Tripterine prodrug compound as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of medicinal chemistry, and particularly relates to a tripterine prodrug compound as well as a preparation method and application thereof. The tripterine prodrug compound provided by the invention has a structure as shown in a formula I. The tripterine prodrug compound designed by utilizing the unique high expression characteristic of nitrate reductase in a tumor microenvironment can be specifically activated by the nitrate reductase at a tumor part, so that accurate release of tripterine at the tumor part is realized; the tripterine prodrug compound is kept relatively stable in normal tissues, so that the toxic effect on tissues such as normal livers is greatly reduced, and an innovative and potential solution is provided for solving the hepatotoxicity problem of the tripterine and improving the anti-tumor treatment effect of the tripterine; and breakthrough is hopefully brought to tumor treatment and the clinical application value is improved. Formula I.

Description

Technical Field

[0001] This invention belongs to the field of medicinal chemistry technology, specifically relating to a triptolide prodrug compound, its preparation method, and its application. Background Technology

[0002] Celastrol, a natural active ingredient extracted from the traditional Chinese medicine Tripterygium wilfordii, possesses broad and significant anti-tumor activity. It exerts its anti-cancer effects through multiple pathways, such as inducing tumor cell apoptosis, inhibiting tumor cell proliferation, regulating the tumor immune microenvironment, and inhibiting tumor angiogenesis. However, celastrol faces a significant challenge in clinical application due to its high hepatotoxicity, which severely limits its further development and application. Summary of the Invention

[0003] The purpose of this invention is to provide a triptolide prodrug compound, its preparation method, and its application. The triptolide prodrug compound provided by this invention can achieve specific activation at the tumor site, maintain relative stability in normal tissues, greatly reduce toxicity to normal tissues such as the liver, and significantly improve the safety and therapeutic index of the drug.

[0004] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a triptolide prodrug compound having the structure shown in Formula I: Formula I, where R is H, C1-5 alkyl, or halogen.

[0005] Preferably, the C1-5 alkyl group is methyl; and the halogen is Cl, Br, or F.

[0006] Preferably, it has any one of the following structures: , , , , , , , , , .

[0007] This invention provides a method for preparing the triptolide prodrug compound described in the above technical solution, comprising the following steps: The compound with the structure shown in Formula 1, the compound with the structure shown in Formula 2, an inorganic base and a first organic solvent were mixed and subjected to an etherification reaction to obtain the triptolide prodrug compound with the structure shown in Formula I. Formula 1, Formula 2.

[0008] Preferably, the method for preparing the compound with the structure shown in Formula 1 includes the following steps: Tripterygium wilfordii, a second organic solvent, sodium bicarbonate, and bromoethane were mixed and subjected to an esterification reaction to obtain a compound with the structure shown in Formula 1.

[0009] Preferably, the first organic solvent includes one or more of N,N-dimethylformamide, acetonitrile, dichloromethane, and toluene; the inorganic base includes one or more of KOH, K2CO3, NaOH, and Na2CO3; and the molar ratio of the compound with the structure shown in Formula 1 to the compound with the structure shown in Formula 2 is 1:1.05~3.

[0010] Preferably, the temperature of the etherification reaction is -20 to 100°C.

[0011] Preferably, after the etherification reaction is completed, an etherification reaction solution is obtained; the process further includes mixing the etherification reaction solution with ice water, then adjusting the pH to neutral with acid to obtain a neutral reaction solution; extracting the neutral reaction solution with ethyl acetate to obtain an organic phase product; washing, drying, and concentrating the organic phase product sequentially to obtain a crude product; purifying the crude product by silica gel column chromatography to obtain a pure product of the triptolide prodrug compound; the eluent used for purification is ethyl acetate and petroleum ether.

[0012] This invention provides the application of the triptolide prodrug compound described in the above-described technical solution or the triptolide prodrug compound prepared by the above-described technical solution in the preparation of anticancer drugs.

[0013] Preferably, the anticancer drug is an anti-liver cancer drug.

[0014] To overcome the hepatotoxicity of triptolide while retaining its antitumor activity, this invention provides a triptolide prodrug compound with the structure shown in Formula I. Based on the high expression of nitrate reductase in the tumor microenvironment, this invention designs a triptolide prodrug compound with the structure shown in Formula I. This invention modifies the chemical structure of triptolide by introducing a nitro group sensitive to nitrate reductase, thus constructing the triptolide prodrug compound. In this invention, a nitrobenzyl ether structure is attached to the hydroxyl structure of triptolide to block this active center, making the entire compound relatively stable and less active after entering the body, thereby reducing toxicity to normal tissues, especially the liver. When this prodrug enters the hypoxic tumor microenvironment, due to the high expression of nitrate reductase in tumor tissue, the nitro group on the prodrug structure becomes a substrate for nitrate reductase. Nitrate reductase can specifically recognize and catalyze the nitro group in the prodrug structure, causing it to undergo a reduction reaction, gradually reducing the nitro group to an amino group. With the reduction of the nitro group, the original structure of triptolide is released, thereby restoring its antitumor activity. The mechanism of drug release, such as Figure 25 As shown (using compound 3-1 as an example).

[0015] Specifically, in the tumor microenvironment, nitrate reductase uses β-nicotinamide adenine dinucleotide (NADH) as an electron donor to provide the necessary electrons for the reduction reaction of nitro groups. During a series of enzymatic reactions, the nitro group is gradually reduced to a nitroso group, then a hydroxylamine group, and finally an amino group. Simultaneously, triptolide is cleaved from the triptolide prodrug compound structure shown in Formula I, achieving specific activation at the tumor site. This mechanism of releasing the active drug in response to nitrate reductase in the tumor microenvironment, as described in this invention, exhibits high targeting specificity. On the one hand, it ensures that triptolide exerts its potent anti-tumor effect in tumor tissue, effectively inhibiting tumor cell growth, inducing apoptosis, and disrupting tumor angiogenesis; on the other hand, because the prodrug remains relatively stable in normal tissues due to the lack of highly expressed nitrate reductase, it significantly reduces toxicity to normal tissues such as the liver, thus significantly improving the drug's safety and therapeutic index. This invention utilizes a clever prodrug design strategy and the unique high expression of nitrate reductase in the tumor microenvironment to achieve precise activation and release of triptolide at the tumor site. This provides an innovative and highly promising solution to address the hepatotoxicity of triptolide and improve its anti-tumor therapeutic effect, and is expected to bring breakthroughs and clinical application value to tumor treatment. Attached Figure Description

