Cycloastragenol carboxylic acid derivative as well as preparation method and application thereof

Abstract

The invention discloses cycloastragenol carboxylic acid derivatives as well as a preparation method and application thereof, and belongs to the field of medicines. Amino acid, salicylic acid, benzoic acid, cinnamic acid and fatty acid are combined with C-3 hydroxyl of CAG through ester bonds, and through in-vitro model screening and structure-function relationship discussion, it is found that all series of compounds have different degrees of inhibiting effects on neuroinflammation; the activity of the compound R2 is the most prominent, and the anti-inflammatory activity of CAG is remarkably improved. In-vivo experiments show that R2 can effectively relieve behavioral and pathological indications of PD and inhibit activation of microglial cells. The invention opens up a new thought for exploration of PD drugs in natural products.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and specifically relates to a cyclococcogenol carboxylic acid derivative, a preparation method thereof, and an application thereof. Background Art

[0002] Parkinson's disease (PD), as the second most common neurodegenerative disease globally, shows an increasing incidence rate of 2% in people over 60 years old according to domestic epidemiological surveys. Currently, clinical treatment mainly relies on chemical drugs. Although levodopa, as the "gold standard" therapy, can effectively relieve symptoms, long-term use inevitably leads to adverse reactions such as reduced efficacy and motor complications, and it cannot prevent the progressive degeneration of dopaminergic neurons. This treatment dilemma highlights the urgent need to develop new therapeutic drugs. Notably, against the backdrop of bottlenecks in chemical drug research and development, natural products, due to their unique multi-target action mechanisms and low toxicity and side effects, are gradually becoming an important resource pool for innovative drug development. Screening for highly efficient and stable candidate drugs from natural products and their derivatives through structure modification strategies is not only the key path to breaking through existing treatment limitations but also an inevitable choice to promote the precision development of PD research, providing innovative solutions for conquering this intractable disease.

[0003] Astragalus membranaceus ( Astragalus membranaceus (Fisch.) Bunge) is a classic traditional Chinese medicine tonic widely used in the treatment of neurodegenerative diseases such as PD. Modern pharmacological studies have shown that the active ingredient in Astragalus membranaceus, cyclococcogenol (CAG), is the glycoside structure of astragaloside IV and has various pharmacological activities such as anti-inflammatory, anti-aging, antioxidant stress, and immunomodulatory effects. In previous studies, we found that CAG can regulate the TLR4 / NF-κB signaling pathway, reduce the expression of inflammatory factors in the brain, inhibit the neuroinflammatory level of microglia, and play a regulatory role in PD. Other studies have shown that CAG can reduce the assembly of NLRP3 inflammasomes and play a neuroinflammatory inhibitory role in in vivo models of PD. Many studies have fully demonstrated that CAG mainly focuses on inhibiting neuroinflammation and improving the pathological state of neurodegenerative diseases. However, in many in vitro and in vivo studies, we found that there is still much room for improvement in the activity of CAG in inhibiting neuroinflammation. Therefore, we hope to modify its structure and discover derivatives with better activity for drug exploration in PD. Summary of the Invention

[0004] The object of the present invention is a cyclocanthol carboxylic acid derivative, its preparation method and application. The present invention uses CAG as a lead compound, introduces fatty acids, amino acids, salicylic acid and cinnamic acid at the C-3 position, and uses PD in vitro cell models and neuroinflammatory cell models to screen for potent derivatives; combined with the PD in vivo model, the in vivo efficacy of the compounds is verified, and the mechanism of action of the compounds is preliminarily explored, providing a scientific basis for the drug exploration of CAG in the treatment of neuroinflammation or neurodegenerative diseases.

[0005] The present invention first provides a cyclocanthol carboxylic acid derivative, and its structural formula is shown in Formula I:

[0006] In Formula I, R is mono-substituted or multi-substituted; R is selected from at least one of H, halogen, nitro, methyl and methoxy.

[0007] In the above cyclocanthol carboxylic acid derivative, R is mono-substituted; said R is H, fluorine, chlorine or bromine.

[0008] In the above cyclocanthol carboxylic acid derivative, when said R is fluorine, chlorine or bromine, the substitution position is the 4'-position.

[0009] The present invention further provides a preparation method of the above cyclocanthol carboxylic acid derivative, including the following steps: reacting cyclocanthol, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, the compound shown in Formula II and 4-dimethylaminopyridine in a solvent to obtain the cyclocanthol carboxylic acid derivative;

[0010] The definition of R in Formula II is the same as the definition of R in the above Formula I.

[0011] In the above preparation method, the solvent is dichloromethane; The molar ratio of cyclocanthol to the volume of the solvent is 2 mmol:5 - 20 mL, specifically 2 mmol:10 mL; The molar ratio of cyclocanthol to 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide is 1:1 - 4, specifically 1:2; The molar ratio of cyclocanthol to the compound shown in Formula II is 1:1 - 4, specifically 1:2; The molar ratio of cyclocanthol to 4-dimethylaminopyridine is 1:1 - 4, specifically 1:1.

[0012] In the above preparation method, the temperature of the reaction is room temperature; The time of the reaction is 2 - 24 h, specifically 24 h.

[0013] The room temperature is well-known to those skilled in the art, generally being 15 - 35°C.

[0014] The above preparation method further has the following steps after the reaction: removing the solvent, redissolving with ethyl acetate, and extracting with water; mixing the obtained ethyl acetate solution with silica gel, then removing the solvent from the obtained solution, purifying by column chromatography, and obtaining a solid powder after removing the solvent.

[0015] Finally, the present invention provides the application of the above cyclocarya alcohol carboxylic acid derivatives in the preparation of drugs for preventing and / or treating Parkinson's disease.

[0016] The application of the above cyclocarya alcohol carboxylic acid derivatives in the preparation of drugs for preventing and / or treating neurodegenerative diseases also belongs to the protection scope of the present invention.

[0017] The present invention further provides the application of the above cyclocarya alcohol carboxylic acid derivatives in the preparation of any one of the following drugs: (1) Drugs for preventing and / or treating neuroinflammation; (2) Drugs for inhibiting IL-1β; (3) Drugs for enhancing the telomerase activity of microglia; (4) Drugs for enhancing TERT expression; (5) Drugs for reducing Iba-1 expression; (6) Drugs for regulating TLR4 expression; (7) Drugs for reducing the phosphorylation level of NF-κB.

[0018] The present invention combines amino acids, salicylic acid, benzoic acid, cinnamic acid, and fatty acids with the C-3 hydroxyl group of CAG through an ester bond. Through in vitro model screening and structure-activity relationship exploration, it is found that each series of compounds has different degrees of inhibitory effects on neuroinflammation; the activity of compound R2 is the most prominent (IC 50 IL-1β = 10.38 ± 0.54 μM), significantly enhancing the anti-inflammatory activity of CAG. In vivo experiments show that R2 can effectively relieve the behavioral and pathological indicators of PD and inhibit the activation of microglia. Further exploration reveals that R2 significantly enhances the decrease in TERT expression caused by MPTP and improves the development of neuroinflammation by regulating the TLR4 / NF-κB signaling pathway. The present invention opens up a new idea for the exploration of PD drugs in natural products. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is the design and synthesis route of the CAG derivative of the present invention.

[0020] Figure 2 It is the in vitro activity evaluation of compound R2; wherein,Figure 2 In which, A is the structure of compound R2; B is the IL-1β inhibition rate of compound R2 and CAG; C is the morphological observation of compound R2 inhibiting the inflammatory response of LPS-induced BV2 cells.

[0021] Figure 3 is for compound R2 to improve the behavioral and pathological indicators of PD; among them, Figure 3 in which, A is the stride frequency test in the gait experiment, LF: left front paw, RF: right front paw, LH: left hind paw, RH: right hind paw; B is the pole climbing experiment; C is the hanging experiment; D is the immunohistochemical experiment to observe the number of TH-positive cells in the substantia nigra area after R2 treatment.

[0022] Figure 4 is for immunofluorescence to detect the expression of TERT and Iba-1 in the compound R2 group.

[0023] Figure 5 is for the release of inflammatory factors in the brain tissue and serum.

[0024] Figure 6 is for the study of the mechanism of action of compound R2 in the in vivo model of PD.

[0025] Figure 7 is for the study of the mechanism of action of compound R2 in the neuroinflammatory cell model. Specific Embodiments

[0026] The present invention will be further described in detail below in conjunction with specific embodiments. The examples given are only for clarifying the present invention, rather than limiting the scope of the present invention.

[0027] The experimental methods in the following examples are all conventional methods unless otherwise specified.

[0028] In the following examples, the quantitative tests are all set with three repeated experiments and the results are averaged unless otherwise specified.

[0029] The materials, reagents, etc. used in the following examples can all be obtained from commercial channels unless otherwise specified.

[0030] The chemical reagents used in the following examples: cycloastragenol (HPLC>98%, CAS: 78574-94-4) was purchased from ABPHYTO (Chengdu, China); MPTP and MPP + were purchased from Sigma-Aldrich (St. Louis, Missouri, USA).

[0031] 1H NMR spectra were measured on a Bruker 600 MHz AV-NEO spectrometer (Bruker, Germany) using tetramethylsilane as an internal standard in CDCl3 solution. High-resolution accurate mass spectrometry (HRMS) determinations of the compounds were obtained on a Waters G2-S QTOF. Melting points were measured on an uncorrected X-4 digital display micro melting point apparatus.

[0032] The designed synthetic route of the CAG derivatives of the present invention is as Figure 1 shown. Among them, a: carboxylic acid group, EDC / DMAP, dichloromethane, reaction at room temperature for 2 - 24 h; b: CF3COOH, dichloromethane, reaction at room temperature for 2 - 4 h.

[0033] Example 1. Preparation of Compounds A1 - A4 The structural formulas of Compounds A1 - A4 are as follows:

[0034] 1. Preparation of Compound A1 (R1 = -CH3) Dissolve CAG (2.0 mmol, 1 equivalent) in 10 mL of dichloromethane, then successively add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC, 4.0 mmol, 2 equivalents), BOC-alanine (4.0 mmol, 2 equivalents) and 4-dimethylaminopyridine (DMAP, 2.0 mmol, 1 equivalent), and stir at room temperature for 2 hours. Remove the solvent under reduced pressure at 40 °C, redissolve in ethyl acetate, extract with water three times to obtain an ethyl acetate solution, add anhydrous sodium sulfate to remove water, filter and mix with silica gel (100 - 200 mesh), and remove the solvent under reduced pressure to obtain the sample to be separated. Purify by column chromatography (silica gel is 300 - 400 mesh), and the separation condition is dichloromethane:methanol volume ratio of 40:1; remove the solvent by rotary evaporation to obtain a white powder, which is Compound A1, melting point: 236 - 238 °C.

