Sporosporine glycoside derivative as well as preparation method and application thereof

Abstract

The invention discloses a staurosporine glucoside derivative as well as a preparation method and application thereof, and belongs to the field of medicine synthesis. C3-NHCH3 of staurosporine is subjected to glycosylation modification, and the staurosporine glycoside derivative shown in the formula (I) is prepared. The glycoside derivative prepared by the invention has a better inhibition effect on human acute myeloid leukemia cells THP-1, human bladder cancer cells 5637, human colon cancer cells HCT-116, human pancreatic cancer cells PATU8988T and human liver cancer cells HuH-7, and the compound 5 also has an excellent inhibition effect on human gastric cancer cells MKN-45. A candidate compound is provided for development of drugs for bladder cancer, colon cancer, pancreatic cancer, liver cancer, stomach cancer or acute myeloid leukemia.

Description

Technical Field

[0001] This invention belongs to the field of drug synthesis, specifically relating to a cruciferous glycoside derivative, its preparation method, and its application. Background Technology

[0002] Discovered in 1977, staurosporine (ST) belongs to the indolecarbazole class of alkaloids and is a microbial metabolite. Staurosporine and its derivatives have been shown to be nanomolar inhibitors of protein kinases, exhibiting significant cytotoxic activity against tumor cells. However, due to their poor selectivity, strong cytotoxic activity against normal cells, and poor solubility, their drug development is limited. To obtain highly effective and low-toxicity active lead compounds, a series of structural modification studies have been conducted on staurosporine.

[0003] Glycosylation modification is widely present in natural products of plants and animals. Glycosylation modification of natural products can improve the physicochemical properties and biological activity of the parent nucleus [Chinese Chemical Letters 2024, 35(7): 108950]. Glycosylated products can achieve targeted anti-tumor effects through sugar transporters and glycosidases, and reduce toxic side effects on normal cells [European Journal of Medicinal Chemistry 2019, 182: 111612]. The research results of the inventors of this patent application on cruciferine in the early stage showed that the introduction of different groups on the nitrogen atom at the 3ʹ-position has a significant impact on its activity. After heteroaromatic formylation modification of cruciferine C3ʹ-NHCH3, the inhibitory effect on human acute myeloid leukemia cells was improved [Chinese Patent, CN114853784A, 2022-08-05], but the activity against solid tumors such as colorectal cancer, liver cancer, and bladder cancer is limited, and the scope of application is narrow. Summary of the Invention

[0004] To address the aforementioned problems, the present invention aims to provide a cruciferous glycoside derivative and its preparation method. By linking cruciferous C3ʹ-NHCH3 to different sugar groups via carbonyl or thiocarbonyl groups, a highly efficient, low-toxicity, and highly selective cruciferous glycoside derivative can be obtained. The invention also discusses the application of this type of compound in the preparation of antitumor drugs.

[0005] The present invention solves its main technical problem by adopting the following technical solution: A cruciferous glycoside derivative, the structure of which is shown in formula (I): (I); in, The sugar group is selected from, but is not limited to, glycosyl groups. D- Lysol, 2-deoxy- D -Glucose, 5-deoxy- D -ribose, D -maltose, D -glucose, L -glucose, D - Xylose, L - Xylose, L -Lessosulfate, 1-thio- D -glucose.

[0006] Furthermore, when both X and Y are O, the glycosyl group is selected from... D- Lysol, 2-deoxy- D -Glucose, 5-deoxy- D -ribose or D -maltose.

[0007] Furthermore, when X is S and Y is O, the glycosyl group is selected from... D -glucose, L -glucose, D - Xylose, L -xylose or L - Lysol.

[0008] Furthermore, when both X and Y are S, the glycosyl group is selected from 1-thio- D -glucose.

[0009] The preparation method of the cruciferous glycoside derivative includes the following steps: ; The acylation reaction of cruciferine with di(trichloromethyl) carbonate to generate an acyl chloride intermediate was followed by a reaction with acetyl-protected 1-deacetylated glycosyl donors T1–T4 catalyzed by 4-dimethylaminopyridine to obtain a glycoside intermediate. Finally, the glycoside end product was obtained by deacetylation with sodium methoxide. Compounds 1–4 were synthesized by this method. Alternatively, cruciferine can be introduced into the thiocarbonyl imidazole fragment by first reacting it with N,Nʹ-thiocarbonyl diimidazole, then reacting it with iodomethane to form a salt, activating the imidazole group to facilitate its departure, and then reacting it with the acetyl-protected 1-deacetylated glycosyl donor T5–T. 10 The reaction yielded a glycoside intermediate, which was then deacetylated with sodium methoxide to obtain the final glycoside product. Compounds 5–10 were synthesized using this method.

[0010] The acetyl-protected 1-deacetylated glycosyl donor T1–T 10 The structure is as follows: .

[0011] The structures of the cruciferous glycoside derivatives 1–10 prepared by the above method are shown below: .

[0012] The application of the aforementioned cruciferous glycoside derivatives is characterized by the use of cruciferous glycoside derivatives 1-10 in the preparation of antitumor drugs for prevention or treatment, including drugs for gastric cancer, bladder cancer, colon cancer, pancreatic cancer, lung cancer, liver cancer, or acute myeloid leukemia. When used as a drug, the compound is used directly or in the form of a pharmaceutical composition containing 0.1-99% of the compound, with the remainder being a pharmaceutical carrier.

[0013] Compared with existing technologies, this invention has significant advantages and beneficial effects: compared with the heteroaromatic formylated derivatives of cruciferous alkaloids, the cruciferous alkaloid glycoside derivatives prepared in this invention show significantly enhanced cytotoxic activity against solid tumor cells such as human bladder cancer cells 5637, human colon cancer cells HCT-116, human pancreatic cancer cells PATU8988T, and human liver cancer cells HuH-7, while significantly reducing toxicity against normal human liver cells L-02. This provides candidate compounds for the development of therapeutic drugs for bladder cancer, colon cancer, pancreatic cancer, liver cancer, gastric cancer, or acute myeloid leukemia. Detailed Implementation

[0014] The present invention will be further described in detail below through specific embodiments.

[0015] Example 1: Preparation of Compound 1 In a 25 mL two-necked flask, cruciferine (200 mg, 0.4 mmol) was dissolved in 10 mL of dichloromethane. Di(trichloromethyl) carbonate (127 mg, 0.4 mmol) and N,N-diisopropylethylamine (100 μL, 0.6 mmol) were slowly added sequentially at −15 °C. The mixture was stirred at low temperature for 5 min, and TLC was used to monitor complete conversion of the starting material. Then, the glycosyl donor T1 (acetyl-protected, 1-deacetylated) was added directly to the reaction mixture at −15 °C. DLysose (165 mg, 0.6 mmol) and 4-dimethylaminopyridine (244 mg, 2 mmol) were reacted at room temperature (25 °C) with stirring for 4 h, and the reaction was monitored by TLC until completion. The reaction solution was poured into ice water, extracted with dichloromethane (50 ml × 2), washed with brine (50 ml × 2), dried over anhydrous sodium sulfate, concentrated, and separated by silica gel column chromatography. Eluent was obtained by dichloromethane:methanol = 100:1 (v / v) to give 68 mg of a pale yellow solid, which was purified by semi-preparative HPLC (YMC-pack ODS-A, 65% MeCN / H2O) to give glycoside intermediate 1a (42 mg, t R =13.3 min, yield 14%, α - D Compound 1a (21 mg, 0.03 mmol) was dissolved in 5 mL of methanol. The pH was adjusted to 9 by adding 100 mg / mL sodium methoxide / methanol solution dropwise at room temperature (25 °C). Stirring continued for 5 min, and TLC was monitored until the reaction was complete. A pretreated acidic cation exchange resin was added to adjust the pH to 7. The reaction solution was evaporated to dryness, and purified by semi-preparative HPLC (YMC-pack ODS-A, 80% MeOH / H2O) to obtain the deacetylated glycoside final product compound 1 (16 mg, t...). R =7.6 min, yield 95%, α - D (Type 1 glycoside), white solid.