[0016] Figure 1 Characterization diagram of the inhibitory effect of compounds 3-4 prepared in the examples on the growth of liver cancer; Figure 2 The results of cytotoxicity tests of compounds 3-4 prepared for the examples; Figure 3 The proton NMR spectrum of compound 1 prepared for example; Figure 4 The carbon spectrum of compound 1 prepared for example; Figure 5 The proton NMR spectrum of compound 3-1 prepared for example; Figure 6 The carbon spectrum of compound 3-1 prepared for example; Figure 7 The proton NMR spectrum of compound 3-2 prepared for example; Figure 8 The carbon spectrum of compound 3-2 prepared for example; Figure 9 The proton NMR spectrum of compound 3-3 prepared for example; Figure 10 Carbon spectra of compound 3-3 prepared for example; Figure 11 The proton NMR spectra of compounds 3-4 prepared for the examples; Figure 12 Carbon spectra of compounds 3-4 prepared for examples; Figure 13 The proton NMR spectra of compounds 3-5 prepared for the examples; Figure 14 Carbon spectra of compounds 3-5 prepared for examples; Figure 15 The proton NMR spectra of compounds 3-6 prepared for the examples; Figure 16 Carbon spectra of compounds 3-6 prepared for examples; Figure 17 The proton NMR spectra of compounds 3-7 prepared for the examples; Figure 18 Carbon spectra of compounds 3-7 prepared for examples; Figure 19 The proton NMR spectra of compounds 3-8 prepared for the examples; Figure 20 Carbon spectra of compounds 3-8 prepared for examples; Figure 21 The proton NMR spectra of compounds 3-9 prepared for the examples; Figure 22 Carbon spectra of compounds 3-9 prepared for examples; Figure 23 The proton NMR spectra of compounds 3-10 prepared for the examples; Figure 24 Carbon spectra of compounds 3-10 prepared for examples; Figure 25 This is a diagram illustrating the mechanism of drug release. Detailed Implementation

[0017] This invention provides a triptolide prodrug compound having the structure shown in Formula I: Formula I, where R is H, C1-5 alkyl, or halogen.

[0018] In this invention, the C1-5 alkyl group can be a straight-chain alkyl group or a branched-chain alkyl group. The C1-5 alkyl group can be methyl, ethyl, propyl, butyl, or pentyl. The halogen can be Cl, Br, or F.

[0019] In this invention, the triptolide prodrug compound preferably has any one of the following structures: Formula 3-1 Equation 3-2 Formula 3-3 Equation 3-4 Formula 3-5 Formula 3-6 Equation 3-7 Formula 3-8 Equation 3-9 Equation 3-10.

[0020] This invention provides a method for preparing the triptolide prodrug compound described in the above technical solution, comprising the following steps: The compound with the structure shown in Formula 1, the compound with the structure shown in Formula 2, an inorganic base and a first organic solvent were mixed and subjected to an etherification reaction to obtain the triptolide prodrug compound with the structure shown in Formula I. Formula 1, Formula 2.

[0021] In this invention, unless otherwise specified, all raw materials / components used in the preparation are commercially available products well known to those skilled in the art.

[0022] In this invention, the method for preparing the compound with the structure shown in Formula 1 preferably includes the following steps: Tripterygium wilfordii, a second organic solvent, sodium bicarbonate, and bromoethane are mixed and subjected to an esterification reaction to obtain a compound with the structure shown in Formula 1. In this invention, the second organic solvent can be N,N-dimethylformamide (DMF). The preferred molar ratio of triptolide to sodium bicarbonate is 2.22:6.67. The preferred molar ratio of triptolide to bromoethane is 2.22:11.1. This invention does not have special requirements on the amount of the second organic solvent, as long as the esterification reaction proceeds smoothly. The esterification reaction is carried out at room temperature and under stirring conditions, and the preferred reaction time is 12-24 hours. After the esterification reaction is completed, an esterification reaction solution is obtained. In this invention, the esterification reaction solution is preferably diluted with water and then neutralized to neutrality with acid to obtain a neutral reaction solution; the neutral reaction solution is extracted with ethyl acetate to obtain an organic phase product; the organic phase product is washed, dried, and concentrated sequentially to obtain a concentrated product; the concentrated product is purified by silica gel column chromatography to obtain a pure product of the compound with the structure shown in Formula 1. The preferred acid is hydrochloric acid, and the preferred molar concentration of the hydrochloric acid is 1 mol / L. The extraction is preferably performed three times. The washing is preferably performed using saturated brine. The washing is preferably performed three times. The drying reagent is preferably anhydrous sodium sulfate. The silica gel used for silica gel column chromatography purification is preferably 200-300 mesh. The eluent used for silica gel column chromatography purification is preferably ethyl acetate and petroleum ether. In this embodiment of the invention, the volume ratio of ethyl acetate to petroleum ether can be gradually increased from 1:10 to 1:1.

[0023] The present invention does not have any special requirements for the preparation method of the compound with the structure shown in Formula 2; any preparation method well known to those skilled in the art can be used.

[0024] In this invention, the first organic solvent preferably includes one or more of N,N-dimethylformamide, acetonitrile, dichloromethane, and toluene; in the examples, acetonitrile may be used. The inorganic base preferably includes one or more of KOH, K₂CO₃, NaOH, and Na₂CO₃; in the examples, Na₂CO₃ may be used. The molar ratio of the compound with the structure shown in Formula 1 to the compound with the structure shown in Formula 2 is preferably 1:1.05~3; in the examples, it may be 1:1.2. The temperature of the etherification reaction is preferably -20~100℃, more preferably 25~90℃, and even more preferably 60~85℃; in the examples, it may be 80℃. The time of the etherification reaction is preferably 2~3 hours. The progress of the esterification reaction is preferably monitored by TLC in this invention.

