Dammarane sapogenin-isatin derivative as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of medicinal chemistry, and particularly relates to a dammarane sapogenin-isatin derivative and application thereof in anti-tumor treatment. The dammarane sapogenin-isatin conjugate is constructed by taking dammarane sapogenin as a mother nucleus, deriving the dammarane sapogenin and chloroacetic acid and then introducing an indolone structural unit, and R1, R2 and R3 are as described in the claims and the specification. According to the invention, isatin fragments and ginsenoside are coupled for the first time, the anti-tumor activity of the compounds is systematically evaluated, and the action mechanism research of the compounds in colorectal cancer and other tumors is further developed. Results show that the obtained compound has relatively good anti-tumor efficacy, tumor cell selectivity, relatively low toxicity and relatively high bioavailability, and shows good patent medicine potential and wide market prospects.

Description

Technical Field

[0001] This invention belongs to the field of medicinal chemistry technology, and relates to a dammarane saponin-indigo derivative, its preparation method and pharmaceutical use, specifically to the dammarane saponin-indigo derivative and its application in the preparation of antitumor drugs. Background Technology

[0002] Ginseng (Panax ginseng CAMeyer) belongs to the Araliaceae family and is a traditional and precious Chinese medicinal herb with extremely high medicinal value. Ginsenosides are the main active substances in ginseng, and are mainly classified into dammarane type, octylene type, and oleanane type according to their skeleton type.

[0003] Indigo (2,3-indolequinone, Isatin) is an aromatic ketone compound that is not only widely found in the traditional Chinese medicine Qingdai, but also plays an important role as an endogenous active substance in mammalian tissues and body fluids.

[0004] Dammarane saponin (PD) has potential applications in anti-tumor therapy, inhibiting the proliferation of tumor cells such as cervical cancer, liver cancer, and colon cancer, and regulating apoptosis and cell cycle-related protein expression, thereby leading to apoptosis or cell cycle arrest. Our team found that PD exhibits good inhibitory effects on triple-negative breast cancer and prostate cancer in vitro and in vivo. In triple-negative breast cancer studies, PD weakened the invasion and metastasis of MDA-MB-231 cells by downregulating MMP-9 expression, induced G1 cell cycle arrest, downregulated the expression of Cyclin D1, CDK2, CDK4, CDK6, p-p38, and MMP9 proteins, and upregulated the expression of p21 and p27 proteins. In xenograft nude mice, an oral dose of 40 mg / kg of PD effectively reduced tumor volume and weight, and its tumor growth inhibition ability was superior to paclitaxel. H&E and vicera index analyses showed that PD has low in vivo toxicity and did not interfere with the normal survival of nude mice. Furthermore, PD significantly inhibits PD-L1 expression at both the protein and mRNA levels and suppresses HIF-1α synthesis via the PI3K and MAPK pathways. Additionally, PD can inhibit STAT3 activation through the JAK1, JAK2, and Src pathways. In T cell and tumor cell co-culture systems, PD pretreatment significantly enhances the activity of cytotoxic T lymphocytes and restores their ability to kill tumor cells. However, current limitations in PD's clinical application stem from its poor water solubility and low bioavailability.

[0005] In recent years, the synthesis and pharmacological activity studies of derivatives with dammarane-type saponins as the parent structure have attracted increasing attention. Given their excellent efficacy from multiple angles and levels, in order to seek lead compounds with higher antitumor activity and stronger targeting, chemical modification methods such as esterification, amidation, and the introduction of polar groups are currently used to prepare a series of derivatives.

[0006] There are no existing reports on the preparation of antitumor drugs by reacting dammarane-type saponins with indigo. Summary of the Invention

[0007] The purpose of this invention is to provide a dammarane saponin-indigo derivative, which has good safety and bioavailability, and significant anti-tumor effects, especially a significant inhibitory effect on colorectal cancer.

[0008] This invention is achieved through the following technical solution:

[0009] This invention provides derivatives of Formula I and their racemates, stereoisomers, tautomers, solvates, polymorphs, metabolites, prodrugs, or pharmaceutically acceptable salts thereof:

[0010]

[0011] in,

[0012] R1 is R2 is OH;

[0013] R3 is H, C1-C6 alkyl, halogen, C1-C6 alkoxy, or halo-C1-C6 alkyl;

[0014] Furthermore, R3 is a monosubstituted substance;

[0015] Furthermore, when R2 is At that time, R1 and R2 have the same R3.

[0016] The present invention preferably uses dammarane saponin-indigo derivatives or pharmaceutically acceptable salts thereof with the following structures:

[0017]

[0018]

[0019] The present invention also provides a method for preparing the dammarane saponin-indigo derivative or a pharmaceutically acceptable salt thereof, comprising the following steps:

[0020]

[0021] (1) Dammarane saponin reacts with N,N'-dicyclohexylcarbodiimide, 4-dimethylaminopyridine and chloroacetic acid to give intermediate 1;

[0022] (2) Dammarane saponin reacts with chloroacetyl chloride to give intermediate 2;

[0023] (3) Intermediate 1 or intermediate 2 reacts with substituted indigo to give dammarane saponin-indigo derivatives or pharmaceutically acceptable salts thereof.

[0024] The present invention provides a pharmaceutical composition comprising a derivative of Formula I and its racemic, stereoisomer, tautomer, solvate, polymorph, metabolite, prodrug, or pharmaceutically acceptable salt thereof.

[0025] The present invention provides the use of compounds of Formula I and their racemates, stereoisomers, tautomers, solvates, polymorphs, metabolites, prodrugs or pharmaceutically acceptable salts thereof or pharmaceutical compositions thereof in the preparation of antitumor drugs.

[0026] The tumors mentioned are colorectal cancer, liver cancer, non-small cell lung cancer, prostate cancer, breast cancer, cervical cancer, ovarian cancer, gastric cancer, skin cancer, melanoma, glioblastoma, testicular cancer, endometrial cancer, pancreatic cancer, osteosarcoma, nasopharyngeal carcinoma, laryngeal squamous cell carcinoma, bile duct cancer, kidney cancer, multiple myeloma, esophageal cancer, and acute myeloid leukemia, preferably colorectal cancer.

[0027] This invention designs and synthesizes a series of conjugates with the active fragment of indigo, and studies their antitumor activity. The results show that the derivatives of this invention, or pharmaceutical compositions containing said derivatives, all exhibit significant antitumor activity and can be used to prepare antitumor drugs. Attached Figure Description

[0028] Figure 1 Survival rate of human embryonic kidney cells HEK293.

[0029] Figure 2 The effect of 4e on colony formation of two types of colon cancer cells.

[0030] Figure 3 The effect of 4e on the cell cycle of HCT-116 cells.

[0031] G01G1: Stationary phase / early DNA synthesis; G2M: Late DNA synthesis / mitosis phase; S: DNA synthesis phase

[0032] Figure 4 The effect of 4e on apoptosis in HCT-116 cells.

[0033] Figure 5 The effect of 4e on the migration ability of HCT-116 cells.

[0034] Figure 6 The effect of 4e on γ-H2AX in HCT-116 cells.

[0035] Figure 7 The effects of 4e on HCT-116 cell cycle and apoptosis-related proteins.

[0036] The samples, from left to right, are Ctrl, 2.5μM, 5μM, and 10μM.

[0037] Figure 8 This shows the molecular docking of 4e with the RalA protein.

[0038] Figure 9 The effect of 4e on RalA protein in HCT-116 cells.

[0039] Figure 10 The effect of 4e on the thermostability binding of RalA protein in HCT-116 cells.

[0040] Figure 11 For in vivo safety evaluation of 4e.

[0041] Figure 12 The effect of 4e on tumor volume in tumor-bearing mice.

[0042] Figure 13 The plasma concentration-time curves of 4e and PD in rats are shown. Detailed Implementation

[0043] To better illustrate the purpose, technical solution, and advantages of this invention, the invention will be further described below with reference to specific embodiments. The purpose of this description is to provide a detailed understanding of the invention, rather than to limit the invention.

[0044] Example 1

[0045]

[0046] Step 1: Weigh an appropriate amount of the starting compound dammarane saponin (1 equiv) and place it in a 100 ml round-bottom flask. Add an appropriate amount of dry dichloromethane and stir at room temperature until completely dissolved. Weigh DCC (2 equiv), DMAP (1 equiv), and chloroacetic acid (1 equiv) and add them to the solution separately. React at room temperature for 4 hours, and monitor the reaction by TLC. After the reaction is complete, filter the reaction mixture twice, and retain the filtrate to remove DCC. Extract the filtrate three times with saturated sodium bicarbonate solution to obtain a dichloromethane extract. Add anhydrous sodium sulfate to the extract, dry it, and let it stand overnight. Filter and concentrate under reduced pressure to obtain the sample to be separated. Purify by column chromatography (petroleum ether / ethyl acetate ratio 5 / 1) to obtain intermediate 1.

[0047] Step 2: Weigh an appropriate amount of intermediate 1 (1 equiv) and place it in a 25 mL round-bottom flask. Add 5 mL of acetonitrile solution and heat with stirring until completely dissolved. Weigh indigo (1 equiv) and add it to the round-bottom flask, stirring until dissolved. The solution changes from colorless and clear to yellow and clear. Add anhydrous potassium carbonate and heat under reflux at 80 °C for 3 h. Monitor the reaction progress by TLC. The development conditions are petroleum ether:ethyl acetate = 3:1. After the reaction stops, remove the solvent under reduced pressure. Add 20 mL of distilled water and 20 mL of dichloromethane solution, respectively, and extract three times to obtain a dichloromethane layer. Add anhydrous sodium sulfate, shake, and let stand for 2 h. Filter the solution and add sample-mixed silica gel (100-200 mesh). Remove the solvent under reduced pressure and dry to obtain the sample to be separated. Separate by silica gel column chromatography under petroleum ether / ethyl acetate (6 / 1) conditions to finally obtain compound 4a.

[0048] The resulting compound was a light yellow powder. 1 H NMR (300MHz, CDCl3, ppm): δ0.72(s,3H),0.80(s,3H),0.87(s,6H),0.97(s,3H),1.18(s,3 H),1.21(s,3H),1.28(s,6H),1.33–1.98(m,21H),3.46–3.56(td,J=5.1,10.3Hz,1H),4.4 0–4.52(m,2H),4.52–4.58(m,1H),6.29(s,1H),6.74–6.80(d,J=7.9Hz,1H),7.11–7.18(t d,J=0.8,7.6Hz,1H),7.55–7.62(td,J=1.4,7.8Hz,1H),7.62–7.67(dd,J=1.3,7.5Hz,1H). 13 C NMR (75MHz, CDCl3) δ15.74,16.23,16.44,17.16,18.25,19.54,23.77,25.27,2 7.26,28.13,30.68,31.25,33.15,34.09,34.85,35.86,36.57,37.11,37.99,38 .54,39.91,41.69,49.26,49.91,51.31,54.83,55.96,69.94,73.25,77.36,83.56,110.24,117.77,124.31,125.73,138.55,150.59,158.17,166.57,182.63.

[0049] Example 2

[0050]

[0051] Step 1: Weigh an appropriate amount of the starting compound dammarane saponin (1 equiv) and place it in a 100 ml round-bottom flask. Add an appropriate amount of dry dichloromethane and stir at room temperature until completely dissolved. Weigh DCC (2 equiv), DMAP (1 equiv), and chloroacetic acid (1 equiv) and add them to the solution separately. React at room temperature for 4 hours, and monitor the reaction by TLC. After the reaction is complete, filter the reaction mixture twice, and retain the filtrate to remove DCC. Extract the filtrate three times with saturated sodium bicarbonate solution to obtain a dichloromethane extract. Add anhydrous sodium sulfate to the extract, dry it, and let it stand overnight. Filter and concentrate under reduced pressure to obtain the sample to be separated. Purify by column chromatography (petroleum ether / ethyl acetate ratio 5 / 1) to obtain intermediate 1.