[0035] Structure confirmation: 1 1H-NMR (CDCl3, 600 MHz, ppm): δ 0.40(d, J J = 4.2 Hz, 1H, H-19b), 0.54(d, J J = 4.2 Hz, 1H, H-19a), 0.95, 1.05, 1.11, 1.13, 1.21, 1.25, 1.29(s, 3H, -CH3×7), 1.44(s, 12H, -CH3×4), 2.34(d, J J = 7.8 Hz, 1H), 2.60(q, J= 10.2 Hz, 1H), 3.52 (t, J 1= 8.4 Hz, J 2= 11.4 Hz, 1H, H-6), 3.77 (t, J 1= 7.2 Hz, J 2= 14.4 Hz, 1H, H-24), 4.30 (t, J 1= 7.2 Hz, J 2= 13.8 Hz, 1H), 4.63 (dd, J 1= 4.2 Hz, J 2= 10.8 Hz, 1H, H-3), 4.70 (q, J = 7.8 Hz, 1H, H-16), 5.09 (d, J = 7.2 Hz, 1H); 13 C-NMR (CDCl3, 150 MHz, ppm): δ 173.17 (- C =O), 155.23 (-O C O-NH-), 87.17 (C-20), 81.42 (C-24), 81.48 (C-3), 79.84 (- C (CH3)3), 73.46 (C-16), 71.95 (C-25), 68.91 (C-6), 57.60 (C-17), 53.80 (C-5), 49.68 (- C H-NH), 47.34 (C-8), 46.65 (C-14), 46.08 (C-15), 45.06 (C-13), 40.69 (C-4), 38.18 (C-7), 34.54 (C-22), 33.01 (C-1), 31.79 (C-12), 31.66 (C-19), 29.41 (C-2), 28.49 (-C( C H3)3), 28.30 (C-10), 28.00 (C-28), 27.81 (C-21), 26.90 (C-11), 26.61 (C-27), 26.01 (C-26), 25.86 (C-23), 21.61 (C-9), 20.93 (C-18), 20.19 (C-30), 19.07 (-CH- C H3), 16.54 (C-29); ESI-HRMS (m / z): calcd for C 38 H 63 O8NNa+ [M+Na] + : 684.4451, found: 684.4451. 2. Preparation of Compound A2 (R1 = ) Dissolve CAG (2.0 mmol, 1 equivalent) in 10 mL of dichloromethane, then successively add EDC (4.0 mmol, 2 equivalents), BOC - phenylalanine (4.0 mmol, 2 equivalents), and DMAP (2.0 mmol, 1 equivalent), and stir at room temperature for 2 hours. Remove the solvent under reduced pressure at 40 °C, redissolve in ethyl acetate, extract three times with water to obtain an ethyl acetate solution. Add anhydrous sodium sulfate to remove water, filter, and mix with silica gel (100 - 200 mesh), then remove the solvent under reduced pressure to obtain the sample to be separated. Purify by column chromatography (silica gel is 300 - 400 mesh), and the separation condition is dichloromethane:methanol volume ratio 40:1; remove the solvent by rotary evaporation to obtain a white powder, which is Compound A2, with a melting point of 242 - 244 °C.

[0036] Structure Confirmation: 1 H-NMR (CDCl3, 600 MHz, ppm): δ 0.37(d, J = 4.2 Hz, 1H, H-19b), 0.52(d, J = 3.6 Hz, 1H, H-19a), 0.95, 1.00, 1.06, 1.13, 1.21, 1.25, 1.29(s, 3H, -CH3×7), 1.39(s, 9H, -CH3×3), 2.34(d, J = 7.8 Hz, 1H), 2.60(q, J = 10.2 Hz, 1H), 3.00 - 3.16(m, 2H), 3.51(t, J 1 = 3.0 Hz, J 2 = 9.6 Hz, 1H, H-6), 3.76(t, J 1 = 6.0 Hz, J 2 = 14.4 Hz, 1H, H-24), 4.55 - 4.59(m, 2H), 4.70(q, J = 7.8 Hz, 1H, H-16), 4.90(d, J = 8.4 Hz, 1H), 7.17(d, J = 8.4 Hz, 2H, AR-H), 7.21 - 7.23(m, 1H, AR-H), 7.28(t, J1 = 7.2 Hz, J 2 = 14.4 Hz, 2H, AR-H); 13 C-NMR (CDCl3, 150 MHz, ppm): δ 173.17(- C =O), 155.22(- C O-NH-), 136.20, 129.51, 128.65, 127.08(AR-C), 87.15(C-20), 81.46(C-24), 81.92(C-3), 79.93(-C(CH3)3), 73.45(C-16), 71.94(C-25), 68.85(C-6), 57.59(C-17), 54.74(-CH2- C H-NH-), 53.76(C-5), 47.31(C-8), 46.64(C-14), 46.07(C-15), 45.03(C-13), 40.54(C-4), 38.54(-C6H5- C H2-CH-), 38.12(C-7), 34.54(C-22), 33.00(C-1), 31.77(C-12), 31.63(C-19), 29.83(C-2), 29.35(C-10), 28.44(-C( C H3)3), 28.02(C-28), 27.80(C-21), 26.76(C-11), 26.60(C-27), 25.99(C-26), 25.89(C-23), 21.59(C-9), 20.91(C-18), 20.19(C-30), 16.56(C-29); ESI-HRMS (m / z): calcd for C 44 H 67 O8NNa + [M + Na] + : 760.4764, found: 760.4761. 3. Preparation of Compound A3 (R1 = H) Dissolve CAG (2.0 mmol, 1 equivalent) in 10 mL of dichloromethane, then successively add EDC (4.0 mmol, 2 equivalents), BOC-glycine (4.0 mmol, 2 equivalents) and DMAP (2.0 mmol, 1 equivalent), and stir at room temperature for 2 hours. Remove the solvent under reduced pressure at 40 °C, redissolve in ethyl acetate, extract three times with water to obtain an ethyl acetate solution, add anhydrous sodium sulfate to remove water, filter and mix with silica gel (100 - 200 mesh), and remove the solvent under reduced pressure to obtain the sample to be separated. Purify by column chromatography (silica gel is 300 - 400 mesh), and the separation condition is dichloromethane:methanol volume ratio 30:1; remove the solvent by rotary evaporation to obtain a white powder, which is compound A3, with a melting point of 231 - 233 °C.

[0037] Structure confirmation: 1 1H-NMR (CDCl3, 600 MHz, ppm): δ 0.39 (d, J J = 4.8 Hz, 1H, H-19b), 0.54 (d, J J = 4.2 Hz, 1H, H-19a), 0.95, 1.04, 1.12, 1.14, 1.23, 1.26, 1.30 (s, 3H, -CH3×7), 1.45 (s, 9H, -CH3×3), 2.33 (d, J J = 7.8 Hz, 1H), 2.59 (q, J J = 10.2 Hz, 1H), 3.53 (t, J J1 = 8.4 Hz, J J2 = 17.4 Hz, 1H, H-6), 3.76 (t, J J1 = 7.2 Hz, J J2 = 14.4 Hz, 1H, H-24), 3.87 - 3.95 (m, 2H, -NH-C H 2-CO-), 4.63 - 4.70 (m, 2H, H-16 and H-3), 5.00 (s, 1H); 13 13C-NMR (CDCl3, 150 MHz, ppm): δ 170.26 (- C C=O), 155.80 (- C O-NH-), 87.20 (C-20), 81.51 (C-24), 81.65 (C-3), 80.05 (- C(CH3)3), 73.47 (C-16), 71.96 (C-25), 68.93 (C-6), 57.59 (C-17), 53.85 (C-5), 47.22 (C-8), 46.59 (C-14), 46.09 (C-15), 45.11 (C-13), 42.78 (-NH- C H2-CO), 40.62 (C-4), 38.12 (C-7), 34.54 (C-22), 33.05 (C-1), 31.80 (C-12), 31.58 (C-19), 29.84 (C-2), 29.35 (C-10), 28.47 (-C( C H3)3), 28.32 (C-28), 27.85 (C-21), 26.88 (C-11), 26.62 (C-27), 26.00 (C-26), 25.77 (C-23), 21.61 (C-9), 20.95 (C-18), 20.18 (C-30), 16.47 (C-29); ESI-HRMS (m / z): calcd for C 37 H 61 O8NNa + [M+Na] + : 670.4295, found: 670.4297. 4. Preparation of Compound A4 (R1 = ) Dissolve CAG (2.0 mmol, 1 equiv) in 10 mL of dichloromethane, then successively add EDC (4.0 mmol, 2 equiv), BOC-valine (4.0 mmol, 2 equiv) and DMAP (2.0 mmol, 1 equiv), and stir at room temperature for 2 hours. Remove the solvent under reduced pressure at 40 °C, redissolve in ethyl acetate, extract three times with water to obtain an ethyl acetate solution, add anhydrous sodium sulfate to remove water, filter, mix with silica gel (100 - 200 mesh), and remove the solvent under reduced pressure to obtain the sample to be separated. Purify by column chromatography (silica gel 300 - 400 mesh), and the separation condition is dichloromethane:methanol volume ratio 30:1; remove the solvent by rotary evaporation to obtain a white powder, which is Compound A4, with a melting point of 235 - 237 °C.

[0038] Structure confirmation: 1 1H-NMR (CDCl3, 600 MHz, ppm): δ 0.39 (d, J J = 4.8 Hz, 1H, H-19b), 0.53 (d, J= 3.6 Hz, 1H, H-19a), 0.87(d, J = 6.6 Hz, 3H, C H 3-CH-CH3), 0.99(d, J = 6.6Hz, 3H, CH3-CH-C H 3), 0.94, 1.04, 1.20, 1.24, 1.28(s, 3H, -CH3×5), 1.12(s, 3H,-CH3×2),1.45(s, 9H, -CH3×3), 1.43(s, 9H, -C(C H 3)3), 2.33(d, J = 7.8 Hz, 1H),2.60(q, J = 10.2 Hz, 1H), 3.51(t, J 1= 8.4 Hz, J 2= 16.8 Hz, 1H, H-6), 3.74(t, J 1=8.4 Hz, J 2= 13.8 Hz, 1H, H-24), 4.23(dd, J 1= 4.2 Hz, J 2= 9.6 Hz, 1H, -NH-C H -CO-),4.61(dd, J 1= 5.4 Hz, J 2= 10.8 Hz, 1H, H-3), 4.69(q, J = 7.8 Hz, 1H, H-16), 5.01(d, J = 9.6 Hz, 1H); 13 C-NMR (CDCl3, 150 MHz, ppm): δ 172.24(- C =O), 155.92(- C O-NH-),87.13(C-20), 81.43(C-24), 81.69(C-3), 79.78(- C (CH3)3), 73.45(C-16), 71.94(C-25), 68.83(C-6), 59.98(-NH- CH-CO-), 57.59 (C-17), 53.75 (C-5), 47.39 (C-8), 46.67 (C-14), 46.05 (C-15), 44.99 (C-13), 40.53 (C-4), 38.15 (C-7), 34.52 (C-22), 32.96 (C-1), 31.79 (C-12), 31.70 (C-19), 31.25 (C-2), 29.41 (C-10), 28.47 (-C( C H3)3), 28.40 (C-28), 28.06 (CH3- C H-CH3), 27.75 (C-21), 26.98 (C-11), 26.57 (C-27), 26.01 (C-26), 25.94 (C-23), 21.58 (C-9), 20.88 (C-18), 20.19 (C-30), 19.52 (( C H3)2CH-), 16.63 (C-29); ESI-HRMS (m / z): calcd for C 40 H 67 O8NNa + [M+Na] + : 712.4764, found: 712.4765. Example 2. Preparation of Compounds A5 - A8 The structural formulas of Compounds A5 - A8 are as follows:

[0039] 1. Preparation of Compound A5 (R1 = -CH3) Dissolve Compound A1 (100 mg) in 10 mL of dichloromethane, then add CF3COOH (100 μL), and stir at room temperature for 4 hours. Remove the solvent under reduced pressure at 40 °C, redissolve in ethyl acetate, extract three times with water to obtain an ethyl acetate solution. Add anhydrous sodium sulfate to remove water, filter, mix with silica gel (100 - 200 mesh), and remove the solvent under reduced pressure to obtain the sample to be separated. Purify by column chromatography (silica gel 300 - 400 mesh), and the separation condition is dichloromethane:methanol volume ratio 30:1; remove the solvent by rotary evaporation to obtain a white powder, which is Compound A5, melting point: 230 - 232 °C.