[0025] In this invention, after the etherification reaction is completed, an etherification reaction solution is obtained. Preferably, the invention further includes mixing the etherification reaction solution with ice water, then adjusting the pH to neutral with acid to obtain a neutral reaction solution; extracting the neutral reaction solution with ethyl acetate to obtain an organic phase product; sequentially washing, drying, and concentrating the organic phase product to obtain a crude product; purifying the crude product by silica gel column chromatography to obtain a pure triptolide prodrug compound. In this invention, the acid is preferably hydrochloric acid, and the molar concentration of the hydrochloric acid is preferably 1 mol / L. The extraction is preferably performed three times. The washing is preferably performed using saturated brine. The washing is preferably performed three times. The drying reagent is preferably anhydrous sodium sulfate. The eluent used for silica gel column chromatography purification is preferably ethyl acetate and petroleum ether. In the embodiments of this invention, the volume ratio of ethyl acetate to petroleum ether can be gradually increased from 1:10 to 1:3.

[0026] This invention provides the application of the triptolide prodrug compound described in the above-described technical solution or the triptolide prodrug compound prepared by the above-described technical solution in the preparation of anticancer drugs.

[0027] In this invention, the anticancer drug is an anti-lung cancer drug.

[0028] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0029] Example 1: Preparation of (2R,4aS,6aS,12bR,14aS,14bR)-10-hydroxy-2,4a,6a,9,12b,14a-hexamethyl-11-oxo-1,2,3,4,4a,5,6,6a,11,12b,13,14,14a,14b-tetradecanoyl-2-carboxylic acid ethyl ester (compound 1, the compound with the structure shown in Formula 1): Formula 1; Tripterygium wilfordii (1.00 g, 2.22 mmol) was dissolved in N,N-dimethylformamide (20 mL), and sodium bicarbonate (0.56 g, 6.67 mmol) was added. After stirring at room temperature for 10 minutes, bromoethane (1.21 g, 11.10 mmol) was added, and the mixture was stirred at room temperature overnight (approximately 16 hours). After the reaction was complete, 100 mL of water was added, and the mixture was neutralized with an appropriate amount of 1N hydrochloric acid. The mixture was extracted three times with ethyl acetate (50 mL), and the organic phases were combined and washed three times with saturated brine (20 mL). The solution was dried over anhydrous sodium sulfate, concentrated, and purified by silica gel column chromatography (silica gel: 200-300 mesh, eluent polarity increased from ethyl acetate / petroleum ether = 1:10 (v:v) to 1:1 (v:v)) to obtain a reddish-brown powder, yield: 94.3%. 1 H NMR (400 MHz, CDCl3) δ 9.62 (s, 1H),7.10 (d, J = 7.1 Hz, 1H), 6.61 (s, 1H), 6.39 (d, J = 7.1 Hz, 1H), 4.04 – 3.91 (m,2H), 2.43 (d, J = 15.7 Hz, 1H), 2.23 (s, 3H), 2.18 (dd, J = 10.5, 4.1 Hz, 2H),2.07 – 2.01 (m, 1H), 1.90 – 1.85 (m, 1H), 1.84 – 1.80 (m, 1H), 1.73 – 1.67(m, 2H), 1.64 (dd, J = 9.4, 3.4 Hz, 1H), 1.58 (d, J = 8.2 Hz, 1H), 1.53 – 1.48(m, 1H), 1.46 (s, 3H), 1.38 (dd, J = 14.1, 4.5 Hz, 1H), 1.27 (s, 3H), 1.25 (d, J = 3.0 Hz, 1H), 1.20 (t, J = 7.2 Hz, 3H), 1.17 (s, 3H), 1.10 (s, 3H), 0.96 (d, J =14.8 Hz, 1H), 0.55 (s, 3H). 13C NMR (151 MHz, CDCl3) δ 178.32, 178.25, 170.36,164.82, 146.04, 134.33, 127.38, 119.52, 118.17, 117.29, 60.29, 45.09, 44.28,42.99, 40.26, 39.45, 38.23, 36.36, 34.78, 33.53, 32.77, 31.60, 30.71, 30.56,29.79, 29.61, 28.67, 21.63, 18.45, 14.02, 10.28. ESI-MS (m / z): 479.3 [M+H] + . Example 2: Preparation of (2R,4aS,6aS,12bR,14aS,14bR)-2,4a,6a,9,12b,14a-hexamethyl-10-[(4-nitrobenzyl)oxy]-11-oxo-1,2,3,4,4a,5,6,6a,11,12b,13,14,14a,14b-tetradecanoyl-2-carboxylic acid ethyl ester (compound 3-1): Equation 3-1; Compound 1 (200 mg, 0.42 mmol) was weighed and dissolved in anhydrous acetonitrile (20 mL). Potassium carbonate (217 mg, 1.57 mmol) was added, and the mixture was stirred at room temperature