3.46(m,2H),3.37(s,3H),3.36-3.29(m ,2H),3.14(d,J=10.0Hz,1H),2.71-2.66(m,1H),2.48-2.42(m,2H),2.27-2 .20(m,3H),2.12-2.03(m,2H),1.73(s,3H),1.55(s,3H),1.40-1.32(m,1H). ESI-MS(m / z):[M+H] + =350.19(calcd:350.19).

[0277] Preparation of compound 75:

[0278] The preparation method of compound 75 was similar to that of compound 64, with a yield of 74%. 1H NMR(500MHz, CDCl3)δ6.20(d,J=3.3Hz,1H),5.48(d,J=3.0Hz,1H),5.06(s,1H), 3.80(t,J=10.1Hz,1H),3.46(t,J=5.8Hz,2H),3.35(s,3H),3.29-3.21(m,2H),3. 14(d,J=10.1Hz,1H),2.71-2.66(m,1H),2.48-2.41(m,2H),2.27-2.20(m,3H),2. 12-2.04(m,2H),1.80-1.76(m,2H),1.73(s,3H),1.53(s,3H),1.40-1.33(m,1H). ESI-MS (m / z): [M+H] + =364.20(calcd:364.20).

[0279] Preparation of compound 76:

[0280] The preparation method of compound 76 was based on compound 64, with a yield of 62%. 1 H NMR (500MHz, CDCl3): δ6.20(d,J=3.5Hz,1H),5.56(s,1H),5.49(d,J=3.0Hz, 1H),3.82(t,J=10.5Hz,1H),3.62-3.58(m,4H),3.36-3.34(m,2H),3.12(d,J= 10.5Hz,1H),2.98-2.87(m,1H),2.71-2.66(m,1H),2.48-2.43(m,2H),2.27- 2.20(m,3H),2.13-1.97(m,3H),1.72(s,3H),1.52(s,3H),1.40-1.32(m,1H). ESI-MS (m / z): [M+H] + =380.20(calcd:380.20).

[0281] Preparation of compound 77:

[0282] The preparation method of compound 77 was based on compound 64, with a yield of 61%. 1H NMR (500MHz, CDCl3): δ6.20(d,J=3.5Hz,1H),5.48(d,J=3.0Hz,1H),5.22(s,1H),3.80(t,J=10.5Hz,1H),3.31-3.22(m,2H),3.16(d,J=10.0H z,1H),2.70-2.67(m,1H),2.48-2.40(m,4H),2.34-2.20(m,3H),2.26( s,6H),2.11-2.04(m,2H),1.72(s,3H),1.55(s,3H),1.39-1.32(m,1H). ESI-MS(m / z):[M+H] + =363.22(calcd:363.22).

[0283] Preparation of compound 78:

[0284] The preparation method of compound 78 was based on compound 64, with a yield of 71%. 1 H NMR (500MHz, CDCl3): δ6.18(d,J=3.5Hz,1H),5.46(d,J=3.0Hz,1H),5.18(s,1H),3.79(t,J=10.5Hz,1H),3.72-3.70(m,4H),3.25-3.23(m,2H), 3.17(d,J=9.5Hz,1H),2.70-2.66(m,1H),2.48-2.37(m,8H),2.26-2.19 (m,3H),2.12-2.07(m,2H),1.71(s,3H),1.53(s,3H),1.39-1.31(m,1H). ESI-MS(m / z):[M+H] + =405.23(calcd:405.23).

[0285] Preparation of compound 79:

[0286] The preparation method of compound 79 was based on compound 64, with a yield of 69%. 1H NMR (500MHz, CDCl3): δ7.35-7.30(m,4H),7.28-7.25(m,1H),6.20(d,J=3.5Hz,1H ),5.48(d,J=3.0Hz,1H),5.13(s,1H),4.43-4.39(m,1H),4.30-4.26(m,1H),3.81 (t,J=10.5Hz,1H),3.18(d,J=9.5Hz,1H),2.68-2.65(m,1H),2.49-2.42(m,2H),2 .28-2.21(m,3H),2.17-2.06(m,2H),1.73(s,3H),1.56(s,3H),1.40-1.32(m,1H). ESI-MS(m / z):[M+H] + =382.19(calcd:382.19).

[0287] Preparation of compound 80:

[0288] The preparation method of compound 80 was based on compound 64, with a yield of 70%. 1 H NMR (500MHz, CDCl3): δ6.20(d,J=3.5Hz,1H),5.48(d,J=3.0Hz,1H),3.83(t,J=10.5Hz,1H),3.07(d,J=10.0Hz,1H),2.96(s,3H),2.89(s,3H) ,2.72-2.66(m,1H),2.54-2.44(m,2H),2.30-2.21(m,3H),2.14-2.09( m,1H),2.03-1.97(m,1H),1.73(s,3H),1.54(s,3H),1.42-1.34(m,1H). ESI-MS(m / z):[M+H] + =320.18(calcd:320.18).

[0289] Preparation of compound 81:

[0290] The preparation method of compound 81 was based on compound 64, with a yield of 69%. 1H NMR (500MHz, CDCl3): δ6.20(d,J=3.5Hz,1H),5.48(d,J=3.0Hz,1H),3.83(t,J=10 .5Hz,1H),3.41-3.35(m,1H),3.30-3.22(m,3H),3.06(d,J=10.0Hz,1H),2.72-2.6 7(m,1H),2.57-2.53(m,1H),2.49-2.44(m,1H),2.30-2.22(m,3H),2.14-2.09(m, 1H),2.04-1.97(m,1H),1.73(s,3H),1.55(s,3H),1.42-1.34(m,1H),1.14(s,6H). ESI-MS(m / z):[M+H] + =348.21(calcd:348.21).

[0291] Preparation of compound 82:

[0292] The preparation method of compound 82 was similar to that of compound 64, with a yield of 71%. 1 H NMR (500MHz, CDCl3): δ6.20(d,J=3.5Hz,1H),5.48(d,J=3.0Hz,1H),3.82(t,J =10.5Hz,1H),3.53-3.49(m,1H),3.37-3.30(m,3H),3.10(d,J=10.0Hz,1H),2. 72-2.66(m,1H),2.53-2.44(m,2H),2.29-2.21(m,3H),2.13-2.08(m,1H),2.07 -2.00(m,1H),1.88-1.83(m,4H),1.73(s,3H),1.56(s,3H),1.42-1.34(m,1H). ESI-MS (m / z): [M+H] + =346.19(calcd:346.19).

[0293] Preparation of compound 83:

[0294] The preparation method of compound 83 was based on compound 64, with a yield of 68%. 1H NMR (500MHz, CDCl3): δ6.20(d,J=3.5Hz,1H),5.47(d,J=3.0Hz,1H),3.82(t,J=10.5Hz,1H),3.51-3.42(m,4H),3.10(d,J=10.0Hz,1H),2. 72-2.68(m,1H),2.52-2.44(m,2H),2.30-2.22(m,3H),2.14-2.01(m, 2H),1.73(s,3H),1.60-1.56(m,6H),1.53(s,3H),1.41-1.35(m,1H). ESI-MS(m / z):[M+H] + =360.21(calcd:360.21).

[0295] Preparation of compound 84:

[0296] The preparation method of compound 84 was based on compound 64, with a yield of 67%. 1 H NMR (500MHz, CDCl3): δ6.19(d,J=3.5Hz,1H),5.47(d,J=3.0Hz,1H),4.24-4.12(m,2H) ,3.81(t,J=10.5Hz,1H),3.08(s,1H),2.83-2.67(m,3H),2.51-2.43(m,2H),2.29-2.2 0(m,3H),2.12-2.09(m,1H),2.05-1.99(m,1H),1.72(s,3H),1.65-1.60(m,2H),1.55- 1.48(m,1H),1.52(s,3H),1.41-1.33(m,1H),1.19-1.04(m,2H),0.94(d,J=6.5Hz,3H). ESI-MS(m / z):[M+H] + =374.23(calcd:374.23).

[0297] Preparation of compound 85:

[0298] The preparation method of compound 85 was similar to that of compound 64, with a yield of 52%. 1H NMR (500MHz, CDCl3): δ6.20(d,J=3.5Hz,1H),5.48(d,J=3.0Hz,1H),3.83(t, J=10.5Hz,1H),3.57-3.40(m,4H),3.08(d,J=10.0Hz,1H),2.71-2.67(m,1H), 2.53-2.46(m,2H),2.43-2.38(m,3H),2.32(s,3H),2.30-2.22(m,4H),2.13- 2.10(m,1H),2.04-2.02(m,1H),1.73(s,3H),1.53(s,3H),1.42-1.37(m,1H). ESI-MS (m / z): [M+H] + =375.22(calcd:375.22).

[0299] Preparation of compound 86:

[0300] The preparation method of compound 86 was based on compound 64, with a yield of 55%. 1 H NMR (500MHz, CDCl3): δ6.20(d,J=3.5Hz,1H),5.48(d,J=3.0Hz,1H),3.82(t,J=10.5Hz,1H),3.71-3.41(m,4H),3.08(d,J=10.0Hz,1H),2.71-2.66(m, 1H),2.52-2.43(m,8H),2.29-2.22(m,3H),2.13-2.10(m,1H),2.07-2.00( m,1H),1.72(s,3H),1.52(s,3H),1.42-1.34(m,1H),1.11(t,J=7.0Hz,3H). ESI-MS(m / z):[M+H] + =389.24(calcd:389.24).

[0301] Preparation of compound 87:

[0302] The preparation method of compound 87 was based on compound 64, with a yield of 71%. 1H NMR (500MHz, CDCl3): δ6.18(d,J=3.5Hz,1H),5.47(d,J=3.0Hz,1H),3.81(t,J=10.5Hz,1H),3.68-3.62(m,5H),3.49-3.41(m,3H),3.06(d . ESI-MS(m / z):[M+H] + =362.19(calcd:362.19).

[0303] Preparation of compound 88:

[0304] The preparation method of compound 88 was based on compound 64, with a yield of 75%. 1 H NMR (500MHz, CDCl3) δ6.17 (d, J = 3.3Hz, 1H), 5.46-5.45 (m, 1H), 4.82 (s, 1H), 3.78 (t,J=10.1Hz,1H),3.13(d,J=9.9Hz,1H),3.02-2.97(m,1H),2.90-2.85(m,1H),2 .71-2.62(m,1H),2.48-2.35(m,2H),2.26-2.17(m,3H),2.12-2.02(m,2H),1.77- 1.71(m,1H),1.70(s,3H),1.51(s,3H),1.38-1.30(m,1H),0.89(d,J=6.7Hz,6H). ESI-MS(m / z):[M+H] + =348.21(calcd:348.21).

[0305] Preparation of compound 89:

[0306] The preparation method of compound 89 was based on compound 64, with a yield of 78%. 1H NMR (500MHz, CDCl3) δ6.20(d,J=3.3Hz,1H),5.83(t,J=56.1Hz,1H),5.48(d,J=2.9Hz,1H),5.16(s,1H),3.80(t,J=10.1Hz,1H),3.62-3.38(m,2H) ,3.11(d,J=10.5Hz,1H),2.73-2.62(m,1H),2.48-2.41(m,2H),2.27-2.2 0(m,3H),2.12-1.99(m,2H),1.72(s,3H),1.53(s,3H),1.38-1.31(m,1H). ESI-MS(m / z):[M+H] + =356.16(calcd:356.16).

[0307] Preparation of compound 90:

[0308] The preparation method of compound 90 was based on compound 64, with a yield of 67%. 1 H NMR (500MHz, CDCl3) δ6.17(d,J=3.2Hz,1H),5.46(d,J=3.0Hz,1H),3.80(t,J=10.0H z,1H),3.37-3.12(m,2H),3.10-3.00(m,1H),2.88(d,J=28.6Hz,3H),2.72-2.62(m, 1H),2.54-2.41(m,2H),2.29-2.18(m,3H),2.11-2.07(m,1H),2.02-1.93(m,1H),1. 71(s,3H),1.63-1.53(m,2H),1.51(s,3H),1.40-1.32(m,1H),0.88(t,J=7.4Hz,3H). ESI-MS(m / z):[M+H] + =348.21(calcd:348.21).

[0309] Preparation of compound 91:

[0310] The preparation method of compound 91 was based on compound 64, with a yield of 68%. 1H NMR(500MHz, CDCl3) δ6.19(d,J=3.3Hz,1H),5.46(d,J=3.0Hz,1H),3.81(t,J =10.1Hz,1H),3.44-3.00(m,5H),2.69-2.66(m,1H),2.59-2.42(m,2H),2.29- 2.20(m,3H),2.12-2.08(m,1H),2.03-1.97(m,1H),1.72(s,3H),1.61-1.54( m,2H),1.53(s,3H),1.42-1.33(m,1H),1.19-1.07(m,3H),0.89-0.87(m,3H). ESI-MS (m / z): [M+H] + =362.23(calcd:362.23).

[0311] Preparation of compound 92:

[0312] The preparation method of compound 92 was based on compound 64, with a yield of 55%. 1 H NMR (500MHz, CDCl3) δ6.20(d,J=3.3Hz,1H),5.72(s,1H),5.48(d,J=3.1Hz,1H),3.81(t,J=10.1Hz,1H),3.73(t,J=4.7Hz,4H),3.30-3.12(m,3H ),2.72-2.65(m,1H),2.47-2.39(m,8H),2.29-2.20(m,3H),2.14-2.05( m,2H),1.72(s,3H),1.69-1.66(m,2H),1.53(s,3H),1.40-1.34(m,1H). ESI-MS(m / z):[M+H] + =419.25(calcd:419.25).

[0313] Preparation of compound 93:

[0314] The preparation method of compound 93 was based on compound 64, with a yield of 47%. 1H NMR (500MHz, CDCl3) δ6.17(d,J=3.3Hz,1H),5.83(s,1H),5.46(d,J=3.0Hz,1H),3.78(t,J=10.1Hz,1H),3.26-3.11(m,3H),2.65(t, J=9.3Hz,1H),2.56-2.36(m,11H),2.26(s,7H),2.12-2.04(m,2H),1.71(s,3H),1.69-1.63(m,2H),1.51(s,3H),1.39-1.32(m,1H). ESI-MS(m / z):[M+H] + =432.28(calcd:432.28).