[0052] Step 2: Weigh an appropriate amount of intermediate 1 (1 equiv) and place it in a 25 mL round-bottom flask. Add 5 mL of acetonitrile solution and heat and stir until completely dissolved. Weigh 5-methylindigo (1 equiv) and add it to the round-bottom flask. Stir and dissolve until the solution changes from colorless and clear to yellow and clear. Add anhydrous potassium carbonate and heat to reflux at 80 °C for 3 h. Monitor the reaction progress by TLC. The development conditions are petroleum ether:ethyl acetate = 3:1. After the reaction stops, remove the solvent under reduced pressure. Add 20 mL of distilled water and 20 mL of dichloromethane solution respectively, and extract three times to obtain a dichloromethane layer. Add anhydrous sodium sulfate, shake, and let stand for 2 h. Filter the solution and add sample-mixed silica gel (100-200 mesh). Remove the solvent under reduced pressure and dry to obtain the sample to be separated. Separate by silica gel column chromatography. The separation conditions are petroleum ether / ethyl acetate (6 / 1), and finally obtain compound 4b.

[0053] The resulting compound was a light yellow powder. 1H NMR (300MHz, CDCl3, ppm) δ0.73(s,3H),0.82(s,4H),0.87(s,6H),0.97(s,3H),1.18(s,3H),1.2 2(s,3H),1.27(s,4H),1.35(m,2H),1.39–1.43(d,J=9.6Hz,2H),1.45–1.88(m,18H),2.34(s,3H) ,3.47–3.56(td,J=5.1,10.2Hz,1H),4.38–4.51(m,2H),4.52–4.57(dd,J=4.2,J2=6.8Hz,1H),6. 24–6.31(s,1H),6.64–6.69(d,J=8.0Hz,1H),7.37–7.41(m,1H),7.43–7.48(d,J=1.8Hz,1H).13C NMR (75MHz, CDCl3) δ15.74,16.24,16.46,17.16,18.25,19.54,20.85,23.77,25. 27,27.26,28.13,30.67,31.25,31.51,33.15,34.85,35.86,36.57,37.11,37.99 ,38.55,39.91,41.70,49.26,49.91,51.31,54.83,55.96,69.96,73.25,77.36,83.46,110.02,117.77,126.04,134.16,138.97,148.44,158.33,166.68,182.90.

[0054] Example 3

[0055]

[0056] Step 1: Weigh an appropriate amount of the starting compound dammarane saponin (1 equiv) and place it in a 100 ml round-bottom flask. Add an appropriate amount of dry dichloromethane and stir at room temperature until completely dissolved. Weigh DCC (2 equiv), DMAP (1 equiv), and chloroacetic acid (1 equiv) and add them to the solution separately. React at room temperature for 4 hours, and monitor the reaction by TLC. After the reaction is complete, filter the reaction mixture twice, and retain the filtrate to remove DCC. Extract the filtrate three times with saturated sodium bicarbonate solution to obtain a dichloromethane extract. Add anhydrous sodium sulfate to the extract, dry it, and let it stand overnight. Filter and concentrate under reduced pressure to obtain the sample to be separated. Purify by column chromatography (petroleum ether / ethyl acetate ratio 5 / 1) to obtain intermediate 1.

[0057] Step 2: Weigh an appropriate amount of intermediate 1 (1 equiv) and place it in a 25 mL round-bottom flask. Add 5 mL of acetonitrile solution and heat with stirring until completely dissolved. Weigh 1 equiv of 4-chloroindigo and add it to the round-bottom flask. Stir and dissolve until the solution changes from colorless and clear to yellow and clear. Add anhydrous potassium carbonate and heat under reflux at 80 °C for 3 h. Monitor the reaction progress by TLC. The development conditions are petroleum ether:ethyl acetate = 3:1. After the reaction stops, remove the solvent under reduced pressure. Add 20 mL of distilled water and 20 mL of dichloromethane solution, respectively, and extract three times to obtain a dichloromethane layer. Add anhydrous sodium sulfate, shake, and let stand for 2 h. Filter the solution and add sample-mixed silica gel (100-200 mesh). Remove the solvent under reduced pressure and dry to obtain the sample to be separated. Separate by silica gel column chromatography. The separation conditions are petroleum ether:ethyl acetate (6:1), finally yielding compound 4c.

[0058] The resulting compound was a light yellow powder. 1 H NMR(300MHz, CDCl3)δ0.75(s,3H),0.81(s,3H),0.85–0.88(m,6H),(s,3H),1.18(s,3H), 1.22(s,3H),1.27(s,4H),1.35(s,1H),1.37–1.97(m,22H),3.47–3.56(td,J=5.1,10.2H z,1H),4.42–4.53(d,J=11.0Hz,2H),4.53–4.59(dd,J=3.8,6.7Hz,1H),6.28(s,1H),6.6 9(dd,J=0.7,8.0Hz,1H),7.08–7.12(dd,J=0.7,8.2Hz,1H),7.47–7.53(t,J=8.1Hz,1H). 13 C NMR (75MHz, CDCl3) δ15.75,16.25,16.41,16.50,17.17,18.27,19.55,23.82,2 5.28,27.28,28.17,30.70,31.26,33.16,34.86,35.87,36.58,37.13,38.03,38 .56,39.92,41.79,49.28,49.93,51.82,54.85,55.98,69.95,73.26,77.58,83.73,108.46,114.94,126.29,133.48,138.61,152.79,157.26,165.54,179.82.

[0059] Example 4

[0060]

[0061] Step 1: Weigh an appropriate amount of the starting compound dammarane saponin (1 equiv) and place it in a 100 mL round-bottom flask. Add an appropriate amount of dry dichloromethane and stir at room temperature until completely dissolved. Weigh DCC (2 equiv), DMAP (1 equiv), and chloroacetic acid (1 equiv) and add them to the solution separately. React at room temperature for 4 hours, and monitor the reaction by TLC. After the reaction is complete, filter the reaction mixture twice, and retain the filtrate to remove DCC. Extract the filtrate three times with saturated sodium bicarbonate solution to obtain a dichloromethane extract. Add anhydrous sodium sulfate to the extract, dry it, and let it stand overnight. Filter and concentrate under reduced pressure to obtain the sample to be separated. Purify by column chromatography (petroleum ether / ethyl acetate ratio 5 / 1) to obtain intermediate 1.

[0062] Step 2: Weigh an appropriate amount of intermediate 1 (1 equiv) and place it in a 25 mL round-bottom flask. Add 5 mL of acetonitrile solution and heat and stir until completely dissolved. Weigh 5-chloroindigo (1 equiv) and add it to the round-bottom flask. Stir and dissolve until the solution changes from colorless and clear to yellow and clear. Add anhydrous potassium carbonate and heat to reflux at 80 °C for 3 h. Monitor the reaction progress by TLC. The development conditions are petroleum ether:ethyl acetate = 3:1. After the reaction stops, remove the solvent under reduced pressure. Add 20 mL of distilled water and 20 mL of dichloromethane solution respectively, and extract three times to obtain a dichloromethane layer solution. Add anhydrous sodium sulfate, shake, and let stand for 2 h. Filter the solution and add sample-mixed silica gel (100-200 mesh). Remove the solvent under reduced pressure and dry to obtain the sample to be separated. Separate by silica gel column chromatography. The separation conditions are petroleum ether:ethyl acetate (6:1). Finally, compound 4d is obtained.

[0063] The resulting compound was a light yellow powder. 1 H NMR(300MHz, CDCl3)δ0.74(s,3H),0.83(s,4H),0.84–0.88(m,6H),0.97(s,3H),0.99 –1.08(m,2H),1.17(s,3H),1.21(s,3H),1.26(s,4H),1.34–1.93(m,20H),3.46–3.55( td,J=5.2,10.3Hz,1H),4.37–4.53(m,2H),4.53–4.59(d,J=11.0Hz,1H),6.30(s,1H), 6.80(d,J=1.6Hz,1H),7.09–7.16(dd,J=1.6,8.0Hz,1H),7.55–7.60(d,J=8.0Hz,1H). 13C NMR (75MHz, CDCl3) δ15.75,16.25,16.41,16.48,17.16,18.27,19.55,23.81,2 5.71,27.27,28.13,30.68,31.66,33.16,34.86,35.87,36.57,37.13,38.03,38 .57,39.92,41.77,49.27,49.92,51.31,54.84,55.98,69.95,73.25,77.36,83.86,112.34,116.08,123.96,126.74,146.39,152.73,159.51,165.84,183.51.

[0064] Example 5

[0065]

[0066] Step 1: Weigh an appropriate amount of the starting compound dammarane saponin (1 equiv) and place it in a 100 ml round-bottom flask. Add an appropriate amount of dry dichloromethane and stir at room temperature until completely dissolved. Weigh DCC (2 equiv), DMAP (1 equiv), and chloroacetic acid (1 equiv) and add them to the solution separately. React at room temperature for 4 hours, and monitor the reaction by TLC. After the reaction is complete, filter the reaction mixture twice, and retain the filtrate to remove DCC. Extract the filtrate three times with saturated sodium bicarbonate solution to obtain a dichloromethane extract. Add anhydrous sodium sulfate to the extract, dry it, and let it stand overnight. Filter and concentrate under reduced pressure to obtain the sample to be separated. Purify by column chromatography (petroleum ether / ethyl acetate ratio 5 / 1) to obtain intermediate 1.

[0067] Step 2: Weigh an appropriate amount of intermediate 1 (1 equiv) and place it in a 25 mL round-bottom flask. Add 5 mL of acetonitrile solution and heat with stirring until completely dissolved. Weigh 7-chloroindigo (1 equiv) and add it to the round-bottom flask. Stir and dissolve until the solution changes from colorless and clear to yellow and clear. Add anhydrous potassium carbonate and heat under reflux at 80 °C for 3 h. Monitor the reaction progress by TLC. The development conditions are petroleum ether:ethyl acetate = 3:1. After the reaction stops, remove the solvent under reduced pressure. Add 20 mL of distilled water and 20 mL of dimethane solution, respectively, and extract three times to obtain a dichloromethane layer. Add anhydrous sodium sulfate, shake, and let stand for 2 h. Filter the solution and add silica gel (100-200 mesh). Remove the solvent under reduced pressure and dry to obtain the sample to be separated. Separate by silica gel column chromatography under petroleum ether:ethyl acetate (6:1) conditions to finally obtain compound 4e.

[0068] The resulting compound was a light yellow powder. 1H NMR(300MHz, CDCl3)δ0.73(s,3H),0.85(s,4H),0.89(s,7H),0.98(s,3H),1.18(s,3H),1. 23(s,4H),1.28(s,5H),1.42–1.88(m,19H),3.47–3.56(td,J=5.1,10.3Hz,1H),4.52–4.59 (dd,J=4.5,11.6Hz,1H),4.83–4.96(d,J=3.1Hz,2H),6.23–6.27(s,1H),7.06–7.13(dd,J1 =7.4,J2=8.2Hz,1H),7.50–7.54(dd,J=1.3,8.2Hz,1H),7.57–7.61(dd,J=1.3,7.4Hz,1H). 13 C NMR (126MHz, CDCl3) δ15.75,16.24,16.41,16.49,17.17,18.26,19.55,23.60, 25.28,27.28,28.14,30.68,31.26,33.15,34.87,35.87,36.58,37.13,37.98,3 8.56, 39.93, 43.58, 49.27, 49.91, 51.32, 54.84, 56.01, 69.97, 73.25, 76.79, 83.45, 117.75, 120.42, 124.34, 125.14, 140.40, 146.19, 158.43, 167.01, 181.86.