[0040] Structure Confirmation: 1 1H-NMR (CDCl3, 600 MHz, ppm): δ 0.39 (d, J = 4.2 Hz, 1H, H-19b), 0.54 (d,J = 4.2 Hz, 1H, H-19a), 0.95, 1.05, 1.11, 1.14, 1.22, 1.25, 1.29(s, 3H, -CH3×7), 1.44(s, 3H, -CH3), 2.34(d, J = 7.8 Hz, 1H), 2.60(q, J = 10.2 Hz, 1H), 3.53(t, J 1= 3.6 Hz, J 2= 10.2 Hz, 1H, H-6), 3.76(t, J 1= 7.8 Hz, J 2= 14.4 Hz, 1H, H-24), 4.30(t, J 1= 6.6 Hz, J 2= 13.8 Hz, 1H), 4.63(dd, J 1= 4.8 Hz, J 2= 10.8 Hz, 1H, H-3), 4.70(q, J = 7.8 Hz, 1H, H-16), 5.09(d, J = 7.2 Hz, 1H); 13 C-NMR (CDCl3, 150MHz, ppm): δ 173.17(- C =O), 87.19(C-20), 81.41(C-24), 81.50(C-3), 73.47(C-16), 71.96(C-25), 68.95(C-6), 57.60(C-17), 53.84(C-5), 49.68(- CH-NH2), 47.27 (C-8), 46.61 (C-14), 46.09 (C-15), 45.10 (C-13), 40.69 (C-4), 38.16 (C-7), 34.54 (C-22), 33.54 (C-1), 31.79 (C-12), 31.60 (C-19), 29.84 (C-2), 29.39 (C-10), 28.29 (C-28), 27.84 (C-21), 26.90 (C-11), 26.60 (C-27), 26.00 (C-26), 25.80 (C-23), 21.61 (C-9), 20.96 (C-18), 20.19 (C-30), 19.07 (-CH-CH3), 16.54 (C-29); ESI-HRMS (m / z): calcd for C 33 H 55 O6NNa + [M+Na] + : 584.3927, found: 584.3920. 2. Preparation of Compound A6 (R1 = ) Dissolve Compound A2 (100 mg) in 10 mL of dichloromethane, then add CF3COOH (100 μL), and stir at room temperature for 4 hours. Remove the solvent under reduced pressure at 40 °C, redissolve in ethyl acetate, extract three times with water to obtain an ethyl acetate solution. Add anhydrous sodium sulfate to remove water, filter, mix with silica gel (100 - 200 mesh), and remove the solvent under reduced pressure to obtain the sample to be separated. Purify by column chromatography (silica gel 300 - 400 mesh), and the separation condition is dichloromethane:methanol volume ratio 30:1; remove the solvent by rotary evaporation to obtain a white powder, which is Compound A6, with a melting point of 240 - 242 °C.

[0041] Structure Confirmation: 1 1H-NMR (CDCl3, 600 MHz, ppm): δ 0.37 (d, J J = 4.2 Hz, 1H, H-19b), 0.53 (d, J J = 3.0 Hz, 1H, H-19a), 0.95, 1.00, 1.06, 1.15, 1.23, 1.26, 1.30 (s, 3H, -CH3×7), 2.34 (d, J J = 7.8 Hz, 1H), 2.60 (q, J J = 10.2 Hz, 1H), 3.00 - 3.16 (m, 2H), 3.50 (t, J1 = 7.8 Hz, J 2 = 16.8 Hz, 1H, H-6), 3.77(t, J 1 = 7.2 Hz, J 2 = 14.4 Hz, 1H, H-24), 4.55 - 4.59(m, 2H), 4.70(q, J = 7.2 Hz, 1H, H-16), 4.90(d, J = 8.4 Hz, 1H), 7.17(d, J = 7.2 Hz, 2H, AR-H), 7.22 - 7.24(m, 1H, AR-H), 7.30(t, J 1 = 7.8 Hz, J 2 = 15.6 Hz, 2H, AR-H); 13 C-NMR (CDCl3, 150 MHz, ppm): δ 171.95(- C = O), 136.21, 129.53, 128.67, 127.10(AR-C), 87.21(C-20), 81.51(C-24), 81.89(C-3), 73.48(C-16), 76.96(C-25), 68.94(C-6), 57.59(C-17), 54.76(-CH2- C H-NH2), 53.86(C-5), 47.17(C-8), 46.58(C-14), 46.09(C-15), 45.13(C-13), 40.55(C-4), 38.56(C6H5- C H2-CH-), 38.11(C-7), 34.55(C-22), 33.06(C-1), 31.77(C-12), 31.51(C-19), 29.85(C-2), 29.32(C-10), 27.92(C-28), 27.86(C-21), 26.77(C-11), 26.62(C-27), 25.98(C-26), 25.75(C-23), 21.61(C-9), 20.96(C-18), 20.18(C-30), 16.57(C-29); ESI-HRMS(m / z): calcd for C 39 H 60 O6NNa + [M + Na] +: 638.4421, found: 638.4414. 3. Preparation of Compound A7 (R1 = H) Dissolve Compound A3 (100 mg) in 10 mL of dichloromethane, then add CF3COOH (100 μL), and stir at room temperature for 4 hours. Remove the solvent under reduced pressure at 40 °C, redissolve in ethyl acetate, extract three times with water to obtain an ethyl acetate solution, add anhydrous sodium sulfate to remove water, filter, mix with silica gel (100 - 200 mesh), and remove the solvent under reduced pressure to obtain the sample to be separated. Purify by column chromatography (silica gel is 300 - 400 mesh), and the separation condition is dichloromethane:methanol volume ratio 30:1; remove the solvent by rotary evaporation to obtain a white powder, which is Compound A7, with a melting point of 226 - 228 °C.

[0042] Structure confirmation: 1 1H-NMR (CDCl3, 600 MHz, ppm): δ 0.38(d, J J = 4.2 Hz, 1H, H-19b), 0.53(d, J J = 4.2 Hz, 1H, H-19a), 0.94, 1.03, 1.11, 1.13, 1.21, 1.25, 1.29(s, 3H, -CH3×7), 2.33(d, J J = 7.8 Hz, 1H), 2.60(q, J J = 10.8 Hz, 1H), 3.51(t, J J1 = 8.4 Hz, J J2 = 16.8 Hz, 1H, H-6), 3.75(t, J J1 = 7.8 Hz, J J2 = 14.4 Hz, 1H, H-24), 3.87 - 3.94(m, 2H, -NH-C H H2-CO-), 4.61 - 4.69(m, 2H, H-16 and H-3), 5.02(s, 1H); 13 13C-NMR (CDCl3, 150 MHz, ppm): δ 170.25(- C=O), 87.15 (C-20), 81.46 (C-24), 81.67 (C-3), 73.45 (C-16), 71.94 (C-25), 68.83 (C-6), 57.59 (C-17), 53.74 (C-5), 47.33 (C-8), 46.63 (C-14), 46.06 (C-15), 45.02 (C-13), 42.76 (NH2- C H2-CO), 40.61 (C-4), 38.13 (C-7), 34.53 (C-22), 32.99 (C-1), 31.79 (C-12), 31.67 (C-19), 29.83 (C-2), 29.38 (C-10), 28.33 (C-28), 27.78 (C-21), 26.87 (C-11), 26.59 (C-27), 26.01 (C-26), 25.89 (C-23), 21.59 (C-9), 20.90 (C-18), 20.18 (C-30), 16.46 (C-29); ESI-HRMS (m / z): calcd for C 32 H 53 O6NNa + [M+Na] + : 570.3771, found: 570.3768. 4. Preparation of Compound A8 (R1 = ). Dissolve Compound A4 (100 mg) in 10 mL of dichloromethane, then add CF3COOH (100 μL), and stir at room temperature for 2 hours. Remove the solvent under reduced pressure at 40 °C, redissolve in ethyl acetate, extract three times with water to obtain an ethyl acetate solution. Add anhydrous sodium sulfate to remove water, filter, mix with silica gel (100 - 200 mesh), and remove the solvent under reduced pressure to obtain the sample to be separated. Purify by column chromatography (silica gel is 300 - 400 mesh), and the separation condition is dichloromethane:methanol volume ratio 30:1; remove the solvent by rotary evaporation to obtain a white powder, which is Compound A8, with a melting point of 234 - 236 °C.

[0043] Structure confirmation: 1 1H-NMR (CDCl3, 600 MHz, ppm): δ 0.39 (d, J J = 4.2 Hz, 1H, H-19b), 0.54 (d, J J = 4.2 Hz, 1H, H-19a), 0.86 - 0.88 (m, 3H, C H3-CH-CH3), 0.99(d, J = 6.6 Hz, 3H, CH3-CH-C H 3), 0.95, 1.05, 1.12, 1.23, 1.21, 1.25, 1.29(s, 3H, -CH3×7), 2.34(d, J = 7.8 Hz, 1H), 2.60(q, J = 10.2 Hz, 1H), 3.52(t, J 1 = 3.0 Hz, J 2 = 9.6 Hz, 1H, H-6), 3.74(t, J 1 = 7.8 Hz, J 2 = 14.4 Hz, 1H, H-24), 4.20 - 4.24(m, 1H), 4.30(t, J 1 = 7.2 Hz, J 2 = 13.8 Hz, 1H), 4.61(dd, J 1 = 4.8 Hz, J 2 = 10.8 Hz, 1H, H-3), 4.70(q, J = 7.8 Hz, 1H, H-16), 5.00(d, J = 9.0 Hz, 1H); 13 C-NMR (CDCl3, 150 MHz, ppm): δ172.23(- C =O), 87.15(C-20), 81.46(C-24), 81.66(C-3), 73.45(C-16), 71.94(C-25), 68.87(C-6), 58.99(NH2- C H-CO-), 57.59(C-17), 53.80(C-5), 47.34(C-8), 46.65(C-14), 46.06(C-15), 45.03(C-13), 40.53(C-4), 38.16(C-7), 34.53(C-22), 32.99(C-1), 31.79(C-12), 31.65(C-19), 31.25(C-2), 29.40(C-10), 28.39(C-28), 28.01(- CH(CH3)2), 27.78 (C-21), 26.98 (C-11), 26.58 (C-27), 26.01 (C-26), 25.89 (C-23), 21.59 (C-9), 20.91 (C-18), 20.18 (C-30), 19.31 (-CH( C H3)2), 16.64 (C-29); ESI-HRMS (m / z): calcd for C 35 H 60 O6N [M] + : 590.4421, found: 590.4420. Example 3. Preparation of Compounds B1 - B4 The structural formulas of Compounds B1 - B4 are as follows:

[0044] 1. Preparation of Compound B1 (R3 = H) Dissolve CAG (2.0 mmol, 1 equivalent) in 10 mL of dichloromethane, then successively add EDC (4.0 mmol, 2 equivalents), benzoic acid (4.0 mmol, 2 equivalents), and DMAP (2.0 mmol, 1 equivalent), and stir at room temperature for 24 hours. Remove the solvent under reduced pressure at 40 °C, redissolve in ethyl acetate, and extract with water three times to obtain an ethyl acetate solution. Add anhydrous sodium sulfate to remove water, filter, and mix with silica gel (100 - 200 mesh), then remove the solvent under reduced pressure to obtain the sample to be separated. Purify by column chromatography (silica gel is 300 - 400 mesh), and the separation condition is dichloromethane:methanol volume ratio of 30:1; remove the solvent by rotary evaporation to obtain a white powder, which is Compound B1, with a melting point of 229 - 231 °C.

