5α,8α-epoxymadrost-4-ene-3-one-3-thiazolidine derivative, its preparation method and application

Abstract

The application belongs to the technical field of pharmaceutical chemistry, and discloses a 5alpha, 8alpha-peroxyl-ergost-3-thiazolidine-dione derivative as well as a preparation method and application thereof. The application takes thiazolidine-dione as raw material, first completes synthesis of the thiazolidine-dione derivative, then introduces a series of side chains containing 5-substituted benzyl-thiazolidine-dione substituents to a 3-hydroxyl site of 5alpha, 8alpha-peroxyl-ergost-3-piperazine formate under molecular hybridization, designs and synthesizes a series of novel 5alpha, 8alpha-peroxyl-ergost-3-thiazolidine-dione derivatives with potential for clinical anti-tumor application. The 5alpha, 8alpha-peroxyl-ergost-3-thiazolidine-dione derivative has inhibitory effect on human lung cancer, liver cancer, breast cancer and other tumor cells in vitro, and has potential for development as a clinical anti-tumor drug.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical chemistry, and in particular to a 5α,8α-peroxyergosterol-3-thiazolidinedione derivative, its preparation method, and its application. Background Technology

[0002] Cancer has become a serious public health problem worldwide and a leading cause of death. Current research indicates that chemotherapy remains one of the effective treatments for cancer, but its adverse side effects severely limit the clinical application of many drugs. Therefore, developing novel, highly effective, and low-toxicity anti-tumor drugs is particularly important for cancer treatment. Natural products have a long history of medicinal use and are one of the main sources of new anti-cancer drugs. Literature review found that of the 136 small-molecule anti-cancer drugs marketed between 1981 and 2014, 113 were closely related to natural products. However, most small-molecule anti-cancer drugs are products of structural modification of the active skeleton of natural products. The probability of developing natural products that can be directly used, such as camptothecin, paclitaxel, and vincristine, is extremely low. Therefore, obtaining highly active natural lead compounds and modifying their structures is an important part of natural drug research. Exploring the development of highly effective and low-toxicity anti-tumor drugs through the structural modification and alteration of natural products is one of the important directions in new drug development.

[0003] Ergosterol peroxide (EP) is a representative 5α,8α-peroxide sterol extracted from the broken-cell spores of Ganoderma lucidum, a traditional Chinese medicine. Literature review indicates that this compound exhibits moderate to high inhibitory effects on various tumor cell lines in vitro, including HepG2 liver cancer cells, DU-145 prostate cancer cells, MCF-7 breast cancer cells, and A547 lung cancer cells. The peroxide bridge structure on the B ring of the steroid is considered to be the main active group responsible for EP's antitumor activity; compared to ergosterol without the peroxide bridge structure, EP shows more significant inhibitory effects on various tumor cell lines. Based on this, rational structural modification and pharmacological studies using EP as a lead compound are of great significance for the development of novel antitumor drugs.

[0004] Thiazolidinediones (TZDs) are five-membered thiazolidinediones with carbonyl groups at positions 2 and 4, possessing high dipole moments and the ability to form hydrogen bonds with target proteins, making them highly advantageous in biomolecular binding. As a multifunctional molecule, TZDs are widely used in drug design, and their derivatives exhibit diverse antitumor activities. Several TZD-containing compounds have entered clinical trials for anticancer treatment, such as S-49076 hydrochloride (Phase I) for treating solid tumors and inolitazone hydrochloride (Phase II) for treating colorectal cancer. Therefore, the synthesis and pharmacological research of compounds containing thiazoles and other multi-target groups are of great significance.

[0005] Therefore, there is an urgent need in this field to develop a novel 5α,8α-peroxyergosterol-3-thiazolidinedione derivative with significant antitumor activity. Summary of the Invention

[0006] The purpose of this invention is to provide a 5α,8α-peroxyergosterol-3-thiazolidinedione derivative, its preparation method and application, in order to solve the problems of the extremely low probability of developing existing natural products that can be directly used as drugs, low preparation efficiency, and poor antitumor activity of existing ergosterol peroxides.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] This invention provides a 5α,8α-peroxyergosterol-3-thiazolidinedione derivative, the structural formula of which is as follows:

[0009]

[0010] R1 is selected from one of -H, -F, -Cl, and -OCH3; R2 is selected from one of -H, -Cl, and -OCH3; and R3 is selected from one of -H, -F, -Cl, -Br, -CH3, -OCH3, and -C(CH3)3.

[0011] The present invention also provides a method for preparing the 5α,8α-peroxyergosterol-3-thiazolidinedione derivative, comprising the following steps:

[0012] (1) Preparation of thiazolidinedione derivatives:

[0013] A. Thiazolidinedione, aromatic aldehyde, piperidine, and ethanol were mixed and refluxed, followed by post-treatment to obtain 5-substituted benzyl-2,4-thiazolidinedione; B. 5-substituted benzyl-2,4-thiazolidinedione, potassium carbonate, potassium iodide, N,N-dimethylformamide, and ethyl bromoacetate were mixed and reacted, followed by post-treatment to obtain 5-substituted benzyl-2,4-thiazolidinedione-3-ethyl acetate; C. 5-substituted benzyl-2,4-thiazolidinedione-3-ethyl acetate, hydrochloric acid, and glacial acetic acid were mixed and refluxed, followed by post-treatment to obtain the thiazolidinedione derivative.