[0016] Compound 1: 1 H NMR (600 MHz, DMSO- d 6) d 9.29 (d, J = 8.0 Hz, 1H), 8.60 (s,1H), 8.06 (d, J = 7.5 Hz, 1H), 7.99 (d, J = 8.4 Hz, 1H), 7.65 (d, J = 8.3 Hz, 1H), 7.49 (t, J = 7.5 Hz, 2H), 7.36 (t, J = 7.1 Hz, 1H), 7.30 (t, J = 7.5 Hz, 1H), 7.02 (t, J = 7.2 Hz, 1H), 5.80 (d, J= 3.4 Hz, 1H) / 5.78 (d, J = 3.4 Hz,1H), 5.45–4.94 (m, 3H), 5.00 (s, 2H), 4.67 (t, J = 13.8 Hz, 1H), 4.33–4.28(m, 1H), 3.89–3.80 (m, 1H), 3.80–3.70 (m, 1H), 3.67 (t, J = 9.1 Hz, 2H),3.63–3.51 (m, 1H), 2.83–2.77 (m, 1H), 2.75 (s, 3H) / 2.74 (s, 3H), 2.74 (s,3H) / 2.71 (s, 3H), 2.39 (s, 3H) / 2.35 (s, 3H), 2.29–2.21 (m, 1H); 13 C NMR (150MHz, DMSO- d 6) d 172.5, 154.6, 139.3, 136.8, 133.2, 129.7, 126.2, 125.9, 125.6, 125.6, 124.3, 123.2, 122.0, 120.9, 120.1, 120.0, 115.7, 114.7, 114.1 / 114.0, 109.5, 95.7, 95.1, 83.7, 82.7, 71.8 / 71.6, 69.4, 67.1, 65.5, 61.0 / 60.9, 50.9 / 50.9, 46.0, 30.7 / 30.4, 30.1 / 29.8, 27.7 / 27.4; the ratio of cis-trans tautomers in solution due to p-π conjugation of tertiary amide bonds is approximately 1:2; HRESIMS m / z 643.2393 [M+H] + (Calculated value: 643.2399).

[0017] Example 2: Preparation of Compound 2 In a 25 mL two-necked flask, cruciferine (200 mg, 0.4 mmol) was dissolved in 10 mL of dichloromethane. Di(trichloromethyl) carbonate (127 mg, 0.4 mmol) and N,N-diisopropylethylamine (100 μL, 0.6 mmol) were slowly added sequentially at −15 °C. The mixture was stirred at low temperature for 5 min, and TLC was used to monitor complete conversion of the starting material. Then, the glycosyl donor T2 (acetyl-protected 1-deacetyl 2-deoxy-) was added directly to the reaction mixture at −15 °C. D - Glucose (174 mg, 0.6 mmol) and 4-dimethylaminopyridine (244 mg, 2 mmol) were reacted at room temperature (25 °C) with stirring for 4 h, and the reaction was monitored by TLC until completion. The reaction solution was poured into ice water, extracted with dichloromethane (50 ml × 2), washed with brine (50 ml × 2), dried over anhydrous sodium sulfate, concentrated, separated by silica gel column chromatography, and eluted with dichloromethane:methanol = 100:1 (v / v) to give 82 mg of a pale yellow solid, which was purified by semi-preparative HPLC (YMC-pack ODS-A, 65% MeCN / H2O) to give glycoside intermediate 2a (43 mg, t R =17.9 min, yield 14%, α - D Compound 2a (20 mg, 0.03 mmol) was dissolved in 5 mL of methanol and the pH was adjusted to 9 by adding 100 mg / mL sodium methoxide / methanol solution dropwise at room temperature (25 °C). Stirring was continued for 5 min, and the reaction was monitored by TLC until complete. A pretreated acidic cation exchange resin was added to adjust the pH to 7, and the reaction solution was evaporated to dryness. The deacetylated glycoside final product, compound 2a (15 mg, t...), was purified by semi-preparative HPLC (YMC-pack ODS-A, 80% MeOH / H2O) to obtain the glycoside final product compound 2 (15 mg, t...). R =7.8 min, yield 90%, α - D (Type 1 glycoside), white solid.

[0018] Compound 2: 1 H NMR (600 MHz, DMSO- d 6) d 9.30 (d, J = 7.9 Hz, 1H), 8.63 (s,1H), 8.06 (t, J = 7.0 Hz, 1H), 7.99 (d, J = 8.5 Hz, 1H) / 7.92 (d,J = 8.5 Hz,1H), 7.66 (d, J = 8.2 Hz, 1H) / 7.65 (d, J = 8.2 Hz, 1H), 7.52 (t, J = 7.4 Hz,2H) / 7.48 (t, J = 7.4 Hz, 2H), 7.35 (t, J = 7.8 Hz, 1H), 7.30 (t, J = 7.4 Hz,1H), 7.07 (t, J = 7.6 Hz, 1H) / 7.02 (t, J = 7.4 Hz, 1H), 6.20 (s, 1H) / 6.14 (s,1H), 5.34 (d, J = 5.6 Hz, 1H) / 5.04 (d, J = 5.6 Hz, 1H), 5.01 (s, 2H), 5.17(d, J = 4.6 Hz, 1H) / 4.96 (d, J = 4.6 Hz, 1H), 4.69 (dt, J = 13.3, 3.1 Hz,1H) / 4.63 (dt, J = 13.3, 3.1 Hz, 1H), 4.82 (t, J = 5.8 Hz, 1H) / 4.61 (t, J =5.8 Hz, 1H), 4.33 (s, 1H) / 4.31 (s, 1H), 3.88–3.83 (m, 1H) 3.77–3.73 (m, 1H) / 3.68–3.65 (m, 1H), 3.65 – 3.62 (m, 1H) / 3.58–3.53 (m, 1H), 3.48–3.43 (m, 1H),3.29–3.23 (m, 1H) / 3.20–3.15 (m, 1H), 2.84 (s, 3H) / 2.75 (s, 3H), 2.80–2.68 (m,1H), 2.74 (s, 3H) / 2.70 (s, 3H), 2.37 (s, 3H) / 2.34 (s, 3H), 2.28–2.18 (m, 1H),2.13 (dd, J= 13.7, 4.9 Hz, 1H) / 1.99 (dd, J = 13.7, 4.9 Hz, 1H), 1.76 (t, J =12.5 Hz, 1H) / 1.64 (t, J = 12.5 Hz, 1H); 13 C NMR (150 MHz, DMSO- d 6) d 172.0, 154.2 / 153.7, 139.0 / 138.9, 136.3, 132.8 / 132.7, 129.2 / 129.0, 125.8, 125.4, 125.1, 124.9, 123.9, 122.7, 121.6 / 121.6, 120.5, 119.6, 119.5, 115.4 / 115.3, 114.2, 113.6 / 113.6, 109.1, 94.7 / 94.5, 93.2 / 92.9, 84.1 / 83.2, 82.3 / 82.2, 76.7 / 75.7, 71.1 / 71.0, 68.2 / 67.8, 61.0 / 60.8, 60.7 / 60.5, 50.3, 45.5, 37.1 / 37.0, 30.1 / 29.9, 29.4, 27.1 / 26.9; the ratio of cis-trans tautomers in solution due to p-π conjugation of tertiary amide bonds is approximately 2:3; HRESIMS m / z 657.2548 [M+H] + (Calculated value: 657.2555).