[0069] Example 6

[0070]

[0071] Step 1: Weigh an appropriate amount of the starting compound dammarane saponin (1 equiv) and place it in a 100 ml round-bottom flask. Add an appropriate amount of dry dichloromethane and stir at room temperature until completely dissolved. Weigh DCC (2 equiv), DMAP (1 equiv), and chloroacetic acid (1 equiv) and add them to the solution separately. React at room temperature for 4 hours, and monitor the reaction by TLC. After the reaction is complete, filter the reaction mixture twice, and retain the filtrate to remove DCC. Extract the filtrate three times with saturated sodium bicarbonate solution to obtain a dichloromethane extract. Add anhydrous sodium sulfate to the extract, dry it, and let it stand overnight. Filter and concentrate under reduced pressure to obtain the sample to be separated. Purify by column chromatography (petroleum ether / ethyl acetate ratio 5 / 1) to obtain intermediate 1.

[0072] Step 2: Weigh an appropriate amount of intermediate 1 (1 equiv) and place it in a 25 mL round-bottom flask. Add 5 mL of acetonitrile solution and heat and stir until completely dissolved. Weigh 5-fluoroindigo (1 equiv) and add it to the round-bottom flask. Stir and dissolve until the solution changes from colorless and clear to yellow and clear. Add anhydrous potassium carbonate and heat to reflux at 80 °C for 3 h. Monitor the reaction progress by TLC. The development conditions are petroleum ether:ethyl acetate = 3:1. After the reaction stops, remove the solvent under reduced pressure. Add 20 mL of distilled water and 20 mL of dichloromethane solution respectively, and extract three times to obtain a dichloromethane layer. Add anhydrous sodium sulfate, shake, and let stand for 2 h. Filter the solution and add sample-mixed silica gel (100-200 mesh). Remove the solvent under reduced pressure and dry to obtain the sample to be separated. Separate by silica gel column chromatography. The separation conditions are petroleum ether:ethyl acetate (6:1), and finally obtain compound 4f.

[0073] The resulting compound was a light yellow powder. 1 H NMR(300MHz, CDCl3)δ0.71(s,3H),0.79(s,4H),0.84–0.88(m,6H),0.96(s ,3H),1.18(s,4H),1.20(s,4H),1.25(s,4H),1.28–1.97(m,20H),3.48–3. 54(dd,J=5.3,10.4Hz,1H),4.40–4.52(m,2H),4.52–4.58(dd,J=4.5,6.2H z,1H),6.26(s,1H),6.71–6.78(dd,J=3.5,8.5Hz,1H),7.27–7.38(m,2H). 13 C NMR (126MHz, CDCl3) δ15.74,16.23,16.39,16.47,17.15,18.25,19.53,23.80,25.26,27.26,28 .14,30.66,31.24,33.14,34.84,35.85,36.56,37.11,38.00,38.54,39.90,41.74,49.25,49.90 ,51.30,54.82,55.95,69.94,73.24,76.77,76.91,83.69,111.45,111.50,112.62,112.81,118.32,118.37,124.85,125.04,146.64,146.65,157.93,158.61,160.57,166.41,182.01,182.03.

[0074] Example 7

[0075]

[0076] Step 1: Weigh an appropriate amount of the starting compound dammarane saponin (1 equiv) and place it in a 100 ml round-bottom flask. Add an appropriate amount of dry dichloromethane and stir at room temperature until completely dissolved. Weigh DCC (2 equiv), DMAP (1 equiv), and chloroacetic acid (1 equiv) and add them to the solution separately. React at room temperature for 4 hours, and monitor the reaction by TLC. After the reaction is complete, filter the reaction mixture twice, and retain the filtrate to remove DCC. Extract the filtrate three times with saturated sodium bicarbonate solution to obtain a dichloromethane extract. Add anhydrous sodium sulfate to the extract, dry it, and let it stand overnight. Filter and concentrate under reduced pressure to obtain the sample to be separated. Purify by column chromatography (petroleum ether / ethyl acetate ratio 5 / 1) to obtain intermediate 1.

[0077] Step 2: Weigh an appropriate amount of intermediate 1 (1 equiv) and place it in a 25 mL round-bottom flask. Add 5 mL of acetonitrile solution and heat and stir until completely dissolved. Weigh 5-fluoroindigo (1 equiv) and add it to the round-bottom flask. Stir and dissolve until the solution changes from colorless and clear to yellow and clear. Add anhydrous potassium carbonate and heat to reflux at 80 °C for 3 h. Monitor the reaction progress by TLC. The development conditions are petroleum ether:ethyl acetate = 3:1. After the reaction stops, remove the solvent under reduced pressure. Add 20 mL of distilled water and 20 mL of dichloromethane solution respectively, and extract three times to obtain a dichloromethane layer solution. Add anhydrous sodium sulfate, shake, and let stand for 2 h. Filter the solution and add sample-mixed silica gel (100-200 mesh). Remove the solvent under reduced pressure and dry to obtain the sample to be separated. Separate by silica gel column chromatography. The separation conditions are petroleum ether:ethyl acetate (6:1). Finally, 4 g of the compound is obtained.

[0078] The resulting compound was a light yellow powder. 1 H NMR(300MHz, CDCl3)δ0.71(s,3H),0.79(s,4H),0.84–0.88(m,6H),0.96(s ,3H),1.18(s,4H),1.20(s,4H),1.25(s,4H),1.28–1.97(m,20H),3.48–3. 54(dd,J=5.3,10.4Hz,1H),4.40–4.52(m,2H),4.52–4.58(dd,J=4.5,6.2H z,1H),6.26(s,1H),6.71–6.78(dd,J=3.5,8.5Hz,1H),7.27–7.38(m,2H). 13C NMR (126MHz, CDCl3) δ15.74,16.23,16.39,16.47,17.15,18.25,19.53,23.80,25.26,27.26,28 .14,30.66,31.24,33.14,34.84,35.85,36.56,37.11,38.00,38.54,39.90,41.74,49.25,49.90 ,51.30,54.82,55.95,69.94,73.24,76.77,76.91,83.69,111.45,111.50,112.62,112.81,118.32,118.37,124.85,125.04,146.64,146.65,157.93,158.61,160.57,166.41,182.01,182.03.

[0079] Example 8

[0080]

[0081] Step 1: Weigh an appropriate amount of the starting compound dammarane saponin (1 equiv) and place it in a 100 ml round-bottom flask. Add an appropriate amount of dry dichloromethane and stir at room temperature until completely dissolved. Weigh DCC (2 equiv), DMAP (1 equiv), and chloroacetic acid (1 equiv) and add them to the solution separately. React at room temperature for 4 hours, and monitor the reaction by TLC. After the reaction is complete, filter the reaction mixture twice, and retain the filtrate to remove DCC. Extract the filtrate three times with saturated sodium bicarbonate solution to obtain a dichloromethane extract. Add anhydrous sodium sulfate to the extract, dry it, and let it stand overnight. Filter and concentrate under reduced pressure to obtain the sample to be separated. Purify by column chromatography (petroleum ether / ethyl acetate ratio 5 / 1) to obtain intermediate 1.

[0082] Step 2: Weigh an appropriate amount of intermediate 1 (1 equiv) and place it in a 25 mL round-bottom flask. Add 5 mL of acetonitrile solution and heat and stir until completely dissolved. Weigh 7-fluoroindigo (1 equiv) and add it to the round-bottom flask. Stir and dissolve until the solution changes from colorless and clear to yellow and clear. Add anhydrous potassium carbonate and heat to reflux at 80 °C for 3 h. Monitor the reaction progress by TLC. The development conditions are petroleum ether:ethyl acetate = 3:1. After the reaction stops, remove the solvent under reduced pressure. Add 20 mL of distilled water and 20 mL of dichloromethane solution respectively, and extract three times to obtain a dichloromethane layer. Add anhydrous sodium sulfate, shake, and let stand for 2 h. Filter the solution and add sample-mixed silica gel (100-200 mesh). Remove the solvent under reduced pressure and dry to obtain the sample to be separated. Separate by silica gel column chromatography. The separation conditions are petroleum ether:ethyl acetate (6:1). The compound was finally obtained after 4 h.

[0083] The resulting compound was a light yellow powder. 1 H NMR(300MHz, CDCl3)δ0.73(s,3H),0.83(s,4H),0.86–0.89(m,6H),0.96(s,3H),1.17(s,3H),1. 21(s,3H),1.24–1.27(d,J=2.3Hz,5H),1.30–1.56(m,11H),1.62–1.93(m,10H),3.49–3.55(m,1H ),4.52–4.60(dd,J=5.1,11.3Hz,1H),4.61–4.67(m,2H),6.23–6.28(s,1H),7.07–7.14(ddd,J=4 .0,7.4,8.3Hz,1H),7.30–7.38(ddd,J=1.1,8.4,11.2Hz,1H),7.45–7.50(dd,J=1.1,7.4Hz,1H). 13 C NMR (126MHz, CDCl3) δ15.75,16.24,16.40,16.46,17.17,18.26,19.55,23.66,25.07,25.28,25.76,27.2 7,28.04,29.84,30.67,31.26,33.15,34.09,34.87,35.87,36.57,37.13,37.98,38.57,39.92,43.54,43 .58,49.27,49.29,49.92,51.32,54.84,56.01,69.97,73.25,76.79,83.44,120.21,120.23,121.60,121.63,124.97,125.02,126.29,126.45,137.00,137.07,147.44,149.40,156.90,157.75,167.03,181.74.

[0084] Example 9

[0085]

[0086] Step 1: Weigh an appropriate amount of the starting compound dammarane saponin (1 equiv) and place it in a 100 mL round-bottom flask. Add an appropriate amount of dry dichloromethane and stir at room temperature until completely dissolved. Weigh DCC (2 equiv), DMAP (1 equiv), and chloroacetic acid (1 equiv) and add them to the solution separately. React at room temperature for 4 hours, and monitor the reaction by TLC. After the reaction is complete, filter the reaction mixture twice, and retain the filtrate to remove DCC. Extract the filtrate three times with saturated sodium bicarbonate solution to obtain a dichloromethane extract. Add anhydrous sodium sulfate to the extract, dry it, and let it stand overnight. Filter and concentrate under reduced pressure to obtain the sample to be separated. Purify by column chromatography (petroleum ether / ethyl acetate ratio 5 / 1) to obtain intermediate 1.

[0087] Step 2: Weigh an appropriate amount of intermediate 1 (1 equiv) and place it in a 25 mL round-bottom flask. Add 5 mL of acetonitrile solution and heat and stir until completely dissolved. Weigh 1 equiv of 4-fluoroindigo and add it to the round-bottom flask. Stir and dissolve until the solution changes from colorless and clear to yellow and clear. Add anhydrous potassium carbonate and heat to reflux at 80 °C for 3 h. Monitor the reaction progress by TLC. The development conditions are petroleum ether:ethyl acetate = 3:1. After the reaction stops, remove the solvent under reduced pressure. Add 20 mL of distilled water and 20 mL of dichloromethane solution respectively, and extract three times to obtain a dichloromethane layer. Add anhydrous sodium sulfate, shake, and let stand for 2 h. Filter the solution and add sample-mixed silica gel (100-200 mesh). Remove the solvent under reduced pressure and dry to obtain the sample to be separated. Separate by silica gel column chromatography. The separation conditions are petroleum ether:ethyl acetate (6:1), and finally obtain compound 4i.

[0088] The resulting compound was a light yellow powder. 1 H NMR(300MHz, CDCl3)δ0.73(s,3H),0.80(s,3H),0.85–0.88(m,7H),0.96(s,4H),1.17(s,3H),1.21( s,4H),1.25–1.26(d,J=1.6Hz,5H),1.28(s,2H),1.33(s,1H),1.47–1.63(m,11H),1.65–1.80(m,5H ),3.47–3.56(td,J=5.1,10.3Hz,1H),4.46–4.53(d,J=10.7Hz,2H),4.53–4.59(m,1H),6.26(s,1H) ,6.68–6.73(dd,J=0.8,7.9Hz,1H),7.27–7.31(dd,J=0.8,8.2Hz,1H),7.37–7.44(t,J=8.0Hz,1H).