[0315] Preparation of compound 94:

[0316] The preparation method of compound 94 was based on compound 64, with a yield of 62%. 1 H NMR(500MHz, CDCl3)δ6.18(d,J=3.2Hz,1H),5.46(d,J=3.0Hz,1H),4.28-4.1 4(m,2H),3.80(t,J=10.1Hz,1H),3.75-3.68(m,4H),3.06(s,1H),2.92-2.63 (m,4H),2.49-2.42(m,2H),2.37-2.18(m,5H),2.14-2.07(m,2H),2.01(d,J= 10.2Hz,1H),1.84-1.82(m,2H),1.71(s,3H),1.49(s,3H),1.46-1.32(m,3H). ESI-MS (m / z): [M+H] + =445.26(calcd:445.26).

[0317] Preparation of compound 95:

[0318] The preparation method of compound 95 was based on compound 64, with a yield of 44%. 1H NMR (500MHz, CDCl3) δ6.17(d,J=3.3Hz,1H),5.45(d,J=3.0Hz,1H),5.22(s,1H),3.78(t,J=10.1Hz,1H),3.31-3.19(m,2H),3.15(d,J=9.5Hz,1H),2.8 3(s,1H),2.67(t,J=9.4Hz,1H),2.48-2.37(m,11H),2.29(s,3H),2.25-2. 18(m,3H),2.11-2.04(m,2H),1.70(s,3H),1.52(s,3H),1.38-1.31(m,1H). ESI-MS(m / z):[M+H] + =418.26(calcd:418.26).

[0319] Preparation of compound 96:

[0320] In a 5 mL reaction flask, compound 83 (100 mg, 0.28 mmol) was dissolved in 1 mL of dichloromethane, and tetrahydropyrrole (197 mg, 2.80 mmol) was added. The mixture was stirred for 3 h and diluted with ethyl acetate (10 mL). The mixture was washed with water (10 mL × 3), saturated sodium bicarbonate solution (10 mL × 3), and saturated brine (10 mL × 3) in that order. After drying over anhydrous sodium sulfate, the mixture was concentrated under reduced pressure and separated by silica gel column chromatography to obtain compound 96 in a yield of 66%. 1 H NMR(500MHz, CDCl3)δ3.80(t,J=10.2Hz,1H),3.57-3.30(m,4H),3.03-2.97(m,1H),2.91-2.81(m,2H),2.59-2.53( m,2H),2.52-2.37(m,5H),2.28-2.14(m,4H),2.14-1.97(m,2H),1.79-1.69(m,8H),1.61-1.53(m,5H),1.52(s,4H). ESI-MS(m / z):[M+H] + =431.28(calcd:431.28).

[0321] Preparation of compound 97:

[0322] Compound 83 and dimethylamine hydrochloride were used as starting materials. The preparation method was the same as that of compound 96. The yield of compound 97 was 85%. 1H NMR (500MHz, CDCl3) δ3.82(t,J=10.1Hz,1H),3.58-3.30(m,4H),3.05-2.96(m,1H),2.74(dd,J=12.9,4.9Hz,1H),2.60(dd,J=12.9,6.6 Hz,1H),2.53-2.35(m,3H),2.26(s,6H),2.28-2.14(m,4H),2.09-1.97(m,2H),1.74-1.65(m,6H),1.59-1.54(m,6H),1.39-1.27(m,1H). ESI-MS(m / z):[M+H] + =405.27(calcd:405.27).

[0323] Preparation of compound 98:

[0324] Compound 78 (404 mg, 1 mmol) was dissolved in dichloromethane (2 mL) and stirred at room temperature for 2 h. Hydrochloric acid solution was then added dropwise to a pH of 4-5. The mixture was filtered and the resulting solid was washed with dichloromethane to obtain a white solid, the hydrochloride salt of compound 78 (compound 98), in a yield of 86%. 1 H NMR(500MHz,Methanol-d4)δ6.15(d,J=6.9Hz,1H),5.62(d,J=14.3Hz,1H),4. 14-4.08(m,2H),4.05-3.89(m,1H),3.88-3.76(m,2H),3.65-3.42(m,4H),3.2 8-3.16(m,3H),2.89-2.71(m,1H),2.57-2.46(m,1H),2.42-2.28(m,4H),2.25 -2.15(m,1H),2.09-2.00(m,3H),1.76(s,3H),1.57(s,3H),1.44-1.37(m,1H). ESI-MS (m / z): [M+H] + =405.23(calcd:405.23).

[0325] Preparation of compound 99:

[0326] Fumaric acid was used instead of hydrochloric acid to prepare the fumarate salt of compound 78, compound 99, referring to the preparation method of the hydrochloride of compound 78. The yield was 71%. 1H NMR (500MHz, Methanol-d4) δ6.74(s,2H),6.14(d,J=10.2Hz,1H),5.61(d,J=10.5Hz,1H),4.01-3.78(m,5H),3.39-3.36(m,2H),3. 19(d,J=11.5Hz,1H),3.11-3.05(m,2H),2.99-2.85(m,4H),2.82-2.72(m,1H),2.52-2.47(m,1H),2.43-2.24(m,4H),2.22-2.13(m, 1H),2.06-2.00(m,1H),1.75(s,3H),1.56(s,3H),1.43-1.35(m,1H). ESI-MS(m / z):[M+H] + =405.23(calcd:405.23).

[0327] Preparation of compound 100:

[0328] Compound 100 was prepared using compound 96 as the starting material by referring to the preparation method of compound 78 hydrochloride with a yield of 91%. 1 H NMR(500MHz,Methanol-d4)δ4.13(t,J=10.0Hz,1H),3.81-3.72(m,2H),3.68( s,1H),3.64-3.56(m,1H),3.51-3.39(m,3H),3.26-3.19(m,2H),3.09(d,J=8. 7Hz,2H),2.54-2.40(m,2H),2.34-2.06(m,8H),2.04-1.94(m,2H),1.75(s,3H ),1.66-1.60(m,2H),1.55-1.51(m,7H),1.49-1.43(m,1H),1.42-1.26(m,1H). ESI-MS (m / z): [M+H] + =431.28(calcd:431.28).

[0329] Preparation of compound 101:

[0330] Fumaric acid was used instead of hydrochloric acid, and the fumarate compound 101 of 96 was prepared according to the preparation method of the hydrochloride of compound 78, with a yield of 75%. 1H NMR(500MHz,Methanol-d4)δ6.72(s,2H),4.17-4.07(m,2H),3.60-3.40(m,9H),3.11-3.01(m,2H),2.52-2.41(m,2H),2.35 -2.21(m,3H),2.18-2.12(m,5H),2.02-1.93(m,2H),1.74(s,3H),1.66-1.61(m,2H),1.55-1.52(m,7H),1.49-1.39(m,1H). ESI-MS(m / z):[M+H] + =431.28(calcd:431.28).

[0331] Preparation of compound 102:

[0332] Fumaric acid was used instead of hydrochloric acid, and the fumarate compound 102 of 97 was prepared according to the preparation method of the hydrochloride of compound 78, with a yield of 71%. 1 H NMR(500MHz,Methanol-d4)δ6.71(s,2H),4.14-4.09(m,1H),3.46-3.40(m,3H), 3.31-3.27(m,1H),3.12-3.01(m,2H),2.90(s,6H),2.52-2.42(m,2H),2.34-2.22(m,3H),2.14(q,J=10.9,10.1Hz ,1H),2.01-1.93(m,2H),1.75(s,3H),1.66-1.61(m,2H),1.55-1.51(m,7H),1.48-1.40(m,1H),1.40-1.29(m,2H). ESI-MS (m / z): [M+H] + =405.27(calcd:405.27).

[0333] Example 2

[0334] Compound water solubility test:

[0335] Accurately weigh 20 μg each of compounds 1-19, 78, 83, Arg, and MCL, and 10 mg each of compounds 20-42 and 98-102, and dissolve completely in 1 mL of deionized water. Saturated solutions were prepared, filtered, and analyzed by HPLC. Injection volumes were 1 μL, 3 μL, 5 μL, 10 μL, 15 μL, and 20 μL, respectively. Standard curves were constructed for the corresponding compounds.

[0336] An unsaturated solution of the above compound was prepared, ultrasonically assisted to dissolve for 4 h, and placed in a 37°C water bath for 1 h. The obtained unsaturated solution was centrifuged, 30 μL of the supernatant was removed, and diluted with 200 μL of deionized water. After filtering, the sample was analyzed by HPLC. After substituting the relevant data into the above-measured standard curve, the solubility of the test compound can be obtained.

[0337] Table 1. Solubility of compounds 1-42, 78, 83, 98-102 in water

[0338] As shown in Table 1, compared with araguebin and michelin, the water solubility of compounds 1-19, 78, 83 and salt compounds 20-42 and 98-102 was improved, and the water solubility of the salt compounds was increased by at least 100 times.

[0339] Example 3

[0340] Pharmaceutically acceptable salts of guaiacyl sesquiterpene derivatives were selected and their conversion rates to Compound 1 and Compound 83 were evaluated in plasma and HEPES. The results are shown in Tables 2-1, 2-2, 3-1, and 3-2.

[0341] Experimental method: Preparation of HEPES 7.4 solution: 1.6 g of NaCl, 0.074 g of KCl, 0.027 g of Na2HPO4, 0.2 g of glucose and 1 g of 4-hydroxyethylpiperazineethanesulfonic acid (HEPES) solution were dissolved in 90 mL of distilled water, adjusted to pH 7.4 with 0.5 M NaOH, and then diluted to 100 mL with distilled water.

[0342] Plasma preparation: Mouse plasma was placed in an EP tube pre-filled with sodium heparin, centrifuged at 8000 rpm for 10 min at 4°C, and the supernatant was collected.

[0343] Sample analysis: Dissolve 0.6 mg of each compound in 250 μl of deionized water. Add 250 μl of mouse serum or HEPES 7.4 solution to the sample and incubate at 37°C. At various time points, sample 20 μl of the sample into an EP tube, add 60 μl of methanol, vortex mix, and centrifuge at 12,000 rpm for 10 min at 4°C. The supernatant was collected at 1, 2, 4, 8, and 12 h for HPLC analysis. Inject 10 μl of the sample and record the corresponding peak areas. Chromatographic conditions were as follows: Hanbang C18 column (4.6 × 250 mm, 5 μm); mobile phase: acetonitrile: 10 mmol / mL ammonium formate solution (60:40); flow rate: 1.0 mL / min; detection wavelength: 210 nm; column temperature: 30°C.

[0344] Table 2-1. Conversion ratios of compounds 20-42 to compound 1 at different time points in HEPES buffer solution

[0345] Table 2-2. Conversion ratios of compounds 100-102 to compound 83 at different time points in HEPES buffer solution

[0346] Table 3-1. Conversion ratio of compounds 20-42 to compound 1 at different time points in mouse plasma

[0347] Table 3-2. Conversion ratios of compounds 100-102 to compound 83 at different time points in mouse plasma

[0348] Experimental Results: As shown in Table 2-1, in HEPES buffer, the salt form of Compound 20-42 can be converted to the prototype Compound 1. Furthermore, the conversion rate of the salt form of Compound 20-42 to the prototype Compound 1 gradually increased with increasing incubation time. Furthermore, as shown in Table 3-1, in mouse plasma, the salt form of Compound 20-42 can also be converted to the prototype Compound 1. Furthermore, the conversion rate of Compound 20-42 to the prototype Compound 1 also gradually increased with increasing incubation time. These experiments demonstrate that Compound 20-42 can function as a prodrug and be converted to the prototype Compound 1 in both HEPES buffer and mouse plasma.

[0349] Similarly, as shown in Tables 2-2 and 3-2, prodrug compounds 100, 101, and 102 can also be converted into the corresponding parent drug compound 83 in plasma and HEPES.

[0350] Example 4

[0351] Activity test of compound 1 and its dimethyl hydrochloride prodrug 20 and fumarate prodrug 21 against sepsis in mice:

[0352] Experimental reagents: the drug of the present invention, prepared according to Example 1; lipopolysaccharide (LPS), Sigma; sodium carboxymethyl cellulose (CMC-Na), Xilong Chemical Plant, Shantou City, Guangdong Province; RNA isolater total RNA extraction reagent, Hiscript Q RT Supermix for qPCR, Nanjing Novezan Biotechnology Co., Ltd.; AceQ qPCR SYBR Green Master Mix, Shanghai Yisheng Biotechnology Co., Ltd.

[0353] Experimental animals: C57BL / 6 male mice, 6-8 weeks old, weighing 18-20 g, provided by Shanghai Slake Animal Breeding Center, production license number: SCXK(Su)2019-002. Animals were fed a standard pelleted diet with free access to food and water at room temperature of 22 ± 2°C and humidity of 45 ± 10%. After acclimation for 7 days, they were used in experiments.

[0354] Experimental methods: establishment of mouse sepsis model and grouped drug administration

[0355] Mice were randomly divided into groups, with one group designated as the normal group and the remaining groups designated as the model group, compound 1 (0.1, 1 mg / kg), compound 20 hydrochloride (1 mg / kg), and compound 21 fumarate (0.1, 1 mg / kg), with six mice in each group. The normal group received an intraperitoneal injection of PBS, while the remaining mice received an intraperitoneal injection of LPS (10 mg / kg) to establish a mouse sepsis model. One hour before model establishment, mice were gavaged with compound 1 (0.1, 1 mg / kg), compound 20 hydrochloride (1 mg / kg), and compound 21 fumarate (0.1, 1 mg / kg) for five consecutive days. Mice in the normal and model groups were gavaged with an equal volume of the vehicle, 0.5% CMC-Na.

[0356] Specimen collection: One hour after the last administration, the mice were killed by cervical dislocation, the abdominal cavity was opened, and the lungs, liver, and kidneys were removed. The mice were rinsed twice with pre-cooled PBS, and the lung tissue, left lobe of the liver, and kidney tissue of the mice were cut and fixed with 4% paraformaldehyde for 24-48 hours. The remaining tissues were frozen at -70℃ for later use.