[0019] Example 3: Preparation of Compound 3 In a 25 mL two-necked flask, cruciferine (200 mg, 0.4 mmol) was dissolved in 10 mL of dichloromethane. Di(trichloromethyl) carbonate (127 mg, 0.4 mmol) and N,N-diisopropylethylamine (100 μL, 0.6 mmol) were slowly added sequentially at −15 °C. The mixture was stirred at low temperature for 5 min, and TLC was used to monitor complete conversion of the starting material. Then, the glycosyl donor T3 (acetyl-protected 1-deacetyl 5-deoxy-) was added directly to the reaction mixture at −15 °C. D-ribose (130 mg, 0.6 mmol) and 4-dimethylaminopyridine (244 mg, 2 mmol) were reacted at room temperature (25 °C) with stirring for 4 h, and the reaction was monitored by TLC until the reaction was complete. The reaction solution was poured into ice water, extracted with dichloromethane (50 ml × 2), washed with brine (50 ml × 2), dried over anhydrous sodium sulfate, concentrated, separated by silica gel column chromatography, and eluted with dichloromethane:methanol = 100:1 (v / v) to give 92 mg of a pale yellow solid, which was purified by semi-preparative HPLC (YMC-pack ODS-A, 65% MeCN / H2O) to give glycoside intermediate 3a (65 mg, t R =12.6 min, yield 26%), white solid. Compound 3a (22 mg, 0.03 mmol) was dissolved in 5 mL of methanol, and the pH was adjusted to 9 by adding 100 mg / mL sodium methoxide / methanol solution dropwise at room temperature (25 °C). Stirring continued for 5 min, and TLC was monitored until the reaction was complete. A pretreated acidic cation exchange resin was added to adjust the pH to 7, and the reaction solution was evaporated to dryness. The deacetylated glycoside final product compound 3 (18 mg, t) was purified by semi-preparative HPLC (YMC-pack ODS-A, 80% MeOH / H2O) to obtain the compound 3 (18 mg, t) R =5.5 min, yield 96%), white solid.

[0020] Compound 3: 1 H NMR (600 MHz, DMSO- d 6) d 9.30 (d, J = 7.8 Hz, 1H), 8.59 (s,1H), 8.05 (t, J = 8.0 Hz, 1H), 8.07(d, J = 8.5 Hz, 1H) / 8.02 (d, J = 8.5 Hz, 1H), 7.64 (t, J = 7.7 Hz, 1H), 7.48 (t, J = 7.0 Hz, 2H), 7.35 (t, J = 7.5,1H), 7.30 (t, J = 7.5 Hz, 1H), 7.02 (t, J = 7.7, 1H), 5.89 (s, 1H) / 5.84 (s,1H), 5.00 (s, 2H), 4.64 (dt, J= 13.1, 3.1 Hz, 1H) / 4.49 (dt, J = 13.1, 3.1Hz, 1H), 4.33 (s, 1H) / 4.31 (s, 1H), 4.21 (d, J = 7.0, 1H) / 3.91 (d, J = 7.0,1H), 4.03 (dd, J = 10.7, 4.8 Hz, 1H) / 3.96 (dd, J = 10.7, 4.8 Hz, 1H), 3.93(d, J = 3.7, 1H), 3.77 (t, J = 4.1, 1H), 2.81–2.80 (m, 1H), 2.78 (s, 3H) / 2.69(s, 3H), 2.69 (s, 3H), 2.39 (s, 3H) / 2.35 (s, 3H), 2.27–2.17 (m, 1H), 1.37 (d, J = 6.0 Hz, 3H) / 1.16 (d, J = 6.0 Hz, 3H); 13 C NMR (150 MHz, DMSO- d 6) d 171.9, 154.3, 138.6, 136.3, 132.7, 129.1, 125.7, 125.4, 125.0, 123.8, 122.7, 121.5, 121.4, 120.4, 119.5, 119.4, 115.2, 114.1, 113.7, 108.9, 101.9 / 101.7, 94.6 / 94.5, 83.9 / 83.1, 82.1, 78.8, 76.0 / 75.8, 74.5 / 74.3, 60.4, 50.4 / 50.3, 45.4, 29.9, 29.4 / 29.2, 27.0 / 26.8, 20.2 / 20.0; the ratio of cis-trans tautomers in solution due to p-π conjugation of tertiary amide bonds is approximately 1:1. ESI-MS m / z 627 [M+H] + .

[0021] Example 4: Preparation of Compound 4 In a 25 mL two-necked flask, cruciferine (200 mg, 0.4 mmol) was dissolved in 10 mL of dichloromethane. Di(trichloromethyl) carbonate (127 mg, 0.4 mmol) and N,N-diisopropylethylamine (100 μL, 0.6 mmol) were slowly added sequentially at −15 °C. The mixture was stirred at low temperature for 5 min, and TLC was used to monitor complete conversion of the starting material. Then, the glycosyl donor T4 (acetyl-protected, 1-deacetylated) was added directly to the reaction mixture at −15 °C. D - Maltose (381 mg, 0.6 mmol) and 4-dimethylaminopyridine (244 mg, 2 mmol) were reacted at room temperature (25 °C) with stirring for 4 h, and the reaction was monitored by TLC until the reaction was complete. The reaction solution was poured into ice water, extracted with dichloromethane (50 ml × 2), washed with brine (50 ml × 2), dried over anhydrous sodium sulfate, concentrated, separated by silica gel column chromatography, and eluted with dichloromethane:methanol = 100:1 (v / v) to give 160 mg of pale yellow solid, which was purified by semi-preparative HPLC (YMC-pack ODS-A, 65% MeCN / H2O) to give glycoside intermediate 4a (66 mg, t R =16.5 min, yield 14%, β - D Compound 4a (30 mg, 0.03 mmol) was dissolved in 5 mL of methanol and the pH was adjusted to 9 by adding 100 mg / mL sodium methoxide / methanol solution dropwise at room temperature (25 °C). Stirring was continued for 5 min, and the reaction was monitored by TLC until complete. A pretreated acidic cation exchange resin was added to adjust the pH to 7, and the reaction solution was evaporated to dryness. The deacetylated glycoside final product, compound 4a (20 mg, t...), was purified by semi-preparative HPLC (YMC-pack ODS-A, 80% MeOH / H2O) to obtain the glycoside final product compound 4a (20 mg, t...). R =5.2 min, yield 92%, β - D (Type 1 glycoside), white solid.