[0089] Example 10

[0090]

[0091] Step 1: Weigh an appropriate amount of the starting compound dammarane saponin (1 equiv) and place it in a 100 mL round-bottom flask. Add an appropriate amount of dry dichloromethane and stir at room temperature until completely dissolved. Weigh DCC (2 equiv), DMAP (1 equiv), and chloroacetic acid (1 equiv) and add them to the solution separately. React at room temperature for 4 hours, and monitor the reaction by TLC. After the reaction is complete, filter the reaction mixture twice, and retain the filtrate to remove DCC. Extract the filtrate three times with saturated sodium bicarbonate solution to obtain a dichloromethane extract. Add anhydrous sodium sulfate to the extract, dry it, and let it stand overnight. Filter and concentrate under reduced pressure to obtain the sample to be separated. Purify by column chromatography (petroleum ether / ethyl acetate ratio 5 / 1) to obtain intermediate 1.

[0092] Step 2: Weigh an appropriate amount of intermediate 1 (1 equiv) and place it in a 25 mL round-bottom flask. Add 5 mL of acetonitrile solution and heat and stir until completely dissolved. Weigh 5-fluoroindigo (1 equiv) and add it to the round-bottom flask. Stir and dissolve until the solution changes from colorless and clear to yellow and clear. Add anhydrous potassium carbonate and heat to reflux at 80 °C for 3 h. Monitor the reaction progress by TLC. The development conditions are petroleum ether:ethyl acetate = 3:1. After the reaction stops, remove the solvent under reduced pressure. Add 20 mL of distilled water and 20 mL of dichloromethane solution respectively, and extract three times to obtain a dichloromethane layer. Add anhydrous sodium sulfate, shake, and let stand for 2 h. Filter the solution and add sample-mixed silica gel (100-200 mesh). Remove the solvent under reduced pressure and dry to obtain the sample to be separated. Separate by silica gel column chromatography. The separation conditions are petroleum ether:ethyl acetate (6:1), and finally obtain compound 4j.

[0093] The resulting compound was a light yellow powder. 1 H NMR(300MHz, CDCl3)δ0.73(s,3H),0.80(s,3H),0.86(s,2H),0.87(s,2H),0.96(s,3H), 1.17(s,3H),1.21(s,3H),1.24–1.27(d,J=2.2Hz,6H),1.35–1.91(m,23H),3.47–3.56(t d,J=5.1,10.3Hz,1H),4.40–4.53(m,2H),4.53–4.59(d,J=8.6Hz,1H),6.26(s,1H),6.6 6–6.71(d,J=8.3Hz,1H),7.68–7.73(dd,J=2.1,8.4Hz,1H),7.74–7.78(d,J=2.0Hz,1H).

[0094] Example 11

[0095]

[0096] Step 1: Weigh an appropriate amount of the starting compound dammarane saponin (1 equiv) and place it in a 100 mL round-bottom flask. Add an appropriate amount of dry dichloromethane and stir at room temperature until completely dissolved. Weigh DCC (2 equiv), DMAP (1 equiv), and chloroacetic acid (1 equiv) and add them to the solution separately. React at room temperature for 4 hours, and monitor the reaction by TLC. After the reaction is complete, filter the reaction mixture twice, and retain the filtrate to remove DCC. Extract the filtrate three times with saturated sodium bicarbonate solution to obtain a dichloromethane extract. Add anhydrous sodium sulfate to the extract, dry it, and let it stand overnight. Filter and concentrate under reduced pressure to obtain the sample to be separated. Purify by column chromatography (petroleum ether / ethyl acetate ratio 5 / 1) to obtain intermediate 1.

[0097] Step 2: Weigh an appropriate amount of intermediate 1 (1 equiv) and place it in a 25 mL round-bottom flask. Add 5 mL of acetonitrile solution and heat and stir until completely dissolved. Weigh 5-methoxyindigo (1 equiv) and add it to the round-bottom flask. Stir and dissolve until the solution changes from colorless and clear to yellow and clear. Add anhydrous potassium carbonate and heat to reflux at 80 °C for 3 h. Monitor the reaction progress by TLC. The development conditions are petroleum ether:ethyl acetate = 3:1. After the reaction stops, remove the solvent under reduced pressure. Add 20 mL of distilled water and 20 mL of dichloromethane solution respectively, and extract three times to obtain a dichloromethane layer. Add anhydrous sodium sulfate, shake, and let stand for 2 h. Filter the solution and add sample-mixed silica gel (100-200 mesh). Remove the solvent under reduced pressure and dry to obtain the sample to be separated. Separate by silica gel column chromatography. The separation conditions are petroleum ether:ethyl acetate (6:1), and finally obtain compound 4K.

[0098] The resulting compound was a light yellow powder. 1 H NMR(300MHz, CDCl3)δ0.71(s,3H),0.79(s,3H),0.86(s,7H),0.96(s,3H),1.17(s,3H),1.21(s,3H),1.26(s,5H),1.38–1.92(m,21H),3.46–3.5 7(td,J=5.1,10.3Hz,1H),3.78–3.84(s,3H),4.37–4.51(m,2H),4.50–4 .57(m,1H),6.25(s,1H),6.67–6.73(d,J=8.4Hz,1H),7.11–7.19(m,2H).

[0099] Example 12

[0100]

[0101] Step 1: Weigh an appropriate amount of the starting compound dammarane saponin (1 equiv) and place it in a 100 mL round-bottom flask. Add an appropriate amount of dry dichloromethane and stir at room temperature until completely dissolved. Weigh DCC (2 equiv), DMAP (1 equiv), and chloroacetic acid (1 equiv) and add them to the solution separately. React at room temperature for 4 hours, and monitor the reaction by TLC. After the reaction is complete, filter the reaction mixture twice, and retain the filtrate to remove DCC. Extract the filtrate three times with saturated sodium bicarbonate solution to obtain a dichloromethane extract. Add anhydrous sodium sulfate to the extract, dry it, and let it stand overnight. Filter and concentrate under reduced pressure to obtain the sample to be separated. Purify by column chromatography (petroleum ether / ethyl acetate ratio 5 / 1) to obtain intermediate 1.

[0102] Step 2: Weigh an appropriate amount of intermediate 1 (1 equiv) and place it in a 25 mL round-bottom flask. Add 5 mL of acetonitrile solution and heat and stir until completely dissolved. Weigh 5-chloroindigo (1 equiv) and add it to the round-bottom flask. Stir and dissolve until the solution changes from colorless and clear to yellow and clear. Add anhydrous potassium carbonate and heat to reflux at 80 °C for 3 h. Monitor the reaction progress by TLC. The development conditions are petroleum ether:ethyl acetate = 3:1. After the reaction stops, remove the solvent under reduced pressure. Add 20 mL of distilled water and 20 mL of dichloromethane solution respectively, and extract three times to obtain a dichloromethane layer. Add anhydrous sodium sulfate, shake, and let stand for 2 h. Filter the solution and add sample-mixed silica gel (100-200 mesh). Remove the solvent under reduced pressure and dry to obtain the sample to be separated. Separate by silica gel column chromatography. The separation conditions are petroleum ether:ethyl acetate (6:1), finally yielding compound 4l.

[0103] The resulting compound was a light yellow powder. 1 H NMR(500MHz,CHCl3)δ0.71(s,3H),0.83(s,3H),0.86(d,J=2.0Hz,6H),0.96(s,3H), 1.17(s,3H),1.21(s,3H),1.25(s,4H),1.33–1.94(m,23H),3.47–3.55(td,J=5.2,1 0.4Hz,1H),4.51–4.58(dd,J=4.5,11.8Hz,1H),4.84–4.94(m,2H),6.27(s,1H),7.0 7–7.11(m,1H),7.50–7.53(dd,J=1.2,8.1Hz,1H),7.57–7.60(dd,J=1.2,7.4Hz,1H).

[0104] Example 13

[0105]

[0106] Step 1: Weigh an appropriate amount of the starting compound dammarane saponin (1 equiv) and place it in a 100 ml round-bottom flask. Add an appropriate amount of dry dichloromethane and stir at room temperature until completely dissolved. Weigh DCC (2 equiv), DMAP (1 equiv), and chloroacetic acid (1 equiv) and add them to the solution separately. React at room temperature for 4 hours, and monitor the reaction by TLC. After the reaction is complete, filter the reaction mixture twice, and retain the filtrate to remove DCC. Extract the filtrate three times with saturated sodium bicarbonate solution to obtain a dichloromethane extract. Add anhydrous sodium sulfate to the extract, dry it, and let it stand overnight. Filter and concentrate under reduced pressure to obtain the sample to be separated. Purify by column chromatography (petroleum ether / ethyl acetate ratio 5 / 1) to obtain intermediate 1.

[0107] Step 2: Weigh an appropriate amount of intermediate 1 (1 equiv) and place it in a 25 mL round-bottom flask. Add 5 mL of acetonitrile solution and heat and stir until completely dissolved. Weigh 6-bromoindigo (1 equiv) and add it to the round-bottom flask. Stir and dissolve until the solution changes from colorless and clear to yellow and clear. Add anhydrous potassium carbonate and heat to reflux at 80 °C for 3 h. Monitor the reaction progress by TLC. The development conditions are petroleum ether:ethyl acetate = 3:1. After the reaction stops, remove the solvent under reduced pressure. Add 20 mL of distilled water and 20 mL of dichloromethane solution respectively, and extract three times to obtain a dichloromethane layer. Add anhydrous sodium sulfate, shake, and let stand for 2 h. Filter the solution and add sample-mixed silica gel (100-200 mesh). Remove the solvent under reduced pressure and dry to obtain the sample to be separated. Separate by silica gel column chromatography. The separation conditions are petroleum ether:ethyl acetate (6:1). Finally, compound 4m is obtained.

[0108] The resulting compound was a light yellow powder. 1 H NMR(500MHz, CDCl3)δ0.72(s,3H),0.81(s,4H),0.86(s,3H),0.87(s,3H),0.96(s,3H),0.99–1.05(m ,2H),1.17(s,3H),1.21(s,4H),1.24–1.26(d,J=3.9Hz,6H),1.39–1.51(m,6H),1.61–1.65(d,J=4.5 Hz,5H),1.71–1.96(m,6H),3.49–3.55(td,J=5.1,10.3Hz,1H),4.40–4.53(m,2H),4.54–4.59(dd,J= 5.1,11.5Hz,1H),6.27(s,1H),6.96(s,1H),7.30–7.33(d,J=8.0Hz,1H),7.49–7.51(d,J=8.0Hz,1H). 13C NMR (126MHz, CDCl3) δ15.75,16.25,16.40,16.47,17.16,18.27,19.55,23.80, 25.28,27.27,28.13,30.67,31.25,33.15,34.86,35.87,36.57,37.13,38.03,3 8.58,39.92,41.77,49.27,49.92,51.31,54.84,55.98,69.95,73.26,76.78,83.87,114.09,116.46,126.65,127.57,133.81,151.31,157.95,166.26,181.35.

[0109] Example 14

[0110]

[0111] Step 1: Weigh an appropriate amount of the starting compound dammarane saponin (1 equiv) and place it in a 100 mL round-bottom flask. Add an appropriate amount of dry dichloromethane and stir at room temperature until completely dissolved. Weigh DCC (2 equiv), DMAP (1 equiv), and chloroacetic acid (1 equiv) and add them to the solution separately. React at room temperature for 4 hours, and monitor the reaction by TLC. After the reaction is complete, filter the reaction mixture twice, and retain the filtrate to remove DCC. Extract the filtrate three times with saturated sodium bicarbonate solution to obtain a dichloromethane extract. Add anhydrous sodium sulfate to the extract, dry it, and let it stand overnight. Filter and concentrate under reduced pressure to obtain the sample to be separated. Purify by column chromatography (petroleum ether / ethyl acetate ratio 5 / 1) to obtain intermediate 1.