[0014] (2) Preparation of 5α,8α-peroxyergosterol-3-piperazine carboxylate:

[0015] a) 5α,8α-peroxyergosterol, p-nitrophenyl chloroformate, dichloromethane, and pyridine were mixed and reacted, followed by post-treatment to obtain 5α,8α-peroxyergosterol-3-p-nitrobenzoate; b) 5α,8α-peroxyergosterol-3-p-nitrobenzoate, piperazine, dichloromethane, and triethylamine were mixed and reacted, followed by post-treatment to obtain 5α,8α-peroxyergosterol-3-piperazine benzoate;

[0016] (3) The thiazolidinedione derivative, 5α,8α-peroxyergosterol-3-piperazine carboxylate, solvent and catalyst were mixed and reacted to obtain the 5α,8α-peroxyergosterol-3-thiazolidinedione derivative.

[0017] The structural formula of the aromatic aldehyde is: Where R is selected from

[0018] One of them;

[0019] The steps (1) and (2) are not in any particular order.

[0020] Preferably, in step A of step (1), the molar volume ratio of thiazolidinedione, aromatic aldehyde, piperidine, and ethanol is 0.0005–0.002 mol: 0.0005–0.002 mol: 0.0005–0.002 mol: 2–3 mL; the reflux reaction temperature is 70–80 °C, and the reflux reaction time is 8–10 h; the post-treatment includes sequentially extracting, washing, combining organic phases, drying, filtering, and reducing pressure on the product obtained from the reflux reaction.

[0021] Preferably, in step B of step (1), the molar volume ratio of 5-substituted benzyl-2,4-thiazolidinedione, potassium carbonate, potassium iodide, N,N-dimethylformamide, and ethyl bromoacetate is 0.002–0.003 mol: 0.0024–0.0036 mol: 0.002–0.003 mol: 9–11 mL: 0.002–0.003 mol; the reaction temperature is 40–50 °C, and the reaction time is 5–7 h; the post-treatment includes sequentially extracting, washing, combining organic phases, drying, filtering, reducing pressure, and purifying the product obtained from the reaction.

[0022] Preferably, in step C of step (1), the molar volume ratio of 5-substituted benzyl-2,4-thiazolidinedione-3-ethyl acetate, hydrochloric acid, and glacial acetic acid is 0.001–0.0021 mol: 1–3 mL: 2–4 mL; the reflux reaction temperature is 70–80 °C, and the reflux reaction time is 5–8 h; the post-treatment includes sequentially extracting, washing, combining organic phases, drying, filtering, and reducing pressure on the product obtained from the reflux reaction.

[0023] Preferably, in step a of step (2), the molar volume ratio of 5α,8α-peroxyergosterol, p-nitrophenyl chloroformate, dichloromethane, and pyridine is 0.003–0.005 mol: 0.007–0.009 mol: 12–18 mL: 0.01–0.015 mol; the reaction includes a first reaction and a second reaction; the temperature of the first reaction is 0–4 °C, and the time of the first reaction is 4–7 min; the temperature of the second reaction is 20–30 °C, and the time of the second reaction is 1–2 h; the post-processing includes sequentially extracting, washing, combining organic phases, drying, filtering, reducing pressure, and purifying the product obtained from the reaction.

[0024] Preferably, in step b of step (2), the molar volume ratio of 5α,8α-peroxyergosterol-3-p-nitrobenzoate, piperazine, dichloromethane, and triethylamine is 0.003–0.005 mol: 0.007–0.009 mol: 12–18 mL: 1–1.5 mL; the reaction temperature is 20–30 °C, and the reaction time is 1–2 h; the post-treatment includes sequentially subjecting the reaction product to reduced pressure and purification.

[0025] Preferably, in step (3), the solvent is dichloromethane or N,N-dimethylformamide; the catalyst is one or more of 1-ethyl-(3-dimethylaminopropyl)carbamate hydrochloride, 4-dimethylaminopyridine, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, and N,N-diisopropylethylamine; the molar volume ratio of thiazolidinedione derivative, 5α,8α-peroxyergosterol-3-piperazine carbamate, solvent, and catalyst is 1-5 mmol: 1 mmol: 50-60 mL: 2-6 mmol.

[0026] Preferably, in step (3), the reaction temperature is 20-30°C and the reaction time is 3-5 hours.

[0027] The present invention also provides the use of the 5α,8α-peroxyergosterol-3-thiazolidinedione derivative in the preparation of tumor prevention drugs or antitumor drugs.