[0045] Structure Confirmation: 1 1H-NMR (CDCl3, 600 MHz, ppm): δ 0.43 (d, J J = 4.2 Hz, 1H, H-19b), 0.59 (d, J J = 4.2 Hz, 1H, H-19a), 0.98, 1.16, 1.21, 1.24, 1.25, 1.28, 1.31 (s, 3H, -CH3×7), 2.34 (d, J J = 7.8 Hz, 1H), 2.58 (q, J J = 10.2 Hz, 1H), 3.57 (td, J J1 = 2.4 Hz, J2 = 9.6 Hz, 1H, H-6), 3.75 (t, J 1 = 7.2 Hz, J 2 = 14.4 Hz, 1H, H-24), 4.70 (q, J = 7.2 Hz, 1H, H-16), 4.82 (dd, J 1 = 4.8 Hz, J 2 = 10.8 Hz, 1H, H-3), 7.45 (t, J 1 = 7.2 Hz, J 2 = 14.4 Hz, 2H, AR-H), 7.55 (t, J 1 = 7.2 Hz, J 2 = 14.4 Hz, 1H, AR-H), 8.06 (d, J = 7.8 Hz, 2H, AR-H); 13 C-NMR (CDCl3, 150 MHz, ppm): δ 166.39 (- C = O), 132.92, 131.00, 129.70, 128.49 (AR-C), 87.24 (C-20), 80.97 (C-24), 81.55 (C-3), 73.51 (C-16), 71.98 (C-25), 69.09 (C-6), 57.61 (C-17), 54.04 (C-5), 47.25 (C-8), 46.60 (C-14), 46.15 (C-15), 45.18 (C-13), 40.94 (C-4), 38.14 (C-7), 34.56 (C-22), 33.11 (C-1), 31.91 (C-12), 31.61 (C-19), 29.85 (C-2), 29.51 (C-10), 28.45 (C-28), 27.89 (C-21), 27.00 (C-11), 26.62 (C-27), 26.03 (C-26), 25.72 (C-23), 21.66 (C-9), 21.02 (C-18), 20.22 (C-30), 16.81 (C-29); ESI-HRMS (m / z): calcd for C 37 H 54 O6Na + [M + Na] + : 617.3818, found: 617.3822. 2. Preparation of Compound B2 (R3 = 4’F) Dissolve CAG (2.0 mmol, 1 equivalent) in 10 mL of dichloromethane, then successively add EDC (4.0 mmol, 2 equivalents), 4-fluorobenzoic acid (4.0 mmol, 2 equivalents) and DMAP (2.0 mmol, 1 equivalent), and stir at room temperature for 24 hours. Remove the solvent under reduced pressure at 40 °C, redissolve in ethyl acetate, extract three times with water to obtain an ethyl acetate solution, add anhydrous sodium sulfate to remove water, filter and mix with silica gel (100 - 200 mesh), and remove the solvent under reduced pressure to obtain the sample to be separated. Purify by column chromatography (silica gel is 300 - 400 mesh), and the separation condition is petroleum ether:acetone volume ratio 5:1; remove the solvent by rotary evaporation to obtain a white powder, which is compound B2, with a melting point of 228 - 230 °C.

[0046] Structure confirmation: 1 1H-NMR (CDCl3, 600 MHz, ppm): δ 0.43 (d, J J = 4.2 Hz, 1H, H-19b), 0.59 (d, J J = 3.6 Hz, 1H, H-19a), 0.97, 1.15, 1.23, 1.27, 1.30 (s, 3H, -CH3×5), 1.19 (s, 3H, -CH3×2), 2.36 (d, J J = 7.8 Hz, 1H), 2.59 (q, J J = 10.8 Hz, 1H), 3.57 (td, J J1 = 3.0 Hz, J J2 = 9.6 Hz, 1H, H-6), 3.76 (t, J J1 = 7.2 Hz, J J2 = 13.8 Hz, 1H, H-24), 4.72 (q, J J = 7.2 Hz, 1H, H-16), 4.81 (dd, J J1 = 3.6 Hz, J J2 = 10.2 Hz, 1H, H-3), 7.11 (t, J J1 = 9.0 Hz, J J2 = 16.8 Hz, 2H, AR-H), 8.05 (t, J J1 = 7.8 Hz, J J2 = 13.2 Hz, 2H, AR-H); 13 13C-NMR (CDCl3, 150 MHz, ppm): δ 165.44 (- C=O), 132.21, 132.15, 127.22, 127.20, 115.68, 115.53(AR-C), 87.20(C-20), 81.20(C-24), 81.50(C-3), 73.51(C-16), 72.01(C-25), 69.02(C-6), 57.60(C-17), 53.94(C-5), 47.33(C-8), 46.58(C-14), 46.13(C-15), 45.11(C-13), 40.92(C-4), 38.18(C-7), 34.56(C-22), 33.05(C-1), 31.89(C-12), 31.69(C-19), 30.50(C-2), 29.52(C-10), 28.45(C-28), 27.81(C-21), 27.00(C-11), 26.60(C-27), 26.03(C-26), 25.83(C-23), 21.64(C-9), 21.00(C-18), 20.21(C-30), 16.80(C-29); ESI-HRMS (m / z): calcd for C 37 H 53 O6FNa + [M+Na] + : 635.3724, found: 635.3728. 3. Preparation of Compound B3 (R3 = 4’Cl) Dissolve CAG (2.0 mmol, 1 equiv) in 10 mL of dichloromethane, then successively add EDC (4.0 mmol, 2 equiv), 4-chlorobenzoic acid (4.0 mmol, 2 equiv) and DMAP (2.0 mmol, 1 equiv), and stir at room temperature for 24 hours. Remove the solvent under reduced pressure at 40 °C, redissolve in ethyl acetate, extract three times with water to obtain an ethyl acetate solution, add anhydrous sodium sulfate to remove water, filter, mix with silica gel (100 - 200 mesh), and remove the solvent under reduced pressure to obtain the sample to be separated. Purify by column chromatography (silica gel 300 - 400 mesh), the separation condition is petroleum ether:acetone volume ratio 5:1; remove the solvent by rotary evaporation to obtain a white powder, which is Compound B3, melting point 228 - 230 °C.

[0047] Structure Confirmation: 1 1H-NMR (CDCl3, 600 MHz, ppm): δ 0.43(d, J = 4.8 Hz, 1H, H-19b), 0.59(d, J= 4.2 Hz, 1H, H-19a), 0.97, 1.15, 1.23, 1.27, 1.31(s, 3H, -CH3×5),1.18(s, 3H, -CH3×2), 2.36(d, J = 8.4 Hz, 1H), 2.61(q, J = 10.2 Hz, 1H), 3.57(td, J 1= 3.0 Hz, J 2= 9.6 Hz, 1H, H-6), 3.76(t, J 1= 7.8 Hz, J 2= 14.4 Hz, 1H, H-24),4.72(q, J = 7.8 Hz, 1H, H-16), 4.81(dd, J 1= 4.8 Hz, J 2= 10.8 Hz, 1H, H-3), 7.41(t, J 1= 7.8 Hz, J 2= 15.6 Hz, 2H, AR-H), 7.97-8.01(m, 2H, AR-H); 13 C-NMR (CDCl3, 150MHz, ppm): δ 165.55(- C=O), 139.55, 131.67, 131.07, 129.42, 128.88, 128.83(AR-C), 87.21(C-20), 81.36(C-24), 81.48(C-3), 73.55(C-16), 72.06(C-25), 69.02(C-6), 57.59(C-17), 53.89(C-5), 47.34(C-8), 46.50(C-14), 46.13(C-15), 45.10(C-13), 40.92(C-4), 38.17(C-7), 34.56(C-22), 33.03(C-1), 31.88(C-12), 31.68(C-19), 29.51(C-2), 28.45(C-10), 28.03(C-28), 27.77(C-21), 26.98(C-11), 26.60(C-27), 26.03(C-26), 25.87(C-23), 21.63(C-9), 20.99(C-18), 20.21(C-30), 16.80(C-29); ESI-HRMS (m / z): calcd for C 37 H 53 O6ClNa + [M+Na] + : 651.3428, found: 651.3431. 4. Preparation of Compound B4 (R3 = 4’Br) Dissolve CAG (2.0 mmol, 1 equiv) in 10 mL of dichloromethane, then sequentially add EDC (4.0 mmol, 2 equiv), 4-bromobenzoic acid (4.0 mmol, 2 equiv) and DMAP (2.0 mmol, 1 equiv), and stir at room temperature for 24 h. Remove the solvent under reduced pressure at 40 °C, redissolve in ethyl acetate, extract three times with water to obtain an ethyl acetate solution, add anhydrous sodium sulfate to remove water, filter and mix with silica gel (100 - 200 mesh), and remove the solvent under reduced pressure to obtain the sample to be separated. Purify by column chromatography (silica gel 300 - 400 mesh), and the separation condition is dichloromethane:methanol volume ratio 30:1; remove the solvent by rotary evaporation to obtain a white powder, which is Compound B4, with a melting point of 234 - 236 °C.