[0112] Step 2: Weigh an appropriate amount of intermediate 1 (1 equiv) and place it in a 25 mL round-bottom flask. Add 5 mL of acetonitrile solution and heat with stirring until completely dissolved. Weigh 7-bromoindigo (1 equiv) and add it to the round-bottom flask. Stir and dissolve until the solution changes from colorless and clear to yellow and clear. Add anhydrous potassium carbonate and heat under reflux at 80 °C for 3 h. Monitor the reaction progress by TLC. The development conditions are petroleum ether:ethyl acetate = 3:1. After the reaction stops, remove the solvent under reduced pressure. Add 20 mL of distilled water and 20 mL of dichloromethane solution, respectively, and extract three times to obtain a dichloromethane layer. Add anhydrous sodium sulfate, shake, and let stand for 2 h. Filter the solution and add sample-mixed silica gel (100-200 mesh). Remove the solvent under reduced pressure and dry to obtain the sample to be separated. Separate by silica gel column chromatography. The separation conditions are petroleum ether:ethyl acetate (6:1), finally yielding compound 4n.

[0113] The resulting compound was a light yellow powder. 1H NMR(500MHz, CDCl3)δ0.71(s,3H),0.83(s,3H),0.86(s,6H),0.96(s,3H),1.17(s ,3H),1.20(s,3H),1.25(s,4H),1.31–1.95(m,23H),3.48–3.55(td,J=5.2,10.3H z,1H),4.51–4.58(dd,J=4.2,12.0Hz,1H),4.91–5.00(m,2H),6.27(s,1H),7.00– 7.04(m,1H),7.60–7.64(dd,J=1.3,7.3Hz,1H),7.68–7.71(dd,J=1.3,8.1Hz,1H).

[0114] Example 15

[0115]

[0116] Step 1: Weigh an appropriate amount of the starting compound dammarane saponin (1 equiv) and place it in a 100 mL round-bottom flask. Add an appropriate amount of dry dichloromethane and stir at room temperature until completely dissolved. Weigh DCC (2 equiv), DMAP (1 equiv), and chloroacetic acid (1 equiv) and add them to the solution separately. React at room temperature for 4 hours, and monitor the reaction by TLC. After the reaction is complete, filter the reaction mixture twice, and retain the filtrate to remove DCC. Extract the filtrate three times with saturated sodium bicarbonate solution to obtain a dichloromethane extract. Add anhydrous sodium sulfate to the extract, dry it, and let it stand overnight. Filter and concentrate under reduced pressure to obtain the sample to be separated. Purify by column chromatography (petroleum ether / ethyl acetate ratio 5 / 1) to obtain intermediate 1.

[0117] Step 2: Weigh an appropriate amount of intermediate 1 (1 equiv) and place it in a 25 mL round-bottom flask. Add 5 mL of acetonitrile solution and heat and stir until completely dissolved. Weigh 7-trifluoromethyl indigo (1 equiv) and add it to the round-bottom flask. Stir and dissolve until the solution changes from colorless and clear to yellow and clear. Add anhydrous potassium carbonate and heat at 80 °C under reflux for 3 h. Monitor the reaction progress by TLC. The development conditions are petroleum ether:ethyl acetate = 3:1. After the reaction stops, remove the solvent under reduced pressure. Add 20 mL of distilled water and 20 mL of dichloromethane solution respectively, and extract three times to obtain a dichloromethane layer solution. Add anhydrous sodium sulfate, shake, and let stand for 2 h. Filter the solution and add sample-mixed silica gel (100-200 mesh). Remove the solvent under reduced pressure and dry to obtain the sample to be separated. Separate by silica gel column chromatography. The separation conditions are petroleum ether:ethyl acetate (6:1), and finally obtain compound 4o.

[0118] The resulting compound was a light yellow powder. 1H NMR (500MHz, CDCl3) δ0.73 (s, 3H), 0.81 (s, 3H), 0.86 (d, J = 2.8Hz, 6H), 0.96 (s, 3H), 1.17(s,3H),1.20(s,3H),1.25(s,4H),1.42(s,13H),1.64–1.96(m,7H),3.48–3.54( td,J=5.2,10.3Hz,1H),4.48–4.53(dd,J=4.8,11.7Hz,1H),4.69–4.79(m,2H),6.25 (s,1H),7.27–7.29(d,J=7.8Hz,1H),7.86–7.87(d,J=1.5Hz,1H),7.87–7.89(m,1H).

[0119] Example 16

[0120]

[0121] Step 1: Weigh an appropriate amount of the starting compound dammarane saponin (1 equiv) into a 100 mL round-bottom flask, add an appropriate amount of dry dichloromethane, and stir at room temperature until completely dissolved. Under ice bath conditions, add chloroacetyl chloride (4-10 equiv) to the solution and react at room temperature for 0.5-3 h. Transfer to an oil bath at 80-100℃ and react for 4-6 h, monitoring with a TCL until the reaction stops. After the reaction stops, extract three times with saturated sodium bicarbonate solution, dry to anhydrous sodium sulfate, and purify by column chromatography to obtain intermediate 2.

[0122] Step 2: Weigh an appropriate amount of intermediate 2 into a 50 mL round-bottom flask, add an appropriate amount of acetonitrile solution, heat in an oil bath to 50 °C, and stir for 10 min until completely dissolved; weigh indigo (2 equiv) into the round-bottom flask and stir to dissolve. The solution changes from colorless and clear to yellow and clear. Add anhydrous potassium carbonate, and heat to reflux at 80 °C for 8 h. Monitor the reaction progress by TLC, with petroleum ether:ethyl acetate = 3:1 as the development conditions. After the reaction stops, remove the solvent under reduced pressure, add 20 mL of distilled water and 20 mL of dichloromethane solution respectively, and extract three times to obtain a dichloromethane layer solution. Add anhydrous sodium sulfate, shake, and let stand for 2 h. Filter the solution, add sample-mixed silica gel (100-200 mesh), remove the solvent under reduced pressure, and dry to obtain the sample to be separated. Separate by silica gel column chromatography, with petroleum ether:ethyl acetate = 3:1 as the separation conditions, finally obtaining compound 5a.

[0123] The resulting compound was a light yellow powder. 1H NMR(300MHz, CDCl3)δ0.68(s,3H),0.77(s,3H),0.80(s,3H),0.90(s,3H),0.92(s,3H),1.13( s,3H),1.17(s,3H),1.21(s,3H),1.29–1.91(m,23H),2.02–2.10(m,1H),4.32–4.52(m,4H),4 .53–4.58(d,J=5.4Hz,1H),4.95–5.05(td,J=5.4,10.5Hz,1H),6.75–6.81(dd,J=2.7,7.9Hz, 2H),7.11–7.19(td,J=4.2,7.6Hz,2H),7.53–7.61(td,J=1.4,7.8Hz,2H),7.62–7.68(m,2H). 13 C NMR (126MHz, CDCl3) δ15.80,16.01,16.45,16.52,18.15,18.39,23.58,25.34,26.54,28.10,28.64, 29.49,29.85,31.15,32.56,34.24,37.05,37.10,37.99,38.43,39.65,41.71,42.40,45.31,49.76, 51.38,52.38,55.80,71.25,75.66,77.24,83.25,110.24,110.67,117.46,117.80,124.22,124.35,125.68,125.78,138.36,138.57,150.58,150.65,158.05,158.20,166.45,166.52,182.61,182.69.

[0124] Example 17

[0125]

[0126] Step 1: Weigh an appropriate amount of the starting compound dammarane saponin (1 equiv) into a 100 mL round-bottom flask, add an appropriate amount of dry dichloromethane, and stir at room temperature until completely dissolved. Under ice bath conditions, add chloroacetyl chloride (4-10 equiv) to the solution and react at room temperature for 0.5-3 h. Transfer to an oil bath at 80-100℃ and react for 4-6 h, monitoring with a TCL until the reaction stops. After the reaction stops, extract three times with saturated sodium bicarbonate solution, dry to anhydrous sodium sulfate, and purify by column chromatography to obtain intermediate 2.

[0127] Step 2: Weigh an appropriate amount of intermediate 2 into a 50 mL round-bottom flask, add an appropriate amount of acetonitrile solution, heat in an oil bath to 50 °C, and stir for 10 min until completely dissolved; weigh 5-methylindigo (2 equiv) into the round-bottom flask and stir to dissolve. The solution changes from colorless and clear to yellow and clear. Add anhydrous potassium carbonate, and heat at 80 °C under reflux for 8 h. Monitor the reaction progress by TLC, with petroleum ether:ethyl acetate = 3:1 as the development conditions. After the reaction stops, remove the solvent under reduced pressure, add 20 mL of distilled water and 20 mL of dichloromethane solution respectively, and extract three times to obtain a dichloromethane layer solution. Add anhydrous sodium sulfate, shake, and let stand for 2 h. Filter the solution, add sample-mixed silica gel (100-200 mesh), remove the solvent under reduced pressure, and dry to obtain the sample to be separated. Separate by silica gel column chromatography, with petroleum ether:ethyl acetate = 3:1 as the separation conditions, finally obtaining compound 5b.

[0128] The resulting compound was a light yellow powder. 1 H NMR(300MHz, CDCl3)δ0.59(s,3H),0.68(s,3H),0.70(s,3H),0.80(s,3H),0.82(s,3H),0.84–1.00(m,3H),1 .03(s,3H),1.07(s,3H),1.11(s,3H),1.15(s,2H),1.29–1.80(m,18H),1.91–2.02(td,J=5.3,8.6,9.1Hz,1H ),2.20–2.27(d,J=2.7Hz,6H),4.19–4.40(m,4H),4.41–4.45(q,J=3.1,4.0Hz,1H),4.83–4.95(td,J=5.4,10 .5Hz, 1H), 6.54–6.61 (dd, J=3.9, 8.0Hz, 2H), 7.24–7.32 (td, J=1.9, 7.6Hz, 2H), 7.32–7.36 (t, J=2.6Hz, 2H). 13C NMR (126MHz, CDCl3) δ15.79,15.96,16.46,16.52,18.15,18.38,20.83,20.84,23.58,25.34,26.52,28. 10,28.64,29.39,29.84,31.17,32.60,33.50,34.24,37.04,37.99,38.43,39.64,41.72,42.43,45.29, 49.76,51.45,52.39,55.80,71.22,75.65,77.41,83.14,110.02,110.48,117.79,117.84,125.93,126.06,134.08,134.20,138.79,138.97,148.42,148.51,158.23,158.36,166.53,166.63,182.87,182.95.

[0129] Example 18

[0130]

[0131] Step 1: Weigh an appropriate amount of the starting compound dammarane saponin (1 equiv) into a 100 ml round-bottom flask, add an appropriate amount of dry dichloromethane, and stir at room temperature until completely dissolved. Add chloroacetyl chloride (4-10 equiv) to the solution under ice bath conditions, and react at room temperature for 0.5-3 h. Transfer to an oil bath at 80-100℃ and react for 4-6 h, monitoring with a TCL until the reaction stops. After the reaction stops, extract three times with saturated sodium bicarbonate solution, dry with anhydrous sodium sulfate, and purify by column chromatography to obtain intermediate 2.

[0132] Step 2: Weigh an appropriate amount of intermediate 2 into a 50 mL round-bottom flask, add an appropriate amount of acetonitrile solution, heat in an oil bath to 50 °C, and stir for 10 min until completely dissolved; weigh 5-methoxyindigo (2 equiv) into the round-bottom flask and stir to dissolve. The solution changes from colorless and clear to yellow and clear. Add anhydrous potassium carbonate and heat to reflux at 80 °C for 8 h. Monitor the reaction progress by TLC, with the development conditions being petroleum ether:ethyl acetate = 3:1. After the reaction stops, remove the solvent under reduced pressure, add 20 mL of distilled water and 20 mL of dichloromethane solution respectively, and extract three times to obtain a dichloromethane layer solution. Add anhydrous sodium sulfate, shake, and let stand for 2 h. Filter the solution, add sample-mixed silica gel (100-200 mesh), remove the solvent under reduced pressure, and dry to obtain the sample to be separated. Separate by silica gel column chromatography, with the separation conditions being petroleum ether:ethyl acetate = 3:1, finally obtaining compound 5c.