[0028] As can be seen from the above technical solution, compared with the prior art, the beneficial effects of the present invention are as follows:

[0029] This invention uses thiazolidinedione (TZD) as a starting material. First, thiazolidinedione derivatives were synthesized. Then, through molecular hybridization, a series of side chains containing 5-substituted benzyl-thiazolidinedione substituents were introduced at the carbon 3 hydroxyl site of 5α,8α-peroxyergosterol-3-piperazine carbamate, designing and synthesizing a series of novel 5α,8α-peroxyergosterol-3-thiazolidinedione derivatives with potential for clinical antitumor applications. Antitumor activity tests showed that the obtained derivatives exhibited varying degrees of inhibitory effects on human lung cancer, liver cancer, and breast cancer cells, and showed a significant inhibitory effect on human lung cancer A549 cells, superior to the lead compound 5α,8α-peroxyergosterol. Detailed Implementation

[0030] This invention provides a 5α,8α-peroxyergosterol-3-thiazolidinedione derivative, the structural formula of which is as follows:

[0031]

[0032] R1 is selected from one of -H, -F, -Cl, and -OCH3; R2 is selected from one of -H, -Cl, and -OCH3; and R3 is selected from one of -H, -F, -Cl, -Br, -CH3, -OCH3, and -C(CH3)3.

[0033] The present invention also provides a method for preparing the 5α,8α-peroxyergosterol-3-thiazolidinedione derivative, comprising the following steps:

[0034] (1) Preparation of thiazolidinedione derivatives:

[0035] A. Thiazolidinedione, aromatic aldehyde, piperidine, and ethanol were mixed and refluxed, followed by post-treatment to obtain 5-substituted benzyl-2,4-thiazolidinedione; B. 5-substituted benzyl-2,4-thiazolidinedione, potassium carbonate, potassium iodide, N,N-dimethylformamide, and ethyl bromoacetate were mixed and reacted, followed by post-treatment to obtain 5-substituted benzyl-2,4-thiazolidinedione-3-ethyl acetate; C. 5-substituted benzyl-2,4-thiazolidinedione-3-ethyl acetate, hydrochloric acid, and glacial acetic acid were mixed and refluxed, followed by post-treatment to obtain the thiazolidinedione derivative.

[0036] (2) Preparation of 5α,8α-peroxyergosterol-3-piperazine carboxylate:

[0037] a) 5α,8α-peroxyergosterol, p-nitrophenyl chloroformate, dichloromethane, and pyridine were mixed and reacted, followed by post-treatment to obtain 5α,8α-peroxyergosterol-3-p-nitrobenzoate; b) 5α,8α-peroxyergosterol-3-p-nitrobenzoate, piperazine, dichloromethane, and triethylamine were mixed and reacted, followed by post-treatment to obtain 5α,8α-peroxyergosterol-3-piperazine benzoate;

[0038] (3) The thiazolidinedione derivative, 5α,8α-peroxyergosterol-3-piperazine carboxylate, solvent and catalyst were mixed and reacted to obtain the 5α,8α-peroxyergosterol-3-thiazolidinedione derivative.

[0039] The structural formula of the aromatic aldehyde is: Where R is selected from

[0040] One of them;

[0041] The steps (1) and (2) are not in any particular order.

[0042] In step A of step (1) of the present invention, the molar volume ratio of thiazolidinedione, aromatic aldehyde, piperidine and ethanol is preferably 0.0005-0.002 mol: 0.0005-0.002 mol: 0.0005-0.002 mol: 2-3 mL, more preferably 0.001-0.0015 mol: 0.001-0.0015 mol: 0.001 mol: 2.5-2.8 mL; the reflux reaction temperature is preferably 70-80 °C, more preferably 75-78 °C; the reflux reaction time is preferably 8-10 h, more preferably 9-9.5 h.

[0043] In step A of step (1) of the present invention, before post-processing, the product obtained from the reflux reaction is sequentially diluted and its pH value is adjusted; the reagent used for dilution is water, and the molar volume ratio of thiazolidinedione to water is preferably 0.0005-0.002 mol: 10-15 mL, more preferably 0.001-0.0015 mol: 12.5-14 mL; the reagent used for pH adjustment is hydrochloric acid, and the mass fraction of hydrochloric acid is preferably 35-40%, more preferably 37-38%; the pH value is adjusted to 7-7.5.

[0044] In step A of step (1) of the present invention, the post-processing includes sequentially extracting, washing, combining organic phases, drying, filtering and reducing pressure on the product obtained from the reflux reaction; the reagent used for extraction is ethyl acetate, and the number of extractions is preferably 3 to 5 times, more preferably 4 times; the reagent used for washing is saturated brine; drying includes drying the organic phase obtained after combining organic phases with anhydrous sodium sulfate; the purpose of reducing pressure is to remove the solvent.