[0357] Q-PCR analysis

[0358] (1) Extraction of total RNA

[0359] Accurately weigh 20 mg of kidney, lung, and liver tissue, rinse with pre-chilled PBS, cut into small pieces with ophthalmic surgical scissors, place in a glass homogenizer, and grind in 1 mL of Trizol reagent to prepare a tissue homogenate. Subsequently, transfer the homogenate to an RNase-free 1.5 mL EP tube and lyse on ice for 10 min. Add 200 μL of chloroform, shake vigorously, let stand on ice for 3-5 min, and centrifuge at 12,000 rpm at 4°C for 15 min. Carefully aspirate the supernatant into a new RNase-free 1.5 mL EP tube, add an equal volume of pre-chilled isopropanol, let stand on ice for 30 min, centrifuge at 12,000 rpm at 4°C for 10 min, and discard the supernatant. Wash the pellet with 75% ethanol-DEPC solution and invert on a flat sheet to air dry. Total RNA is then obtained. Subsequently, dissolve the pellet in 10 μL of DEPC-treated water. Take 1 μL of the total RNA solution, dilute it 100-fold, and measure the OD values ​​at 260 nm and 280 nm using a full-wavelength microplate reader. When the OD260 / OD280 ratio is between 1.8 and 2.0, the extracted RNA is of high purity and can be used for subsequent experiments.

[0360] (2) cDNA synthesis

[0361] According to the method described in the kit instructions, 20 μL reverse transcription reaction system was used for subsequent experiments:

[0362] Use a pipette to gently mix the above reagents. The reaction conditions are as follows:

[0363] The synthesized cDNA was stored at -80°C or used immediately in subsequent Q-PCR reactions.

[0364] (3) Primer design

[0365] Sequences were designed and primers were synthesized based on the mouse nucleotide sequence in GenBank. The detailed information is as follows:

[0366] (4) PCR amplification

[0367] According to the method described in the kit instructions, a 20 μL reaction system was used for subsequent experiments:

[0368] After adding the above reagents to the micro-reaction tube, seal it with sealing film, place it in the Q-PCR instrument, set the parameters according to the following reaction conditions, and perform the amplification experiment. The reaction conditions are as follows:

[0369] Record the threshold cycle (Ct) value of each group, using 2 -ΔΔCtThe expression level of the control gene Gapdh was set to 1 to correct the expression level of the target gene.

[0370] Data statistics: All data are expressed as mean ± SEM. Analysis of variance was used to analyze the significance of differences. Significant differences were further compared between groups using one-way ANOVA and Dunnett's test. A P value of less than 0.05 was considered significant.

[0371] Experimental results

[0372] Effects on the inflammatory response of lung in septic mice

[0373] As shown in Table 4, compared with the control group, the mRNA expression levels of inflammatory factors (Tnf, Il6, and Il1) in the lung tissue of septic mice were significantly increased, indicating a significant inflammatory response in the lungs of septic mice. After intervention with compound 1 (1 mg / kg), hydrochloride 20 (1 mg / kg), and fumarate 21 (1 mg / kg), Tnf, Il6, and Il1 mRNA expression in mouse lung tissue was significantly downregulated. Notably, at the same dose, hydrochloride 20 was more active than compound 1 and fumarate 21. These results demonstrate that compound 1 and its prodrug salt can inhibit the expression of inflammatory factors and improve lung damage in septic mice.

[0374] Table 4. Pneumonia factor mRNA expression

[0375] Note: Compared with the Normal group ## P<0.01; compared with LPS group * P<0.05, ** P<0.01.

[0376] Effects of septic mice on hepatic inflammatory response

[0377] As shown in Table 5, compared with the control group, the mRNA expression levels of inflammatory factors (Tnf, Il6, and Il1) in the liver tissue of septic mice were significantly increased, indicating a significant inflammatory response in the liver of septic mice. After intervention with compound 1 (1 mg / kg), hydrochloride 20 (1 mg / kg), and fumarate 21 (1 mg / kg), the expression of Tnf, Il6, and Il1 mRNA in mouse liver tissue was significantly downregulated. Notably, at the same dose, hydrochloride 20 was significantly more active than compound 1 and fumarate 21. These results demonstrate that compound 1 and its prodrug salt can inhibit the expression of inflammatory factors and improve liver damage in septic mice.

[0378] Table 5. mRNA expression of hepatic inflammatory factors

[0379] Note: Compared with the Normal group ## P<0.01; compared with LPS group ** P<0.01.

[0380] Effects of septic mice on renal inflammatory response

[0381] As shown in Table 6, compared with the control group, the mRNA expression levels of inflammatory factors (Tnf, Il6, and Il1) in the kidney tissue of septic mice were significantly increased, indicating a significant inflammatory response in the kidneys of septic mice. After intervention with compound 1 (1 mg / kg), hydrochloride 20 (1 mg / kg), and fumarate 21 (1 mg / kg), the expression of Tnf, Il6, and Il1 mRNA in the mouse kidney tissue was significantly downregulated. Notably, at the same dose, the activity of hydrochloride 20 was significantly stronger than that of compound 1 and fumarate 21. These results demonstrate that compound 1 and its prodrug salt can inhibit the expression of inflammatory factors and improve renal damage in septic mice.

[0382] Table 6. Renal inflammatory factor mRNA expression

[0383] Note: Compared with the Normal group ## P<0.01; compared with LPS group * P<0.05, ** P<0.01.

[0384] Example 5

[0385] Anti-acute lung injury activity test of compound 1 and its dimethyl hydrochloride prodrug 20 and fumarate prodrug 21:

[0386] Experimental reagents: All drugs used were prepared according to Example 2; LPS was purchased from Sigma, USA; dexamethasone (Dex) was purchased from Sigma, USA; ELISA kits were purchased from Dakoway Biotech Co., Ltd.

[0387] Experimental animals: Male C57BL / 6 mice, 6-8 weeks old, weighing 18-20 g, were purchased from Jiangsu Jicui Pharmaceutical Technology Co., Ltd. [Production License No.: SCXK(Su)2018-0008]. Animals were housed at 22 ± 2°C and 45 ± 10% humidity with free access to food and water. After acclimation for 7 days, they were used in subsequent experiments.

[0388] Experimental methods: Establishment of acute lung injury model in mice and grouped drug administration

[0389] Treatment of acute lung injury with oral administration of 1, 20, and 21: Male C57BL / 6J mice, 6-8 weeks old and weighing 18-20 g, were randomly divided into a normal group, a model group, a group receiving 1, 20, and 21 (0.1 and 1 mg / kg), and a positive control drug, Dex (5 mg / kg), with 8 mice per group. Except for the normal group, which received an oral administration of PBS, the remaining mice received an intraperitoneal injection of LPS (7.5 mg / kg) to establish a mouse acute lung injury model. Three days prior to modeling, compound 1 (0.1 and 1 mg / kg), 20 hydrochloride (0.1 and 1 mg / kg), 21 fumarate (0.1 and 1 mg / kg), and the positive control drug, Dex (5 mg / kg), were administered orally. On the third day, LPS (7.5 mg / kg) was injected intraperitoneally half an hour after drug administration. Lung tissue was collected 12 hours after modeling, and the effects of 1, 20, and 21 on the expression of inflammatory factors in the lungs of mice with acute lung injury were assessed by ELISA.

[0390] Specimen collection: 12 hours after the last administration, the mice were killed by cervical dislocation, the abdominal cavity was opened, the lungs were removed, and the lungs were rinsed twice with pre-cooled PBS. The lung tissues of the mice were cut and fixed with 4% paraformaldehyde for 24-48 hours. The remaining tissues were frozen at -70℃ for later use.

[0391] ELISA analysis: extraction of total protein

[0392] Accurately weigh 20 mg of lung tissue, rinse with pre-cooled PBS solution, cut into small pieces with ophthalmic surgical scissors, place in a glass homogenizer, add 180 μL PBS (containing 1 mM PMSF), homogenize at 60 Hz for 180 s, centrifuge at 3000 rpm / min, 4°C for 20 min, take 1 μL of supernatant, measure the protein concentration by Nanodrop, and level it for use in subsequent experiments.

[0393] Data: All data are presented as means ± SEM. Statistical differences between groups were analyzed using one-way ANOVA and t-tests in SPSS software. P values ​​less than 0.05 were considered significant.

[0394] Experimental results

[0395] Effects of oral administration of 1, 20, and 21 mg of therapeutic drugs on acute lung injury in mice

[0396] As shown in Table 7, compared with the control group, the protein expression levels of inflammatory factors (IL-1β, IL-6) in the lung tissue of mice were significantly increased, indicating a significant inflammatory response in the mouse lungs. After intervention with the original drug 1 (0.1, 1 mg / kg), the hydrochloride salt 20 (0.1, 1 mg / kg), and the fumarate salt 21 (0.1, 1 mg / kg), the expression of IL-1β and IL-6 proteins in the mouse lung tissue was significantly downregulated. Notably, at the same dose, the hydrochloride salt 20 was more active than the compound 1 and the fumarate salt 21. These results demonstrate that compound 1 and its prodrug salt can inhibit the expression of inflammatory factors and improve acute lung injury in mice.

[0397] Table 7. Expression of inflammatory factors (IL-1β and IL-6) in the serum of mice treated with intraperitoneal injection for 1, 20, and 21 days

[0398] Note: *p<0.05, **p<0.01, ***p<0.001 (compared with LPS group)

[0399] Example 6

[0400] Anti-hepatic injury activity test of compound 1 and its dimethyl hydrochloride prodrug 20 and fumarate prodrug 21:

[0401] Experimental reagents: the drug of the present invention, prepared according to Example 2; silybin, Shanghai Yuanye Biotechnology Co., Ltd.; olive oil, Beijing Kelipecui Olive Oil Development Center; analytical grade carbon tetrachloride (CCl4), Shantou Xilong Chemical Plant; sodium carboxymethyl cellulose (CMC-Na), Xilong Chemical Plant, Shantou City, Guangdong Province; RNA isolater total RNA extraction reagent, Hiscript Q RT Supermix for qPCR, Nanjing Novezan Biotechnology Co., Ltd.; AceQ qPCR SYBR Green Master Mix, Shanghai Yisheng Biotechnology Co., Ltd.; aspartate aminotransferase (AST / GOT) colorimetric test kit, alanine aminotransferase (ALT / GPT) colorimetric test kit, Wuhan Yilai Ruite Biotechnology Co., Ltd.

[0402] Experimental animals: C57BL / 6 male mice, 6-8 weeks old, weighing 18-20 g, provided by Shanghai Slake Animal Breeding Center, production license number: SCXK(Su)2019-002. Animals were fed a standard pelleted diet with free access to food and water at room temperature of 22 ± 2°C and humidity of 45 ± 10%. After acclimation for 7 days, they were used in experiments.

[0403] Experimental methods: Establishment of acute liver injury model in mice and grouped drug administration

[0404] Mice were randomly divided into groups: one normal group, the other groups designated as the model group, the compound 1, the hydrochloride salt of 20, the fumarate salt of 21, and the silibinin group, with six mice in each group. Except for the normal group, which received an injection of 10 mL of olive oil per kg, the remaining mice were intraperitoneally injected with 0.5 mL of CCl₄ + 9.5 mL of olive oil per kg of body weight, for a CCl₄ to olive oil ratio of 1:19. Starting on day 1 of modeling, the original compound 1, the hydrochloride salt of 20, and the fumarate salt of 21 were administered orally for three consecutive days. Mice in the normal and model groups were also administered an equal volume of the vehicle, 0.5% CMC-Na, by gavage.

[0405] Specimen Collection: One hour after the last dose, blood was collected from the fundus venous plexus, allowed to stand at room temperature for 2 hours, and centrifuged at 3000 rpm for 20 minutes. Serum was collected and aliquoted, then frozen at -70°C until further use. Following blood collection, mice were sacrificed by cervical dislocation. The peritoneal cavity was opened, and the liver was removed. The liver was rinsed twice with pre-chilled PBS, and the left lobe of the liver was excised and fixed with 4% paraformaldehyde for 24-48 hours. The remaining tissue was frozen at -70°C until further use.

[0406] Serum AST and ALT Assays: 10 μL of each sample was collected for testing. Mouse serum AST and ALT levels were measured according to the manufacturer's instructions. Absorbance was measured at 510 nm using a microplate reader. Sample concentrations (U / L) were calculated using a standard curve.

[0407] Q-PCR analysis: The method for total RNA extraction from tissues, cDNA synthesis, and PCR amplification were the same as in Example 4. The additional primer information is as follows:

[0408] Data statistics: All data are expressed as mean ± SEM. Analysis of variance was used to analyze the significance of differences. Significant differences were further compared between groups using one-way ANOVA and Dunnett's test. A P value of less than 0.05 was considered significant.

[0409] Experimental results

[0410] Effects on serum ALT and AST levels

[0411] Changes in serum ALT and AST levels are important indicators of liver injury. To determine whether CCl₄ indeed induced acute liver injury in mice, this study first measured serum ALT and AST levels in model mice. The results, shown in Table 8, showed significant increases in serum ALT and AST levels in mice treated with CCl₄ compared to the normal olive oil group. This suggests that the mouse model of acute liver injury was successfully established. Furthermore, oral administration of compound 1 (1 mg / kg), hydrochloride 20 (1 mg / kg), and fumarate 21 (1 mg / kg) significantly reduced serum ALT and AST levels in mice. Notably, at equivalent doses, hydrochloride 20 exhibited greater activity than the original compound 1 and fumarate 21.

[0412] Table 8. ALT and AST levels

[0413] Note: Compared with the Normal group ## P<0.01; compared with CCl4 group * P<0.05, ** P<0.01.