[0048] Structure confirmation: 1 H-NMR (CDCl3, 600 MHz, ppm): δ 0.43(d, J = 4.2 Hz, 1H, H-19b),0.59(d, J= 4.2 Hz, 1H, H-19a), 0.97, 1.14, 1.22, 1.27, 1.30(s, 3H, -CH3×5),1.18(s, 3H, -CH3×2), 2.36(d, J = 7.8 Hz, 1H), 2.61(q, J = 10.2 Hz, 1H), 3.57(td, J 1= 3.6 Hz, J 2= 10.2 Hz, 1H, H-6), 3.75(t, J 1= 7.8 Hz, J 2= 14.4 Hz, 1H, H-24),4.72(q, J = 7.8 Hz, 1H, H-16), 4.81(dd, J 1= 4.2 Hz, J 2= 10.8 Hz, 1H, H-3), 7.58(d, J = 8.4 Hz, 2H, AR-H), 7.90(d, J = 8.4 Hz, 2H, AR-H); 13 C-NMR (CDCl3, 150 MHz,ppm): δ 165.67(- C =O), 131.83, 131.22, 129.88, 128.00(AR-C), 87.20(C-20),81.40(C-24), 81.49(C-3), 73.53(C-16), 72.04(C-25), 69.00(C-6), 57.59(C-17),53.88(C-5), 47.35(C-8), 46.54(C-14), 46.12(C-15), 45.09(C-13), 40.91(C-4),38.18(C-7), 34.56(C-22), 33.03(C-1), 31.88(C-12), 31.70(C-19), 29.51(C-2),28.45(C-10), 28.02(C-28), 27.78(C-21), 26.97(C-11), 26.60(C-27), 26.03(C-26),25.87(C-23), 21.63(C-9), 20.99(C-18), 20.21(C-30), 16.80(C-29); ESI-HRMS (m / z): calcd for C 37 H53 O6BrNa + [M+Na] + : 695.2923, found: 695.2927. Example 4, Preparation of Compounds C1 - C11 The structural formulas of Compounds C1 - C11 are as follows:

[0049] 1. Preparation of Compound C1 (R4 = H) Dissolve CAG (2.0 mmol, 1 equivalent) in 10 mL of dichloromethane, then successively add EDC (4.0 mmol, 2 equivalents), salicylic acid (4.0 mmol, 2 equivalents) and DMAP (2.0 mmol, 1 equivalent), and stir at room temperature for 24 hours. Remove the solvent under reduced pressure at 40 °C, redissolve in ethyl acetate, extract with water three times to obtain an ethyl acetate solution, add anhydrous sodium sulfate to remove water, filter and mix with silica gel (100 - 200 mesh), and remove the solvent under reduced pressure to obtain the sample to be separated. Purify by column chromatography (silica gel is 300 - 400 mesh), and the separation condition is dichloromethane:methanol volume ratio 40:1; remove the solvent by rotary evaporation to obtain a white powder, which is Compound C1, with a melting point of 240 - 242 °C.

[0050] Structure Confirmation: 1 H-NMR (CDCl3, 600 MHz, ppm): δ 0.44(d, J = 3.6 Hz, 1H, H-19b),0.60(d, J = 3.6 Hz, 1H, H-19a), 0.98, 1.51, 1.23, 1.28, 1.31(s, 3H, -CH3×5),1.21(s, 3H, -CH3×2), 2.35(d, J = 7.8 Hz, 1H), 2.61(q, J = 10.8 Hz, 1H), 3.57(td, J 1= 9.0 Hz, J 2= 18.0 Hz, 1H, H-6), 3.76(t, J 1= 7.2 Hz, J 2= 14.4 Hz, 1H, H-24),4.71(q, J = 7.2 Hz, 1H, H-16), 4.86(dd, J 1= 4.2 Hz, J2 = 10.8 Hz, 1H, H-3), 6.89(t, J 1 = 7.2 Hz, J 2 = 14.4 Hz, 1H, AR-H), 6.99(d, J = 8.4 Hz, 1H, AR-H), 7.45(t, J 1 = 7.8Hz, J 2 = 15.0 Hz, 1H, AR-H), 7.86(d, J = 7.8 Hz, 1H, AR-H), 10.92(s, 1H, AR-H); 13 C-NMR (CDCl3, 150 MHz, ppm): δ 170.03(- C =O), 161.88, 135.67, 129.88, 119.25, 117.74, 113.11(AR-C), 87.20(C-20), 81.51(C-24), 81.72(C-3), 73.84(C-16), 71.98(C-25), 68.97(C-6), 57.61(C-17), 53.92(C-5), 47.30(C-8), 46.62(C-14), 46.11(C-15), 45.12(C-13), 40.91(C-4), 38.21(C-7), 34.55(C-22), 33.05(C-1), 31.85(C-12), 31.65(C-19), 29.43(C-2), 28.46(C-10), 27.96(C-28), 27.86(C-21), 26.95(C-11), 26.62(C-27), 26.04(C-26), 25.80(C-23), 21.64(C-9), 21.04(C-18), 20.22(C-30), 16.77(C-29); ESI-HRMS (m / z): calcd for C 37 H 54 O7Na + [M+Na] + : 633.3767, found: 633.3771. 2. Preparation of Compound C2 (R4 = 5’CH3) Dissolve CAG (2.0 mmol, 1 equiv.) in 10 mL of dichloromethane, then successively add EDC (4.0 mmol, 2 equiv.), 5-methylsalicylic acid (4.0 mmol, 2 equiv.) and DMAP (2.0 mmol, 1 equiv.), and stir at room temperature for 24 h. Remove the solvent under reduced pressure at 40 °C, redissolve in ethyl acetate, extract three times with water to obtain an ethyl acetate solution, add anhydrous sodium sulfate to remove water, filter, mix with silica gel (100 - 200 mesh), and remove the solvent under reduced pressure to obtain the sample to be separated. Purify by column chromatography (silica gel 300 - 400 mesh), and the separation condition is dichloromethane:methanol volume ratio 50:1; remove the solvent by rotary evaporation to obtain a white powder, which is compound C2, with a melting point of 246 - 248 °C.

[0051] Structure confirmation: 1 1H-NMR (CDCl3, 600 MHz, ppm): δ 0.44 (d, J J = 4.2 Hz, 1H, H-19b), 0.60 (d, J J = 4.2 Hz, 1H, H-19a), 0.98, 1.15, 1.24, 1.28, 1.31 (s, 3H, -CH3×5), 1.20 (s, 3H, -CH3×2), 2.27 (s, 3H, -CH3, AR-CH3), 2.35 (d, J J = 7.8 Hz, 1H), 2.61 (q, J J = 10.2 Hz, 1H), 3.57 (td, J J1 = 2.4 Hz, J J2 = 9.6 Hz, 1H, H-6), 3.76 (t, J J1 = 7.8 Hz, J J2 = 15.0 Hz, 1H, H-24), 4.71 (q, J J = 7.2 Hz, 1H, H-16), 4.86 (dd, J J1 = 4.2 Hz, J J2 = 10.8 Hz, 1H, H-3), 6.79 (t, J J1 = 7.8 Hz, J J2 = 15.6 Hz, 1H, AR-H), 7.32 (d, J J = 7.2 Hz, 1H, AR-H), 7.71 (d, J J = 8.4 Hz, 1H, AR-H), 11.17 (s, 1H, AR-OH); 1313C-NMR (CDCl3, 150 MHz, ppm): δ 170.47 (- C =O), 160.30, 136.41, 127.39, 126.78, 118.57, 112.37 (AR-C), 87.22 (C-20), 81.53 (C-24), 81.59 (C-3), 73.48 (C-16), 71.98 (C-25), 68.99 (C-6), 57.61 (C-17), 53.96 (C-5), 47.25 (C-8), 46.61 (C-14), 46.12 (C-15), 45.14 (C-13), 40.92 (C-4), 38.19 (C-7), 34.56 (C-22), 33.08 (C-1), 31.86 (C-12), 31.62 (C-19), 29.43 (C-2), 28.44 (C-10), 27.92 (C-28), 27.87 (C-21), 26.94 (C-11), 26.63 (C-27), 26.03 (C-26), 25.76 (C-23), 21.65 (C-9), 21.05 (C-18), 20.22 (C-30), 16.77 (C-29), 15.83 (AR-CH3); ESI-HRMS (m / z): calcd for C 38 H 56 O7Na + [M + Na] + : 647.3924, found: 647.3927. 3. Preparation of Compound C3 (R4 = 4’CH3) Dissolve CAG (2.0 mmol, 1 equiv) in 10 mL of dichloromethane, then successively add EDC (4.0 mmol, 2 equiv), 4-methylsalicylic acid (4.0 mmol, 2 equiv) and DMAP (2.0 mmol, 1 equiv), and stir at room temperature for 24 hours. Remove the solvent under reduced pressure at 40 °C, redissolve in ethyl acetate, extract with water three times to obtain an ethyl acetate solution, add anhydrous sodium sulfate to remove water, filter and mix with silica gel (100 - 200 mesh), and remove the solvent under reduced pressure to obtain the sample to be separated. Purify by column chromatography (silica gel 300 - 400 mesh), the separation condition is dichloromethane:methanol volume ratio 50:1; remove the solvent by rotary evaporation to obtain a white powder, which is Compound C3, melting point 238 - 240 °C.

[0052] Structure confirmation: 1H-NMR (CDCl3, 600 MHz, ppm): δ 0.44 (d, J J = 4.2 Hz, 1H, H-19b), 0.60 (d, J J = 4.2 Hz, 1H, H-19a), 0.98, 1.16, 1.24, 1.28, 1.31 (s, 3H, -CH3×5), 1.20 (s, 3H, -CH3×2), 2.34 (s, 3H, AR-CH3), 2.60 (q, J J = 9.6 Hz, 1H), 3.57 (td, J J1 = 3.0 Hz, J J2 = 9.6 Hz, 1H, H-6), 3.77 (t, J J1 = 7.2 Hz, J J2 = 15.0 Hz, 1H, H-24), 4.71 (q, J J = 7.8 Hz, 1H, H-16), 4.84 (dd, J J1 = 4.2 Hz, J J2 = 10.8 Hz, 1H, H-3), 6.70 (d, J J = 7.8 Hz, 1H, AR-H), 6.79 (s, 1H, AR-H), 7.73 (d, J J = 7.8 Hz, 1H, AR-H), 10.86 (s, 1H, AR-OH); 13 C-NMR (CDCl3, 150 MHz, ppm): δ 170.04 (- C=O), 161.85, 146.98, 129.69, 120.52, 117.87, 112.15, 110.52(AR-C), 87.23(C-20), 81.40(C-24), 81.54(C-3), 73.49(C-16), 71.99(C-25), 69.02(C-6), 57.61(C-17), 53.98(C-5), 47.22(C-8), 46.85(C-14), 46.13(C-15), 45.17(C-13), 40.91(C-4), 38.18(C-7), 34.56(C-22), 33.09(C-1), 31.85(C-12), 31.59(C-19), 29.42(C-2), 28.44(C-10), 27.89(C-28), 27.36(C-21), 26.97(C-11), 26.63(C-27), 26.03(C-26), 25.72(C-23), 22.00(AR-CH3), 21.65(C-9), 21.05(C-18), 20.21(C-30), 16.77(C-29); ESI-HRMS (m / z): calcd for C 38 H 56 O7Na + [M+Na] + : 647.3924, found: 647.3920. 4. Preparation of Compound C4 (R4 = 3’CH3) Dissolve CAG (2.0 mmol, 1 equiv.) in 10 mL of dichloromethane, then successively add EDC (4.0 mmol, 2 equiv.), 3-methylsalicylic acid (4.0 mmol, 2 equiv.) and DMAP (2.0 mmol, 1 equiv.), and stir at room temperature for 8 hours. Remove the solvent under reduced pressure at 40 °C, redissolve in ethyl acetate, extract with water three times to obtain an ethyl acetate solution, add anhydrous sodium sulfate to remove water, filter and mix with silica gel (100 - 200 mesh), and remove the solvent under reduced pressure to obtain the sample to be separated. Purify by column chromatography (silica gel 300 - 400 mesh), and the separation condition is dichloromethane:methanol volume ratio 50:1; remove the solvent by rotary evaporation to obtain a white powder, which is Compound C4, with a melting point of 241 - 243 °C.