[0022] Compound 4: 1 H NMR (600 MHz, DMSO- d 6) d 9.29 (d, J = 8.0 Hz, 1H), 8.60 (s,1H), 8.06 (t, J = 6.6 Hz, 1H), 8.02 (d, J = 8.5 Hz, 1H) / 7.98 (d, J= 8.5 Hz,1H), 7.63 (d, J = 8.2 Hz, 1H), 7.52 (t, J = 7.9 Hz, 1H), 7.48 (t, J = 7.6 Hz,1H), 7.37 (t, J = 7.5 Hz, 1H), 7.30 (t, J = 7.5 Hz, 1H), 7.01 (t, J = 7.6 Hz,1H), 5.88 (s, 1H) / 5.85 (s, 1H), 5.67 (s, 1H), 5.48 (d, J = 8.2 Hz, 1H) / 5.40(d, J = 8.2 Hz, 1H), 5.11 (d, J = 3.7 Hz, 1H) / 5.05 (d, J = 3.4 Hz, 1H), 5.00(s, 2H), 4.97 (t, J = 5.9 Hz, 1H) / 4.57 (t, J = 5.9 Hz, 1H), 4.70 (dt, J =13.1, 3.1 Hz, 1H) / 4.54 (dt, J = 13.1, 3.1 Hz, 1H), 4.66 (t, J = 5.4 Hz, 1H) / 4.61 (t, J = 5.4 Hz, 1H), 4.43 (s, 1H) / 4.34 (s, 1H), 3.75 (dd, J = 11.5, 3.8Hz, 1H) / 3.69 (dd, J = 11.5, 3.8 Hz, 1H), 3.66 (d, J = 10.1 Hz, 1H), 3.63 (d, J = 8.8 Hz, 1H), 3.59–3.53 (m, 1H), 3.51 (d, J = 6.9 Hz, 1H), 3.49 (d, J =5.3 Hz, 1H), 3.44 (d, J = 9.5 Hz, 2H), 3.31 (dd,J = 8.3, 3.8 Hz, 1H), 3.28–3.12 (m, 1H), 3.14–3.05 (m, 1H), 2.84–2.77 (m, 1H), 2.75 (s, 3H) / 2.71 (s,3H), 2.70 (s, 3H) / 2.67 (s, 3H), 2.37 (s, 3H) / 2.36 (s, 3H), 2.33–2.17 (m, 1H); 13 C NMR (150 MHz, DMSO- d 6) d 172.0, 154.5 / 153.8, 138.8 / 138.7, 136.3 / 136.3, 132.8 / 132.7, 129.3 / 129.2, 125.8, 125.5, 125.3 / 125.2, 125.1 / 125.0, 123.8, 122.7, 121.7 / 121.6, 120.4, 119.6, 119.5, 115.3 / 115.2, 114.2, 113.5 / 113.4, 109.1 / 109.0, 100.9 / 100.9, 95.9 / 95.6, 94.7 / 94.6, 83.7 / 83.3, 82.3 / 82.1, 79.2 / 79.1, 76.5 / 76.1, 76.0, 73.6 / 73.6, 73.3, 72.5, 72.4, 70.0 / 70.0, 60.9 / 60.6, 60.3 / 60.3, 50.6 / 50.5, 45.5, 30.3 / 29.9, 29.2 / 29.0, 27.0; In solution, the ratio of cis-trans tautomers caused by p-π conjugation of tertiary amide bonds is approximately 1:2; HRESIMS m / z 835.3042 [M+H] + (Calculated value 835.3032).

[0023] Example 5: Preparation of Compound 5 Preparation of compound ST2: Cruciferine and N,Nʹ-thiocarbonyldiimidazole were dissolved in dry dichloromethane and reacted under triethylamine catalysis. After the reaction was completed, the mixture was extracted with dichloromethane and water. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The mixture was purified by normal-phase silica gel column chromatography to obtain intermediate ST1, in which the C3ʹ-NHCH3 group of cruciferine is linked to the imidazole group via a CN bond. At room temperature (25°C), compound ST1 was stirred with excess iodomethane in dry acetonitrile for 24 h. After the reaction was completed, the reaction solution was concentrated under reduced pressure and evaporated to dryness. The resulting pale yellow solid was washed repeatedly with petroleum ether / dichloromethane (v / v, 1:1) until small polar impurities disappeared, yielding intermediate compound ST2, which was converted to an alkyl iodide salt.

[0024] In a 10 mL two-necked reaction flask, compound ST2 (60 mg, 0.1 mmol) and glycosyl donor T5 (acetyl-protected, 1-deacetylated) were dissolved in N,N-dimethylformamide (2 mL). D - Glucose, 60 mg, 0.2 mmol), was slowly added dropwise with triethylamine (30 μL, 0.2 mmol), stirred at room temperature (25 ℃) for 24 h, and monitored by TLC until the reaction was complete. Extraction was performed with ethyl acetate (10 ml × 2), followed by washing with 1N HCl (5 ml × 2) and brine (5 ml × 2). The organic phases were combined, dried over anhydrous sodium sulfate, and then evaporated under vacuum to remove the solvent. The compound 5a (32 mg, t) was purified by semi-preparative HPLC (YMC-pack ODS-A, 75% MeCN / H2O) to obtain the compound. R =5.2 min, yield 42%, α - D Compound 5a (20 mg, 0.05 mmol) was dissolved in 5 mL of methanol. The pH was adjusted to 9 by adding 100 mg / mL sodium methoxide / methanol solution dropwise at room temperature (25 °C). Stirring continued for 5 min, and TLC was monitored until the reaction was complete. A pretreated acidic cation exchange resin was added to adjust the pH to 7. The reaction solution was evaporated to dryness, and purified by semi-preparative HPLC (YMC-pack ODS-A, 80% MeOH / H2O) to obtain the deacetylated glycoside final product compound 5a (15 mg, t...). R =5.5 min, yield 92%, α - D (Type 1 glycoside), white solid.

[0025] Compound 5: 1 H NMR (600 MHz, DMSO- d 6) d 9.31 (d,J = 8.4 Hz, 1H) / 9.29 (d, J = 8.4 Hz, 1H), 8.62 (s, 1H), 8.08 (d, J = 7.8 Hz, 1H) / 8.06 (d, J = 7.8 Hz,1H), 8.01 (d, J = 8.5 Hz, 1H) / 8.00 (d, J = 8.5 Hz, 1H), 7.70 (d, J = 8.4 Hz,1H) / 7.68 (d, J = 8.4 Hz, 1H), 7.55–7.46 (m, 2H), 7.37 (t, J = 7.6 Hz, 1H) / 7.36 (t, J = 7.6 Hz, 1H), 7.30 (t, J = 7.8 Hz, 1H), 7.10 (t, J = 7.5 Hz, 1H) / 7.00 (t, J = 7.5 Hz, 1H), 6.63 (d, J = 3.5 Hz, 1H), 5.59 (dt, J = 13.4, 3.3Hz, 1H) / 5.40 (dt, J = 13.4, 3.3 Hz, 1H), 5.45 (d, J = 5.8 Hz, 1H) / 5.22 (d, J = 5.8 Hz, 1H), 5.28 (d, J = 6.0 Hz, 1H) / 5.22 (d, J = 6.0 Hz, 1H), 5.08 (d, J = 6.0 Hz, 1H) / 5.05 (d, J = 6.0 Hz, 1H), 5.01 (s, 2H), 4.71 (t, J = 2.7 Hz,1H) / 4.60 (t, J = 2.7 Hz, 1H), 4.61 (d, J= 6.3 Hz, 1H), 3.76–3.57 (m, 2H), 3.56–3.40 (m, 2H), 3.35–3.28 (m, 1H), 3.27–3.21 (m, 1H), 3.12 (s, 3H) / 2.98(s, 3H), 2.86 (s, 3H) / 2.83 (s, 3H), 2.83–2.78 (m, 1H), 2.41 (s, 3H) / 2.37 (s,3H), 2.34–2.28 (m, 1H); 13 C NMR (150 MHz, DMSO- d 6) d 187.3, 171.9, 139.1 / 138.6, 136.3 / 136.3, 132.8 / 132.8, 129.0, 125.8 / 125.7, 125.4, 125.1 / 125.1, 124.8, 123.8, 122.7, 121.5, 120.4, 119.6, 119.5, 115.4 / 115.3, 114.3, 113.9, 109.1, 99.0, 94.7 / 94.5, 82.1 / 82.1, 81.7, 75.8 / 75.6, 73.4 / 72.2, 70.9, 69.2, 60.7, 60.6 / 60.4, 55.6, 45.5, 32.7, 29.6 / 29.0, 27.0; the ratio of cis-trans tautomers in solution due to p-π conjugation of tertiary amide bonds is approximately 2:1; HRESIMS m / z 689.2265 [M+H] + (Calculated value: 689.2276).