[0414] Effects on mRNA expression of liver fibrosis-related factors

[0415] The effects of compound 1, hydrochloride 20 and fumarate 21 on liver proinflammatory factors in mice were evaluated by detecting changes in the mRNA expression levels of fibrosis-related factors (Acta2, Col1a1, Tgfb1) in the liver tissue of each group of mice. As shown in Table 9, compared with the Normal group, the mRNA levels of Acta2, Col1a1, and Tgfb1 in the liver tissue were significantly increased 72 hours after intraperitoneal injection of CCl4, and their expression was significantly reduced in the treatment groups of compound 1 (1 mg / kg), hydrochloride 20 (1 mg / kg) and fumarate 21 (1 mg / kg). It is worth noting that the activity of hydrochloride 20 was stronger than that of compound 1 and fumarate 21 at the same dose. The above results indicate that compound 1 and its prodrug salt can inhibit CCl4-induced liver damage to a certain extent.

[0416] Table 9. mRNA expression of liver fibrosis-related factors

[0417] Note: Compared with the Normal group ## P<0.01; compared with CCl4 group ** P<0.01.

[0418] Example 7

[0419] Effects of compound 1 and its dimethyl hydrochloride prodrug 20 and fumarate prodrug 21 on renal injury induced by folic acid and unilateral ureteral occlusion (UUO) in mice:

[0420] Experimental reagents: the drug of the present invention, prepared according to Example 2; folic acid (FA), MP Biomedicals; sodium carboxymethyl cellulose (CMC-Na), Xilong Chemical Plant, Shantou City, Guangdong Province; RNA isolater total RNA extraction reagent, Hiscript Q RT Supermix for qPCR, Nanjing Novezan Biotechnology Co., Ltd.; AceQ qPCR SYBR Green Master Mix, Shanghai Yisheng Biotechnology Co., Ltd.; urea colorimetric test kit, creatinine colorimetric test kit, Wuhan Yilai Ruite Biotechnology Co., Ltd.

[0421] Experimental animals: C57BL / 6 male mice, 6-8 weeks old, weighing 18-20 g, provided by Shanghai Slake Animal Breeding Center, production license number: SCXK(Su)2019-002. Animals were fed a standard pelleted diet with free access to food and water at room temperature of 22 ± 2°C and humidity of 45 ± 10%. After acclimation for 7 days, they were used in experiments.

[0422] Experimental methods: Establishment of mouse renal injury model and grouped drug administration

[0423] Acute Kidney Injury: Mice were randomly divided into groups: one normal group, the other groups designated as the model group, and the other groups designated as the compound 1 (0.1 and 1 mg / kg), the hydrochloride of 20 (1 mg / kg), and the fumarate of 21 (0.1 and 1 mg / kg), with six mice in each group. Except for the normal group, the other mice were intraperitoneally injected with folic acid (250 mg / kg) dissolved in 0.9% NaHCO₃ solution to establish an acute kidney injury model. Compound 1 (0.1 and 1 mg / kg), the hydrochloride of 20 (1 mg / kg), and the fumarate of 21 (0.1 and 1 mg / kg) were administered orally 1 hour before model establishment for three consecutive days. Mice in the normal and model groups were also orally administered with an equal volume of the vehicle, 0.5% CMC-Na.

[0424] Chronic kidney injury: Mice were randomly divided into five groups. One group was randomly selected as the normal group, and the remaining four groups were divided into model groups, compound 1 (0.1, 1 mg / kg), compound 20 hydrochloride (1 mg / kg), and compound 21 fumarate (0.1, 1 mg / kg) groups, with 6 mice in each group. Except for the normal group mice, which underwent sham surgery (i.e., only the left ureter was exposed without other procedures), the remaining mice were treated with unilateral ureteral ligation to establish a mouse chronic kidney injury model as follows: the abdominal skin was disinfected with iodine and then with alcohol, and the abdominal skin was incised and separated layer by layer. The left ureter was fully exposed and separated, and then double-ligated at the proximal bladder end with digestible sutures, followed by suture. Compound 1 (0.1, 1 mg / kg), compound 20 hydrochloride (1 mg / kg), and compound 21 fumarate (0.1, 1 mg / kg) were administered orally on the first day of model establishment and continued for 14 consecutive days. The mice in the normal group and the model group were gavaged with an equal volume of 0.5% CMC-Na solvent.

[0425] Specimen Collection: One hour after the last dose, blood was collected from the fundus venous plexus, allowed to stand at room temperature for 2 hours, and centrifuged at 3000 rpm for 20 minutes. Serum was collected and aliquoted, then frozen at -70°C until further use. Following blood collection, mice were sacrificed by cervical dislocation. The abdominal cavity was opened, and the kidneys were removed. The perirenal fat and capsule were removed. After rinsing twice with pre-chilled PBS, the kidneys were fixed with 4% paraformaldehyde for 24-48 hours, embedded in paraffin, and stained with hematoxylin and eosin. The remaining tissue was frozen at -70°C until further use.

[0426] Serum creatinine (Scr) and urea (BUN) levels were determined: 10 μL of serum was collected from each sample for creatinine and urea level determination. Samples were added in the order specified in the kit instructions. After incubation at 37°C, absorbance was measured using a microplate reader. The levels of each assay were calculated using the standard curve.

[0427] Q-PCR analysis: Total RNA extraction from animal tissues, cDNA synthesis, and PCR amplification were performed as in the previous example. The primer addition information is shown in Table 10:

[0428] Table 10. Q-PCR primer information

[0429] Experimental results

[0430] Effects on serum creatinine and urea levels

[0431] As shown in Tables 11 and 12, compared with the Normal group, the levels of urea and creatinine in the serum of mice were significantly increased 48 hours after folic acid administration, while the levels of urea and creatinine in the groups treated with compound 1 (1 mg / kg), hydrochloride 20 (1 mg / kg), and fumarate 21 (1 mg / kg) were significantly lower than those in the model group, with statistically significant differences (P<0.05). It is worth noting that the activity of hydrochloride 20 was stronger than that of compound 1 and fumarate 21 at the same dose. These results indicate that compound 1 and its prodrug salt have a good protective effect against acute and chronic renal injury induced by folic acid or unilateral ureteral ligation.

[0432] Table 11. Serum Creatinine and Urea Levels - Acute Model

[0433] Note: Compared with the Normal group ## P<0.01; compared with FA group * P<0.05, ** P<0.01.

[0434] Table 12. Serum Creatinine and Urea Levels - Chronic Model

[0435] Note: Compared with the Normal group ## P<0.01; compared with the UUO group * P<0.05, ** P<0.01.

[0436] Effects on mRNA expression of renal injury markers

[0437] The effects of the original drug 1, hydrochloride 20, and fumarate 21 on the renal function of mice were evaluated by detecting the changes in the mRNA expression levels of renal injury markers (Ngal, Havcr1) in the renal tissue of each group of mice. As shown in Tables 13 and 14, compared with the Normal group, the mRNA levels of Ngal and Havcr1 in the renal tissue of mice were significantly increased after 48 hours of folic acid administration or unilateral ureteral ligation, and the mRNA levels of Ngal and Havcr1 in the treatment groups of compound 1 (1 mg / kg), hydrochloride 20 (1 mg / kg), and fumarate 21 (1 mg / kg) were significantly reduced. It is worth noting that the activity of hydrochloride 20 was stronger than that of compound 1 and fumarate 21 at the same dose. The above results show that compound 1 and its prodrug salt can inhibit the increase of renal injury markers caused by folic acid or unilateral ureteral ligation.

[0438] Table 13. Renal Injury Marker mRNA Expression - Acute Model

[0439] Note: Compared with the Normal group## P<0.01; compared with FA group * P<0.05, ** P<0.01.

[0440] Table 14. Renal injury marker mRNA expression - chronic model

[0441] Note: Compared with the Normal group ## P<0.01; compared with the UUO group * P<0.05, ** P<0.01.

[0442] Effects on the mRNA expression of inflammatory factors in renal tissue

[0443] The effects of compound 1, hydrochloride 20, and fumarate 21 on renal proinflammatory factors in mice were evaluated by detecting changes in the mRNA expression levels of inflammatory factors (Tnf, Il6, Il1) in the renal tissue of each group of mice. As shown in Tables 15 and 16, compared with the Normal group, the levels of Tnf, Il6, and Il1 mRNA in renal tissue were significantly increased 48 hours after folic acid administration or unilateral ureteral ligation, while the expression of these mRNAs was significantly reduced in the treatment groups with compound 1 (1 mg / kg), hydrochloride 20 (1 mg / kg), and fumarate 21 (1 mg / kg). It is worth noting that at the same dose, hydrochloride 20 was more active than compound 1 and fumarate 21. These results indicate that compound 1 and its prodrug salt can reduce the renal inflammatory response induced by folic acid or unilateral ureteral ligation to a certain extent.

[0444] Table 15. Renal inflammatory factor mRNA expression - acute model

[0445] Note: Compared with the Normal group ## P<0.01; compared with FA group * P<0.05, ** P<0.01.

[0446] Table 16. Renal inflammatory factor mRNA expression - chronic model

[0447] Note: Compared with the Normal group ## P<0.01; compared with the UUO group * P<0.05, ** P<0.01.

[0448] Effects on the mRNA expression of renal fibrosis-related factors

[0449] The effects of compound 1, hydrochloride 20, and fumarate 21 on renal fibrosis in mice were evaluated by measuring changes in mRNA expression levels of fibrosis-related factors (α-Sma, Col1a1, Tgfb1) in the renal tissue of each group of mice. As shown in Tables 17 and 18, compared with the Normal group, Acta2, Col1a1, and Tgfb1 mRNA levels were significantly increased in the renal tissue after 48 hours of folic acid administration or unilateral ureteral ligation. Compound 1 (1 mg / kg), hydrochloride 20 (1 mg / kg), and fumarate 21 (1 mg / kg) treatment groups significantly reduced their expression. Notably, at the same dose, hydrochloride 20 was more active than compound 1 and fumarate 21. These results indicate that compound 1 and its prodrug salt can reduce folic acid-induced renal fibrosis to a certain extent.

[0450] Table 17. Renal fibrosis-related factor mRNA expression - acute model

[0451] Note: Compared with the Normal group ## P<0.01; compared with folic acid group * P<0.05, ** P<0.01.

[0452] Table 18. mRNA expression of renal fibrosis-related factors - chronic model

[0453] Note: Compared with the Normal group ## P<0.01; compared with the UUO group * P<0.05, ** P<0.01.

[0454] Example 8

[0455] The therapeutic effect of compound 20 in lupus nephritis:

[0456] Experimental reagents: MCC950 (NLRP3 inhibitor), Shanghai Taoshu Biotechnology Co., Ltd.; sodium carboxymethyl cellulose (CMC-Na), Xilong Chemical Plant, Shantou City, Guangdong Province; RNA isolater total RNA extraction reagent, Hiscript QRT Supermix for qPCR, Nanjing Novezan Biotechnology Co., Ltd.; urea colorimetric test kit, creatinine colorimetric test kit, Nanjing Jiancheng Biotechnology Co., Ltd.; urine Kim-1 and β-MG content determination ELISA kit, Abcam.

[0457] Experimental animals: MRL / MpJ and MRL / lpr female mice, 10 weeks old, were purchased from Jiangsu Jicui Pharmaceutical Kang Biotechnology Co., Ltd., production license number: SCXK(Su)2023-0009. Animals were fed a standard pelleted diet with free access to food and water at room temperature of 22 ± 2°C and humidity of 45 ± 10%. After acclimation for 7 days, they were used in experiments.

[0458] Experimental methods

[0459] (1) Grouping and administration of mice:

[0460] The normal group (MRL / MpJ), the model group (MRL / lpr), the positive drug MCC950 (30 mg / kg) group, and the compound 20 (30 mg / kg) group, with 6 mice in each group, were administered by gavage for 8 consecutive weeks. Mice in the normal and model groups were gavaged with an equal volume of the vehicle, 0.5% CMC-Na.

[0461] Specimen Collection: One hour after the last dose, blood was collected from the eyeballs. The blood was allowed to stand at room temperature for 2 hours and then centrifuged at 3000 rpm for 20 minutes. The serum was collected and aliquoted, then frozen at -70°C until further use. After blood collection, the mice were sacrificed by cervical dislocation. The abdominal cavity was opened, the kidneys were removed, and the perirenal fat and capsule were removed. The kidneys were rinsed twice with pre-chilled PBS and fixed with 4% paraformaldehyde for 24-48 hours. The remaining tissue was frozen at -70°C until further use.

[0462] Serum creatinine (Scr) and urea (BUN) levels were determined: 10 μL of serum was collected from each sample for creatinine and urea level determination. Samples were added in the order specified in the kit instructions. After incubation at 37°C, absorbance was measured using a microplate reader. The levels of each assay were calculated using the standard curve.

[0463] Q-PCR analysis: Total RNA extraction from animal tissues, cDNA synthesis, and PCR amplification were performed as in the previous example. The primers were added as follows:

[0464] Table 19. Q-PCR primer information

[0465] Experimental results

[0466] Effect on urinary protein: As shown in Table 20, compared with the MpJ group mice, the 24-hour urinary protein level of lpr mice was significantly increased, while the 24-hour urinary protein content of the compound 20 (30 mg / kg) treatment group was significantly lower than that of the model group, and the proteinuria reduction effect was better than that of the MCC950 group, and the difference was statistically significant (P<0.05).

[0467] Table 20. Urine protein content

[0468] Note: Compared with MpJ group ##P<0.01; compared with the lpr group * P<0.05, ** P<0.01.

[0469] Effects on Serum Renal Function Indicators: As shown in Table 21, compared with the MpJ group, the serum urea and creatinine levels of lpr mice were significantly elevated. However, the urea and creatinine levels in the Compound 20 (30 mg / kg) treatment group were significantly lower than those in the model group, and the overall effect was superior to that of the MCC950 group, with statistically significant differences (P < 0.05). This indicates that Compound 20 has a good protective effect on the renal function of mice with lupus nephritis.

[0470] Table 21. Serum creatinine and urea levels

[0471] Note: Compared with MpJ group ## P<0.01; compared with the lpr group * P<0.05, ** P<0.01.