[0053] Structure Confirmation: 1 1H-NMR (CDCl3, 600 MHz, ppm): δ 0.44(d, J = 4.2 Hz, 1H, H-19b), 0.60(d,J = 4.2 Hz, 1H, H-19a), 0.98, 1.15, 1.21, 1.24, 1.25, 1.28, 1.31(s, 3H,-CH3×7), 2.30(s, 3H, AR-CH3), 2.35(d, J = 7.8 Hz, 1H), 2.60(q, J = 10.2 Hz, 1H),3.60(td, J 1= 2.4 Hz, J 2= 9.6 Hz, 1H, H-6), 3.77(t, J 1= 7.8 Hz, J 2= 14.4 Hz, 1H, H-24), 4.71(q, J = 7.2 Hz, 1H, H-16), 4.86(dd, J 1= 4.2 Hz, J 2= 10.8 Hz, 1H, H-3),6.88(d, J = 8.4 Hz, 1H, AR-H), 7.53(q, J = 3.6 Hz, 1H, AR-H), 7.61(s, 1H, AR-H),10.74(s, 1H, AR-OH); 13 C-NMR (CDCl3, 150 MHz, ppm): δ 169.92(- C=O), 159.66,136.52, 132.46, 130.90, 128.82, 117.39(AR-C), 87.08(C-20), 81.39(C-24), 81.39(C-3), 73.35(C-16), 71.84(C-25), 68.86(C-6), 57.48(C-17), 53.84(C-5), 47.15(C-8), 46.48(C-14), 45.99(C-15), 45.01(C-13), 40.08(C-4), 38.08(C-7), 34.43(C-22), 32.94(C-1), 31.74(C-12), 31.51(C-19), 29.72(C-2), 28.31(C-10), 27.80(C-28), 27.74(C-21), 26.85(C-11), 26.50(C-27), 25.90(C-26), 25.64(C-23),21.53(C-9), 20.92(C-18), 20.52(AR-CH3), 20.09(C-30), 16.70(C-29); ESI-HRMS(m / z): calcd for C 38 H 56 O7Na + [M+Na] + : 647.3924, found: 647.3928. 5. Preparation of Compound C5 (R4 = 5’NO2) Dissolve CAG (2.0 mmol, 1 equiv) in 10 mL of dichloromethane, then successively add EDC (4.0 mmol, 2 equiv), 5-nitrosalicylic acid (4.0 mmol, 2 equiv) and DMAP (2.0 mmol, 1 equiv), and stir at room temperature for 8 hours. Remove the solvent under reduced pressure at 40 °C, redissolve in ethyl acetate, extract with water three times to obtain an ethyl acetate solution, add anhydrous sodium sulfate to remove water, filter and mix with silica gel (100 - 200 mesh), and remove the solvent under reduced pressure to obtain the sample to be separated. Purify by column chromatography (silica gel 300 - 400 mesh), and the separation condition is dichloromethane:methanol volume ratio 50:1; remove the solvent by rotary evaporation to obtain a white powder, which is Compound C5, with a melting point of 226 - 228 °C.

[0054] Structure confirmation: 1 H-NMR (CDCl3, 600 MHz, ppm): δ 0.44(d, J = 4.2 Hz, 1H, H-19b),0.61(d,J = 4.2 Hz, 1H, H-19a), 0.98, 1.16, 1.20, 1.25, 1.28, 1.31, 1.34(s, 3H,-CH3×7), 2.35(d, J = 7.8 Hz, 1H), 2.60(q, J = 10.2 Hz, 1H), 3.59(td, J 1 = 3.0 Hz, J 2 = 9.6 Hz, 1H, H-6), 3.78(t, J 1 = 7.8 Hz, J 2 = 15.0 Hz, 1H, H-24), 4.71(q, J = 7.8Hz, 1H, H-16), 4.91(dd, J 1 = 4.2 Hz, J 2 = 11.4 Hz, 1H, H-3), 6.97 - 7.02(m, 1H, AR-H), 8.11 - 8.16(m, 2H, AR-H), 12.19(s, 1H, AR-OH); 13 C-NMR (CDCl3, 150 MHz, ppm):δ 170.00(- C =O), 167.91, 156.06, 132.59, 131.03, 128.95, 118.42(AR-C), 87.21(C-20), 81.53(C-24), 83.31(C-3), 73.46(C-16), 72.00(C-25), 68.95(C-6), 57.61(C-17), 53.85(C-5), 47.20(C-8), 46.55(C-14), 46.11(C-15), 45.17(C-13), 40.91(C-4), 38.28(C-7), 34.56(C-22), 33.06(C-1), 31.80(C-12), 31.75(C-19), 29.85(C-2), 28.46(C-10), 27.90(C-28), 27.58(C-21), 26.87(C-11), 26.65(C-27), 26.04(C-26), 25.69(C-23), 21.66(C-9), 21.14(C-18), 20.22(C-30), 16.71(C-29); ESI-HRMS (m / z): calcd for C37 H 53 NO9Na + [M+Na] + : 678.3618, found: 678.3619. 6. The preparation of compounds C6-C11 is similar to that of compound C5. The structural formulas of C6-C11 are shown in Figure 1 .

[0055] Example 5. Preparation of Compounds R1-R4 The structural formulas of compounds R1-R4 are as follows:

[0056] 1. Preparation of Compound R1 (R = H) Dissolve CAG (2.0 mmol, 1 equivalent) in 10 mL of dichloromethane, then sequentially add EDC (4.0 mmol, 2 equivalents), cinnamic acid (4.0 mmol, 2 equivalents) and DMAP (2.0 mmol, 1 equivalent), and stir at room temperature for 24 hours. Remove the solvent under reduced pressure at 40 °C, redissolve in ethyl acetate, extract with water three times to obtain an ethyl acetate solution, add anhydrous sodium sulfate to remove water, filter and mix with silica gel (100-200 mesh), and remove the solvent under reduced pressure to obtain the sample to be separated. Purify by column chromatography (silica gel is 300-400 mesh), and the separation condition is dichloromethane:methanol volume ratio 30:1; remove the solvent by rotary evaporation to obtain a white powder, which is compound R1, with a melting point of 234-236 °C.

[0057] Structure confirmation: 1 H-NMR (CDCl3, 600 MHz, ppm): δ 0.42(d, J = 4.2 Hz, 1H, H-19b),0.58(d, J = 4.2 Hz, 1H, H-19a), 0.97, 1.13, 1.15, 1.18, 1.24, 1.28, 1.31(s, 3H,-CH3×7), 2.35(d, J = 7.8 Hz, 1H), 2.60(q, J = 10.2 Hz, 1H), 3.57(t, J 1= 3.0 Hz, J 2=10.2 Hz, 1H, H-6), 3.77(t, J 1= 7.2 Hz, J2 = 14.4 Hz, 1H, H-24), 4.68 - 4.73(m, 2H, H-3 and H-16), 6.47(d, J = 16.2 Hz, 1H, -CH=C H -), 7.38 - 7.39(m, 3H), 7.53 - 7.55(m, 2H, AR-H), 7.67(d, J = 15.6 Hz, AR-H); 13 C-NMR (CDCl3, 150 MHz, ppm): δ166.91(- C =O), 144.56, 118.90(- C H= C H-), 134.66, 130.31, 129.00, 128.21(AR-C), 87.22(C-20), 80.40(C-24), 81.53(C-3), 73.50(C-16), 71.96(C-25), 69.06(C-6), 57.60(C-17), 54.00(C-5), 47.25(C-8), 46.61(C-14), 46.14(C-15), 45.15(C-13), 40.78(C-4), 38.10(C-7), 34.56(C-22), 33.10(C-1), 31.92(C-12), 31.60(C-19), 29.50(C-2), 28.34(C-10), 27.92(C-28), 27.87(C-21), 27.03(C-11), 26.61(C-27), 26.02(C-26), 25.75(C-23), 21.64(C-9), 20.97(C-18), 20.21(C-30), 16.67(C-29); ESI-HRMS (m / z): calcd for C 39 H 56 O6Na + [M + Na] + : 643.3975, found: 643.3978. 2. Preparation of Compound R2 (R = 4’F) Dissolve CAG (2.0 mmol, 1 equiv) in 10 mL of dichloromethane, then successively add EDC (4.0 mmol, 2 equiv), 4-fluorocinnamic acid (4.0 mmol, 2 equiv) and DMAP (2.0 mmol, 1 equiv), and stir at room temperature for 24 h. Remove the solvent under reduced pressure at 40 °C, redissolve in ethyl acetate, extract three times with water to obtain an ethyl acetate solution, add anhydrous sodium sulfate to remove water, filter and mix with silica gel (100 - 200 mesh), and remove the solvent under reduced pressure to obtain the sample to be separated. Purify by column chromatography (silica gel 300 - 400 mesh), and the separation condition is petroleum ether:acetone volume ratio 5:1; remove the solvent by rotary evaporation to obtain a white powder, which is compound R2, with a melting point of 233 - 235 °C.

[0058] Structure confirmation: 1 1H-NMR (CDCl3, 600 MHz, ppm): δ 0.42 (d, J J = 4.2 Hz, 1H, H-19b), 0.57 (d, J J = 4.2 Hz, 1H, H-19a), 0.97, 1.12, 1.14, 1.17, 1.23, 1.30, 1.33 (s, 3H, -CH3×7), 2.35 (d, J J = 7.8 Hz, 1H), 2.61 (q, J J = 10.2 Hz, 1H), 3.56 (t, J J1 = 3.0 Hz, J J2 = 9.6 Hz, 1H, H-6), 3.76 (t, J J1 = 7.2 Hz, J J2 = 14.4 Hz, 1H, H-24), 4.67 - 4.72 (m, 2H, H-3 and H-16), 6.38 (d, J J = 10.2 Hz, 1H, -CH=C H -), 7.08 (t, J J1 = 8.4 Hz, J J2 = 17.4 Hz, 2H, AR-H), 7.51 - 7.53 (m, 2H, AR-H), 7.64 (d, J J = 16.2 Hz, 1H, -C H =CH-); 13 13C-NMR (CDCl3, 150 MHz, ppm): δ 166.78 (- C =O), 143.24, 118.64 (- CH= C H-), 163.14, 130.91, 130.08, 130.02, 116.21, 116.07(AR-C), 87.19(C-20), 80.50(C-24), 81.50(C-3), 73.48(C-16), 71.95(C-25), 69.00(C-6), 57.61(C-17), 53.94(C-5), 47.35(C-8), 46.66(C-14), 46.11(C-15), 45.09(C-13), 40.77(C-4), 38.14(C-7), 34.55(C-22), 33.05(C-1), 31.91(C-12), 31.86(C-19), 29.52(C-2), 28.35(C-10), 27.99(C-28), 27.83(C-21), 27.02(C-11), 26.60(C-27), 26.03(C-26), 25.85(C-23), 21.63(C-9), 20.95(C-18), 20.21(C-30), 16.66(C-29); ESI-HRMS (m / z): calcd for C 39 H 55 O6FNa + [M+Na] + : 661.3880, found: 661.3883. 3. Preparation of Compound R3 (R = 4’Cl) Dissolve CAG (2.0 mmol, 1 equivalent) in 10 mL of dichloromethane, then successively add EDC (4.0 mmol, 2 equivalents), 4-chlorocinnamic acid (4.0 mmol, 2 equivalents) and DMAP (2.0 mmol, 1 equivalent), and stir at room temperature for 24 hours. Remove the solvent under reduced pressure at 40 °C, redissolve in ethyl acetate, extract with water three times to obtain an ethyl acetate solution, add anhydrous sodium sulfate to remove water, filter and mix with silica gel (100 - 200 mesh), and remove the solvent under reduced pressure to obtain the sample to be separated. Purify by column chromatography (silica gel is 300 - 400 mesh), and the separation condition is petroleum ether:acetone volume ratio 5:1; remove the solvent by rotary evaporation to obtain a white powder, which is Compound R3, with a melting point of 236 - 238 °C.