[0045] In step B of step (1) of the present invention, the preferred molar volume ratio of 5-substituted benzyl-2,4-thiazolidinedione, potassium carbonate, potassium iodide, N,N-dimethylformamide and ethyl bromoacetate is 0.002-0.003 mol: 0.0024-0.0036 mol: 0.002-0.003 mol: 9-11 mL: 0.002-0.003 mol, more preferably 0.0022-0.0029 mol: 0.0025-0.003 mol: 0.0025-0.0028 mol: 10 mL: 0.0021-0.0025 mol; the preferred reaction temperature is 40-50 °C, more preferably 45-48 °C; the preferred reaction time is 5-7 h, more preferably 5.5-6.5 h.

[0046] In step B of step (1) of the present invention, the post-processing includes sequentially extracting, washing, combining organic phases, drying, filtering, reducing pressure and purifying the product obtained from the reaction; the reagent used for extraction is dichloromethane, and the number of extractions is preferably 3 to 5 times, more preferably 4 times; the reagent used for washing is saturated brine; drying includes drying the organic phase obtained after combining the organic phases with anhydrous sodium sulfate; the purpose of reducing pressure is to remove the solvent; purification includes purifying the residue by silica gel column chromatography (PE:EA = 20:1, v / v).

[0047] In step C of step (1) of the present invention, the mass fraction of hydrochloric acid is preferably 35-40%, more preferably 37-38%; the molar volume ratio of 5-substituted benzyl-2,4-thiazolidinedione-3-ethyl acetate, hydrochloric acid and glacial acetic acid is preferably 0.001-0.0021 mol: 1-3 mL: 2-4 mL, more preferably 0.0013-0.0015 mol: 2-2.5 mL: 2.5-3 mL; the reflux reaction temperature is preferably 70-80°C, more preferably 75-77°C; the reflux reaction time is preferably 5-8 h, more preferably 6-7 h.

[0048] In step C of step (1) of the present invention, the post-processing includes sequentially extracting, washing, combining organic phases, drying, filtering and reducing pressure on the product obtained from the reflux reaction; the reagent used for extraction is ethyl acetate, and the number of extractions is preferably 3 to 5 times, more preferably 4 times; the reagent used for washing is saturated brine; drying includes drying the organic phase obtained after combining the organic phases with anhydrous sodium sulfate; the purpose of reducing pressure is to remove the solvent.

[0049] The reaction equation for step (1) of this invention is as follows:

[0050]

[0051] Where R is selected from

[0052] One of them.

[0053] In step a of step (2) of the present invention, the preferred molar volume ratio of 5α,8α-peroxyergosterol, p-nitrophenyl chloroformate, dichloromethane, and pyridine is 0.003–0.005 mol: 0.007–0.009 mol: 12–18 mL: 0.01–0.015 mol, more preferably 0.004–0.0045 mol: 0.0075–0.008 mol: 15–16 mL: 0.012–0.014 mol; the reaction includes a first reaction and a second reaction; the preferred temperature of the first reaction is 0–4 °C, more preferably 1–2 °C; the preferred time of the first reaction is 4–7 min, more preferably 5–6 min; the preferred temperature of the second reaction is 20–30 °C, more preferably 25–28 °C; the preferred time of the second reaction is 1–2 h, more preferably 1.5 h.

[0054] In step a of step (2) of the present invention, the post-processing includes sequentially extracting, washing, combining organic phases, drying, filtering, reducing pressure and purifying the product obtained from the reaction; the reagent used for extraction is saturated sodium carbonate, and the number of extractions is preferably 3 to 5 times, more preferably 4 times; the reagent used for washing is saturated brine; drying includes drying the organic phase obtained after combining the organic phases with anhydrous sodium sulfate; the purpose of reducing pressure is to remove the solvent; purification includes purifying the residue by silica gel column chromatography.

[0055] In step b of step (2) of the present invention, the molar volume ratio of 5α,8α-peroxyergosterol-3-p-nitrobenzoate, piperazine, dichloromethane and triethylamine is preferably 0.003-0.005 mol: 0.007-0.009 mol: 12-18 mL: 1-1.5 mL, more preferably 0.0035-0.004 mol: 0.008-0.0085 mol: 13-15 mL: 1.1-1.4 mL; the reaction temperature is preferably 20-30°C, more preferably 25-28°C; the reaction time is preferably 1-2 h, more preferably 1.5 h.

[0056] In step b of step (2) of the present invention, the post-processing includes sequentially subjecting the reaction product to reduced pressure and purification; the purpose of reduced pressure is to remove the solvent; the purification includes purification of the residue by silica gel column chromatography (DCM:MeOH = 80:1, v / v).

[0057] The reaction equation for step (2) of this invention is:

[0058]

[0059] In step (3) of the present invention, the solvent is dichloromethane or N,N-dimethylformamide; the catalyst is one or more of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 4-dimethylaminopyridine, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, and N,N-diisopropylethylamine; the molar volume ratio of thiazolidinedione derivative, 5α,8α-peroxyergosterol-3-piperazine carbamate, solvent and catalyst is preferably 1-5 mmol:1 mmol:50-60 mL:2-6 mmol, more preferably 2-4 mmol:1 mmol:55-58 mL:4-5 mmol;

[0060] When the solvent is dichloromethane, the catalysts are 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 4-dimethylaminopyridine; when the solvent is N,N-dimethylformamide, the catalysts are 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate and N,N-diisopropylethylamine.