[0472] Effects on Urinary Indicators of Renal Injury: The effects of the hydrochloride compound 20 on mouse renal function were evaluated by measuring changes in the expression levels of Kim-1 and β-MG in the urine of each group of mice. As shown in Table 22, compared with the control group, compound 20 inhibited the elevation of kidney injury markers in the urine of lupus mice, and the overall effect was superior to that of the MCC950 group, with statistically significant differences (P < 0.05).

[0473] Table 22. Urinary levels of kidney injury markers

[0474] Note: Compared with MpJ group ## P<0.01; compared with the lpr group * P<0.05, ** P<0.01.

[0475] Effect on Renal Inflammatory Factor mRNA Expression: The effect of Compound 20 on renal pro-inflammatory factors in mice was evaluated by measuring changes in the mRNA expression levels of inflammatory factors (Tnf, Il6, and Il1) in the renal tissue of each group of mice. As shown in Table 23, compared with the control group (MpJ mice), the mRNA levels of Tnf, Il1, and Il6 in the renal tissue of the lupus nephritis model lpr mice were significantly increased. Compound 20 (30 mg / kg) treatment significantly reduced their expression, with statistically significant differences (P < 0.05). This suggests that Compound 20 can reduce renal inflammatory responses in mice with lupus nephritis to a certain extent.

[0476] Table 23. Renal inflammatory factor mRNA expression

[0477] Note: Compared with MpJ group ## P<0.01; compared with the lpr group * P<0.05, ** P<0.01.

[0478] Example 9

[0479] The therapeutic effect of compound 20 in diabetic nephropathy:

[0480] Experimental reagents: sodium carboxymethyl cellulose (CMC-Na), Xilong Chemical Plant, Shantou City, Guangdong Province; streptozocin (STZ), Sigma-Aldrich (Shanghai) Trading Co., Ltd.; RNA isolater total RNA extraction reagent, Hiscript Q RT Supermix for qPCR, Nanjing Novezan Biotechnology Co., Ltd.; urea colorimetric test kit, creatinine colorimetric test kit, Nanjing Jiancheng Biotechnology Co., Ltd.; urine Kim-1 and β-MG content determination ELISA kit, Abcam.

[0481] Experimental animals: Male C57 / BL 6J mice, 10 weeks old, purchased from Jiangsu Jicui Pharmaceutical Kang Biotechnology Co., Ltd., production license number: SCXK(Su)2023-0009. Animals were fed a standard pelleted diet with free access to food and water at room temperature of 22 ± 2°C and humidity of 45 ± 10%. After acclimation for 7 days, they were used in experiments.

[0482] Experimental methods

[0483] (1) Mouse modeling and grouping and drug administration:

[0484] Mice were selected for model development after intraperitoneal injection of streptozotocin (50 mg / kg) for 5 consecutive days. Fasting blood glucose levels were greater than 16.7 mmol / L 72 hours later. These mice were divided into a model group (DKD), a compound 20 (1 mg / kg) group, and a compound 20 (10 mg / kg) group, with 6 mice in each group. The model mice were fed a high-fat diet (HFD) containing 60% fat for 8 weeks. After 8 weeks, oral administration of the compound was initiated. Mice in the control group (Ctrl) and the model group (DKD) were given an equal volume of the vehicle, 0.5% CMC-Na, by oral administration for 8 consecutive weeks.

[0485] Specimen Collection: One hour after the last dose, blood was collected from the eyeballs. The blood was allowed to stand at room temperature for 2 hours and then centrifuged at 3000 rpm for 20 minutes. The serum was collected and aliquoted, then frozen at -70°C until further use. After blood collection, the mice were sacrificed by cervical dislocation. The abdominal cavity was opened, the kidneys were removed, and the perirenal fat and capsule were stripped. The tissue was then frozen at -70°C until further use.

[0486] Serum creatinine (Scr) and urea (BUN) levels were determined: 10 μL of serum was collected from each sample for creatinine and urea level determination. Samples were added in the order specified in the kit instructions. After incubation at 37°C, absorbance was measured using a microplate reader. The levels of each assay were calculated using the standard curve.

[0487] Q-PCR analysis: Total RNA extraction from animal tissues, cDNA synthesis, PCR amplification, and primer information are as in the previous example. Additional primer information is as follows:

[0488] Table 24. Q-PCR primer addition information

[0489] Experimental results:

[0490] Effects on Key Indicators of Renal Function: As shown in Table 25, compared with the Ctrl group, the serum urea and creatinine levels of DKD mice were significantly elevated, while the urea and creatinine levels in the Compound 20 (1 mg / kg) and Compound 20 (10 mg / kg) treatment groups were significantly lower than those in the model group in a dose-dependent manner, with statistically significant differences (P < 0.05). This indicates that Compound 20 has a good protective effect on the renal function of DKD mice.

[0491] Table 25. Serum creatinine and urea levels

[0492] Note: Compared with the Ctrl group ## P<0.01; compared with DKD group ** P<0.01.

[0493] Effect on mRNA expression of key factors of renal injury: The effect of compound 20 on renal proinflammatory factors in mice was evaluated by detecting changes in the mRNA expression levels of renal injury factors (Kim-1, Ngal) in the renal tissue of each group of mice. As shown in Table 26, compared with the control group Ctrl mice, the mRNA levels of Kim-1 and Ngal in the renal tissue of the DKD model lpr mice were significantly increased. The compound 20 (1 mg / kg) and compound 20 (10 mg / kg) treatment groups significantly reduced their expression in a dose-dependent manner, and the differences were statistically significant (P < 0.05). It can be seen that compound 20 has the effect of improving renal injury in DKD mice.

[0494] Table 26. mRNA expression levels of key factors in renal injury

[0495] Note: Compared with the Ctrl group ## P<0.01; compared with DKD group ** P<0.01.

[0496] Effect on mRNA expression of inflammatory factors in renal tissue: The effect of compound 20 on the expression of renal pro-inflammatory factors in mice was evaluated by detecting the changes in the mRNA expression levels of inflammatory factors (Mcp-1, Tnf-α) in the renal tissue of each group of mice. As shown in Table 27, compared with the control group Ctrl mice, the mRNA levels of Mcp-1 and Tnf-α in the renal tissue of DKD model mice were significantly increased. The compound 20 (1 mg / kg) and compound 20 (10 mg / kg) treatment groups significantly reduced their expression in a dose-dependent manner, and the differences were statistically significant (P < 0.05). It can be seen that compound 20 can reduce the renal inflammatory response of DKD mice to a certain extent.

[0497] Table 27. mRNA expression levels of inflammatory factors in renal tissue

[0498] Note: Compared with the Ctrl group ## P<0.01; compared with DKD group ** P<0.01.

[0499] Example 10

[0500] Activity testing of compound 1 in treating atopic dermatitis

[0501] 1. Effect of Compound 1 Ointment on Atopic Dermatitis in Mice

[0502] Preparation of blank ointment:

[0503] (1) Preparation of oil phase: Weigh white vaseline, light liquid paraffin, glyceryl monostearate, ethylparaben, and cetyl alcohol, heat to 65°C, stir to dissolve, slowly add to the dissolved API, continue stirring, mix evenly, and set aside;

[0504] (2) Preparation of aqueous phase: Weigh purified water and Tween 80, heat to 70°C, stir and dissolve;

[0505] (3) Emulsification: slowly add the water phase to the oil phase, maintain at 65°C, homogenize, and continue stirring for 30 min;

[0506] (3) Cool down, stop heating, continue stirring, gradually cool down to room temperature and cool into paste, and fill.

[0507] Preparation of 0.01%, 0.1%, 1% Compound 1 ointment and 1% dexamethasone ointment

[0508] 0.005 g, 0.05 g, and 0.5 g of compound 1 and 0.5 g of dexamethasone were respectively weighed and dissolved in a small amount of propylene glycol. The mixture was then placed in the above-mentioned condensed blank ointment base. Finally, water was added to 10 g and the mixture was ground in a mortar to obtain 0.01%, 0.1%, and 1% compound 1 ointments and 1% dexamethasone ointment.

[0509] Establishment and administration of MC903-induced atopic dermatitis (AD) mouse model

[0510] Establishment and Dosing of Mouse Ear AD Model: 36 mice were randomly divided into a normal group (Normal), a model group (MC903), and 0.01%, 0.1%, and 1% compound 1, and dexamethasone (Dex), with 6 mice in each group. All groups except the normal group were treated with an AD model: 20 μL of MC903 (also known as calcipotriol, purchased from Shanghai Yuanye Biotechnology Co., Ltd., which can be used to induce atopic dermatitis) solution was applied to each ear of the mice once daily for 14 consecutive days to induce atopic dermatitis in mice. Seven days after modeling, 20 mg of 0.01%, 0.1%, and 1% compound 1 and dexamethasone ointment were evenly applied to the corresponding mice daily. Mice in the model and normal groups were treated with blank ointment daily. Changes in ear thickness were measured every two days using an electronic vernier caliper before modeling and after dosing. Skin lesions were photographed 24 hours after modeling on day 14, and skin and blood samples were collected.

[0511] Establishment and dosing of AD model on the back of mice: 36 mice were randomly divided into normal group (Normal), model group (MC903), 0.01%, 0.1%, and 1% compound 1, and dexamethasone (Dex), with 6 mice in each group. Except for the normal group, AD models were established in all other groups: 48 hours before the experiment, the hair on the back of the mouse neck was carefully removed using a trimmer and depilatory cream. After the start of modeling, 20 μL of MC903 solution was applied to the back of the mouse neck once a day for 14 consecutive days to induce the mouse AD model. After 7 days of modeling, 0.01%, 0.1%, and 1% compound 1 and 20 mg of dexamethasone ointment were evenly applied to the corresponding mice every day. Mice in the model group and normal group were applied with blank ointment every day. 24 hours after the completion of modeling on the 14th day, photos of the mouse skin lesions were taken, and skin lesions and blood samples were collected from the mice.

[0512] Lesion severity scoring: The mice were scored daily for skin lesion severity using the following criteria, which mainly include four symptoms: 1) erythema / hemorrhage; 2) dryness; 3) exfoliation / erosion; and 4) edema. Each symptom was scored from 0 to 3 points depending on the severity of the lesion, with no symptom scored 0, mild 1, moderate 2, and severe 3. The sum of all symptom scores is the lesion severity. The higher the score, the more severe the lesion, with a maximum score of 12. The data were statistically analyzed. On the 14th day of the experiment, the AD-like lesions, such as erythema, edema, dryness, exudation, and scabs, were compared among the groups and photographed and recorded with a digital camera.

[0513] Q-PCR analysis

[0514] Total RNA extraction from animal tissues, cDNA synthesis, and PCR amplification were performed as described above. The primers added are as follows:

[0515] Table 28. Q-PCR analysis

[0516] Experimental results: Compared with the normal group, the model group mice showed obvious AD-like skin lesions such as erythema, scaling, scabs, and edema in the ear area. However, after applying 0.01%, 0.1%, and 1% compound 1 ointment, the above symptoms were significantly alleviated.

[0517] As shown in Table 29, ear thickness increased significantly in the AD group over time, but the rate of increase was significantly slower in the Compound 1-treated group. On the final day of the experiment, ear thickness in the AD group increased 2.4-fold compared to baseline. Compound 1 ointment at 0.01%, 0.1%, and 1% concentrations inhibited this increase by 71%, 71%, and 80%, respectively. This suggests that Compound 1 can alleviate the severity of ear lesions in mice.

[0518] Table 29. Compound 1 significantly reduced the thickness of the auricle in AD mice

[0519] As shown in Table 30, compared with normal mice, AD mice developed skin lesion-like symptoms in the ears, manifested as obvious erythema, scales, scabs and edema, and continuous application of 0.01%, 0.1%, and 1% compound 1 ointment for 7 days significantly alleviated the skin lesion symptoms of mice, as manifested by a significant decrease in dermatitis scores.

[0520] Table 30. Compound 1 significantly down-regulated the ear dermatitis score of AD mice

[0521] As shown in Table 31, compared with normal mice, the mRNA expression of Th2-related factors Il4, Il13, Il31 and inflammatory factors Tslp, Tnf, Il13, Il1 secreted by keratinocytes in the ears of mice in the AD group was significantly increased, but the administration of compound 1 significantly inhibited the expression of the above genes.

[0522] Compared with the normal group, the model group mice showed obvious AD-like skin lesions on their backs, including erythema, scaling, scabs, and edema. However, after application of 0.01%, 0.1%, and 1% compound 1 ointment, the above symptoms were significantly alleviated.

[0523] As shown in Table 32, on the final day of the experiment, the back thickness of mice in the AD group increased 2.4-fold compared to baseline values, while the inhibition rates of 0.01%, 0.1%, and 1% Compound 1 ointment were 66%, 72%, and 83%, respectively. This suggests that Compound 1 can alleviate the severity of back lesions in mice.

[0524] Table 31. Compound 1 inhibits mRNA expression of inflammatory factors in the ears of AD mice

[0525] Table 32. Compound 1 significantly reduces the back thickness of AD mice

[0526] As shown in Table 33, compared with normal mice, AD mice developed skin lesion-like symptoms on the back, manifested as obvious erythema, scales, scabs and edema, and continuous application of 0.01%, 0.1%, and 1% compound 1 ointment for 7 days significantly alleviated the skin lesion symptoms of mice, as manifested by a significant decrease in dermatitis scores.

[0527] Table 33. Compound 1 significantly down-regulated the back dermatitis score of AD mice

[0528] 2. Effect of oral administration of compound 20 on atopic dermatitis in mice

[0529] A mouse atopic dermatitis model was established as described in Part 1, and compound 20 (0.1, 1, and 10 mg / kg) was administered orally for 7 consecutive days. The results are shown in Table 34. The skin tissue structure of the lesions on the ears of normal mice was normal, with clear layers, and no typical inflammatory reactions such as vascular dilation were observed. The skin tissue of the left ear of the mice in the MC903 group began to become red, swollen, and hardened on the 7th day, and was accompanied by epidermal dryness and desquamation on the 14th day. As the experimental time prolonged and the number of stimulations increased, the inflammatory symptoms of the skin tissue gradually worsened, as manifested by a significantly increased inflammation score. Oral administration of compound 20 (1 and 10 mg / kg) significantly alleviated MC903-induced AD-like lesions in the ears of mice and reduced the atopic dermatitis score.