[0059] Structure Confirmation: 1 H-NMR (CDCl3, 600 MHz, ppm): δ 0.42(d, J = 4.2 Hz, 1H, H-19b), 0.57(d,J = 4.2 Hz, 1H, H-19a), 0.97, 1.12, 1.16, 1.17, 1.24, 1.28, 1.31(s, 3H,-CH3×7), 2.34(d, J = 7.8 Hz, 1H), 2.60(q, J = 10.2 Hz, 1H), 3.52 - 3.58(m, 1H, H-6), 3.76(t, J 1= 7.2 Hz, J 2= 14.4 Hz, 1H, H-24), 4.67 - 4.73(m, 2H, H-3 and H-16),6.43(d, J = 15.6 Hz, 1H, -CH=C H -), 7.36(d, J = 8.4 Hz, 2H, AR-H), 7.47(d, J = 8.4Hz, 2H, AR-H), 7.63(d, J = 15.6 Hz, 1H, -C H =CH-); 13 C-NMR (CDCl3, 150 MHz, ppm):δ 166.65(- C =O), 143.10, 119.48(- C H= CH-), 136.21, 133.15, 129.36, 129.29(AR-C), 87.21(C-20), 80.58(C-24), 81.53(C-3), 73.49(C-16), 71.96(C-25), 69.04(C-6), 57.61(C-17), 53.98(C-5), 47.28(C-8), 46.63(C-14), 46.12(C-15), 45.13(C-13), 40.76(C-4), 38.13(C-7), 34.56(C-22), 33.08(C-1), 31.91(C-12), 31.62(C-19), 29.85(C-2), 29.51(C-10), 28.35(C-28), 27.94(C-21), 27.01(C-11), 26.61(C-27), 26.03(C-26), 25.77(C-23), 21.63(C-9), 20.97(C-18), 20.21(C-30), 16.66(C-29); ESI-HRMS (m / z): calcd for C 39 H 55 O6ClNa + [M+Na] + : 677.3585, found: 677.3586. 4. Preparation of Compound R4 (R = 4’Br) Dissolve CAG (2.0 mmol, 1 equiv.) in 10 mL of dichloromethane, then successively add EDC (4.0 mmol, 2 equiv.), 4-bromocinnamic acid (4.0 mmol, 2 equiv.) and DMAP (2.0 mmol, 1 equiv.), and stir at room temperature for 24 h. Remove the solvent under reduced pressure at 40 °C, redissolve in ethyl acetate, extract three times with water to obtain an ethyl acetate solution, add anhydrous sodium sulfate to remove water, filter, mix with silica gel (100 - 200 mesh), and remove the solvent under reduced pressure to obtain the sample to be separated. Purify by column chromatography (silica gel 300 - 400 mesh), and the separation condition is dichloromethane:methanol volume ratio 40:1; remove the solvent by rotary evaporation to obtain a white powder, which is Compound R4, with a melting point of 238 - 240 °C.

[0060] Structure Confirmation: 1 1H-NMR (CDCl3, 600 MHz, ppm): δ 0.42(d, J = 4.2 Hz, 1H, H-19b),0.57(d, J= 3.6 Hz, 1H, H-19a), 0.97, 1.12, 1.14, 1.17, 1.23, 1.27, 1.30(s, 3H,-CH3×7), 2.35(d, J = 7.8 Hz, 1H), 2.61(q, J = 10.8 Hz, 1H), 3.56(t, J 1= 3.0 Hz, J 2=10.2 Hz, 1H, H-6), 3.77(t, J 1= 7.2 Hz, J 2= 13.8 Hz, 1H, H-24), 4.67-4.72(m, 2H,H-3 and H-16), 6.45(d, J = 16.2 Hz, 1H, -CH=C H -), 7.40(d, J = 7.2 Hz, 2H, AR-H),7.52(d, J = 7.8 Hz, 2H, AR-H), 7.61(d, J = 16.2 Hz, 1H, -C H =CH-); 13 C-NMR (CDCl3,150 MHz, ppm): δ 166.64(- C =O), 143.17, 119.59(- C H= CH-), 133.58, 132.24, 129.59, 124.55 (AR-C), 87.19 (C-20), 80.62 (C-24), 81.51 (C-3), 73.48 (C-16), 71.96 (C-25), 69.01 (C-6), 57.61 (C-17), 53.95 (C-5), 47.32 (C-8), 46.65 (C-14), 46.12 (C-15), 45.11 (C-13), 40.76 (C-4), 38.15 (C-7), 34.55 (C-22), 33.06 (C-1), 31.91 (C-12), 31.66 (C-19), 29.84 (C-2), 29.50 (C-10), 28.36 (C-28), 27.84 (C-21), 27.01 (C-11), 26.61 (C-27), 26.03 (C-26), 25.85 (C-23), 21.63 (C-9), 20.96 (C-18), 20.21 (C-30), 16.66 (C-29); ESI-HRMS (m / z): calcd for C 39 H 55 O6BrNa + [M+Na] + : 721.3080, found: 721.3082. Example 6, Preparation of Compounds S1 and S2 The structural formulas of Compounds S1 and S2 are as follows:

[0061] 1. Preparation of Compound S1 (R2 = ) Dissolve CAG (2.0 mmol, 1 equivalent) in 10 mL of dichloromethane, then successively add EDC (4.0 mmol, 2 equivalents), trifluoroacetic acid (4.0 mmol, 2 equivalents) and DMAP (2.0 mmol, 1 equivalent), and stir at room temperature for 2 hours. Remove the solvent under reduced pressure at 40 °C, redissolve in ethyl acetate, extract with water three times to obtain an ethyl acetate solution, add anhydrous sodium sulfate to remove water, filter and mix with silica gel (100 - 200 mesh), and remove the solvent under reduced pressure to obtain the sample to be separated. Purify by column chromatography (silica gel is 300 - 400 mesh), and the separation condition is dichloromethane:methanol volume ratio 40:1; remove the solvent by rotary evaporation to obtain a white powder, which is Compound S1, with a melting point of 244 - 246 °C.

[0062] Structure Confirmation: 1H-NMR (CDCl3, 600 MHz, ppm): δ 0.42 (d, J J = 4.2 Hz, 1H, H-19b), 0.58 (d, J J = 4.2 Hz, 1H, H-19a), 0.96, 1.10, 1.16, 1.17, 1.24, 1.25, 1.31 (s, 3H, -CH3×7), 2.34 (d, J J = 7.8 Hz, 1H), 2.59 (q, J J = 10.2 Hz, 1H), 3.57 (t, J J1 = 3.0 Hz, J J2 = 10.2 Hz, 1H, H-6), 3.77 (t, J J1 = 7.2 Hz, J J2 = 14.4 Hz, 1H, H-24), 4.71 (q, J J = 10.2 Hz, 1H, H-16), 4.78 (dd, J J1 = 4.2 Hz, J J2 = 11.4 Hz, 1H, H-3); 13 C-NMR (CDCl3, 150 MHz, ppm): δ 167.87 (- C C=O), 115.79 (- C CF3), 87.22 (C-20), 81.52 (C-24), 85.57 (C-3), 73.46 (C-16), 72.01 (C-25), 68.85 (C-6), 57.60 (C-17), 53.60 (C-5), 47.10 (C-8), 46.50 (C-14), 46.08 (C-15), 45.16 (C-13), 40.74 (C-4), 38.24 (C-7), 34.55 (C-22), 33.05 (C-1), 31.64 (C-12), 31.54 (C-19), 29.85 (C-2), 29.10 (C-10), 28.10 (C-28), 27.90 (C-21), 27.87 (C-11), 26.66 (C-27), 26.02 (C-26), 25.68 (C-23), 21.63 (C-9), 21.11 (C-18), 20.19 (C-30), 16.28 (C-29); ESI-HRMS (m / z): calcd for C 32 H49 O6F3Na + [M+Na] + : 609.3379, found: 609.3384. 2. R2 in compound S2 = , and its preparation method is similar to that of compound S1.

[0063] Example 7 I. Experimental method 1. Cell culture SH-SY5Y and BV2 cells were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The cells were cultured in DMEM medium supplemented with 10% FBS in an environment containing 5% CO2 at 37 °C. There are mainly two types of cell models, an in vitro cell model of PD: MPP + induced SH-SY5Y cells; an in vitro model of neuroinflammation: LPS-induced BV2 cells.

[0064] 2. In vitro experiments Cell viability: SH-SY5Y cells were seeded in 96-well plates (cell density 1*10 5 cells / mL), a blank group was set, a model group (MPP + group, dissolved in PBS buffer and treated with cells at a final concentration of 3 mM), compound (dissolved in DMSO) + model group [compound (treated with cells at a final concentration of 10 μM) co-administered with MPP + , incubated in a 37 °C cell incubator for 24 hours, and 6 parallel replicates were set for each compound. Then, CCK-8 solution was added to each well, incubated at 37 °C for 1 hour, and the absorbance was measured at 450 nm using a microplate reader. LDH determination: According to the detection requirements of the LDH detection kit, the cell supernatant of each group was collected, added to the detection reagent, the absorbance value was measured at 450 nm, and the LDH content in each group was calculated.

[0065] IL-1β inhibition assay: BV2 cells were seeded in 96-well plates (cell density 1*10 5 cells / mL), a blank group was set, a model group (LPS group, dissolved in PBS buffer and treated with cells at a final concentration of 1 μg / mL), compound (dissolved in DMSO) + model group [compound (treated with cells at a final concentration of 10 μM) co-administered with LPS], after incubating in a 37 °C cell incubator for 24 hours, the cell supernatant was collected, 6 parallel replicates were set for each compound, and the IL-1β content of different groups was measured according to the experimental requirements of the IL-1β kit. Morphological observation: The neuroinflammation model (LPS-induced BV2) was used to evaluate the morphological changes of cells after compound treatment. The morphological changes of cells after drug administration were observed in 6-well plates, and the efficacy of the compound was analyzed by taking pictures.

[0066] 3. Animal experiments All C57BL / 6 mice were purchased from Vital River Company (Beijing, China). All animal procedures were approved by the Animal Experiment Ethics Committee of Shanxi University of Traditional Chinese Medicine (AWE202307367). A total of 60 C57BL / 6 mice (male, 6 - 8 weeks old, body weight 20 ± 2 g) were used in this study and divided into 6 groups (n = 10): control group, MPTP group, CAG group (30 mg / kg), R2-L group (10 mg / kg), R2-H group (30 mg / kg), and L-DOPA group (70 mg / kg). A mouse PD model was established by intraperitoneal injection of MPTP once a day for one week, with a concentration of 15 mg / kg on the first day, 20 mg / kg on the second day, and 30 mg / kg from the third to the seventh day. Starting from the first day of preparation in the MPTP group, the control group was gavaged with 0.5% CMC-Na once a day; the MPTP group was injected with MPTP (dissolved in normal saline, and the administration volume was calculated according to 10 mL / kg of mouse body weight) according to the above model establishment concentration requirements for 7 days, and gavaged with 0.5% CMC-Na once a day; the CAG group was gavaged with CAG once a day according to the grouped concentration; the R2-L group was gavaged with R2 once a day according to the grouped concentration; the R2-H group was gavaged with R2 once a day according to the grouped concentration; the L-DOPA group was gavaged with L-DOPA once a day according to the grouped concentration for 14 days. During the study, the mice had free access to water, food, and activities. The solvents for CAG and R2 were both 0.5% CMC-Na, and the solvent for L-DOPA was normal saline, and the administration volume was calculated according to 10 mL / kg of mouse body weight.