[0061] In step (3) of the present invention, the reaction temperature is preferably 20-30°C, more preferably 25-28°C; the reaction time is preferably 3-5 h, more preferably 4-4.5 h.

[0062] The present invention also provides the use of the 5α,8α-peroxyergosterol-3-thiazolidinedione derivative in the preparation of tumor prevention drugs or antitumor drugs.

[0063] 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.

[0064] Example 1

[0065] (1) Preparation of 5α,8α-peroxyergosterol-3-piperazine-(5-(benzylidene)-2,4-thiazolidinedione):

[0066] A. 5-(benzylene)-2,4-thiazolidinedione Preparation:

[0067] Thiazolidinedione (TZD, 0.004 mol, 0.5 g), benzaldehyde (0.004 mol), and piperidine (0.004 mol) were co-dissolved in 8 mL of anhydrous ethanol and refluxed at 80 °C for 10 h. After the reaction was completed, the mixture was poured into H2O (50 mL), and the pH was adjusted to 7 with HCl (37% by mass). The mixture was extracted three times with ethyl acetate (50 mL), washed with saturated brine, and the organic phases were separated and combined. The organic phases were dried over anhydrous NaSO4, filtered, and the solvent was removed under reduced pressure to obtain a white solid product, 5-(benzylidene)-2,4-thiazolidinedione, with a yield of 89.5%.

[0068] 1 H NMR (600MHz, Chloroform-d) δ7.75 (d, J = 2.0Hz, 2H, Ar-H), 7.74 (d, J = 2.3Hz, 1H, CH2 = CH2), 7.45 (d, J = 14.9Hz, 2H, Ar-H), 7.35-7.31 (m, 1H, Ar-H). 13 C NMR (150MHz, Chloroform-d) δ167.75,164.79,133.47,130.54,130.23,130.22,129.75,123.67.

[0069] B. 5-(benzylene)-2,4-thiazolidinedione-3-ethyl acetate Preparation:

[0070] 5-(benzylene)-2,4-thiazolidinedione (0.0024 mol, 0.49 g), K₂CO₃ (0.0029 mol), and KI (0.0024 mol) were dissolved in 10 mL of N,N-dimethylformamide (DMF), followed by the addition of ethyl bromoacetate (0.0024 mol). The mixture was stirred at 50 °C for 5 h. After the reaction was complete, the mixture was extracted three times with dichloromethane (50 mL), washed with saturated brine, and the organic phases were separated and combined. The organic phases were dried over anhydrous Na₂SO₄, filtered, and the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (PE:EA = 20:1, v / v) to give a white solid product, 5-(benzylene)-2,4-thiazolidinedione-3-ethyl acetate, in 82.7% yield.

[0071] 1 H NMR(600MHz,Chloroform-d)δ7.84(s,1H,CH2=CH2),7.74(dd,J=7.7,2.4Hz,2H,Ar-H),7 .45(t,J=7.4Hz,2H,Ar-H),7.35-7.32(m,1H,Ar-H),4.56(s,2H,CH2),2.19(s,3H,CH3). 13 C NMR (150MHz, Chloroform-d) δ201.63,174.76,168.27,134.54,131.98,130.48,130.23,129.75,121.12,48.49,27.09.

[0072] C. 5-(benzylene)-2,4-thiazolidinedione-3-acetic acid Preparation:

[0073] 0.0021 mol of ethyl 5-(benzylene)-2,4-thiazolidinedione-3-acetic acid, 2 mL of 37% HCl, and 3 mL of glacial acetic acid were added to a 100 mL round-bottom flask. The system was refluxed at 70 °C for 8 h, and the reaction progress was monitored by TLC. After the reaction was completed, the system was extracted with 50 mL of ethyl acetate, washed with saturated brine, and the organic phases were separated and combined. The organic phases were dried over anhydrous NaSO4, filtered, and the solvent was removed under reduced pressure to finally obtain a white solid product, 5-(benzylene)-2,4-thiazolidinedione-3-acetic acid, in 78.7% yield.

[0074] 1H NMR (600MHz, DMSO-d6) δ13.53(s,1H,OH),7.99(s,1H,CH2=CH2),7.65(d,J=7.3Hz,2 H, Ar-H), 7.56 (t, J = 7.3Hz, 2H, Ar-H), 7.51 (t, J = 7.3Hz, 1H, Ar-H), 4.38 (s, 2H, CH2). 13 C NMR (150MHz, DMSO-d6) δ168.5,167.4,165.5,134.4,133.3,131.4,130.7,129.90,42.9.