[0530] Table 34. Oral administration of compound 20 significantly reduced the atopic dermatitis score in mice

[0531] As shown in Table 35, compared with the normal group of mice, the ear tissue thickness of the MC903 group of mice was significantly increased, and oral administration of compound 20 (1, 10 mg / kg) could significantly reduce the ear thickness of AD mice.

[0532] Table 35. Oral administration of compound 20 significantly reduced ear thickness in dermatitis mice

[0533] As shown in Table 36, serum IgE levels in mice in the MC903 group were significantly elevated, while oral administration of Compound 20 (1 and 10 mg / kg) reduced IgE levels in a concentration-dependent manner. Finally, compared with mice in the Normal group, mRNA expression of the inflammatory cytokines Tslp and Il6, as well as the Th2-related factors Il4, Il13, and Il31, secreted by keratinocytes in the ear skin of mice in the MC903 group was significantly increased. However, oral administration of Compound 20 (1 and 10 mg / kg) significantly inhibited the expression of these genes.

[0534] Table 36. Compound 20 inhibits mRNA expression of inflammatory factors in the ear of AD mice

[0535] Example 11 Test on the skin soothing function of compound 1

[0536] (1) Testing of hyaluronic acid and hyaluronidase levels in human keratinocytes

[0537] Human keratinocytes (Hacat) were cultured in DMEM medium containing 10% FBS in a cell culture incubator containing 5% CO2. When the cells grew to 70-80% confluence, 1×10 5 The cells were evenly seeded in 6-well plates with 2 mL per well and divided into Normal, UVB, and compound 1 (0.01, 0.1, 1, and 10 μM) groups. 2 After irradiation with different doses, the above-mentioned compound 1 at different concentrations was added, and after incubation for 24 hours, the levels of hyaluronic acid and hyaluronidase were detected by kit method.

[0538] As shown in Table 37, compared to the Normal group, hyaluronic acid levels were significantly decreased and hyaluronidase levels were significantly increased after UVB irradiation. After 24 hours of treatment with Compound 1 (0.1, 1, and 10 μM), hyaluronic acid levels were significantly restored and hyaluronidase levels were significantly decreased. This suggests that Compound 1 may exert a soothing effect by increasing hyaluronic acid levels.

[0539] Table 37. Compound 1 downregulates hyaluronidase and upregulates hyaluronic acid levels in human keratinocytes

[0540] (2) Testing for mast cell degranulation

[0541] Rat mast cells (RBL-2H3) were cultured in DMEM medium containing 10% FBS in a cell culture incubator containing 5% CO2. When the cells grew to 70-80% confluence, 1×10 5 The cells were evenly seeded in a 6-well plate at a density of 2 mL per well and divided into Normal, IgE+HSA, and compound 1 (0.01, 0.1, 1, 10 μM) groups. Mast cells were sensitized with 500 μg / L anti-DNP IgE for 12 h, washed with PBS, and cultured for a further 12 h with 100 μg / L DNP-HSA. Then, the above-mentioned different concentrations of compound 1 were added and incubated for 4 h. The level of β-hexosaminidase was detected by a kit method.

[0542] As shown in Table 38, compared to the Normal group, mast cell degranulation was significantly enhanced after antigen-antibody stimulation, as evidenced by significantly elevated β-hexosaminidase levels. β-hexosaminidase levels were significantly decreased after 4-hour treatment with Compound 1 (0.1, 1, and 10 μM), suggesting that Compound 1 may exert a soothing effect by inhibiting mast cell degranulation.

[0543] Table 38. Compound 1 significantly inhibits mast cell degranulation

[0544] Example 12 Compounds inhibit MDSCs and promote T cell proliferation experiment

[0545] Mice were sacrificed by cervical dislocation and immersed in 75% ethanol for 5 minutes. Under sterile conditions, the tibiae of the mice were removed, the ends of the bones cut, and culture medium was aspirated with a 1mL syringe. The bone marrow was repeatedly flushed into a 15mL centrifuge tube. Bone marrow cells were collected by centrifugation at 1600rpm for 5 minutes, washed twice with 1×PBS, and resuspended in RPMI 1640 complete medium containing the cytokines GM-CSF (10ng / mL) and IL-4 (10ng / mL) in a cell culture dish to stimulate the bone marrow cells to differentiate into MDSCs. MDSCs were then treated with DMSO (negative control) and 1μM of the compound in Table 39 for 12 hours. At the same time, the connective tissue beneath the spleen was separated with forceps, and the spleen was removed and placed in a sterile culture dish containing 5 mL of RPMI 1640 medium. The spleen was placed on a 200-mesh cell screen and gently crushed with a 10 mL syringe needle. A small amount of RPMI 1640 medium was simultaneously drawn up to rinse the cell screen. The spleen cell suspension was rinsed into a 15 mL centrifuge tube. T cells were collected by centrifugation at 1600 rpm for 5 min, washed twice with 1× PBS, and resuspended in 1 mL of 10 μM CFDA-SE solution in the centrifuged T cells. The cells were incubated at room temperature for 8 min. After the incubation period, 10 mL of PRMI 1640 complete medium was added to terminate CFDA-SE staining. The cells were centrifuged at 1600 rpm for 5 min and washed twice with 1× PBS. The cells were resuspended in RPMI 1640 complete medium containing concanavalin A (5 μg / mL) in a cell culture dish and cultured in a 37°C, 5% CO2 incubator for 12 h to stimulate T cell activation. After the administration is completed, the MDSCs cells are collected into a centrifuge tube, centrifuged at 1600 rpm for 5 minutes, the supernatant is removed, and 1 mL of fresh culture medium is added to resuspend the cell, placed in a plate, and then 1 mL of stimulated and activated T cells (1:1) are added to co-culture for 48 hours. After the incubation is completed, the cells are collected into a centrifuge tube, washed twice with PBS, and Fc antibody is added and incubated at room temperature in the dark for 10 minutes; CD3-labeled flow cytometry antibody is added and incubated in the dark at 4°C for 30 minutes. After the incubation is completed, the cells are washed with PBS, resuspended with 300 μL PBS, and detected by flow cytometry.

[0546] As shown in Table 39, all compounds promoted T cell proliferation to varying degrees in the MDSCs and T cell co-culture model, suggesting that these compounds have strong anti-tumor activity in tumor models with T lymphocyte infiltration (such as hepatocellular carcinoma, colorectal cancer, and lymphoma). Compounds 78, 83, and 1 significantly promoted T cell proliferation, with results similar to those of the positive compound Arg.

[0547] Table 39. Effects of compounds on T cell proliferation in MDSCs and T cell co-culture model

[0548] Note: “+”, compared with the control group, the T cell proliferation rate is in the range of 0-20%; “++”, compared with the control group, the T cell proliferation rate is in the range of 20-40%; “+++”, compared with the control group, the T cell proliferation rate is >40%.

[0549] Example 13 Compounds 78, 83, and 1 inhibit chemotaxis of MDSCs

[0550] Hepa1-6 mouse liver cancer cells were evenly spread in a 6-well plate. After the cells adhered to the wall and grew fully, the supernatant was aspirated and 1-1.5 mL of fresh culture medium was added to each well. After culturing for 24 hours, the supernatant was collected and centrifuged at 1600 rpm for 5 minutes. The supernatant was aspirated and filtered through a 0.22 μM filter membrane to obtain tumor-conditioned culture medium for later use. The mice were killed by cervical dislocation and soaked in 75% ethanol solution for 5 minutes. The tibia of the mouse was removed under a sterile environment, the two ends of the bone were cut open, the culture medium was aspirated with a 1 mL syringe, and the bone marrow was repeatedly flushed into a 15 mL centrifuge tube. Bone marrow cells were collected by centrifugation at 1600 rpm for 5 minutes, washed twice with 1×PBS, and resuspended in RPMI 1640 complete culture medium containing cytokines GM-CSF (10 ng / mL) and IL-4 (10 ng / mL) to stimulate the bone marrow cells to differentiate into MDSCs. The cells were counted and the cell density was adjusted to 1×10 6 Cells were evenly plated in a 24-well plate at a concentration of 1 μg / mL. 300 μL was added to each well. MDSCs were treated with DMSO (negative control), 1 μM, and 10 μM Arg, 78, 83, and 1 for 12 hours. The cells were centrifuged at 1600 rpm for 5 minutes, the supernatant removed, and 300 μL of fresh culture medium was added to resuspend the cells. The lower chamber was filled with 600 μL of normal culture medium (control group) and tumor-conditioned medium (model group) and cultured for 24 hours. After incubation, the chamber was removed and the number of cells entering the lower chamber was counted to investigate the inhibitory effect of Arg, 78, 83, and 1 on MDSC chemotaxis.

[0551] As shown in Table 40, in the MDSCs co-culture model with tumor-conditioned medium, 78, 83, and 1 all significantly inhibited MDSC chemotaxis compared to the control group in a concentration-dependent manner. At a concentration of 10 μM, the inhibitory rates of 83 and 1 on MDSC chemotaxis were superior to those of the positive compound Arg.

[0552] Table 40. Inhibitory effects of Arg, 78, 83, and 1 on MDSCs chemotaxis

[0553] Example 14 Compounds 78, 83, and 1 down-regulate the level of CCR2 mRNA

[0554] Mice were killed by cervical dislocation and immersed in 75% ethanol solution for 5 minutes. The tibia of the mouse was removed under sterile conditions, the two ends of the bone were cut open, and the culture medium was aspirated with a 1mL syringe. The bone marrow was repeatedly flushed into a 15mL centrifuge tube. Bone marrow cells were collected by centrifugation at 1600rpm for 5 minutes, washed twice with 1×PBS, and resuspended in a cell culture dish with RPMI 1640 complete medium containing GM-CSF (10ng / mL) + IL-4 (10ng / mL) to stimulate the differentiation of bone marrow cells into MDSCs. Cell count and complete culture medium were used to adjust the cell density to 1×10 6 Cells were plated evenly in a 6-well plate at a concentration of 1 μg / mL. 2 mL was added to each well. MDSCs were then treated with DMSO (negative control), 1 μM, and 10 μM of Arg, 78, 83, and 1, respectively, for 2 hours. After incubation, cells were collected into centrifuge tubes and centrifuged at 1600 rpm for 5 minutes. Washed once with 1× PBS, 500 μL of RNA extraction reagent was added to each tube. After centrifugation, 20 μL of DEPC water was added to each tube to dissolve the precipitate. RNA concentration was determined and reverse transcribed into cDNA. The cDNA, primers, and reverse transcriptase were added to a 96-well plate and centrifuged at 4000 rpm for 5 minutes. The plate was then placed in a PCR instrument to determine the inhibitory effects of Arg, 78, 83, and 1 on CCR2 mRNA expression.

[0555] As shown in Table 41, compounds Arg, 78, 83, and 1 can downregulate CCR2 mRNA levels in a concentration-dependent manner.

[0556] Table 41. Compounds 78, 83, and 1 inhibit CCR2 mRNA levels

[0557] Example 15 Antitumor Effects of Compounds 20, 99, and 102 in a Hepa1-6 Mouse Hepatocellular Carcinoma Subcutaneous Transplant Tumor Model Hepa1-6 cells were cultured and expanded in vitro. An appropriate amount of cells in the logarithmic growth phase were resuspended in serum-free DMEM medium and Matrigel (1:1) suspension and prepared under sterile conditions to 2.5×10 6 100 μL of cell suspension was inoculated into the subcutaneous tissue of the left axilla of male C57BL / 6 mice using a syringe. 3 At the time of the experiment, animals with tumors of appropriate size were randomly divided into groups of 6 per group. They were solvent control group, 40 μmol / kg / d Arg group, compound 20 group, compound 99 group, and compound 102 group. The drugs were administered by gavage every day for 2 weeks. During the administration period, the body weight and tumor diameter of the mice were measured every day. After the experiment, the mice were killed by cervical dislocation and the tumors were weighed. The formula for calculating tumor volume (TV) is: TV = 1 / 2 × a × b 2, a represents the long diameter of the tumor; b represents the short diameter of the tumor.

[0558] As shown in Table 42, in the Hepa1-6 mouse liver cancer subcutaneous transplant tumor model, Arg, 20, 99, and 102 all had significant tumor inhibitory effects after continuous administration for 2 weeks.

[0559] Table 42. Antitumor effects of compounds 20, 99, and 102 in the Hepa1-6 mouse liver cancer transplant model

[0560] *, p<0.05; **, p<0.01; ***, p<0.001 (compared with solvent control).

[0561] Example 16 Antitumor effects of compounds 20, 99, and 102 in H22 mouse subcutaneous liver cancer transplantation tumor model

[0562] H22 cells were cultured and expanded in vitro. An appropriate amount of cells in the logarithmic growth phase were resuspended in serum-free RPMI 1640 medium and Matrigel (1:1) suspension and prepared into 3×10 5 100 μL of cell suspension was inoculated into the subcutaneous tissue of the left axilla of male BALB / c mice using a syringe. 3 At the time of the experiment, animals with tumors of appropriate size were randomly divided into groups of 6 per group. They were solvent control group, 40 μmol / kg / d Arg group, compound 20 group, compound 99 group, and compound 102 group. The drugs were administered by gavage every day for 2 weeks. During the administration period, the body weight and tumor diameter of the mice were measured every day. After the experiment, the mice were killed by cervical dislocation and the tumors were weighed. The formula for calculating tumor volume (TV) is: TV = 1 / 2 × a × b 2 , a represents the long diameter of the tumor; b represents the short diameter of the tumor.