[0067] Behavioral tests (test on the 15th day of the experiment): Gait determination: The DigiGait animal gait detection system was used to evaluate the gait behavior indicators of mice. The speed was set at 15 cm / s, and the frequency of the mouse's paws in different groups was recorded continuously for 10 seconds. Pole climbing test: The mouse was placed on the experimental rod (diameter 1 cm, length 60 cm), and the time the mouse stayed on the rod and the time it took to descend to the bottom were recorded. The number of repeated experiments was 3 times. Hanging test: The mouse's front paws were hung on the horizontal wire, and the score was based on the number of the mouse's hind paws hanging on the wire at the end. The rules were as follows: two hind paws (3 points); one hind paw (2 points); zero hind paws (1 point); the mouse landed (0 points). All experiments were repeated three times, with an interval of 10 minutes between each iteration.

[0068] ELISA analysis: On the 16th day of the experiment, brain tissue and blood samples from different groups were collected and the supernatant was extracted. ELISA analysis of inflammatory factors (IL-1β, IL-6, and TNF-α) was performed respectively, and the absorbance value was measured at 450 nm to analyze the expression level of inflammatory factors.

[0069] Immunohistochemistry and immunofluorescence staining: Three mouse brain tissues were dehydrated using a sucrose solution gradient and embedded in OCT reagent. The brain tissues were frozen in liquid nitrogen and coronal sections (10 μm) were cut using a cryostat. The sections were sliced and dried for 24 hours and then stored at -80 °C. The sections were washed with PBS to remove the OCT embedding agent. The sections were immersed in PBS containing 0.3% Triton X-100 and incubated at room temperature for 30 minutes. Immunohistochemistry staining: An appropriate amount of the first antibody (TH) was added to the sections, incubated overnight, washed with PBS, and the secondary antibody was evenly distributed. Finally, counterstaining was performed with hematoxylin staining solution, and the results were observed and analyzed. Immunofluorescence staining: The first antibodies (Iba-1 and TERT) were evenly added to the sections and incubated overnight. Then, incubation was performed with the secondary fluorescent antibody. Finally, DAPI was added dropwise and incubated for 5 - 10 minutes. Confocal laser microscopy was used to observe and capture images, and Image-J software was used to analyze the fluorescence intensity or cell count.

[0070] Western blotting: Brain tissue and cell samples from different groups were collected, added to RIPA lysis buffer, and total protein was extracted. Proteins were separated by SDS-PAGE and then transferred to a membrane. 5% skim milk was used to block the polyvinylidene fluoride membrane. Subsequently, the membrane was incubated with anti-NF-κB, anti-p-NF-κB, anti-TLR4, and anti-β-actin (volume ratio: 1:1000) at 4 °C overnight. TBST was used to wash the membrane. The membrane was incubated with the anti-rabbit secondary antibody (volume ratio: 1:5000). Finally, the developed signal was captured using a gel imaging system.

[0071] Statistical analysis: One-way analysis of variance was used for statistical comparison between groups, followed by Tukey's post hoc test. The data are expressed as the mean ± standard deviation of three independently performed repeated experiments. A p-value < 0.05 was considered statistically significant.

[0072] II. Experimental results 1. Twenty-nine cyclocanthol carboxylic acid derivatives were synthesized in the above examples. Using MPP +Two models, namely, SH-SY5Y cells induced (in vitro PD model) and BV2 cells induced by LPS (in vitro neuroinflammation model), were used to investigate the cell viability, cytotoxicity, and IL-1β inhibitory effect of the compound-treated cells. Comprehensive different experimental results showed that among the amino acid derivatives (A1 - A8), the deprotected derivatives had significantly stronger activity than the prototype compounds; among benzoic acids (B1 - B4) and cinnamic acids (R1 - R4), due to the extension of the group carbon chain combined with the introduction of double bonds, the anti-inflammatory activity and cell viability of cinnamic acid derivatives were significantly stronger than those of benzoic acid derivatives; in the case of salicylic acid derivatives (C1 - C11), the toxicity increased and the LPS-induced inflammatory level was exacerbated (Table 1). Considering the activity of different series of derivatives, we found that compound R2 had the best biological activity and could effectively reverse the risks brought by MPP + and LPS to the cells. In the further activity confirmation, the IC 50 value of compound R2 for inhibiting IL-1β was 10.38 μM ( Figure 2 B). Therefore, in the cell morphology screening, compound R2 could significantly inhibit the activated morphology such as the enlargement of the cell body caused by LPS ( Figure 2 C). Therefore, compound R2 was selected for subsequent in vivo activity evaluation and mechanism research.

[0073] Table 1 In vitro activity evaluation of CAG derivatives

[0074] *: The test concentration of the compound was 10 μM.

[0075] 2. Compound R2 improves the behavioral and pathological indicators of PD. In the in vivo experimental evaluation of the PD model, compound R2 could effectively improve the behavioral disorders induced by MPTP. In the gait experiment, R2 could significantly slow down the stride frequency of mice; in the pole climbing experiment, the pole climbing speed of the R2 group was significantly faster, especially the staying time at the top was shortened, indicating that the mice got rid of the bradykinesia caused by MPTP; in the hanging experiment, the mice in the R2 group could also complete the climbing action better. The behavioral experiments fully demonstrated that compound R2 could significantly improve the behavioral symptoms of mice, which was comparable to the effect of the positive drug levodopa. Similarly, in the pathological immunohistochemical analysis, MPTP could reduce the number of TH-positive cells in the substantia nigra region. After treatment with R2, the number of TH-positive cells increased significantly, inhibiting the progressive damage of neurons ( Figure 3 ).

[0076] 3. Compound R2 can enhance the telomerase activity of microglia and inhibit neuroinflammation. Since CAG has a unique effect of enhancing telomerase activity, and the change of telomerase activity has a regulatory effect on the level of neuroinflammation, improving the senescent state of cells. Therefore, this invention attempts to explore the expression of TERT in the PD model by compound R2, and detect the secretion of neuroinflammatory mediators in the brain tissue and serum. The experimental results show that immunofluorescence staining analysis reveals that after MPTP induction, the expression of TERT in the substantia nigra region of mice is significantly decreased, accompanied by an increase in the expression of Iba-1, indicating that after MPTP induction, the microglia in the substantia nigra region are in an activated state, and the decrease in the level of TERT reflects the senescent state of cells and their tendency to undergo programmed cell death. After treatment with compound R2, the expression of TERT is significantly increased, and at the same time, the expression of Iba-1 is significantly decreased, indicating that R2 can effectively relieve the neuroinflammatory effect ( Figure 4 ). In addition, we detected the release of inflammatory factors in the brain tissue and serum of the R2 group and obtained the same conclusion ( Figure 5 ).

[0077] 4. Compound R2 regulates the TLR4 / NF-κB signaling pathway to improve neuroinflammation. Since in previous studies, we found that CAG can regulate the NF-κB signaling pathway to inhibit the progression of neuroinflammation, therefore, in order to further explore the mechanism of action of compound R2 in inhibiting neuroinflammation and improving PD, we investigated the effect of compound R2 on the TLR4 / NF-κB signaling pathway. The results show that compound R2 can regulate the expression of TLR4, effectively reduce the phosphorylation level of NF-κB, and inhibit the classical inflammatory activation pathway of NF-κB ( Figure 6 ). In addition, we selected LPS-induced BV2 cells to construct a neuroinflammatory cell model and investigated the effect of compound R2 on the TLR4 / NF-κB signaling pathway, and obtained the same results as those of tissue samples ( Figure 7 ).

[0078] In summary, this invention takes CAG as the research object, through the form of structural modification, introduces different forms of carboxylic acid groups at the C-3 hydroxyl group, aiming to increase the anti-neuroinflammatory activity of CAG for the exploration and development of PD drugs. Through in vitro activity screening and structure-activity relationship exploration, compound R2 has the best inhibitory effect on IL-1β. In in vivo experiments, it can effectively relieve the PD behavioral and pathological symptoms induced by MPTP. Furthermore, at the mechanism level, we found that compound R2 can effectively increase the telomerase activity (increase in TERT expression) in the model, and regulate the TLR4 / NF-κB signaling pathway to improve neuroinflammation, thereby playing a regulatory role in PD, providing new ideas for the innovative drug research and development of CAG applied to PD.

Claims

1. A cyclococcidiol carboxylic acid derivative, the structural formula of which is shown in Formula I: In Formula I, R is mono-substituted or multi-substituted; R is selected from at least one of H, halogen, nitro, methyl and methoxy.

2. The cyclocanthol carboxylic acid derivative according to claim 1, wherein: R is mono-substituted; said R is H, fluorine, chlorine or bromine.

3. The cyclocanthol carboxylic acid derivative according to claim 3, characterized in that: When said R is fluorine, chlorine or bromine, the substitution position is the 4'-position.

4. The preparation method of the cyclococcidiol carboxylic acid derivative according to any one of claims 1-3, comprising the following steps: reacting cyclococcidiol, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, the compound shown in Formula II and 4-dimethylaminopyridine in a solvent to obtain the cyclococcidiol carboxylic acid derivative; In Formula II, the definition of R is the same as the definition of R in Formula I in claims 1-3.

5. The preparation method according to claim 4, characterized in that: Said solvent is dichloromethane; The molar ratio of said cyclococcidiol to 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide is 1:1-4 The molar ratio of said cyclococcidiol to the compound shown in Formula II is 1:1-4; The molar ratio of said cyclococcidiol to 4-dimethylaminopyridine is 1:1-4.

6. The preparation method according to claim 4 or 5, characterized in that: The temperature of said reaction is room temperature; The time of said reaction is 2-24 h.

7. The preparation method according to any one of claims 4-6, characterized in that: After said reaction, there are also the following steps: removing the solvent, redissolving with ethyl acetate and extracting with water; mixing the obtained ethyl acetate solution with silica gel, then removing the solvent from the obtained solution, purifying by column chromatography, and obtaining a solid powder after removing the solvent.

8. The application of the cyclococcidiol carboxylic acid derivative according to any one of claims 1-3 in the preparation of a drug for preventing and / or treating Parkinson's disease.

9. The application of the cyclococcidiol carboxylic acid derivative according to any one of claims 1-3 in the preparation of a drug for preventing and / or treating neurodegenerative diseases.

10. The application of the cyclococcidiol carboxylic acid derivative according to any one of claims 1-3 in the preparation of any one of the following drugs: (1) A drug for preventing and / or treating neuroinflammation; (2) A drug for inhibiting IL-1β; (3) A drug for enhancing the telomerase activity of microglia; (4) A drug for enhancing TERT expression; (5) A drug for reducing Iba-1 expression; (6) A drug for regulating TLR4 expression; (7) A drug for reducing the phosphorylation level of NF-κB.

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