for 10 minutes. Then, 4-nitrobenzyl bromide (340 mg, 1.57 mmol) was added, and the mixture was stirred at room temperature overnight (approximately 16 hours). After the reaction was complete, 100 mL of water was added, and the mixture was neutralized with an appropriate amount of 1N hydrochloric acid. The mixture was extracted three times with ethyl acetate (50 mL), and the organic phases were combined and washed three times with saturated brine (20 mL). The mixture was dried over anhydrous sodium sulfate, concentrated, and purified by silica gel column chromatography (silica gel: 200-300 mesh, eluent polarity increased from ethyl acetate / petroleum ether = 1:10 (v:v) to 1:3 (v:v)) to obtain 191 mg of reddish-brown powder, yield: 74.6%. 1 H NMR (400 MHz, CDCl3) δ 8.22 (d, J = 8.5Hz, 2H), 7.65 (d, J = 8.6 Hz, 2H), 6.99 (d, J = 7.0 Hz, 1H), 6.47 (s, 1H), 6.32(d, J= 7.2 Hz, 1H), 5.23 (s, 2H), 4.03 – 3.93 (m, 2H), 2.44 (d, J = 15.3 Hz,1H), 2.24 – 2.20 (m, 1H), 2.18 (s, 3H), 2.15 (s, 1H), 2.08 – 2.01 (m, 1H),1.88 – 1.84 (m, 1H), 1.82 (d, J = 12.1 Hz, 1H), 1.72 – 1.67 (m, 2H), 1.66 (d, J =4.1 Hz, 1H), 1.58 (d, J = 7.0 Hz, 1H), 1.51 (d, J = 5.8 Hz, 1H), 1.46 (s, 3H),1.37 – 1.33 (m, 1H), 1.28 (s, 1H), 1.26 (s, 3H), 1.25 (s, 1H), 1.21 (t, J = 7.1Hz, 3H), 1.17 (s, 3H), 1.09 (s, 3H), 0.97 (d, J = 13.7 Hz, 1H), 0.58 (s, 3H). 13 13C NMR (151 MHz, CDCl3) δ 178.24, 170.81, 162.72, 148.56, 147.46, 145.58,132.15, 128.40, 126.92, 123.57, 117.97, 72.36, 60.29, 45.16, 44.27, 42.47,40.25, 39.28, 38.17, 36.35, 34.77, 33.54, 32.77, 31.59, 30.67, 30.56, 29.78,29.57, 28.64, 21.91, 18.57, 14.05, 11.10. ESI-MS (m / z): 614.4 [M+H] + . Example 3: Preparation of (2R,4aS,6aS,12bR,14aS,14bR)-10-[(3-chloro-4-nitrobenzyl)oxy]-2,4a,6a,9,12b,14a-hexamethyl-11-oxo-1,2,3,4,4a,5,6,6a,11,12b,13,14,14a,14b-tetradecylhydrobenzyl-2-carboxylic acid ethyl ester (compound 3-2): Equation 3-2; The preparation method is the same as in Example 2, except that 4-nitrobenzyl bromide in Example 2 is replaced with 4-(bromomethyl)-2-chloro-1-nitrobenzene, yielding a reddish-brown powder with a yield of 84.3%. 1 H NMR (400 MHz, CDCl3) δ 8.16 (s, 1H), 8.10(d, J = 8.7 Hz, 1H), 7.42 (dd, J = 8.7, 2.4 Hz, 1H), 7.03 (d, J = 7.1 Hz, 1H), 6.49(s, 1H), 6.33 (d, J = 7.1 Hz, 1H), 5.41 (s, 2H), 3.98 (dtd, J = 21.3, 7.1, 3.6Hz, 2H), 2.44 (d, J = 15.8 Hz, 1H), 2.25 (s, 3H), 2.20 (dt, J = 8.7, 2.6 Hz, 1H),2.18 – 2.11 (m, 1H), 2.09 – 2.01 (m, 1H), 1.86 (d, J = 7.8 Hz, 1H), 1.82 (d, J =11.4 Hz, 1H), 1.72 – 1.70 (m, 1H), 1.68 (s, 1H), 1.63 (s, 1H), 1.59 – 1.57(m, 1H), 1.48 (s, 3H), 1.39 (dd, J = 14.2, 4.6 Hz, 1H), 1.33 (s, 1H), 1.28 (s,1H), 1.27 (s, 3H), 1.25 (s, 1H), 1.21 (t, J = 7.2 Hz, 3H), 1.17 (s, 3H), 1.10(s, 3H), 0.96 (d, J= 10.4 Hz, 1H), 0.58 (s, 3H). 13 C NMR (151 MHz, CDCl3) δ178.22, 170.89, 162.74, 148.64, 144.78, 140.82, 137.35, 132.56, 129.14,127.97, 126.94, 126.11, 123.49, 117.96, 69.70, 60.31, 45.16, 44.29, 42.49,40.25, 39.28, 38.18, 36.37, 34.76, 33.58, 32.76, 31.60, 30.67, 30.57, 29.81,29.71, 29.59, 28.66, 21.92, 18.54, 14.05, 11.04. ESI-MS (m / z): 648.3 [M+H] + . Example 4: Preparation of (2R,4aS,6aS,12bR,14aS,14bR)-2,4a,6a,9,12b,14a-hexamethyl-10-[(2-nitrobenzyl)oxy]-11-oxo-1,2,3,4,4a,5,6,6a,11,12b,13,14,14a,14b-tetradecanoyl-2-carboxylic acid ethyl ester (compound 3-3): Equation 3-3; The preparation method is the same as in Example 2, except that 4-nitrobenzyl bromide in Example 2 is replaced with 2-nitrobenzyl bromide, yielding a reddish-brown powder with a yield of 78.2%. 1 H NMR (400 MHz, CDCl3) δ 8.14 – 8.09 (m, 2H), 7.70 (t, J =7.6 Hz, 1H), 7.46 (t, J = 7.8 Hz, 1H), 7.00 (d, J = 7.1 Hz, 1H), 6.49 (s, 1H), 6.32 (d, J = 7.1 Hz, 1H), 5.43 (s, 2H), 4.04 – 3.94 (m, 2H), 2.44 (d, J = 15.2Hz, 1H), 2.22 (s, 3H), 2.17 (d, J= 16.5 Hz, 1H), 2.08 – 2.02 (m, 1H), 1.89 –1.85 (m, 1H), 1.81 (d, J = 11.8 Hz, 1H), 1.73 – 1.69 (m, 2H), 1.68 – 1.63 (m,2H), 1.58 (d, J = 7.9 Hz, 2H), 1.52 (d, J = 6.2 Hz, 1H), 1.48 (s, 3H), 1.41 –1.34 (m, 1H), 1.27 (s, 3H), 1.25 (s, 1H), 1.21 (t, J = 7.2 Hz, 3H), 1.17 (s,3H), 1.10 (s, 3H), 0.97 (d, J = 15.2 Hz, 1H), 0.58 (s, 3H). 