[0133] The resulting compound was a light yellow powder. 1H NMR(300MHz, CDCl3)δ0.70(s,3H),0.78(s,3H),0.80(s,3H),0.90(s,3H),0.92(s,3H),1.01(s,1H),1.13 (s,3H),1.17(s,3H),1.20(s,3H),1.25(s,5H),1.39–1.53(m,8H),1.57(s,6H),1.73–1.91(m,3H),2.01– 2.08(m,1H),3.78–3.84(d,J=2.5Hz,6H),4.28–4.50(m,4H),4.51–4.56(m,1H),4.94–5.05(td,J=5.3,10 .5Hz,1H),6.67–6.70(d,J=1.9Hz,1H),6.70–6.74(d,J=1.7Hz,1H),7.09–7.16(m,2H),7.16–7.19(m,2H). 13 CNMR (126MHz, CDCl3) δ15.80,15.99,16.47,16.51,18.15,18.38,23.57,25.33,26.52,28.10,28.64,29 .44,29.91,31.15,32.56,33.54,34.24,37.04,37.10,37.99,38.43,39.64,41.73,42.46,45.29,49.76 ,51.40,52.37,55.80,56.14,71.23,75.65,77.37,83.16,109.61,109.72,111.25,111.70,118.17,118.21,124.89,125.08,144.51,144.60,156.83,156.89,158.23,158.35,166.56,166.65,182.96,183.04.

[0134] Example 19

[0135]

[0136] Step 1: Weigh an appropriate amount of the starting compound dammarane saponin (1 equiv) into a 100 ml round-bottom flask, add an appropriate amount of dry dichloromethane, and stir at room temperature until completely dissolved. Add chloroacetyl chloride (4-10 equiv) to the solution under ice bath conditions, and react at room temperature for 0.5-3 h. Transfer to an oil bath at 80-100℃ and react for 4-6 h, monitoring with a TCL until the reaction stops. After the reaction stops, extract three times with saturated sodium bicarbonate solution, dry with anhydrous sodium sulfate, and purify by column chromatography to obtain intermediate 2.

[0137] Step 2: Weigh an appropriate amount of intermediate 2 into a 50 mL round-bottom flask, add an appropriate amount of acetonitrile solution, heat in an oil bath to 50 °C, and stir for 10 min until completely dissolved; weigh 2 equiv of 4-chloroindigo and add it to the round-bottom flask, stirring to dissolve. The solution changes from colorless and clear to yellow and clear. Add anhydrous potassium carbonate, and heat under reflux at 80 °C for 8 h. Monitor the reaction progress by TLC, with petroleum ether:ethyl acetate = 3:1 as the development conditions. After the reaction stops, remove the solvent under reduced pressure, add 20 mL of distilled water and 20 mL of dichloromethane solution respectively, and extract three times to obtain a dichloromethane layer solution. Add anhydrous sodium sulfate, shake, and let stand for 2 h. Filter the solution, add silica gel (100-200 mesh), remove the solvent under reduced pressure, and dry to obtain the sample to be separated. Separate by silica gel column chromatography, with petroleum ether:ethyl acetate = 3:1 as the separation conditions, finally obtaining compound 5d.

[0138] The resulting compound was a light yellow powder. 1 H NMR(300MHz, CDCl3)δ0.72(s,3H),0.79(s,3H),0.82(s,3H),0.90(s,3H),0.93(s,3H), 1.13(s,3H),1.16(s,3H),1.18(s,3H),1.25(s,3H),1.40–1.91(dd,J=8.7,22.1Hz,21H ),4.35–4.52(m,4H),4.52–4.57(m,1H),4.94–5.06(td,J=5.3,10.4Hz,1H),6.65–6.72 (dd,J=1.4,7.9Hz,2H),7.06–7.14(dd,J=4.3,8.2Hz,2H),7.43–7.54(q,J=7.8Hz,2H). 13 C NMR (126MHz, CDCl3) δ15.83,16.03,16.49,18.15,18.38,23.60,25.29,26.55,28.13,28.62,29.65, 31.11,32.46,33.70,34.24,37.04,37.09,38.02,38.43,39.65,41.79,42.50,45.31,49.75,51.23, 52.35,53.57,55.80,71.27,75.66,77.35,83.39,108.47,108.89,114.93,114.97,125.76,125.91,134.21,134.32,138.44,138.64,151.59,151.68,157.15,157.28,166.19,166.27,179.42,179.52.

[0139] Example 20

[0140]

[0141] Step 1: Weigh an appropriate amount of the starting compound dammarane saponin (1 equiv) into a 100 ml round-bottom flask, add an appropriate amount of dry dichloromethane, and stir at room temperature until completely dissolved. Add chloroacetyl chloride (4-10 equiv) to the solution under ice bath conditions, and react at room temperature for 0.5-3 h. Transfer to an oil bath at 80-100℃ and react for 4-6 h, monitoring with a TCL until the reaction stops. After the reaction stops, extract three times with saturated sodium bicarbonate solution, dry with anhydrous sodium sulfate, and purify by column chromatography to obtain intermediate 2.

[0142] Step 2: Weigh an appropriate amount of intermediate 2 into a 50 mL round-bottom flask, add an appropriate amount of acetonitrile solution, heat in an oil bath to 50 °C, and stir for 10 min until completely dissolved; weigh 5-chloroindigo (2 equiv) into the round-bottom flask and stir to dissolve. The solution changes from colorless and clear to yellow and clear. Add anhydrous potassium carbonate, and heat to reflux at 80 °C for 8 h. Monitor the reaction progress by TLC, with the development conditions being petroleum ether:ethyl acetate = 3:1. After the reaction stops, remove the solvent under reduced pressure, add 20 mL of distilled water and 20 mL of dichloromethane solution respectively, and extract three times to obtain a dichloromethane layer solution. Add anhydrous sodium sulfate, shake, and let stand for 2 h. Filter the solution, add sample-mixed silica gel (100-200 mesh), remove the solvent under reduced pressure, and dry to obtain the sample to be separated. Separate by silica gel column chromatography, with the separation conditions being petroleum ether:ethyl acetate = 3:1, finally obtaining compound 5e.

[0143] The resulting compound was a light yellow powder. 1 H NMR(300MHz, CDCl3)δ0.72(s,3H),0.79(s,3H),0.82(s,3H),0.90(s,3H),0.93(s ,3H),1.13(s,3H),1.16(s,3H),1.20(s,3H),1.36–1.90(m,24H),4.34–4.51(m,4 H),4.52–4.56(m,1H),4.95–5.05(td,J=5.4,10.5Hz,1H),6.72–6.77(dd,J=1.6, 8.4Hz, 2H), 7.52–7.58 (td, J=2.2, 8.0Hz, 2H), 7.59–7.65 (dd, J=2.2, 3.9Hz, 2H). 13C NMR (126MHz, CDCl3) δ15.83,16.00,16.51,18.15,18.38,23.61,25.20,26.54,28.14,28.65,29.71, 29.83,31.09,32.43,33.82,34.23,37.04,37.10,38.02,38.44,39.65,41.74,42.51,45.31,49.74, 51.19,52.32,55.80,71.27,75.65,77.31,83.40,111.57,112.04,118.57,118.62,125.51,125.65,130.09,130.20,137.73,137.93,148.81,148.93,157.51,157.62,166.20,166.27,181.58,181.68.

[0144] Example 21

[0145]

[0146] Step 1: Weigh an appropriate amount of the starting compound dammarane saponin (1 equiv) into a 100 ml round-bottom flask, add an appropriate amount of dry dichloromethane, and stir at room temperature until completely dissolved. Add chloroacetyl chloride (4-10 equiv) to the solution under ice bath conditions, and react at room temperature for 0.5-3 h. Transfer to an oil bath at 80-100℃ and react for 4-6 h, monitoring with a TCL until the reaction stops. After the reaction stops, extract three times with saturated sodium bicarbonate solution, dry with anhydrous sodium sulfate, and purify by column chromatography to obtain intermediate 2.

[0147] Step 2: Weigh an appropriate amount of intermediate 2 into a 50 mL round-bottom flask, add an appropriate amount of acetonitrile solution, heat in an oil bath to 50 °C, and stir for 10 min until completely dissolved; weigh 6-chloroindigo (2 equiv) into the round-bottom flask and stir to dissolve. The solution changes from colorless and clear to yellow and clear. Add anhydrous potassium carbonate and heat at 80 °C under reflux for 8 h. Monitor the reaction progress by TLC, with the development conditions being petroleum ether:ethyl acetate = 3:1. After the reaction stops, remove the solvent under reduced pressure, add 20 mL of distilled water and 20 mL of dichloromethane solution respectively, and extract three times to obtain a dichloromethane layer solution. Add anhydrous sodium sulfate, shake, and let stand for 2 h. Filter the solution, add sample-mixed silica gel (100-200 mesh), remove the solvent under reduced pressure, and dry to obtain the sample to be separated. Separate by silica gel column chromatography, with the separation conditions being petroleum ether:ethyl acetate = 3:1, finally obtaining compound 5f.

[0148] The resulting compound was a light yellow powder. 1H NMR(300MHz, CDCl3)δ0.71(s,3H),0.81(s,3H),0.82(s,3H),0.91(s,3H),0.93(s,3H),0.95–1. 06(m,3H),1.13(s,3H),1.14(s,3H),1.21(s,3H),1.25(s,3H),1.36–1.54(m,10H),1.61–1.91(m ,8H),4.35–4.52(m,4H),4.52–4.59(q,J=4.9Hz,1H),4.99–5.09(td,J=5.5,10.4Hz,1H),6.77–6 .81(d,J=1.6Hz,2H),7.10–7.16(ddd,J=1.6,4.6,8.0Hz,2H),7.56–7.61(dd,J=3.1,8.0Hz,2H). 13 C NMR (126MHz, CDCl3) δ15.57,15.71,16.23,16.26,17.90,18.03,23.32,24.68,26.19,27.85,28.38, 29.53,29.58,30.69,32.10,33.96,36.76,36.87,37.75,38.15,39.38,41.52,42.30,44.99,49.42, 50.99, 51.99, 55.54, 71.00, 75.31, 77.04, 83.28, 110.98, 111.41, 115.83, 115.86, 124.19, 124.32, 126.40, 126.50, 144.55, 144.81, 151.26, 151.31, 157.63, 157.82, 165.83, 165.95, 180.79, 180.92.

[0149] Example 22

[0150]

[0151] Step 1: Weigh an appropriate amount of the starting compound dammarane saponin (1 equiv) into a 100 ml round-bottom flask, add an appropriate amount of dry dichloromethane, and stir at room temperature until completely dissolved. Add chloroacetyl chloride (4-10 equiv) to the solution under ice bath conditions, and react at room temperature for 0.5-3 h. Transfer to an oil bath at 80-100℃ and react for 4-6 h, monitoring with a TCL until the reaction stops. After the reaction stops, extract three times with saturated sodium bicarbonate solution, dry with anhydrous sodium sulfate, and purify by column chromatography to obtain intermediate 2.

[0152] Step 2: Weigh an appropriate amount of intermediate 2 into a 50 mL round-bottom flask, add an appropriate amount of acetonitrile solution, heat in an oil bath to 50 °C, and stir for 10 min until completely dissolved; weigh 5-fluoroindigo (2 equiv) into the round-bottom flask and stir to dissolve. The solution changes from colorless and clear to yellow and clear. Add anhydrous potassium carbonate, and heat to reflux at 80 °C for 8 h. Monitor the reaction progress by TLC, with the development conditions being petroleum ether:ethyl acetate = 3:1. After the reaction stops, remove the solvent under reduced pressure, add 20 mL of distilled water and 20 mL of dichloromethane solution respectively, and extract three times to obtain a dichloromethane layer solution. Add anhydrous sodium sulfate, shake, and let stand for 2 h. Filter the solution, add sample-mixed silica gel (100-200 mesh), remove the solvent under reduced pressure, and dry to obtain the sample to be separated. Separate by silica gel column chromatography, with the separation conditions being petroleum ether:ethyl acetate = 3:1, finally obtaining 5 g of the compound.