[0026] Example 6: Preparation of Compound 6 In a 10 mL two-necked reaction flask, compound ST2 (60 mg, 0.1 mmol) and glycosyl donor T6 (acetyl-protected, 1-deacetylated) were dissolved in N,N-dimethylformamide (2 mL). LGlucose (60 mg, 0.2 mmol) was slowly added dropwise with triethylamine (30 μL, 0.2 mmol). The mixture was stirred at room temperature (25 °C) for 24 h, and the reaction was monitored by TLC until completion. The mixture was extracted with ethyl acetate (10 ml × 2), washed with 1N HCl (5 ml × 2), and washed with brine (5 ml × 2). The organic phases were combined, dried over anhydrous sodium sulfate, and then evaporated under vacuum to remove the solvent. The mixture was separated by silica gel column chromatography, eluting with dichloromethane:methanol = 100:1 (v / v) to give 48 mg of a pale yellow solid. The obtained pale yellow powder was dissolved in methanol (5 mL), and the pH was adjusted to 8 by adding 100 mg / mL sodium methoxide / methanol solution at room temperature (25 °C). Continue stirring for 5 min, monitor the reaction by TLC until complete, add pretreated acidic cation exchange resin to adjust pH to 7, evaporate the reaction solution to dryness, and purify by semi-preparative HPLC (YMC-packODS-A, 80% MeOH / H2O) to obtain the deacetylated glycoside end product compound 6 (25 mg, t R =5.6 min, yield 36%), white solid.

[0027] Compound 6: 1 H NMR (600 MHz, DMSO- d 6) d 9.30 (d, J = 7.9 Hz, 1H), 8.61 (s,1H), 8.06 (d, J = 7.8 Hz, 1H), 8.02 (d, J = 8.6 Hz, 1H), 7.70 (d, J = 8.2 Hz, 1H), 7.49 (t, J = 8.0 Hz, 2H), 7.36 (t, J = 7.5 Hz, 1H), 7.31 (t, J = 7.5 Hz, 1H), 7.11 (t, J = 8.3 Hz, 1H) / 7.10 (t, J = 8.3 Hz, 1H), 5.65 (d, J = 4.3 Hz, 1H) / 5.61 (d, J = 4.3 Hz, 1H), 5.39 (d, J = 4.5 Hz, 1H) / 5.30 (d, J = 4.5 Hz, 1H), 5.34 (d,J = 5.6 Hz, 1H) / 5.23 (d, J = 5.6 Hz, 1H), 5.02 (d, J = 8.8 Hz,1H), 5.01 (s, 2H), 4.92 (d, J = 2.3 Hz, 1H) / 4.63 (d, J = 2.3 Hz, 1H), 4.69(t, J = 3.1 Hz, 1H) / 4.54 (t, J = 3.1 Hz, 1H), 3.76–3.69 (m, 1H), 3.68–3.63(m, 1H), 3.57–3.51 (m, 1H), 3.50–3.45 (m, 1H), 3.35–3.28 (m, 1H), 3.27–3.19(m, 1H), 3.12 (s, 3H) / 2.98 (s, 3H), 3.06 (s, 3H) / 2.80 (s, 3H), 2.89–2.81 (m, 1H), 2.38 (s, 3H) / 2.32 (s, 3H), 2.37–2.16 (m, 1H); 13 C NMR (150 MHz, DMSO- d 6) d 187.4 / 187.2, 171.9, 139.5, 139.0, 136.4 / 136.3, 133.1 / 132.8, 129.1 / 128.5, 125.7, 125.4, 125.1, 124.9, 123.8 / 123.8, 122.7, 121.5 / 121.5, 120.4, 119.6, 119.5 / 119.4, 115.6, 115.4, 114.3, 113.8, 109.1, 108.9, 99.0 / 98.6, 94.7 / 94.6, 82.1 / 81.9, 81.9 / 81.6, 75.8 / 75.6, 73.5 / 72.4, 71.0 / 70.8, 69.3 / 69.2, 60.9 / 60.8, 60.6 / 60.3, 55.6, 45.5, 36.8, 32.7 / 31.2, 29.4 / 29.4, 27.0 / 26.9; the ratio of cis-trans tautomers in solution due to p-π conjugation of tertiary amide bonds is approximately 2:1; HRESIMS m / z 689.2278 [M+H] +(Calculated value: 689.2276).

[0028] Example 7: Preparation of Compound 7 In a 10 mL two-necked reaction flask, compound ST2 (60 mg, 0.1 mmol) and glycosyl donor T7 (acetyl-protected, 1-deacetylated) were dissolved in N,N-dimethylformamide (2 mL). D Xylose (55 mg, 0.2 mmol) was slowly added dropwise with triethylamine (30 μL, 0.2 mmol) at room temperature (25 ℃) and stirred for 24 h. The reaction was monitored by TLC until completion. The mixture was extracted with ethyl acetate (10 ml × 2), washed with 1N HCl (5 ml × 2), washed with brine (5 ml × 2), and the organic phases were combined. After drying with anhydrous sodium sulfate, the solvent was removed by vacuum evaporation. The compound 7a (32 mg, t) was purified by semi-preparative HPLC (YMC-pack ODS-A, 75% MeCN / H2O) to obtain compound 7a (32 mg, t). R =10.1 min, yield 41%, α - D Compound 7a (20 mg, 0.03 mmol) was dissolved in 5 mL of methanol and the pH was adjusted to 8 by adding 100 mg / mL sodium methoxide / methanol solution dropwise at room temperature (25 °C). Stirring was continued for 5 min, and the reaction was monitored by TLC until complete. A pretreated acidic cation exchange resin was added to adjust the pH to 7, and the reaction solution was evaporated to dryness. The deacetylated glycoside final product, compound 7a (15 mg, t...), was purified by semi-preparative HPLC (YMC-pack ODS-A, 80% MeOH / H2O) to obtain the glycoside final product compound 7a (15 mg, t...). R =8.3 min, yield 92%, α - D (Type 1 glycoside), white solid.