13 C NMR (151 MHz, CDCl3) δ 178.23, 170.62, 162.63, 148.91, 146.92, 134.82, 133.85, 132.54,129.42, 128.00, 127.04, 124.56, 123.48, 117.96, 70.19, 60.31, 45.13, 44.28,42.44, 40.25, 39.27, 38.16, 36.37, 34.76, 33.57, 32.76, 31.60, 30.67, 30.57,29.80, 29.59, 28.65, 21.91, 18.53, 14.05, 10.99. ESI-MS (m / z): 614.4 [M+H] + . Example 5: Preparation of (2R,4aS,6aS,12bR,14aS,14bR)-10-[(5-chloro-2-nitrobenzyl)oxy]-2,4a,6a,9,12b,14a-hexamethyl-11-oxo-1,2,3,4,4a,5,6,6a,11,12b,13,14,14a,14b-tetradecylhydrocarboxylate-2-carboxylic acid ethyl ester (compounds 3-4): Equation 3-4; The preparation method is the same as in Example 2, except that 4-nitrobenzyl bromide in Example 2 is replaced with 2-bromomethyl-4-chloro-1-nitrobenzene, yielding an orange-yellow powder with a yield of 44.5%.1 H NMR (400 MHz, CDCl3) δ 8.16 (s, 1H), 8.10 (d, J = 8.7 Hz, 1H), 7.42 (dd, J = 8.6, 2.4 Hz, 1H), 7.03 (d, J = 7.0 Hz, 1H), 6.46 (s,1H), 6.33 (d, J = 7.0 Hz, 1H), 5.41 (s, 2H), 3.99 (ddt, J = 17.8, 7.1, 3.6 Hz,2H), 2.44 (d, J = 15.8 Hz, 1H), 2.24 (s, 3H), 2.16 (d, J = 12.8 Hz, 1H), 2.08 –2.02 (m, 1H), 1.88 (d, J = 5.8 Hz, 1H), 1.84 (d, J = 9.6 Hz, 1H), 1.80 (s, 1H),1.71 (d, J = 8.2 Hz, 2H), 1.67 (d, J = 7.5 Hz, 2H), 1.58 (d, J = 8.0 Hz, 1H), 1.52(d, J = 6.4 Hz, 1H), 1.48 (s, 3H), 1.41 (dd, J = 6.9, 3.2 Hz, 1H), 1.37 – 1.33(m, 1H), 1.27 (s, 1H), 1.25 (s, 3H), 1.21 (t, J = 7.2 Hz, 3H), 1.17 (s, 3H),1.10 (s, 3H), 0.96 (d, J = 10.5 Hz, 1H), 0.58 (s, 3H). 13C NMR (151 MHz, CDCl3) δ178.23, 170.93, 162.78, 148.65, 144.78, 140.81, 137.35, 132.60, 129.13,127.97, 126.94, 126.11, 123.50, 117.96, 69.70, 60.32, 45.16, 44.29, 42.50,40.25, 39.29, 38.19, 36.37, 34.75, 33.58, 32.76, 31.60, 30.67, 30.57, 29.80,29.71, 29.58, 28.66, 21.91, 18.54, 14.05, 11.04. ESI-MS (m / z): 648.3 [M+H] + . Example 6: Preparation of (2R,4aS,6aS,12bR,14aS,14bR)-10-[(4-bromo-2-nitrobenzyl)oxy]-2,4a,6a,9,12b,14a-hexamethyl-11-oxo-1,2,3,4,4a,5,6,6a,11,12b,13,14,14a,14b-tetradecylhydrocarboxylate-2-carboxylic acid ethyl ester (compounds 3-5): Equation 3-5; The preparation method is the same as in Example 2, except that 4-nitrobenzyl bromide in Example 2 is replaced with 4-bromo-2-nitrobenzyl bromide, yielding an orange-yellow powder with a yield of 62.4%. 1 H NMR (400 MHz, CDCl3) δ 8.25 (d, J = 2.0 Hz, 1H), 8.03(d, J = 8.4 Hz, 1H), 7.81 (dd, J = 8.3, 2.1 Hz, 1H), 7.02 (d, J = 6.9 Hz, 1H), 6.47(s, 1H), 6.33 (d, J = 7.0 Hz, 1H), 5.37 (s, 2H), 3.99 (dqd, J = 17.3, 7.2, 3.6Hz, 2H), 2.44 (d, J = 15.6 Hz, 1H), 2.21 (s, 3H), 2.15 (dd, J= 11.3, 3.8 Hz, 1H), 2.08 – 2.01 (m, 1H), 1.89 (t, J = 5.7 Hz, 1H), 1.86 – 1.83 (m, 1H), 1.82(s, 1H), 1.70 (d, J = 8.2 Hz, 1H), 1.66 (d, J = 7.9 Hz, 1H), 1.58 (d, J = 8.2 Hz, 1H), 1.53 (t, J = 6.7 Hz, 1H), 1.47 (s, 3H), 1.41 (dd, J = 7.2, 3.4 Hz, 1H), 1.35(dd, J = 13.7, 4.2 Hz, 1H), 1.27 (s, 3H), 1.25 (s, 2H), 1.21 (t, J = 7.1 Hz, 3H), 1.17 (s, 3H), 1.09 (s, 3H), 0.96 (d, J = 10.8 Hz, 1H), 0.58 (s, 3H). 13 C NMR (151 MHz, CDCl3) δ 178.23, 170.96, 162.77, 148.74, 147.18, 136.79, 134.09, 132.54, 130.99, 127.41, 126.93, 123.44, 121.11, 117.98, 69.77, 60.31, 45.17, 44.28, 42.50, 40.25, 39.29, 38.19, 36.37, 34.76, 33.58, 32.76, 31.60, 30.67, 30.57, 29.80, 29.71, 29.58, 28.66, 21.91, 18.54, 14.05, 11.01. ESI-MS (m / z): 692.3 [M+H] + . Example 7: Preparation of (2R,4aS,6aS,12bR,14aS,14bR)-2,4a,6a,9,12b,14a-hexamethyl-10-[(3-methyl-4-nitrobenzyl)oxy]-11-oxo-1,2,3,4,4a,5,6,6a,11,12b,13,14,14a,14b-tetradecanoyl-2-carboxylic acid ethyl ester (compounds 3-6): Equation 3-6; The preparation method is the same as in Example 2, except that 4-nitrobenzyl bromide in Example 2 is replaced with 3-methyl-4-nitrobenzyl bromide, yielding an orange-yellow color with a yield of 69.1%. 