[0563] Tumor-bearing mice were killed by cervical dislocation and placed in a beaker containing 75% alcohol. The tumors of the mice were removed and then immersed in 100 mm flasks containing RPMI 1640. 3After all tumor cells have been removed from the culture dish, cut fingernail-sized pieces of the tumor into small pieces and transfer them to a 1.5mL EP tube. Use scissors to mince the tumor. Transfer the minced tumor tissue suspension to a 15mL centrifuge tube containing 3-5mL of digestive enzymes (Collagenase IV 1mg / mL, Hyaluronidase 1mg / mL, DNase I 20U / mL) and incubate at 200rpm, 37°C, for 1 hour. After digestion, filter through a 200-mesh sieve to obtain a tumor cell suspension. Centrifuge the resulting tumor suspension at 1600rpm for 5 minutes, aspirate the supernatant, and resuspend the cells in 1mL of 1× PBS per tube. Transfer the cell suspension to a 1.5mL EP tube and centrifuge at 1600rpm for 5 minutes. Discard the supernatant and resuspend the cells in 100μL of 1× PBS per tube. Add 1μL of anti-vital dye to each tube, mix thoroughly, and incubate at room temperature in the dark for 15 minutes. At the same time, remove the spleen with forceps and place it in a sterile culture dish containing 5 mL of RPMI 1640 medium. Place the spleen on a 200-mesh cell sieve and gently crush the spleen with a 10 mL syringe needle. Rinse the sieve with a small amount of RPMI 1640 medium. Transfer the spleen cell suspension to a 15 mL centrifuge tube and centrifuge at 1600 rpm for 5 minutes to collect the cells. Wash once with 1× PBS. Add 3 mL of red blood cell lysis buffer (diluted with ddH2O) to each tube and lyse for 5 minutes. After lysis, add 10 mL of 1× PBS to each tube to terminate lysis. Centrifuge at 1600 rpm for 5 minutes. Add 1 mL of 1× PBS to each 15 mL centrifuge tube, transfer the tube to a 1.5 mL EP tube, and centrifuge at 1600 rpm for 5 minutes. Wash each tube of stained tumor cells and lysed spleen cells with 1 mL of 1× PBS, centrifuge at 1600 rpm for 5 minutes, and discard the supernatant. Resuspend the tumor cells and spleen cells separately with 100 μL 1× PBS, and add FcX TM 1 μL of antibody was added and incubated at room temperature in the dark for 10 min. After the incubation, 1 μL of CD45, CD11b, and Gr1 antibodies were added to each tube in turn and incubated in the dark at 4°C for 30 min. After the incubation, the cells were washed once with 1× PBS, the supernatant was discarded, and the cells were resuspended with 300 μL of 1× PBS. The cells were filtered through the membrane into the flow tube and detected by the flow cytometer.

[0564] Tumor-bearing mice were killed by cervical dislocation and placed in a beaker containing 75% alcohol. The tumors of the mice were removed and then immersed in 100 mm flasks containing RPMI 1640. 3After all tumor cells have been removed from the culture dish, fingernail-sized pieces of the tumor are transferred to a 1.5mL EP tube. The tumor is then minced with scissors. The minced tumor tissue suspension is transferred to a 15mL centrifuge tube containing 3-5mL of digestive enzymes (Collagenase IV 1mg / mL, Hyaluronidase 1mg / mL, DNase I 20U / mL) and incubated in a shaker at 200rpm, 37°C for 1 hour. After digestion, the cells are filtered through a 200-mesh sieve to obtain a tumor cell suspension. The resulting tumor suspension is centrifuged at 1600rpm for 5 minutes, the supernatant aspirated, and 1mL of 1× PBS is added to each tube to resuspend the cells. The cell suspension is transferred to a 1.5mL EP tube and centrifuged at 1600rpm for 5 minutes. The supernatant is discarded, and the cells are resuspended in 100μL of 1× PBS per tube. 1μL of anti-vital dye is added to each tube, mixed, and incubated at room temperature in the dark for 15 minutes. At the same time, remove the spleen with forceps and place it in a sterile culture dish containing 5 mL of RPMI 1640 medium. Place the spleen on a 200-mesh cell sieve and gently crush the spleen with a 10 mL syringe needle. Rinse the sieve with a small amount of RPMI 1640 medium. The spleen cell suspension is then transferred to a 15 mL centrifuge tube and centrifuged at 1600 rpm for 5 minutes to collect the cells. Wash once with 1× PBS. Add 3 mL of red blood cell lysis buffer (diluted with ddH2O) to each tube and lyse for 5 minutes. After lysis, add 10 mL of 1× PBS to each tube to terminate lysis. Centrifuge at 1600 rpm for 5 minutes. Add 1 mL of 1× PBS to each 15 mL centrifuge tube, transfer the cells to a 1.5 mL EP tube, and centrifuge at 1600 rpm for 5 minutes. Wash each tube of stained tumor cells and lysed spleen cells with 1× PBS, centrifuge at 1600 rpm for 5 minutes, and discard the supernatant. Resuspend the tumor cells and spleen cells in 100 μL of 1× PBS, and add FcX to each tube. TM Incubate with 1 μL of antibody at room temperature in the dark for 10 minutes. After incubation, add 1 μL each of CD45, CD3, and CD8 antibodies to each tube in sequence and incubate at 4°C in the dark for 30 minutes. After staining, wash once with 1 mL of PBS and centrifuge at 1600 rpm for 5 minutes. Discard the supernatant and resuspend the cells in 300 μL of 1× PBS. Filter the cell suspension and transfer it to a flow cytometer for analysis.

[0565] The results are shown in Table 43. In the H22 mouse liver cancer subcutaneous transplant tumor model, Arg, 20, 99, and 102 all had tumor inhibitory effects after continuous administration for 2 weeks.

[0566] Table 43. Antitumor effects of compounds 20, 99, and 102 in the H22 mouse liver cancer transplant model

[0567] *, p<0.05; **, p<0.01; ***, p<0.001 (compared with solvent control).

[0568] As shown in Table 44, after the experiment, flow cytometry was used to detect the proportion of MDSCs in the tumor tissue and spleen of the H22 mouse subcutaneous transplanted tumor model. Compared with the solvent control group, the proportion of MDSCs in the tumor tissue and spleen of the Arg, 20, 99, and 102 treatment groups was significantly reduced.

[0569] Table 44. Inhibitory effects of compounds 20, 99, and 102 on MDSCs in the H22 mouse liver cancer transplant model

[0570] *, p<0.05; **, p<0.01; ***, p<0.001 (compared with solvent control).

[0571] As shown in Table 45, after the experiment, flow cytometry was used to detect the proportion of T cells in the tumor tissue and spleen in the H22 mouse subcutaneous transplant tumor model. Compared with the solvent control group, the proportion of T cells in the tumor tissue and spleen of the Arg, 20, 99, and 102 administration groups was significantly increased.

[0572] Table 45. Effects of Compounds 20, 99, and 102 on T cells in the H22 mouse liver cancer transplant model

[0573] *, p < 0.05; **, p < 0.01; ***, p < 0.001 (compared with solvent control)

[0574] Example 17 Antitumor Effects of Arg and Compound 20 in a Mouse Transplanted Tumor Model of CT26 Colon Cancer Cells

[0575] CT26 cells were cultured and expanded in vitro. An appropriate amount of cells in the logarithmic growth phase were resuspended in serum-free RPMI 1640 medium and prepared into 5×10 5 100 μL of cell suspension was inoculated into the subcutaneous tissue of the left axilla of male BALB / c mice using a syringe. 3 At the time of the experiment, animals with tumors of appropriate size were randomly divided into 5 groups. They were the solvent control group, the 80 μmol / kg / d Arg group, and the compound 20 group. The drug was administered by gavage every day for 3 weeks. During the administration period, the body weight and tumor diameter of the mice were measured every day. After the experiment, the mice were killed by cervical dislocation and the tumors were weighed. The tumor volume (TV) was calculated as follows: TV = 1 / 2 × a × b 2 , a represents the long diameter of the tumor; b represents the short diameter of the tumor.

[0576] As shown in Table 46, in the CT26 mouse subcutaneous transplant tumor model, Arg and Compound 20 both had tumor inhibitory effects after continuous administration for 3 weeks.

[0577] Table 46. Antitumor effects of Arg and compound 20 in CT26 mouse transplant tumor model

[0578] Example 18 Antitumor effects of compounds 20, 99, and 102 in a 4T1 mouse breast cancer transplant model

[0579] 4T1 cells were cultured and expanded in vitro. An appropriate amount of cells in the logarithmic growth phase were resuspended in serum-free RPMI 1640 medium and Matrigel (1:1) suspension and prepared into 4×10 5 100 μL of cell suspension was inoculated into the subcutaneous tissue of the left axilla of female BALB / c mice using a syringe. 3 At the time of the experiment, animals with tumors of appropriate size were randomly divided into groups of 5 each. They were solvent control group, 40 μmol / kg / d Arg group, compound 20 group, compound 99 group, and compound 102 group. The drugs were administered by gavage every day for 3 weeks. During the administration period, the body weight and tumor diameter of the mice were measured every day. After the experiment, the mice were killed by cervical dislocation and the tumors were weighed. The formula for calculating tumor volume (TV) is: TV = 1 / 2 × a × b 2 , a represents the long diameter of the tumor; b represents the short diameter of the tumor.

[0580] As shown in Table 47, in the 4T1 mouse breast cancer subcutaneous transplant tumor model, Arg, 20, 99, and 102 had no significant tumor inhibitory effect after continuous administration for 3 weeks.

[0581] Table 47. Antitumor effects of compounds 20, 99, and 102 in 4T1 mouse breast cancer xenograft model

[0582] *, p<0.05; **, p<0.01; ***, p<0.001 (compared with solvent control).

[0583] Example 19 Antitumor effect of compound 20 combined with anti-PD-L1 monoclonal antibody in Hepa1-6 mouse liver cancer subcutaneous transplant tumor model

[0584] Hepa1-6 cells were cultured and expanded in vitro. An appropriate amount of cells in the logarithmic growth phase were resuspended in serum-free DMEM medium and Matrigel (1:1) suspension and prepared into 2.5×10 6100 μL of cell suspension was inoculated into the subcutaneous tissue of the left axilla of male C57BL / 6 mice using a syringe. 3 When the tumor size was moderate, animals with moderately sized tumors were randomly divided into groups of 6 in each group. They were solvent control group, compound 20 (1 mg / kg / d) group, anti-PD-L1 monoclonal antibody 5 mg / kg group, and compound 20 combined with anti-PD-L1 monoclonal antibody group. Compound 20 was intraperitoneally injected every day, and anti-PD-L1 monoclonal antibody was intraperitoneally injected once every three days for 2 weeks. During the administration period, the weight and tumor diameter of the mice were measured every day. After the experiment, the mice were killed by cervical dislocation and the tumors were weighed. The calculation formula for tumor volume (TV) is: TV = 1 / 2 × a × b 2 , a represents the long diameter of the tumor; b represents the short diameter of the tumor.

[0585] In the Hepa1-6 mouse liver cancer subcutaneous transplant tumor model, after continuous administration for 2 weeks, the tumor inhibition rate of the combined administration group was significantly higher than that of the compound 20 and anti-PD-L1 monoclonal antibody group, showing a synergistic effect (Table 48).

[0586] Table 48. Antitumor effect of compound 20 combined with anti-PD-L1 monoclonal antibody in the Hepa1-6 mouse liver cancer transplant tumor model

[0587] *, p<0.05; **, p<0.01; ***, p<0.001 (compared with solvent control).

[0588] Example 20 Antitumor effect of compound 20 combined with anti-PD-L1 monoclonal antibody in H22 mouse liver cancer subcutaneous transplant tumor model

[0589] H22 cells were cultured and expanded in vitro. An appropriate amount of cells in the logarithmic growth phase were resuspended in serum-free RPMI 1640 medium and Matrigel (1:1) suspension and prepared into 2×10 6 100 μL of cell suspension was inoculated into the subcutaneous tissue of the left axilla of male Balb / c mice using a syringe. 3 When the tumor size was moderate, animals with moderately sized tumors were randomly divided into groups of 5 each. They were solvent control group, compound 20 (2.5 mg / kg / d) monotherapy group, anti-PD-L1 monoclonal antibody (5 mg / kg) monotherapy group, and compound 20 combined with anti-PD-L1 monoclonal antibody group. Compound 57 was injected intraperitoneally every day, and anti-PD-L1 monoclonal antibody was injected intraperitoneally once every three days for 2 weeks. During the administration period, the weight and tumor diameter of the mice were measured every day. After the experiment, the mice were killed by cervical dislocation and the tumors were weighed. The calculation formula for tumor volume (TV) is: TV = 1 / 2 × a × b2 , a represents the long diameter of the tumor; b represents the short diameter of the tumor.

[0590] In the H22 mouse liver cancer subcutaneous transplant tumor model, after continuous administration for 2 weeks, the tumor inhibition rate of the combined administration group was significantly higher than that of the compound 20 and anti-PD-L1 monoclonal antibody group, showing a synergistic effect (Table 49).

[0591] Table 49. Antitumor effect of compound 20 combined with anti-PD-L1 monoclonal antibody in H22 mouse liver cancer transplant tumor model

[0592] *, p<0.05; **, p<0.01; ***, p<0.001 (compared with solvent control).

[0593] Example 21 Safety Study of Compound 20

[0594] 1 Experimental reagents: Compound 20 (purity >98%), prepared according to Example 1; sodium carboxyl methyl cellulose (CMC-Na), Sinopharm Chemical Reagent Co., Ltd.; all other reagents were of analytical grade.

[0595] 2 Experimental methods

[0596] 2.1 Mouse Grouping and Dosing: To investigate the toxicity of Compound 20 in mice, male and female C57BL / 6 mice weighing 18-22 g were randomly divided into three groups based on body weight: a Normal group and a Compound 20 (500 and 1000 mg / kg) group, with 10 mice per group, half male and half female. Compound 20 was administered orally once daily for 30 days at a dose volume of 0.1 mL / 10 g. The Normal group received an equal volume of vehicle (normal saline).

[0597] 2.2 Specimen Collection: After the last dose, blood was collected from the fundus venous plexus, allowed to stand at room temperature for 2 hours, and centrifuged at 3500 rpm at 4°C for 15-20 minutes. The upper serum layer was carefully aspirated and aliquoted, then frozen at -80°C until further use. After chloral hydrate anesthesia, mice were sacrificed by cervical dislocation. The abdominal skin of the mice was carefully cut open and fixed with pins. The abdominal and thoracic cavities of the mice were then cut open, and the liver and kidneys were removed with forceps. After repeated washing with pre-chilled PBS solution, small pieces of tissue were cut and fixed in 10% neutral formalin solution (4% formaldehyde solution). The remaining tissue was frozen at -80°C until further use.