[0153] The resulting compound was a light yellow powder. 1 H NMR(300MHz, CDCl3)δ0.71(s,3H),0.79(s,3H),0.82(s,3H),0.90(s,3H),0.92–0.94(d,J=2.4Hz,3H), 1.13(s,3H),1.16(s,3H),1.20(s,3H),1.25(s,3H),1.39–1.54(m,9H),1.61(s,10H),1.80–1.92(m,2H ),4.15–4.27(m,1H),4.41–4.55(m,4H),4.94–5.05(td,J=5.5,10.5Hz,1H),6.70–6.79(ddd,J=1.8,3. 5,8.6Hz,2H),7.29–7.36(m,3H),7.50–7.56(dd,J=3.4,5.7Hz,1H),7.68–7.72(dd,J=3.3,5.7Hz,1H). 13C NMR (126MHz, CDCl3) δ15.82,16.01,18.14,18.38,25.26,26.55,28.12,28.63,29.06,29.67,29.83,30.49,31.10,32.44,3 3.74,34.23,37.04,37.09,38.00,38.43,38.86,39.64,41.74,42.48,45.31,49.75,51.20,52.33,55.79,68.29,71.27,75 .66,83.35,111.45,111.51,111.89,111.95,112.51,112.66,112.70,112.85,118.33,118.37,118.39,118.43,124.65,124.85,124.86,125.06,146.61,146.73,157.84,157.94,158.58,158.63,160.54,160.60,166.31,166.37,182.00,182.09.

[0154] Example 23

[0155]

[0156] Step 1: Weigh an appropriate amount of the starting compound dammarane saponin (1 equiv) into a 100 ml round-bottom flask, add an appropriate amount of dry dichloromethane, and stir at room temperature until completely dissolved. Add chloroacetyl chloride (4-10 equiv) to the solution under ice bath conditions, and react at room temperature for 0.5-3 h. Transfer to an oil bath at 80-100℃ and react for 4-6 h, monitoring with a TCL until the reaction stops. After the reaction stops, extract three times with saturated sodium bicarbonate solution, dry with anhydrous sodium sulfate, and purify by column chromatography to obtain intermediate 2.

[0157] Step 2: Weigh an appropriate amount of intermediate 2 into a 50 mL round-bottom flask, add an appropriate amount of acetonitrile solution, heat in an oil bath to 50 °C, and stir for 10 min until completely dissolved; weigh 7-fluoroindigo (2 equiv) into the round-bottom flask and stir to dissolve. The solution changes from colorless and clear to yellow and clear. Add anhydrous potassium carbonate and heat to reflux at 80 °C for 8 h. Monitor the reaction progress by TLC, with the development conditions being petroleum ether:ethyl acetate = 3:1. After the reaction stops, remove the solvent under reduced pressure, add 20 mL of distilled water and 20 mL of dichloromethane solution respectively, and extract three times to obtain a dichloromethane layer solution. Add anhydrous sodium sulfate, shake, and let stand for 2 h. Filter the solution, add sample-mixed silica gel (100-200 mesh), remove the solvent under reduced pressure, and dry to obtain the sample to be separated. Separate by silica gel column chromatography, with the separation conditions being petroleum ether:ethyl acetate = 3:1, finally obtaining the compound after 5 h.

[0158] The resulting compound was a light yellow powder. 1 H NMR(300MHz, CDCl3)δ0.72(s,3H),0.81(s,3H),0.83(s,3H),0.91(s,3H),0.94(s,3H),0.96–1.09 (m,3H),1.13(s,3H),1.15(s,3H),1.20(s,3H),1.25(s,3H),1.35–1.55(m,10H),1.61–1.92(m,7H) ,2.04–2.15(q,J=9.0Hz,1H),4.35–4.51(m,4H),4.52–4.58(m,1H),4.97–5.07(td,J=5.5,10.6Hz, 1H), 6.46–6.54 (dd, J=2.1, 8.6Hz, 2H), 6.78–6.86 (m, 2H), 7.64–7.73 (ddd, J=3.9, 5.5, 8.4Hz, 2H). 13 C NMR (126MHz, CDCl3) δ15.83,15.97,16.49,18.16,18.35,23.59,25.08,26.51,28.13,28.65,29.76,29.84,3 1.03,32.40,33.94,34.23,37.04,37.11,38.02,38.41,39.66,41.80,42.56,45.30,49.73,51.18,52.29,55. 80,71.27,75.61,77.36,83.51,99.17,99.40,99.64,99.86,111.13,111.27,111.32,111.45,114.28,128.39,128.49,128.53,128.62,158.21,158.37,166.15,166.23,168.10,168.22,170.18,170.30,180.40,180.51.

[0159] Example 24

[0160]

[0161] Step 1: Weigh an appropriate amount of the starting compound dammarane saponin (1 equiv) into a 100 ml round-bottom flask, add an appropriate amount of dry dichloromethane, and stir at room temperature until completely dissolved. Add chloroacetyl chloride (4-10 equiv) to the solution under ice bath conditions, and react at room temperature for 0.5-3 h. Transfer to an oil bath at 80-100℃ and react for 4-6 h, monitoring with a TCL until the reaction stops. After the reaction stops, extract three times with saturated sodium bicarbonate solution, dry with anhydrous sodium sulfate, and purify by column chromatography to obtain intermediate 2.

[0162] Step 2: Weigh an appropriate amount of intermediate 2 into a 50 mL round-bottom flask, add an appropriate amount of acetonitrile solution, heat in an oil bath to 50 °C, and stir for 10 min until completely dissolved; weigh 4-bromoindigo (2 equiv) into the round-bottom flask and stir to dissolve. The solution changes from colorless and clear to yellow and clear. Add anhydrous potassium carbonate and heat to reflux at 80 °C for 8 h. Monitor the reaction progress by TLC, with the development conditions being petroleum ether:ethyl acetate = 3:1. After the reaction stops, remove the solvent under reduced pressure, add 20 mL of distilled water and 20 mL of dichloromethane solution respectively, and extract three times to obtain a dichloromethane layer solution. Add anhydrous sodium sulfate, shake, and let stand for 2 h. Filter the solution, add sample-mixed silica gel (100-200 mesh), remove the solvent under reduced pressure, and dry to obtain the sample to be separated. Separate by silica gel column chromatography, with the separation conditions being petroleum ether:ethyl acetate = 3:1, finally obtaining compound 5i.

[0163] The resulting compound was a light yellow powder. 1 H NMR(300MHz, CDCl3)δ0.71(s,3H),0.79(s,3H),0.82(s,3H),0.90(s,3H),0.93(s,3H),1.13(s,3H) ,1.16(s,3H),1.20(s,3H),1.23–1.27(d,J=2.4Hz,3H),1.40–1.54(m,9H),1.59(s,5H),1.62–1.92 (m,6H),2.03–2.12(m,1H),4.34–4.51(m,4H),4.52–4.57(d,J=9.6Hz,1H),4.95–5.04(td,J=5.4,1 0.5Hz,1H),6.69–6.75(dt,J=1.0,7.8Hz,2H),7.26–7.31(m,2H),7.35–7.44(td,J=6.9,8.0Hz,2H). 13C NMR (126MHz, CDCl3) δ15.82,16.02,16.47,18.13,18.37,23.58,25.27,26.53,28.12,28.61,29.63, 29.81,31.10,32.44,33.68,34.22,37.02,37.08,38.00,38.41,39.63,41.66,42.38,45.29,49.74, 51.21,52.34,55.78,71.25,75.64,77.34,83.37,109.02,109.43,116.59,116.61,121.93,122.03,128.90,129.05,138.33,138.53,152.02,152.11,157.09,157.22,166.18,166.26,179.94,180.04.

[0164] Example 25 Cell Viability Assay

[0165] Tumor cells in the logarithmic growth phase were collected separately, and the cell density was adjusted to 2.5 × 10⁻⁶. 4 Cell suspension was added to 96-well plates at a volume of 100 μL per well and incubated overnight at 37°C with 5% CO2. The next day, all test compounds were diluted twofold to an initial concentration of 100 μM, setting six concentration points. All culture medium in the wells was discarded, and 100 μL of drug-containing medium was added, and the plates were incubated for another 48 h. 10 μL of 0.5% MTT solution was added to each well and incubated for 4 h. The supernatant was then carefully aspirated, and 150 μL of DMSO was added to each well. The plates were shaken in the dark to completely dissolve the precipitate at the bottom. The absorbance (OD) value of each well was measured at 490 nm using a multi-mode microplate reader, with DMSO without drug as a control. Data were analyzed using Excel software, and the IC50 of the test compounds was calculated using GraphPad Prism 9.0.2 software. 50 value.

[0166] Calculation formula: Cell inhibition rate = [1 - (OD value of drug-treated group - OD value of blank group) / (OD value of control group - OD value of blank group)] × 100%.

[0167] The antiproliferative activity of the 24 PD-indigo derivatives against six different human tumor cell lines (lung cancer A549, prostate cancer PC-3, breast cancer MCF-7, liver cancer HepG-2, colon cancer HCT-116, and cervical cancer HeLa) was detected by the MTT assay. The results showed that, compared with the parent compound PD, the inhibitory activity of compounds with a single hydroxyl substitution at the C-3 position (compounds 4a-4o) was superior to that of compounds with simultaneous substitution at both the C-3 and C-12 positions (5a-5i). Among these, the 5a-5i series compounds did not significantly enhance the antitumor activity of the parent compound, while several compounds in the 4a-4o series exhibited good broad-spectrum inhibitory activity against the above tumor cell lines, as shown in Table 1.

[0168] Table 1. Evaluation of the in vitro antitumor activity of PD-indigo derivatives

[0169]

[0170]

[0171] All data were calculated using the mean ± standard deviation of three independent experiments.

[0172] Example 26

[0173] The toxic effects of the two series of PD indigo derivatives on normal human embryonic kidney cells (HEK293 cells) were further evaluated using the MTT assay. The experimental method is described in Example 25. The results are as follows: Figure 1 As shown, at a treatment concentration of 10 μM, the derivatives of the 4a, 4c, 4e, 4h, 4i, 4n, and 5a-5i series all exhibited low toxicity to normal cells. Figure 1 ).

[0174] Because derivative 4e exhibited relatively high anti-HCT-116 activity and low toxicity, it was further evaluated for tumor selectivity in different colon cancer cells. As shown in Table 2, derivative 4e significantly improved the therapeutic window, with tumor selectivity ranging from 8.35 to 16.92 times.

[0175] Table 2.4 Selectivity indices of e and PD for colorectal cancer cells

[0176]

[0177] a SI: Selectivity Index = IC 50 (HEK293) / IC 50 (Colorectal cancer cell line)

[0178] Example 27 Cell plate colony formation of derivative 4e

[0179] Logarithmic growth phase HCT-116 and CT-26 cells were seeded at 3300 cells / well in 6-well plates and incubated overnight at 37°C with 5% CO2. The next day, different concentrations of derivative 4e were added, and the culture medium was replaced after 48 days. The culture was continued for 14 days. When clear colony formation was observed under a microscope, the cells were fixed with 4% paraformaldehyde, stained with crystal violet, and photographed. The number of colonies was counted using ImageJ.

[0180] Experimental results showed that derivative 4e had a strong anti-proliferative effect, inhibiting colony formation of two types of colon cancer cells in a dose-dependent manner. Figure 2 ).

[0181] Example 28 Cell cycle detection of derivative 4e

[0182] Cell density 1×10 6 HCT-116 cells were seeded at 1 / mL in 6-well plates and cultured overnight at 37°C in a 5% CO2 cell culture incubator. After treatment with different concentrations of derivative 4e in each well for 48 h, the cells were digested, centrifuged, and fixed overnight with 75% ethanol. After resuspending the cells by centrifugation, they were stained with propidium iodide and analyzed by flow cytometry.