[0075] (2) Preparation of 5α,8α-peroxyergosterol-3-piperazine carboxylate:

[0076] a, 5α,8α-peroxyergosterol-3-p-nitrobenzoate Preparation:

[0077] Under nitrogen protection, 5α,8α-peroxyergosterol (EP, 0.004 mol, 1.7 g) and p-nitrophenyl chloroformate (0.008 mol, 1.6 g) were co-dissolved in 15 mL of dichloromethane. Pyridine (0.012 mol, 0.95 mL) was slowly added dropwise to the system at 0 °C. After the addition was complete, the reaction was carried out at 0 °C for 5 min, then at room temperature for 1 h. The reaction progress was monitored by TLC. After the reaction was complete, the mixture was extracted with saturated Na₂CO₃ and washed with saturated brine. The organic phases were separated and combined. The organic phases were dried over anhydrous Na₂SO₄, filtered, and the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (DCM) to give a white solid, 5α,8α-peroxyergosterol-3-p-nitrobenzoate, in 92.5% yield.

[0078] 1H NMR(600MHz,Chloroform-d)δ8.20(d,J=9.2Hz,2H,Ar-H),7.31(d,J=9.2Hz,2H,Ar-H),6.47(d,J=8.5Hz,1H,CH2=CH2),6.19(d,J=8.5Hz,1H,CH2=CH2),5 .15(dd,J=15.2,7.7Hz,1H,CH2=CH2),5.07(dd,J=15.2,8.3Hz,1H,CH2=CH2) ,4.95-4.91(m,1H),2.24(ddd,J=13.6,5.2,2.0Hz,1H),2.10(d,J=11.8Hz,1 H),2.04(d,J=9.7Hz,1H),1.96(s,1H),1.91-1.88(m,1H),1.78(d,J=7.1Hz, 1H),1.70(dd,J=13.4,3.2Hz,2H),1.55-1.49(m,3H),1.46(d,J=8.0Hz,1H), 1.40(d,J=6.6Hz,1H),1.34-1.31(m,1H),1.29(d,J=10.2Hz,1H),1.17(s,4H ),0.93(d,J=6.7Hz,3H,CH3),0.86-0.83(m,6H,CH3),0.77-0.74(m,9H,CH3). 13 C NMR(150MHz,Chloroform-d)δ155.68,151.58,145.43,135.26,134.84,132.49,131.28,125.40,121.89,81.85,79.69,75.50,56.30,51.7 0,51.10,44.70,42.89,39.84,39.38,37.05,34.27,33.18,29.82,28 .75,26.21,23.51,20.99,20.72,20.07,19.76,18.15,17.69,13.01.

[0079] b, 5α,8α-peroxyergosterol-3-piperazine carboxylate Preparation:

[0080] 5α,8α-peroxyergosterol-3-p-nitrobenzoate (0.004 mol, 2.2 g), piperazine (0.008 mol, 0.7 g), dichloromethane (15 mL), and triethylamine (1.1 mL) were placed together in a 100 mL round-bottom flask and reacted at room temperature for 1 h. After the reaction was completed, the solvent was removed under reduced pressure, and the residue was purified by silica gel column chromatography (DCM:MeOH = 80:1, v / v) to give a white solid product, 5α,8α-peroxyergosterol-3-piperazine carbamate, in 63.2% yield.

[0081] 1 H NMR(600MHz,Chloroform-d)δ6.50(d,J=8.5Hz,1H,CH2=CH2),6.23(d,J=8.5Hz,1H,CH2=CH2),5.22(dd,J=15.2,7.6Hz,1H,CH2=CH2),5.14(dd,J=15.2 ,8.4Hz,1H,CH2=CH2),4.89(dt,J=11.7,6.2Hz,1H),3.44(s,4H,,C31,C32) ,2.83(s,4H,C30,C33),2.17(ddd,J=13.8,5.3,1.8Hz,1H),2.01(dt,J=11. 8,7.4Hz,4H),1.95(dd,J=9.4,2.8Hz,1H),1.85(q,J=6.8Hz,1H),1.78-1.7 3(m,1H),1.71-1.68(m,1H),1.63-1.55(m,3H),1.51(d,J=4.2Hz,1H),1.48 -1.44(m,1H),1.43-1.39(m,1H),1.37-1.33(m,1H),1.27-1.22(m,4H),1.0 0(d,J=6.6Hz,3H,CH3),0.91(d,J=7.5Hz,6H,CH3),0.84-0.81(m,9H,CH3). 13 C NMR(150MHz,Chloroform-d)δ154.81,135.32,132.41,130.95,81.96,79.46,70.53,56.27,51.72,51.10,45.78,44.65, 42.88,39.84,39.42,37.06,34.40,33.70,33.17,28.74,26.94,23.48,20.98,20.74,20.06,19.74,18.24,17.67,12.97.