[0029] Compound 7: 1 H NMR (600 MHz, DMSO- d 6) d 9.32 (d, J = 7.9 Hz, 1H) / 9.29 (d, J = 7.9 Hz, 1H), 8.65 (s, 1H) / 8.63 (s, 1H), 8.09 (d, J = 8.3 Hz, 1H) / 8.05 (d, J = 8.3 Hz, 1H), 8.07 (d, J= 8.9 Hz, 1H) / 8.01 (d, J = 8.9 Hz, 1H), 7.70 (d, J = 8.3 Hz, 1H) / 7.56 (d, J = 8.3 Hz, 1H), 7.54–7.47(m, 2H), 7.40 (t, J = 7.8Hz, 1H) / 7.36 (t, J = 7.8 Hz, 1H), 7.30 (t, J = 7.8 Hz, 1H), 7.10 (t, J = 7.7Hz, 1H) / 6.99 (t, J = 7.7 Hz, 1H), 6.62 (d, J = 3.5 Hz, 1H) / 6.53 (d, J = 3.5Hz, 1H), 5.59 (dt, J = 13.4, 3.3 Hz, 1H) / 5.20 (dt, J = 13.1, 3.2 Hz, 1H),5.47 (d, J = 6.0 Hz, 1H) / 5.25 (d, J = 6.0 Hz, 1H), 5.40 (d, J = 12.8 Hz, 1H) / 5.12 (d, J = 12.8 Hz, 1H), 5.00 (s, 2H), 4.58 (s, 1H) / 4.46 (s, 1H), 3.68–3.51(m, 2H), 3.50–3.34 (m, 1H), 3.30 (t, J = 10.1 Hz, 2H), 3.11 (s, 3H) / 2.99 (s,3H), 2.96 (s, 3H) / 2.84 (s, 3H), 2.77–2.64 (m, 1H), 2.37 (s, 3H) / 2.28 (s, 3H),2.34–2.23 (m, 1H); 13 C NMR (150 MHz, DMSO- d 6) d187.2, 171.9, 139.2 / 139.0, 136.3 / 136.2, 132.8 / 132.4, 129.7 / 129.0, 126.1, 125.8 / 125.7, 125.5 / 125.4, 125.1 / 124.8, 123.9 / 123.8, 122.7 / 122.6, 121.8 / 121.4, 120.8 / 120.4, 119.8 / 119.7, 119.6 / 119.5, 115.3 / 114.9, 114.2, 113.8 / 112.6, 109.1 / 109.0, 99.2 / 98.8, 94.7 / 94.6, 82.1 / 82.0, 81.8 / 80.9, 74.7 / 73.8, 70.9, 69.8 / 69.2, 64.7 / 64.1, 60.6 / 59.9, 53.4 / 52.1, 45.4 / 45.4, 32.6 / 30.6, 30.5 / 29.5, 27.8 / 26.9; the ratio of cis-trans tautomers in solution due to p-π conjugation of tertiary amide bonds is approximately 2:1; HRESIMS m / z 659.2195 [M+H] + (Calculated value: 659.2170).

[0030] Example 8: Preparation of Compound 8 In a 10 mL two-necked reaction flask, compound ST2 (60 mg, 0.1 mmol) and glycosyl donor T8 (acetyl-protected, 1-deacetylated) were dissolved in N,N-dimethylformamide (2 mL). L Xylose (55 mg, 0.2 mmol) was slowly added dropwise with triethylamine (30 μL, 0.2 mmol) at room temperature (25 ℃) and stirred for 24 h. The reaction was monitored by TLC until completion. The mixture was extracted with ethyl acetate (10 ml × 2), washed with 1N HCl (5 ml × 2), washed with brine (5 ml × 2), and the organic phases were combined. After drying with anhydrous sodium sulfate, the solvent was removed by vacuum evaporation. The compound 8a (35 mg, t) was purified by semi-preparative HPLC (YMC-pack ODS-A, 75% MeCN / H2O) to obtain compound 8a (35 mg, t). R =11.2 min, yield 45%, α - LCompound 8a (20 mg, 0.03 mmol) was dissolved in 5 mL of methanol and the pH was adjusted to 8 by adding 100 mg / mL sodium methoxide / methanol solution dropwise at room temperature (25 °C). Stirring was continued for 5 min, and the reaction was monitored by TLC until complete. A pretreated acidic cation exchange resin was added to adjust the pH to 7. The reaction solution was evaporated to dryness, and the deacetylated glycoside final product, compound 8a (16 mg, t...), was purified by semi-preparative HPLC (YMC-pack ODS-A, 80% MeOH / H2O) to obtain the deacetylated glycoside final product compound 8a (16 mg, t...). R =8.3 min, yield 93%, α - L (Type 1 glycoside), white solid.

[0031] Compound 8: 1 H NMR (600 MHz, DMSO- d 6) d 9.29 (d, J = 8.4 Hz, 1H) / 9.27 (d, J = 8.4 Hz, 1H), 8.61 (s, H), 8.05 (d, J = 8.6 Hz, 1H), 8.04 (d, J = 8.6 Hz, 1H) / 7.97 (d, J = 8.6 Hz, 1H), 7.72 (d, J = 8.3 Hz, 1H) / 7.69 (d, J = 8.3 Hz,1H), 7.57–7.47 (m, 2H), 7.37 (d, J = 7.4 Hz, 1H) / 7.36 (d, J = 7.4 Hz, 1H), 7.31 (t, J = 7.5 Hz, 1H), 7.11 (d, J = 7.1 Hz, 1H) / 7.09 (d, J = 7.1 Hz, 1H), 6.65 (d, J = 3.2 Hz, 1H) / 6.55 (d, J = 3.5 Hz, 1H), 5.65 (dt, J = 13.4, 3.3Hz, 1H) / 4.95 (dt, J= 13.4, 3.3 Hz, 1H), 5.52–5.03 (m, 2H), 5.01 (s, 2H), 4.90 (s, 1H) / 4.60 (s, 1H), 3.73 (dd, J = 9.7, 4.6 Hz, 1H) / 3.45 (dd, J = 9.7, 4.6 Hz, 1H), 3.66 (d, J = 4.7 Hz, 1H), 3.57 (t, J = 5.6 Hz, 1H), 3.31 (t, J =6.5 Hz, 1H), 3.23 (d, J = 10.2 Hz, 1H), 3.12 (s, 3H) / 2.99 (s, 3H), 3.04 (s,3H) / 2.77 (s, 3H), 2.89–2.81 (m, 1H), 2.40 (s, 3H) / 2.32 (s, 3H), 2.39–2.32 (m,1H); 13 C NMR (150 MHz, DMSO- d 6) d 187.3 / 187.1, 171.9 / 171.9, 139.5 / 138.9, 136.4 / 136.3, 133.0 / 132.8, 129.2 / 128.5, 125.8 / 125.7, 125.5 / 125.4, 125.1 / 125.0, 124.8 / 124.4, 123.8, 122.7 / 122.7, 121.6 / 121.5, 120.4 / 120.3, 119.6 / 119.6, 119.5 / 119.4, 115.6 / 115.4, 114.3, 114.0 / 113.7, 109.1 / 108.9, 99.0 / 98.6, 94.7 / 94.6, 82.1 / 82.0, 81.9 / 81.8, 74.5 / 73.8, 71.0 / 70.9, 69.8 / 69.3, 64.4 / 64.0, 60.8 / 60.5, 55.5 / 52.7, 45.5, 32.6 / 31.1, 29.2, 27.0; the ratio of cis-trans tautomers in solution due to p-π conjugation of tertiary amide bonds is approximately 1:1; HRESIMS m / z 659.2169 [M+H] + (Calculated value: 659.2170).

[0032] Example 9: Preparation of Compound 9 In a 10 mL two-necked reaction flask, compound ST2 (60 mg, 0.1 mmol) and glycosyl donor T9 (acetyl-protected, 1-deacetylated) were dissolved in N,N-dimethylformamide (2 mL). L Lysose (55 mg, 0.2 mmol) was slowly added dropwise with triethylamine (30 μL, 0.2 mmol) at room temperature (25 ℃) and stirred for 24 h. The reaction was monitored by TLC until completion. The mixture was extracted with ethyl acetate (10 ml × 2), washed with 1N HCl (5 ml × 2), washed with brine (5 ml × 2), and the organic phases were combined. After drying with anhydrous sodium sulfate, the solvent was removed by vacuum evaporation. Compound 9a (33 mg, t) was purified by semi-preparative HPLC (YMC-pack ODS-A, 75% MeCN / H2O) to obtain compound 9a (33 mg, t). R =11.5 min, yield 42%, α - L Compound 9a (19 mg, 0.03 mmol) was dissolved in 5 mL of methanol and the pH was adjusted to 8 by adding 100 mg / mL sodium methoxide / methanol solution dropwise at room temperature (25 °C). Stirring was continued for 5 min, and the reaction was monitored by TLC until complete. A pretreated acidic cation exchange resin was added to adjust the pH to 7. The reaction solution was evaporated to dryness, and the deacetylated glycoside final product, compound 9a (19 mg, t...), was purified by semi-preparative HPLC (YMC-pack ODS-A, 80% MeOH / H2O) to obtain the deacetylated glycoside final product compound 9a (19 mg, t...). R =7.6 min, yield 93%, α - L (Type 1 glycoside), white solid.