1 H NMR (400 MHz, CDCl3) δ 7.98 (d, J = 8.2 Hz, 1H), 7.47 (s,1H), 7.44 (d, J = 8.6 Hz, 1H), 6.99 (d, J = 7.0 Hz, 1H), 6.48 (s, 1H), 6.32 (d, J =7.1 Hz, 1H), 5.16 (s, 2H), 3.98 (ddq, J = 17.3, 7.3, 3.4 Hz, 2H), 2.62 (s, 3H), 2.44 (d, J = 15.7 Hz, 1H), 2.24 – 2.20 (m, 1H), 2.18 (s, 3H), 2.14 (d, J = 7.0Hz, 1H), 2.08 – 2.01 (m, 1H), 1.89 (d, J = 8.0 Hz, 1H), 1.84 (d, J = 6.0 Hz, 1H), 1.79 (d, J = 5.1 Hz, 1H), 1.71 (d, J = 7.7 Hz, 1H), 1.68 – 1.65 (m, 1H), 1.58 (d, J = 7.1 Hz, 1H), 1.51 (d, J = 6.4 Hz, 1H), 1.46 (s, 3H), 1.38 (dd, J = 14.0, 4.6Hz, 1H), 1.30 (d, J= 19.2 Hz, 1H), 1.26 (s, 3H), 1.25 (s, 1H), 1.21 (t, J = 7.1Hz, 3H), 1.17 (s, 3H), 1.09 (s, 3H), 0.97 (d, J = 15.4 Hz, 1H), 0.58 (s, 3H). 13 CNMR (151 MHz, CDCl3) δ 178.26, 170.82, 162.74, 148.41, 143.69, 133.82,132.23, 131.95, 126.95, 126.07, 124.87, 123.44, 117.98, 72.38, 60.29, 45.16,44.27, 42.48, 40.26, 39.29, 38.18, 36.35, 34.77, 33.54, 32.77, 31.60, 30.67,30.57, 29.78, 29.71, 29.57, 28.65, 21.91, 20.65, 18.57, 14.05, 11.11. ESI-MS(m / z): 628.4 [M+H] + . Example 8: Preparation of (2R,4aS,6aS,12bR,14aS,14bR)-10-[(2-chloro-6-nitrobenzyl)oxy]-2,4a,6a,9,12b,14a-hexamethyl-11-oxo-1,2,3,4,4a,5,6,6a,11,12b,13,14,14a,14b-tetradecylhydro-2-carboxylic acid ethyl ester (compounds 3-7): Equation 3-7; The preparation method is the same as in Example 2, except that 4-nitrobenzyl bromide in Example 2 is replaced with 6-chloro-2-nitrobenzyl bromide, yielding a reddish-brown powder with a yield of 50.4%. 1 H NMR (400 MHz, CDCl3) δ 7.67 (dd, J = 16.1, 8.0 Hz, 2H), 7.41 (t, J = 8.1 Hz, 1H), 6.91 (d, J = 7.0 Hz, 1H), 6.42 (s, 1H), 6.27 (d, J= 7.1Hz, 1H), 5.63 – 5.55 (m, 2H), 4.04 – 3.94 (m, 2H), 2.44 (d, J = 15.7 Hz, 1H),2.23 – 2.18 (m, 1H), 2.15 – 2.10 (m, 1H), 2.04 (d, J = 3.6 Hz, 1H), 2.00 (s,3H), 1.87 – 1.83 (m, 1H), 1.81 – 1.77 (m, 1H), 1.69 (d, J = 8.5 Hz, 1H), 1.65(s, 1H), 1.63 – 1.59 (m, 1H), 1.56 (dd, J = 10.4, 5.2 Hz, 2H), 1.50 (d, J = 4.9Hz, 1H), 1.44 (s, 3H), 1.40 – 1.35 (m, 1H), 1.30 (d, J = 19.8 Hz, 1H), 1.25 (s,3H), 1.21 (t, J = 7.1 Hz, 3H), 1.17 (s, 3H), 1.09 (s, 3H), 0.95 (d, J = 14.6 Hz,1H), 0.57 (s, 3H). 13 C NMR (151 MHz, CDCl3) δ 178.23, 169.98, 162.36, 151.81,148.43, 137.34, 134.26, 133.70, 131.82, 129.89, 129.63, 127.11, 123.51,122.70, 117.88, 66.03, 60.32, 45.05, 44.29, 42.30, 40.25, 39.22, 38.10,36.38, 34.77, 33.44, 32.77, 31.60, 30.67, 30.57, 29.82, 29.71, 29.59, 28.63,21.94, 18.54, 14.05, 10.68. ESI-MS (m / z): 648.3 [M+H] + . Example 9: Preparation of (2R,4aS,6aS,12bR,14aS,14bR)-2,4a,6a,9,12b,14a-hexamethyl-10-[(4-methyl-2-nitrobenzyl)oxy]-11-oxo-1,2,3,4,4a,5,6,6a,11,12b,13,14,14a,14b-tetradecanoyl-2-carboxylic acid ethyl ester (compounds 3-8): Equation 3-8; The preparation method is the same as in Example 2, except that 4-nitrobenzyl bromide in Example 2 is replaced with 1-(bromomethyl)-4-methyl-2-nitrobenzene, resulting in a reddish-brown powder with a yield of 59.2%. 1 H NMR (400 MHz, CDCl3) δ 7.96 (d, J = 7.9 Hz,1H), 7.91 (s, 1H), 7.49 (d, J = 9.9 Hz, 1H), 6.99 (d, J = 7.0 Hz, 1H), 6.46 (s,1H), 6.31 (d, J = 7.1 Hz, 1H), 5.39 (s, 2H), 4.03 – 3.94 (m, 2H), 2.47 (d, J =7.6 Hz, 1H), 2.44 (s, 3H), 2.43 – 2.40 (m, 1H), 2.22 (s, 1H), 2.19 (s, 3H), 2.14 (t, J = 7.3 Hz, 1H), 2.08 – 2.01 (m, 1H), 1.89 (t, J = 6.9 Hz, 1H), 1.84 (d, J = 10.0 Hz, 1H), 1.79 (d, J = 4.5 Hz, 1H), 1.70 (d, J = 7.4 Hz, 1H), 1.67 – 1.65(m, 1H), 1.58 (d, J = 7.6 Hz, 1H), 1.52 (t, J = 6.6 Hz, 1H), 1.47 (s, 3H), 1.35(dd, J = 14.1, 4.7 Hz, 1H), 1.26 (s, 3H), 1.25 (s, 1H), 1.21 (t, J= 7.1 Hz, 3H), 1.17 (s, 3H), 1.09 (s, 3H), 0.96 (d, J = 13.0 Hz, 1H), 0.58 (s, 3H). 13 C NMR (151MHz, CDCl3) δ 178.23, 170.46, 162.59, 149.00, 146.88, 138.45, 134.58, 132.48,131.72, 129.50, 127.09, 124.84, 123.53, 117.94, 70.17, 60.31, 45.11, 44.28,42.41, 40.25, 39.26, 38.16, 36.38, 34.76, 33.55, 32.76, 31.59, 30.67, 30.57,29.81, 29.59, 28.65, 21.91, 20.85, 18.53, 14.05, 10.96. ESI-MS (m / z): 628.4[M+H] + . Example 10: Preparation of (2R,4aS,6aS,12bR,14aS,14bR)-2,4a,6a,9,12b,14a-hexamethyl-10-[(2-methyl-4-nitrobenzyl)oxy]-11-oxo-1,2,3,4,4a,5,6,6a,11,12b,13,14,14a,14b-tetradecanoyl-2-carboxylic acid ethyl ester (compounds 3-9): Equation 3-9; The preparation method is the same as in Example 2, except that 4-nitrobenzyl bromide in Example 2 is replaced with 1-bromomethyl-2-methyl-4-nitrobenzene, yielding an orange-yellow powder with a yield of 77.8%. 