[0598] 3 Experimental results

[0599] 3.1 Effect on Mouse Body Weight: As shown in Table 50, during the experiment, the body weight of the experimental mice fluctuated within the normal range. Compared with the Normal group, 20 (500, 1000 mg / kg) had no significant effect on the body weight of both female and male C57BL / 6 mice (P>0.05).

[0600] Table 50. Effect of Compound 20 on Mouse Body Weight

[0601] 3.2 Effect on the activity state of mice: Compared with the Normal group, the mice in the Compound 20 (500, 1000 mg / kg) group showed relatively less activity and poor spirits after each administration, but returned to normal after 1 to 2 hours. During the 30-day continuous observation, there was no significant change in the shiny fur, behavioral activity, and mental state of the mice compared with the Normal group.

[0602] 3.3 Effect on mouse survival rate:

[0603] Table 51. Effect of Compound 20 on the Survival Rate of Mice

[0604] As shown in Table 51, compound 20 (500, 1000 mg / kg) had no significant effect on the survival rate of mice after continuous administration for 30 days.

Claims

1. Use of a guaiacyl sesquiterpene derivative or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating tumors or inflammatory diseases or soothing the skin; the structure of the guaiacyl sesquiterpene derivative is shown in Formula I; in, R1 and R2 together form a double bond; or R1 is hydrogen or deuterium, and R2 is wherein R3 and R4 are alkyl groups, or R3, R4 and the N atom to which they are connected form a 3-10-membered ring structure; R5 is selected from hydroxy, C1-C6 alkoxy, C1-C6 alkyl ester or halogen, and -OCONR6R7, wherein R6 is hydrogen or C1-C6 alkyl; R7 is C1-C6 alkyl, fluorine-substituted C1-C6 alkyl, C1-C6 cycloalkyl, hydroxy-substituted C1-C6 alkyl, C1-C6 alkoxy-substituted C1-C6 alkyl, OH-(CH2) n -O-substituted C1~C6 alkyl, amine-substituted C1~C6 alkyl, 5~6-membered nitrogen-containing heterocyclic ring substituted C1~C6 alkyl or benzene ring substituted C1~C6 alkyl; the 5~6-membered nitrogen-containing heterocyclic ring or benzene ring is optionally substituted by C1~C6 alkyl; or R6, R7 and the N atom to which they are connected form a 3-10-membered cyclic structure; or the 3rd carbon atom is connected to the 4th carbon atom to form a double bond; or the 4th carbon atom is connected to the 5th carbon atom to form a double bond; R8 is hydrogen or hydroxyl; R9 is C1-C6 alkyl, R 10 With R 11 connected to form cyclopropane; or R9 is a C1-C6 alkyl group, and the carbon atom at position 1 is connected to the carbon atom at position 10 to form a double bond; When R9 is a C1-C6 alkyl group, the carbon atom at position 1 is connected to the carbon atom at position 10 to form a double bond, and R5 is not a hydroxyl group; n is selected from 1, 2, 3, 4, 5 or 6; The tumor is selected from: hepatocellular carcinoma, colorectal cancer or lymphoma; The inflammatory disease is selected from (1) acute lung injury; (2) hepatitis; (3) sepsis caused by bacterial and / or viral infection; (4) acute and chronic kidney disease; (5) renal fibrosis; (6) lupus nephritis; (7) diabetic nephropathy; (8) atopic dermatitis; Preferably, the skin soothing is by increasing skin hyaluronic acid levels and / or inhibiting mast cell degranulation.

2. The use according to claim 1, wherein the structure of the guaiacyl sesquiterpene derivative is as shown in Formula II; R1 and R2 together form a double bond; or R1 is hydrogen or deuterium, R2 is wherein R3 and R4 are alkyl groups, or R3, R4 and N atoms form a 3-10 membered ring structure, R5 is hydroxyl, C1-C6 alkoxy, C1-C6 alkyl ester or halogen; or the carbon atom at position 3 is connected to the carbon atom at position 4 to form a double bond; or the carbon atom at position 4 is connected to the carbon atom at position 5 to form a double bond; R8 is hydrogen or hydroxy.

3. The use according to claim 1, wherein the structure of the guaiacyl sesquiterpene derivative is as shown in Formula III; R1 and R2 together form a double bond; or R1 is hydrogen or deuterium, R2 is wherein R3 and R4 are alkyl groups, or R3, R4 and N atoms form a 3-10 membered ring structure; R5 is -OCONR6R7, wherein R6 is hydrogen or C1-C6 alkyl; R7 is C1-C6 alkyl, C1-C6 alkyl substituted with fluorine, C1-C6 cycloalkyl, C1-C6 alkyl substituted with hydroxyl, C1-C6 alkyl substituted with C1-C6 alkoxy, OH-(CH2) n -O-substituted C1~C6 alkyl, amine-substituted C1~C6 alkyl, 5~6-membered nitrogen-containing heterocycle-substituted C1~C6 alkyl, or benzene ring-substituted C1~C6 alkyl; the 5~6-membered nitrogen-containing heterocycle or benzene ring is optionally substituted by C1~C6 alkyl; or R6, R7 and the N atom to which they are connected form a 3-10 membered ring structure.

4. The use according to claim 1, wherein the structure of the guaiacyl sesquiterpene derivative is as shown in Formula IV; in, R1 and R2 together form a double bond; or R1 is hydrogen or deuterium, R2 is Wherein R3 and R4 are alkyl groups respectively, or R3, R4 and the N atom to which they are connected form a 3-10 membered ring structure.

5. The method according to any one of claims 1 to 4, wherein R1 and R2 together form a double bond.

6. The use according to any one of claims 1 to 4, wherein R1 is hydrogen, R2 is R3 and R4 are C1~C 10 Alkyl; preferably, R3 and R4 are respectively C1~C6 alkyl.

7. The use according to any one of claims 1 to 4, wherein R1 is hydrogen, R2 is The R3, R4 and the N atom to which they are connected form a 4-6 membered ring structure.

8. The use according to any one of claims 1 to 4, wherein R1 is hydrogen, R2 is The cyclic structure formed by R3, R4 and the N atom to which they are connected is selected from the following structures: azetidine, tetrahydropyrrole, piperidine, piperazine, morpholine, azocyclooctane, 6-azaspiro[2,5]octane, 8-azaspiro[4,5]decane and octahydrocyclopenta[c]pyrrole.

9. The method according to any one of claims 1 to 3, wherein R5 is selected from hydroxyl.

10. The use according to any one of claims 1 or 3, wherein R5 is selected from -OCONR6R7, wherein R6 is hydrogen; R7 is a C1-C6 alkyl substituted with a 5- to 6-membered nitrogen-containing heterocycle; the 5- to 6-membered nitrogen-containing heterocycle is selected from tetrahydropyrrole, piperidine, piperazine, morpholine; and optionally the 5- to 6-membered nitrogen-containing heterocycle is substituted with a C1-C6 alkyl.

11. The use according to any one of claims 1 or 3, wherein R5 is selected from -OCONR6R7, wherein R6, R7 and the N atom to which they are connected form a 4-6 membered cyclic structure.

12. The use according to any one of claims 1 or 3, wherein R5 is selected from -OCONR6R7, wherein the cyclic structure formed by R6, R7 and the N atom to which they are connected is selected from the following structures: azetidine, tetrahydropyrrole, piperidine, piperazine, morpholine.

13. The use according to claim 1, wherein R1 and R2 together form a double bond; or R1 is hydrogen or deuterium, and R2 is wherein R3 and R4 are C1-C3 alkyl groups, or R3, R4 and the N atom form a 5-6 membered ring structure, and the 5-6 membered ring structure is selected from pyrrole, proline, piperidine, piperazine, and morpholine; R5 is hydroxy, methoxy, methyl ester, halogen, and -OCONR6R7, wherein R6 is hydrogen, C1-C6 alkyl; R7 is C1-C6 alkyl, fluorine-substituted C1-C3 alkyl, cyclopropane, hydroxy-substituted C1-C6 alkyl, methoxy-substituted C1-C6 alkyl, dimethylaminoethyl, morpholine-substituted C1-C6 alkyl, piperazine-substituted C1-C6 alkyl, benzene ring-substituted methyl; or R6, R7 and N atom form a substituted 5-6 membered ring structure; or the 3rd carbon atom is connected to the 4th carbon atom to form a double bond; or the 4th carbon atom is connected to the 5th carbon atom to form a double bond; R8 is hydrogen or hydroxy; R9 is methyl, R 10 With R 11 Connected to form cyclopropane; or the carbon atom at position 1 is connected to the carbon atom at position 10 to form a double bond.

14. The use according to claim 2, wherein R1 and R2 together form a double bond; or R1 is hydrogen or deuterium, and R2 is Wherein R3 and R4 are C1-C3 alkyl groups respectively, or R3, R4 and N atom form a 5-6 membered ring structure, and the 5-6 membered ring structure is selected from pyrrole, proline, piperidine, piperazine and morpholine; R5 is hydroxyl, methoxy, methyl ester and halogen; or the carbon atom at position 3 is connected to the carbon atom at position 4 to form a double bond; or the carbon atom at position 4 is connected to the carbon atom at position 5 to form a double bond; R8 is hydrogen or hydroxyl.

15. The use according to claim 3, wherein R1 and R2 together form a double bond; or R1 is hydrogen or deuterium, and R2 is Wherein R3 and R4 are C1~C3 alkyl groups respectively, or R3, R4 and N atom form pyrrole; R5 is -OCONR6R7, wherein R6 is hydrogen or C1~C6 alkyl group; R7 is C1~C6 alkyl group, fluorine-substituted C1~C3 alkyl group, cyclopropane group, hydroxyl-substituted C1~C6 alkyl group, methoxy-substituted C1~C6 alkyl group, dimethylaminoethyl group, morpholine-substituted C1~C6 alkyl group, piperazine-substituted C1~C6 alkyl group, or methyl group substituted by a benzene ring; or R6, R7 and N atom form a substituted 5-6-membered ring structure.

16. The use according to claim 4, wherein: R1 and R2 together form a double bond; or R1 is hydrogen or deuterium, and R2 is wherein R3 and R4 are C1-C4 alkyl and isopropyl respectively; or R3, R4 and N atom form a 4-6 membered monocyclic structure, the 4-6 membered monocyclic structure is selected from azetidine, pyrrole, morpholine, piperidine and piperazine, and the substituents on the ring are selected from ethyl, ethoxy, morpholine and piperidine; or R3, R4 and N atom form an 8-membered cyclic structure, the 8-membered cyclic structure is selected from azacyclooctane; or R3, R4 and N atom form an 8-membered spirocyclic structure, the 8-membered spirocyclic structure is selected from 6-azaspiro[2,5]octane; or R3, R4 and N atom form a 10-membered spirocyclic structure, the 10-membered spirocyclic structure is selected from 8-azaspiro[4,5]decane; or R3, R4 and N atom form an 8-membered condensed ring structure, the 8-membered spirocyclic structure is selected from octahydrocyclopenta[c]pyrrole.

17. The use according to any one of claims 1 to 16, wherein the cyclic structure formed by R3, R4 and N atom or the cyclic structure formed by R6, R7 and N atom is substituted by a substituent selected from C1 to C6 alkyl, a 5-6 membered nitrogen-containing heterocyclic group, and a C1 to C6 alkoxycarbonyl group; further preferably, the 5-6 membered nitrogen-containing heterocyclic group is selected from tetrahydropyrrolyl, piperidinyl, piperazinyl or morpholinyl.

18. The use according to any one of claims 1 to 17, wherein the pharmaceutically acceptable salt of the guaiacyl sesquiterpene derivative is selected from hydrochloride, sulfate, phosphate, maleate, fumarate or citrate.

19. The use according to any one of claims 1 to 18, wherein the guaiacyl sesquiterpene derivative or a pharmaceutically acceptable salt thereof is selected from the following structures:

20. Use of a pharmaceutical composition in preparing a drug for treating tumors or inflammatory diseases or soothing skin; the pharmaceutical composition comprises the guaiacyl sesquiterpene derivative or a pharmaceutically acceptable salt thereof according to claims 1 to 19 and a pharmaceutically acceptable carrier; the tumor is selected from: hepatocellular carcinoma, colorectal cancer or lymphoma; The inflammatory disease is selected from (1) acute lung injury; (2) hepatitis; (3) sepsis caused by bacterial and / or viral infection; (4) acute and chronic kidney disease; (5) renal fibrosis; (6) lupus nephritis; (7) diabetic nephropathy; (8) atopic dermatitis; Preferably, the skin soothing is by increasing skin hyaluronic acid levels and / or inhibiting mast cell degranulation.

21. The use according to claim 20, wherein the pharmaceutical composition further comprises an immune checkpoint inhibitor.

22. The use according to claim 21, characterized in that The immune checkpoint inhibitor is an anti-PD-L1 / PD-1 monoclonal antibody or an anti-CTLA-4 monoclonal antibody.

23. Use of a guaiacyl sesquiterpene derivative or a pharmaceutically acceptable salt, prodrug, solvate, stereoisomer thereof in the preparation of a drug for treating inflammatory diseases or tumors or soothing the skin; the guaiacyl sesquiterpene derivative is compound 1 The tumor is selected from: hepatocellular carcinoma, colorectal cancer or lymphoma; the inflammatory disease is selected from: (1) acute lung injury; (2) hepatitis; (3) sepsis caused by bacterial and / or viral infection; (4) acute and chronic kidney disease; (5) Renal fibrosis; (6) lupus nephritis; (7) diabetic nephropathy; (8) atopic dermatitis; Preferably, the skin soothing is by increasing skin hyaluronic acid levels and / or inhibiting mast cell degranulation.

24. The use according to claim 23, wherein the guaiacyl sesquiterpene derivative prodrug is selected from the following structures:

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