[0033] Compound 9: 1 H NMR (600 MHz, DMSO- d 6) d 9.31 (d, J = 7.8 Hz, 1H) / 9.28 (d, J = 7.8 Hz, 1H), 8.62 (s, 1H), 8.05 (d, J = 8.2 Hz, 1H), 8.03 (d, J = 8.6 Hz, 1H) / 7.96 (d, J = 8.6 Hz, 1H), 7.68 (d, J= 7.7 Hz, 1H), 7.53–7,47 (m, 2H),7.37 (t, J = 7.3 Hz, 1H) / 7.36 (t, J = 7.3 Hz, 1H), 7.31 (t, J = 7.3 Hz, 1H),7.10 (t, J = 7.3 Hz, 1H) / 7.08 (t, J = 7.3 Hz, 1H), 6.48 (d, J = 3.1 Hz, 1H) / 6.44 (d, J = 3.1 Hz, 1H), 5.62 (dt, J = 13.4, 3.3 Hz, 1H) / 4.97 (dt, J = 13.4,3.3 Hz, 1H), 5.33 (d, J = 4.8 Hz, 1H) / 5.27 (d, J = 4.8 Hz, 1H), 5.22 (d, J =4.5 Hz, 1H) / 4.94 (d, J = 5.6 Hz, 1H), 5.01 (s, 2H), 5.17 (d, J = 4.5 Hz, 1H) / 4.91 (d, J = 5.7 Hz, 1H), 4.60 (s, 1H) / 4.52 (s, 1H), 3.83 (dt, J = 11.4, 3.2Hz, 1H) / 3.71 (dt, J = 11.4, 3.2 Hz, 1H), 3.80 (t, J = 2.4 Hz, 1H) / 3.69 (t, J = 2.4 Hz, 1H), 3.79 (d, J = 4.8 Hz, 1H) / 3.67 (d, J = 4.8 Hz, 1H), 3.57–3,53(m, 1H), 3.28 (t, J= 8.3 Hz, 1H), 3.12 (s, 3H) / 2.96 (s, 3H), 2.92 (s, 3H) / 2.78 (s, 3H), 2.88–2.79 (m, 1H), 2.40 (s, 3H) / 2.34 (s, 3H), 2.34–2.22 (m,1H); 13 C NMR (150 MHz, DMSO- d 6) d 186.5 / 186.2, 171.9, 139.2 / 138.9, 136.3 / 136.3, 132.9 / 132.8, 129.1 / 128.8, 125.7, 125.5 / 125.4, 125.1 / 124.9, 124.6, 123.8, 122.7, 121.5, 120.4, 119.6, 119.5, 115.5 / 115.4, 114.3, 113.7, 109.0 / 108.9, 100.1 / 100.0, 94.7 / 94.5, 82.9 / 82.1, 81.9 / 81.8, 71.8 / 71.2, 68.8 / 68.7, 66.2, 65.3, 60.7 / 60.5, 55.7 / 52.5, 45.5, 36.7 / 32.6, 30.1 / 29.3, 27.0 / 26.9; the ratio of cis-trans tautomers in solution due to p-π conjugation of tertiary amide bonds is approximately 2:1; HRESIMS m / z 659.2174 [M+H] + (Calculated value: 659.2170).

[0034] Example 10: Preparation of Compound 10 In a 10 mL two-necked reaction flask, compound ST2 (60 mg, 0.1 mmol) and glycosyl donor T were dissolved in N,N-dimethylformamide (2 mL). 10 (acetyl-protected 1-deacetyl- 1-thio- D - Glucose, 73 mg, 0.2 mmol), triethylamine (30 μL, 0.2 mmol) was slowly added dropwise, and the mixture was stirred at room temperature (25 ℃) for 24 h. The reaction was monitored by TLC until completion. The mixture was extracted with ethyl acetate (10 ml × 2), washed with 1N HCl (5 ml × 2), washed with brine (5 ml × 2), and the organic phases were combined. After drying with anhydrous sodium sulfate, the solvent was removed by vacuum evaporation. The compound 10a (39 mg, t) was purified by semi-preparative HPLC (YMC-pack ODS-A, 75% MeCN / H2O) to obtain compound 10a (39 mg, tR =13.6 min, yield 45%, β - D Compound 10a (25 mg, 0.03 mmol) was dissolved in 5 mL of methanol and the pH was adjusted to 8 by adding 100 mg / mL sodium methoxide / methanol solution dropwise at room temperature (25 °C). Stirring was continued for 5 min, and the reaction was monitored by TLC until complete. A pretreated acidic cation exchange resin was added to adjust the pH to 7, and the reaction solution was evaporated to dryness. The deacetylated glycoside final product, compound 10 (16 mg, t...), was purified by semi-preparative HPLC (YMC-pack ODS-A, 80% MeOH / H2O) to obtain the glycoside final product compound 10 (16 mg, t...). R =5.6 min, yield 80%, β - D (Type 1 glycoside), white solid.

[0035] Compound 10: 1 H NMR (600 MHz, DMSO- d 6) d 9.29 (d, J = 7.9 Hz, 1H), 8.62(s, 1H), 8.05 (d, J = 7.6 Hz, 1H), 8.04 (d, J = 8.6 Hz, 1H), 7.66 (d, J = 8.3Hz, 1H), 7.51–7.47 (m, 2H), 7.36 (t, J = 7.6 Hz, 1H), 7.30 (t, J = 7.6 Hz, 1H), 7.10 (t, J = 7.2 Hz, 1H), 5.53 (d, J = 10.0 Hz, 1H), 5.48 (d, J = 5.8Hz, 1H), 5.21 (d, J = 4.8 Hz, 1H), 5.00 (d, J = 4.1 Hz, 1H), 5.00 (s, 2H), 4.61 (s, 1H), 4.45 (t, J = 5.8 Hz, 1H), 3.65 (dd, J= 11.3, 5.4 Hz, 1H),3.55–3.48 (m, 1H), 3.32–3.29 (m, 1H), 3.27 (d, J = 1.4 Hz, 1H), 3.23–3.12 (m,2H), 3.17 (s, 3H), 2.89–2.82 (m, 1H), 2.74 (s, 3H), 2.40 (s, 3H), 2.39–2.35(m, 1H); 13 C NMR (150 MHz, DMSO- d 6) d 196.4, 171.9, 138.8, 136.3, 132.7, 129.1,125.7, 125.4, 125.1, 125.0, 123.8, 122.7, 121.5, 120.4, 119.6, 119.5, 115.3,114.2, 113.7, 109.1, 94.9, 89.8, 82.2 / 81.8, 81.5 / 81.4, 78.7, 76.5, 71.2,69.4, 60.6, 60.4, 57.1, 45.5, 36.3, 29.2, 27.1; HRESIMS m / z 705.2056 [M+H] + (Calculated value 705.2047).