1 H NMR (400 MHz, CDCl3) δ 8.08 – 8.05 (m, 1H),8.04 (d, J = 2.3 Hz, 1H), 7.73 (d, J = 8.3 Hz, 1H), 6.99 (d, J = 6.9 Hz, 1H), 6.50(s, 1H), 6.32 (d, J= 7.1 Hz, 1H), 5.19 (s, 2H), 4.03 – 3.94 (m, 2H), 2.45 (s,3H), 2.42 (s, 1H), 2.23 – 2.19 (m, 1H), 2.16 (s, 3H), 2.08 – 2.01 (m, 1H),1.88 (d, J = 7.1 Hz, 1H), 1.84 (d, J = 8.6 Hz, 1H), 1.80 (s, 1H), 1.71 (d, J = 8.3Hz, 1H), 1.66 (d, J = 4.4 Hz, 1H), 1.58 (d, J = 7.1 Hz, 2H), 1.51 (s, 1H), 1.47(s, 3H), 1.45 – 1.39 (m, 1H), 1.35 (dd, J = 14.0, 4.4 Hz, 1H), 1.26 (s, 3H),1.25 (s, 1H), 1.21 (t, J = 7.1 Hz, 3H), 1.17 (s, 3H), 1.10 (s, 3H), 0.97 (d, J =14.8 Hz, 1H), 0.58 (s, 3H). 13 C NMR (151 MHz, CDCl3) δ 178.26, 170.91, 162.76,148.70, 147.24, 143.75, 137.55, 132.35, 128.64, 126.93, 124.66, 123.42,121.07, 117.99, 70.62, 60.29, 45.17, 44.27, 42.49, 40.26, 39.29, 38.17,36.35, 34.76, 33.55, 32.77, 31.59, 30.67, 30.57, 29.78, 29.57, 28.64, 21.90,18.89, 18.56, 14.05, 11.04. ESI-MS (m / z): 628.4 [M+H] + . Example 11: Preparation of (2R,4aS,6aS,12bR,14aS,14bR)-10-[(2-chloro-4-nitrobenzyl)oxy]-2,4a,6a,9,12b,14a-hexamethyl-11-oxo-1,2,3,4,4a,5,6,6a,11,12b,13,14,14a,14b-tetradecanoyl-2-carboxylic acid ethyl ester (compound 3-10): Equation 3-10; The preparation method is the same as in Example 2, except that 4-nitrobenzyl bromide in Example 2 is replaced with 1-(bromomethyl)-2-chloro-4-nitrobenzene, yielding an orange-yellow powder with a yield of 74.1%. 1 H NMR (400 MHz, CDCl3) δ 8.24 (d, J = 2.4 Hz, 1H), 8.17 (dd, J = 8.4, 2.4 Hz, 1H), 7.98 (d, J = 8.5 Hz, 1H), 7.03 (d, J = 6.9 Hz,1H), 6.48 (s, 1H), 6.33 (d, J = 7.1 Hz, 1H), 5.26 (s, 2H), 4.04 – 3.93 (m, 2H), 2.44 (d, J = 15.6 Hz, 1H), 2.21 (s, 3H), 2.16 (d, J = 7.4 Hz, 1H), 2.08 – 2.01(m, 1H), 1.88 (d, J = 5.8 Hz, 1H), 1.84 (d, J = 6.0 Hz, 1H), 1.76 (s, 2H), 1.71(d, J = 8.1 Hz, 1H), 1.66 (d, J = 5.1 Hz, 1H), 1.58 (d, J = 7.7 Hz, 1H), 1.53 (d, J =6.7 Hz, 1H), 1.47 (s, 3H), 1.39 (dd, J = 14.1, 4.5 Hz, 1H), 1.34 – 1.30 (m,1H), 1.27 (s, 3H), 1.25 (s, 1H), 1.21 (t, J= 7.2 Hz, 3H), 1.17 (s, 3H), 1.10(s, 3H), 0.96 (d, J = 9.0 Hz, 1H), 0.58 (s, 3H). 13 C NMR (151 MHz, CDCl3) δ178.28, 171.30, 162.91, 148.57, 147.50, 143.52, 132.87, 132.58, 129.42,128.13, 126.84, 124.17, 123.35, 121.84, 118.02, 70.00, 60.31, 45.21, 44.27,42.57, 40.26, 39.31, 38.18, 36.35, 34.76, 33.57, 32.76, 31.59, 30.67, 30.57,29.78, 29.56, 28.65, 21.89, 18.56, 14.05, 11.05. ESI-MS (m / z): 648.4 [M+H] + . Example 12: Log-phase HepG2 cells (human liver cancer cells) were selected to evaluate the responsiveness (ability to release the original drug to kill tumor cells) and toxicity of triptolide prodrug to nitroreductase. The specific procedure was as follows: Log-phase cells were harvested and spaced at approximately 2 × 10⁶ cells per well. 4 Cells were evenly seeded at a density of 1,000 cells per well in a 96-well cell culture plate and incubated at 37°C with 5% CO2 for 24 h to allow for cell adhesion and growth. After incubation, the original culture medium was discarded, and fresh culture medium containing different concentrations of the test compound was added to each well. The cells were then incubated for 8 h under normal oxygen (21% O2) and hypoxic (0.1% O2) conditions, respectively. After incubation, the drug-containing culture medium was discarded, and the cells were gently washed three times with phosphate-buffered saline (PBS, pH 7.4). Fresh DMEM complete culture medium was added to each well, and the cells were cultured for another 24 h. 10 μL of MTT solution (5 mg / mL) was added to each well, and the cells were incubated at 37°C with 5% CO2 for another 4 h. After incubation, the culture medium and MTT mixture in each well was carefully aspirated, and 100 μL of dimethyl sulfoxide (DMSO) was added to each well. The cells were then shaken on a shaker (80-100 rpm) for 10-15 seconds. The absorbance (OD value) of each well was measured at 490 nm using a microplate reader, with wells containing only culture medium and MTT but no cells serving as blank controls. Cell viability was calculated. The results are shown in Table 1.