[0082] (3) 5α,8α-peroxyergosterol-3-piperazine-(5-(benzylidene)-2,4-thiazolidinedione) Preparation:

[0083] 5α,8α-peroxyergosterol-3-piperazincarbamate (0.092 mmol) and 5-(benzylene)-2,4-thiazolidinedione-3-acetic acid (0.139 mmol) were placed in 5 mL of dichloromethane and reacted under the catalysis of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 0.139 mmol) and 4-dimethylaminopyridine (DMAP, 0.092 mmol) at room temperature for 3 h. The reaction was monitored by TLC until the reaction was complete. The solvent was evaporated, and the crude product was purified by silica gel column chromatography (DCM:MeOH = 160:1, v / v) to give the target compound as a white solid with a yield of 67.8%.

[0084] 1 H NMR(600MHz,Chloroform-d)δ7.93(s,1H,CH2=CH2),7.52(d,J=7.7Hz,2H,Ar-H), 7.49-7.46(m,2H,Ar-H),7.45(d,J=7.2Hz,1H,Ar-H),6.51(d,J=8.4Hz,1H,CH2=C H2),6.24(d,J=8.4Hz,1H,CH2=CH2),5.22(dd,J=15.1,7.7Hz,1H,CH2=CH2),5.14 (dd,J=15.2,8.4Hz,1H,CH2=CH2),4.95-4.90(m,1H),4.56(s,2H,CH2),3.55(d,J= 60.6Hz,8H,4×CH3),2.19(dd,J=13.6,5.2Hz,1H),2.06-2.01(m,4H),1.95(d,J=7 .7Hz,1H),1.87-1.83(m,1H),1.73(d,J=8.4Hz,2H),1.62-1.54(m,3H),1.53-1.50 (m,1H),1.49-1.45(m,1H),1.44-1.41(m,1H),1.39-1.36(m,1H),1.26-1.22(m,4 H),1.00(d,J=6.6Hz,3H,CH3),0.91(d,J=4.9Hz,6H,CH3),0.84-0.81(m,9H,CH3). 13C NMR(150MHz,Chloroform-d)δ168.02,166.10,163.34,154.52,135.29,135.18,134.62, 133.26,132.39,131.02,130.77,130.35,129.36,121.39,81.92,79.4971.16,56.23,51 .68,51.05,44.63,42.85,42.44,42.12,39.82,39.38,37.03,34.36,33.62,33.14,29.7 9,28.73,26.86,23.46,20.97,20.72,20.05,19.73,18.21,17.66,12.96.MS(ESI)[M+Na] + 808.4.

[0085] Example 2

[0086] 5α,8α-Peroxyergosterol-3-piperazine-(5-(4-fluorobenzyl)-2,4-thiazolidinedione) Preparation:

[0087] Let R in Example 1 be... The aromatic aldehyde (benzaldehyde) is replaced with R. The aromatic aldehydes were used in the same manner as in Example 1, and the final product was a yellow solid, 5α,8α-peroxyergosterol-3-piperazine-(5-(4-fluorobenzyl)-2,4-thiazolidinedione), with a yield of 72.5%.

[0088] 1H NMR(600MHz,Chloroform-d)δ7.91(d,J=17.7Hz,1H,CH2=CH2),7.55-7.50(m,2H,Ar-H),7.21-7.17(m,2H,Ar-H),6.53(d,J=11.4Hz,1H,CH2=CH2),6.25(d ,J=11.4Hz,1H,CH2=CH2),5.22(dd,J=11.6,7.7Hz,1H,CH2=CH2),5.15(dd,J =11.6,8.4Hz,1H,CH2=CH2),4.97-4.91(m,1H),4.57(d,J=17.6Hz,2H,CH2),3 .56(d,J=58.1Hz,8H,4×CH3),2.23-2.15(m,1H),2.07-2.00(m,4H),1.98(s, 1H),1.87-1.84(m,1H),1.77-1.69(m,2H),1.62-1.58(m,3H),1.54-1.53(m,1 H),1.44-1.49(m,1H),1.39-1.34(m,1H),1.28(s,1H),1.25(s,4H),1.01(d, J=12.9Hz,3H,CH3),0.92(d,J=16.2Hz,6H,CH3),0.83(d,J=11.5Hz,9H,CH3). 13 C NMR(150MHz,Chloroform-d)δ167.77,166.04,163.27,154.54,135.32,135.28,135. 20,133.35,132.43,131.05,129.60,121.10,116.80,116.66,81.94,71.21,56.27,51 .71,51.08,44.70,42.88,42.51,39.86,39.40,37.06,34.38,33.65,33.17,29.80,2 8.76,26.89,23.49,20.99,20.74,20.07,19.76,18.24,17.68,12.98.MS(ESI)[M+Na] + 826.4.

[0089] Example 3

[0090] 5α,8α-Peroxyergosterol-3-piperazine-(5-(4-chlorobenzyl)-2,4-thiazolidinedione) Preparation:

[0091] Let R in Example 1 be... The aromatic aldehyde (benzaldehyde) is replaced with R. The aromatic aldehydes were used in the same manner as in Example 1, and the final product was a pink solid, 5α,8α-peroxyergosterol-3-piperazine-(5-(4-chlorobenzyl)-2,4-thiazolidinedione), with a yield of 56.8%.