[0183] Experimental results showed that derivative 4e induced G0 / G1 cell cycle arrest in HCT-116 cells in a dose-dependent manner, manifested as the accumulation of G0G1 cells during the cell cycle and a decrease in the G2 / M and S phases. Figure 3 ).

[0184] Example 29 Derivative 4e Cell Apoptosis Detection

[0185] HCT-116 cells were treated with derivative 4e for 48 h, then digested with EDTA-free trypsin and centrifuged at 3000 rpm for 10 min. After washing with PBS, the cell suspension was collected, and negative control, positive control, and single-stain groups were set up. The cells were incubated with Annexin V-FITC and propidium iodide at room temperature in the dark for 15 min, and the apoptosis level was detected by flow cytometry.

[0186] Experimental results showed that derivative 4e induced apoptosis in HCT-116 cells in a dose-dependent manner, with the total apoptosis rate reaching 83.6% in both the early and late stages at a dose of 10 μM. Figure 4 ).

[0187] Example 30 Cell scratch assay of derivative 4e

[0188] 5×10 5HCT-116 cells were seeded in 6-well plates and cultured overnight. After the cells reached confluence, two intersecting cross-shaped scratches were made on the bottom of the plates using a 200 μL pipette tip. After removing detached cells twice with PBS, a blank control group and a drug-treated group (serum-free medium diluted 4e to the final concentration) were set up, and images were captured at 0 h using an inverted microscope. Subsequently, at 48 h, the convergence of scratches at the same location was observed, the scratch healing width was measured using ImageJ software, and the migration distance for each group was calculated.

[0189] Experimental results showed that derivative 4e significantly inhibited the migration of HCT-116 cells in a dose-dependent manner. Figure 5 ).

[0190] Example 31 DNA damage detection of derivative 4e

[0191] HCT-116 cells were seeded in 24-well plates containing a climbing slide and cultured overnight. After treatment with derivative 4e for 48 hours, the liquid in the plate was aspirated, and paraformaldehyde was added for fixation for 10 minutes. After washing three times with PBS, immunostaining blocking solution was added, and the cells were blocked at room temperature for 20 minutes. Rabbit γ-H2AX monoclonal antibody was then added, and the cells were incubated overnight at 4°C. The next day, residual primary antibody was washed off, and fluorescent secondary antibody was added and incubated at room temperature in the dark for 1 hour. After washing three times, nuclear staining solution (DAPI) was added, and staining was performed at room temperature for 5 minutes. After washing three more times, anti-fluorescence quenching mounting solution was added, and the cells were mounted with coverslips and observed under a fluorescence microscope. γ-H2AX showed green fluorescence in the field of view, and DAPI staining of the cell nuclei showed blue fluorescence.

[0192] Phosphorylated H2AX Ser139 product (γ-H2AX) can indirectly assess the degree of DNA damage and is widely used in apoptosis research, becoming an important DNA damage marker. Immunofluorescence detection of γ-H2AX showed that derivative 4e dose-dependently induced DNA damage in HCT-116 cells. Figure 6 ).

[0193] Example 32 Western blot detection of derivative 4e

[0194] Logarithmically growing HCT-116 cells were seeded in 6-well plates and cultured overnight. Cells were incubated for 48 hours with 2.5, 5, and 10 μM of derivative 4e. After protein lysis, extraction, quantification, and denaturation, proteins were separated by 10% SDS-PAGE and transferred to an NC membrane. The membrane was blocked with 5% skim milk at room temperature for 1 hour, and anti-Bcl-2, Bax, CDK2, CDK4, CDK6, and Cyclin D1 antibodies were added. The protein membrane was then incubated overnight at 4°C, washed with TBST, and incubated with secondary antibody at room temperature for 1 hour. Images were acquired using an ECL gel imaging system.

[0195] This study investigated the effects of derivative 4e on apoptosis and cyclin expression in HCT-116 cells. The results showed that with increasing derivative 4e concentration, Bax protein expression was upregulated, while Bcl-2, CDK2, CDK4, CDK6, and Cyclin D1 expression were downregulated, suggesting that derivative 4e altered the expression of cell cycle-related proteins in HCT-116 cells and induced HCT-116 cell apoptosis. Figure 7 ).

[0196] Example 33 Molecular docking of derivative 4e

[0197] The three-dimensional structure of protein RalA (2BOV) was obtained from the Protein Data Bank (PDB) database. Molecular docking studies were conducted using Discovery Studio 2019. The binding mode of derivative 4e to RalA was investigated through model preparation and energy minimization. The location of the active docking center and the sphere radius parameters were optimized, and docking was performed using the LibDock algorithm to obtain a series of specific ligand-receptor binding conditions. Finally, the results were processed and analyzed.

[0198] like Figure 8 As shown, molecular docking results indicate that derivative 4e exhibits good binding performance with RalA (ΔG = -145.67 kJ / mol). Extensive π-alkyl interactions between derivative 4e and the amino acid residues surrounding the RAL-GDP allosteric sites (TYR A:75, ARG A:79, PHE A:83, GLN A:110, VAL A:20, PHE A:107) contribute to enhanced intermolecular packaging and binding stability. These residues can form a hydrophobic bag around the hydrophobic group of 4e. Furthermore, the indigo-2-carbonyl oxygen in the ligand can form a CH bond with GLU A:73. The π-σ interaction between derivative 4e and the PHE A:83 residues further stabilizes the structure of the complex.

[0199] Example 34: Inhibitory effect of derivative 4e on RalA protein

[0200] Following the same procedure as in Example 32 above, this example demonstrated the inhibitory effect of derivative 4e on RalA protein using Western blotting detection. Figure 9 As shown, the results indicate that derivative 4e inhibits the activation of RalA protein in a dose-dependent manner.

[0201] Example 35: Detection of the cell thermal stability of derivative 4e

[0202] HCT-116 cells (1×10⁻⁶) 6Cells were seeded in 10 cm culture dishes and cultured overnight. Then, the cells were treated with derivative 4e (10 μM) and DMSO for 2 h each. After washing with PBS, the cells were collected and divided into 6 aliquots. Each aliquot was heated for 3 min at different temperatures (37, 45, 55, 65, 75, 85 °C). After centrifugation of the soluble lysis buffer, the expression of RalA in the protein supernatant was detected as described in Example 32.

[0203] like Figure 10 As shown, cell thermostability assays demonstrated the binding stability of derivative 4e to RalA. Compared to the DMSO control, derivative 4e remained stable for RalA protein within a higher denaturation temperature range, indicating a strong interaction between derivative 4e and RalA in HCT-116 cells.

[0204] Safety evaluation of derivative 4e in Example 36

[0205] Male BALB / c mice were randomly divided into a control group, an administration group, and a positive control group. All mice were intraperitoneally injected with 4e or 5-Fu at 50 mg / kg for two consecutive weeks. Additionally, the effects of 4e or PD on mouse survival were observed over one week at a high dose (100 mg / kg). Daily changes in mouse body weight and time of death were recorded, and curves showing changes in body weight and survival were plotted.

[0206] The results showed that, compared with PD, high-dose 4e did not induce acute systemic toxicity in mice, and body weight remained stable throughout the study. The 5-FU treatment group experienced a 100% mortality rate within 3-8 days after administration and exhibited severe treatment-related toxicities, while the 4e treatment group showed significantly improved tolerability, indicating its high safety profile. Figure 11 ).

[0207] Example 37: In vivo antitumor activity of derivative 4e

[0208] A CT-26 subcutaneous xenograft model was constructed using male BALB / c mice. The xenograft volume was increased to 50 mm². 3 Mice bearing tumors were randomly divided into 6 groups (n=8). The experimental groups were as follows: carrier solvent control group (Vehicle, 5% DMSO + 35% PEG-400 + 60% saline), low-dose 4e group (4e-L, 5 mg / kg), high-dose 4e group (4e-H, 10 mg / kg), PD group (10 mg / kg), and positive control 5-fluorouracil group (5-Fu, 10 mg / kg). Mice were administered intraperitoneally every two days for three weeks. All mice were euthanized, and tumor tissue was harvested for weighing and measurement. Serum was used to detect various liver and kidney function indicators.

[0209] The results showed that the tumor volume in the derivative 4e group generally exhibited a significant decreasing trend. Figure 12 Compared with PD and the positive control 5-Fu, the 4e-H treatment group showed a significant tumor-suppressing effect. Furthermore, derivative 4e had no significant effect on organ indices in mice, and its effects on various liver and kidney function indicators fluctuated within the normal range. In conclusion, derivative 4e exhibits excellent antitumor activity in the in vivo CT-26 colon cancer model.

[0210] Pharmacokinetic characteristics of derivative 4e in Example 38

[0211] Pharmacokinetic studies were conducted in two groups of adult male SD rats (180-220g). Compounds 4e and PD were administered intraperitoneally at a dose of 20 mg / kg. Blood samples (400 μL) were collected from the retro-orbital venous plexus at 5, 10, 30, 30, 1, 4, 6, 8, 10, 12, and 24 hours post-administration. After appropriate treatment, plasma concentrations of the compounds were determined using ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS / MS). Concentration-time curves were plotted using GraphPadPrism 9, and pharmacokinetic parameters were calculated using DAS software. Pharmacokinetic results showed that the area under the plasma concentration-time curve (AUC) of 4e was [missing value]. 0-t The peak plasma concentration (Cmax), peak concentration (Tmax), and concentration of drug 4e were all higher than those of PD, indicating that compound 4e significantly improved the bioavailability of PD. Figure 13 ).

[0212] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. Derivatives of Formula I and their racemates, stereoisomers, tautomers, solvates, polymorphs, metabolites, prodrugs, or pharmaceutically acceptable salts thereof: in, R1 is R2 is OH; R3 can be H, C1-C6 alkyl, halogen, C1-C6 alkoxy, or halogenated C1-C6 alkyl.

2. The derivatives of claim 1 and their racemates, stereoisomers, tautomers, solvates, polymorphs, metabolites, prodrugs, or pharmaceutically acceptable salts thereof, characterized in that, R3 is a single substitution.

3. The derivatives of claim 1 and their racemates, stereoisomers, tautomers, solvates, polymorphs, metabolites, prodrugs, or pharmaceutically acceptable salts thereof, characterized in that, R3 is H, 5-CH3, 4-Cl, 5-Cl, 6-Cl, 7-Cl, 5-F, 6-F, 7-F, 4-Br, 5-Br, 6-Br, 7-Br, 5-OCH3, 7-CF3.

4. The derivative of claim 1 and its racemic mixtures, stereoisomers, tautomers, solvates, polymorphs, metabolites, prodrugs, or pharmaceutically acceptable salts thereof, characterized in that, R2 is At that time, R1 and R2 have the same R3.

5. The following derivatives and their racemates, stereoisomers, tautomers, solvates, polymorphs, metabolites, prodrugs or pharmaceutically acceptable salts thereof:

6. A method for preparing the derivative shown in claim 1 or a pharmaceutically acceptable salt thereof, characterized in that, Includes the following steps: R3 is as described in claim 1.

7. A pharmaceutical composition comprising the derivative of any one of claims 1-6 and its racemic, stereoisomer, tautomer, solvate, polymorph, metabolite, prodrug, or pharmaceutically acceptable salt thereof.

8. The use of the derivatives of any one of claims 1-6 and their racemates, stereoisomers, tautomers, solvates, polymorphs, metabolites, prodrugs or pharmaceutically acceptable salts thereof in the preparation of antitumor drugs.

9. The use of the pharmaceutical composition of claim 7 in the preparation of an antitumor drug.

10. The application according to claim 8 or 9, characterized in that, The tumors mentioned are colorectal cancer, liver cancer, non-small cell lung cancer, prostate cancer, breast cancer, cervical cancer, ovarian cancer, gastric cancer, skin cancer, melanoma, glioblastoma, testicular cancer, endometrial cancer, pancreatic cancer, osteosarcoma, nasopharyngeal carcinoma, laryngeal squamous cell carcinoma, bile duct cancer, kidney cancer, multiple myeloma, esophageal cancer, and acute myeloid leukemia, preferably colorectal cancer.

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