[0030] Table 1. Test results of compounds 3-1 to 3-10

[0031] As shown in Table 1, compounds 3-2 and 3-4 exhibited strong cytotoxicity against HepG2 cells under hypoxic conditions (cell viability was 23.6% and 21.1%, respectively). Compound 3-4 was selected as an example for the following studies.

[0032] Example 13: Establishment of the C57BL / 6 hepatocellular carcinoma mouse model: HepG2 hepatocellular carcinoma cells seeded in 10 cm dishes were digested, collected, resuspended in serum-free cell culture medium, and counted. The cells were then seeded into the left axilla of C57BL / 6 mice (adjusting the cell density to 1×10⁶ cells / cm²). 6 (100 μL / mouse). After establishing the tumor animal model, mice were randomly divided into three groups of five mice each: a blank control group (receiving only the same volume of physiological saline), a positive control group (oxaliplatin (Oxa) group (5 mg / kg), and a treatment group (compounds 3-4, 5 mg / kg). Intraperitoneal injections were administered every two days. The weight of the tumor-bearing mice was recorded daily, and tumor volume was measured using calipers. Two weeks later, the mice were sacrificed, and the tumors and major organs were removed for subsequent experiments. The formula for calculating tumor volume is: V (cm²). 3 )= a 2 × b / 2 (a: short diameter, b: long diameter, V: tumor volume) Tumor inhibition rate % = (average tumor weight in control group - average tumor weight in treatment group) / average tumor weight in control group × 100%. Results are as follows: After 2 weeks of treatment, the tumor inhibition effect of compounds 3-4 was comparable to that of oxaliplatin, which is widely used clinically. Figure 1 ), and has no obvious organ toxicity ( Figure 2 This demonstrates its good potential for clinical translation.

[0033] As can be seen from the above embodiments, the triptolide prodrug compound provided by the present invention can achieve specific activation at the tumor site, maintain relative stability in normal tissues, greatly reduce toxic effects on normal liver and other tissues, and significantly improve the safety and therapeutic index of the drug.

[0034] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. Other embodiments can be obtained based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A triptolide prodrug compound, characterized in that, It has the structure shown in Equation I: Formula I, where R is H, C1-5 alkyl, or halogen.

2. The triptolide prodrug compound according to claim 1, characterized in that, The C1-5 alkyl group is methyl; the halogen is Cl, Br or F.

3. The triptolide prodrug compound according to claim 1 or 2, characterized in that, It has any of the following structures: 、 、 、 、 、 、 、 、 、 。 4. The method for preparing the triptolide prodrug compound according to any one of claims 1 to 3, characterized in that, Includes the following steps: The compound with the structure shown in Formula 1, the compound with the structure shown in Formula 2, an inorganic base and a first organic solvent were mixed and subjected to an etherification reaction to obtain the triptolide prodrug compound with the structure shown in Formula I. Formula 1, Formula 2.

5. The preparation method according to claim 4, characterized in that, The preparation method of the compound with the structure shown in Formula 1 includes the following steps: Tripterygium wilfordii, a second organic solvent, sodium bicarbonate, and bromoethane were mixed and subjected to an esterification reaction to obtain a compound with the structure shown in Formula 1.

6. The preparation method according to claim 4, characterized in that, The first organic solvent includes one or more of N,N-dimethylformamide, acetonitrile, dichloromethane and toluene; the inorganic base includes one or more of KOH, K2CO3, NaOH and Na2CO3; the molar ratio of the compound with the structure shown in Formula 1 to the compound with the structure shown in Formula 2 is 1:1.05~3.

7. The preparation method according to claim 4 or 6, characterized in that, The temperature for the etherification reaction is -20 to 100°C.

8. The preparation method according to claim 4, characterized in that, The etherification reaction is completed to obtain an etherification reaction solution; the process also includes mixing the etherification reaction solution with ice water, then adjusting the pH to neutral with acid to obtain a neutral reaction solution; and extracting the neutral reaction solution with ethyl acetate to obtain an organic phase product. The organic phase product was washed, dried, and concentrated sequentially to obtain a crude product; the crude product was purified by silica gel column chromatography to obtain a pure product of the triptolide prodrug compound; the eluent used for purification was ethyl acetate and petroleum ether.

9. The use of the triptolide prodrug compound according to any one of claims 1 to 3 or the triptolide prodrug compound prepared by the preparation method according to any one of claims 4 to 8 in the preparation of anticancer drugs.

10. The application according to claim 9, characterized in that, The anticancer drug mentioned is an anti-liver cancer drug.

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