[0036] Example of effect 1: To further verify the beneficial effects of the compounds synthesized in this invention, antitumor activity tests were conducted on the synthesized compounds, and the specific experiments are as follows: 1. Experimental Methods (1) Preparation of the sample solution: Accurately weigh the sample, add cell-grade DMSO to dissolve and mix well, with a stock solution concentration of 10 mM. Dilute the cell culture medium to 10 times the concentration to be tested.

[0037] (2) Cell seeding: Take cells that have grown to the logarithmic phase, dilute the cells to a concentration of 5×104 cells / mL with culture medium containing 10% fetal bovine serum, and seed them into 96-well plates with a seeding volume of 90 μL per well. Incubate for 24 h at 5% CO2 and 37℃.

[0038] (3) Add the sample solution to be tested: Three parallel replicates were set up for each concentration, and 10 μL of culture medium was added to each well to dilute the sample to different concentrations (the initial screening concentration was 1 μM, IC50). 50Eight concentration gradients were set up for each sample, and the samples were incubated in an incubator for 48 h. The experiment included a blank group, a control group, and a drug group.

[0039] (4) Color development: Remove the old culture medium and drug solution from the adherent cells (add 10 μL of original CCK-8 solution directly to the suspension cells), add 100 μL of CCK-8 solution diluted 10 times to each well, and continue to culture at 37℃ and 5% CO2 for 1–4 h (operate in the dark and observe in real time).

[0040] (5) Detection: Use an enzyme-linked immunosorbent assay (ELISA) reader to measure the absorbance at 450 nm and record the raw data results.

[0041] (6) Use Excel software to standardize the raw data. Calculate the cell proliferation inhibition rate based on the OD value of each well during the initial screening (formula = (OD value of each well)). Control -OD Drug ) / (OD Control -OD Blank ( ) ×100%), statistical inhibition rate. IC 50 The calculations were performed using GraphPad Prism 8 software, and the experimental results are expressed as ±SD.

[0042] Positive control: Doxorubicin hydrochloride (Dox).

[0043] 2. The experimental results are shown in Table 1 and Table 2.

[0044] Table 1. Inhibitory activity of compounds against human leukemia cell proliferation (IC50) 50 )

[0045] a ND indicates unmeasured. b IC 50 > 1 μM.

[0046] Table 2. Inhibitory activity of compounds against solid tumor cell proliferation in vitro (IC50) 50 )

[0047] a ND indicates unmeasured. b IC 50 > 1 μM.

[0048] As shown in Table 1, compound 5 exhibits strong inhibitory activity against human acute myeloid leukemia cells THP-1, twice that of the positive control drug doxorubicin, with a half-maximal inhibitory concentration (IC50) of [missing value].50 The concentration reached 88.4 nM, which can be used to prepare drugs for the prevention or treatment of acute myeloid leukemia.

[0049] Table 2 shows that compounds 4 and 5 exhibit strong inhibitory activity against human lung cancer cells A549, human pancreatic cancer cells PATU8988T, human liver cancer cells HuH-7, human colon cancer cells HCT-116, and human bladder cancer cells 5637, which is 2.8 to 20 times more potent than the positive control drug doxorubicin. 50 It reaches 12~89 nM and can be used for the development of drugs for the prevention or treatment of human lung cancer, pancreatic cancer, liver cancer, colon cancer and bladder cancer.

[0050] Compounds 1, 2, and 3 exhibited significantly higher inhibitory activity against human hepatocellular carcinoma cells (HuH-7) than the positive control drug doxorubicin, ranging from 2.5 to 16 times higher, with an IC50 value of [missing value]. 50 It can reach 34~59 nM, but its inhibitory activity on normal human hepatocytes L-02 is lower than that of doxorubicin. Its selectivity index (SI) is 7.2, 12.3, 8.0 and 13.2, respectively, and it can be used for the development of drugs for the prevention or treatment of liver cancer.

[0051] Compounds 1–10 exhibited strong inhibitory activity against human bladder cancer cells 5637, which was 2.5–16 times that of the positive control drug doxorubicin, with an IC50 value of [missing value]. 50 It reached 15~99 nM, which can be used for the development of drugs for the prevention or treatment of human bladder cancer.

[0052] Compounds 2 and 3 exhibited significantly higher inhibitory activity against human colon cancer cells HCT-116 than the positive control drug doxorubicin, reaching 2.1–3.5 times the activity. The IC50 value was [not specified in the original text]. 50 It can reach 57~95 nM, but its inhibitory activity on normal human hepatocytes L-02 is lower than that of doxorubicin. It can be used in the development of drugs for the prevention or treatment of colon cancer.

[0053] Compound 2 exhibits 4.6 times the inhibitory activity against human pancreatic cancer cells PATU8988T compared to the positive control drug doxorubicin, but its inhibitory activity against normal human liver cells L-02 is lower than that of doxorubicin. Its selectivity index (SI) is 5.1, making it suitable for the development of drugs for the prevention or treatment of pancreatic cancer.

[0054] Compound 5 exhibited 7.1 times the cytotoxic activity against human gastric cancer cells MKN-45 compared to the positive control drug doxorubicin, with an IC50 value of [missing value]. 50 The concentration was 68.8 nM. However, its cytotoxic activity against normal human hepatocytes L-02 was relatively weak (IC50). 50 With a molecular weight of 474.6 nM and a selectivity index (SI) of 6.9, it can be used for the development of drugs for the prevention or treatment of gastric cancer.

[0055] The above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A cruciferous glycoside derivative, the structure of which is shown in formula (I): （I）； in, Represents a glycosyl group, selected from... D- Lysol, 2-deoxy- D -Glucose, 5-deoxy- D -ribose, D -maltose, D -glucose, L -glucose, D - Xylose, L - Xylose, L -Lessosulfate, 1-thio- D - One of the components of glucose.

2. A cruciferous glycoside derivative as described in claim 1, characterized in that, When X is O and Y is O, the glycosyl group is selected from... D- Lysol, 2-deoxy- D -Glucose, 5-deoxy- D -ribose or D -maltose.

3. A cruciferous glycoside derivative as described in claim 1, characterized in that, When X is S and Y is O, the glycosyl group is selected from... D -glucose, L -glucose, D - Xylose, L -xylose or L - Lysol.

4. A cruciferous glycoside derivative as described in claim 1, characterized in that, When X is S and Y is S, the glycosyl group is selected from 1-thio- D -glucose.

5. A method for preparing a cruciferous glycoside derivative as described in any one of claims 1-4, characterized in that, The process includes the following steps: Acylation of cruciferine with di(trichloromethyl) carbonate to generate an acyl chloride intermediate, followed by a reaction with an acetyl-protected 1-deacetyl glycosyl donor catalyzed by 4-dimethylaminopyridine to obtain a glycoside intermediate, and finally deacetylation with sodium methoxide to obtain the final glycoside product; or, reacting cruciferine with N,Nʹ-thiocarbonyldiimidazole to introduce a thiocarbonyl imidazole fragment, followed by a reaction with iodomethane to form a salt, activating the imidazole group to facilitate its removal, and then reacting with an acetyl-protected 1-deacetyl glycosyl donor to obtain a glycoside intermediate, and finally deacetylation with sodium methoxide to obtain the final glycoside product.

6. The use of the cruciferoside derivative as described in any one of claims 1–4 in the preparation of antitumor drugs.

7. The use of the cruciferous glycoside derivative as described in any one of claims 1–4 in the preparation of a drug for the prevention or treatment of gastric cancer, bladder cancer, colon cancer, pancreatic cancer, lung cancer, liver cancer, or acute myeloid leukemia.

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