[0092] 1 H NMR(600MHz,Chloroform-d)δ7.86(s,1H,CH2=CH2),7.45(s,4H,Ar-H),6.51(d,J=8.5Hz,1H,CH2=CH2),6.23(d,J=8.5Hz,1H,CH2=CH2),5.22(dd, J=15.2,7.7Hz,1H,CH2=CH2),5.14(dd,J=15.2,8.7Hz,1H,CH2=CH2),4.95-4.90(m,1H),4.56(s,2H,CH2),3.49(s,8H,4×CH3),2.19(dd,J=13.7,4 .9Hz,1H),2.06-1.99(m,4H),1.95(d,J=9.2Hz,1H),1.88-1.1(m,1H),1. 71(d,J=14.8Hz,2H),1.61-1.54(m,3H),1.52-1.50(m,1H),1.48-1.45(m ,1H),1.40-1.37(m,1H),1.35(d,J=11.3Hz,1H),1.26-1.21(m,4H),1.00 (d,J=6.6Hz,3H,CH3),0.91(d,J=5.3Hz,6H,CH3),0.84-0.80(m,9H,CH3). 13 CNMR(150MHz,Chloroform-d)δ169.93,168.29,165.59,156.88,136.89,135.30,135.1 9,133.11,132.42,131.74,131.45,131.04,129.71,122.05,81.93,79.51,73.54,56.2 6,51.70,51.07,44.65,42.87,42.54,39.85,39.40,37.05,34.38,33.64,33.16,29.80 ,28.75,26.88,23.48,20.99,20.73,20.06,19.75,18.23,17.68,12.97.MS(ESI)[M+Na] + 842.3.

[0093] Example 4

[0094] 5α,8α-Peroxyergosterol-3-piperazine-(5-(4-bromobenzyl)-2,4-thiazolidinedione) Preparation:

[0095] Let R in Example 1 be... The aromatic aldehyde (benzaldehyde) is replaced with R. The aromatic aldehydes were used in the same manner as in Example 1, and the final product was a pale yellow solid, 5α,8α-peroxyergosterol-3-piperazine-(5-(4-bromobenzyl)-2,4-thiazolidinedione), with a yield of 62.5%.

[0096] 1 H NMR (600MHz, Chloroform-d) δ7.84(s,1H,CH2=CH2),7.61(d,J=8.2Hz,2H,Ar-H),7.37(d,J=8.2Hz,2H,Ar-H),6.51(d,J=8.5Hz,1H,CH2=CH2),6.23(d,J=8. 5Hz,1H,CH2=CH2),5.22(dd,J=15.2,7.7Hz,1H,CH2=CH2),5.14(dd,J=15.2,8 .5Hz,1H,CH2=CH2),4.95-4.90(m,1H),4.55(s,2H,CH2),3.49(s,8H,4×CH3),2 .19(dd,J=13.5,5.2Hz,1H),2.06-1.99(m,4H),1.95(d,J=7.7Hz,1H),1.87-1 .81(m,H),1.73-1.70(m,2H),1.60-1.54(m,3H),1.51(d,J=6.6Hz,1H),1.47(d ,J=6.7Hz,1H),1.44-1.39(m,1H),1.37(d,J=10.3Hz,1H),1.26-1.21(m,4H), 1.00(d,J=6.5Hz,3H,CH3),0.91(d,J=5.8Hz,6H,CH3),0.84-0.81(m,9H,CH3). 13C NMR(150MHz,Chloroform-d)δ167.53,165.91,163.20,154.50,135.28,135.17,133.15 ,132.66,132.40,132.14,131.58,131.03,125.32,122.18,81.92,79.49,71.17,56.24 ,51.69,51.05,44.64,42.85,42.53,39.83,39.38,37.03,34.37,33.63,33.15,29.79, 28.73,26.86,23.46,20.97,20.72,20.05,19.74,18.22,17.67,12.96.MS(ESI)[M+Na] + 886.3.

[0097] Example 5

[0098] 5α,8α-Peroxyergosterol-3-piperazine-(5-(4-methylbenzyl)-2,4-thiazolidinedione) Preparation:

[0099] Let R in Example 1 be... The aromatic aldehyde (benzaldehyde) is replaced with R. The aromatic aldehydes were used in the same manner as in Example 1, and the final product was a white solid, 5α,8α-peroxyergosterol-3-piperazine-(5-(4-methylbenzyl)-2,4-thiazolidinedione), with a yield of 69.2%.

[0100] 1H NMR(600MHz,Chloroform-d)δ7.90(s,1H,CH2=CH2),7.41(d,J=8.0Hz,2H,Ar-H),7.28(d,J=8.0Hz,2H,Ar-H),6.51(d,J=8.4Hz,1H,CH2=CH2),6.24(d,J=8.5Hz ,1H,CH2=CH2),5.22(dd,J=15.2,7.7Hz,1H,CH2=CH2),5.14(dd,J=15.2,8.5Hz ,1H,CH2=CH2),4.95-4.90(m,1H),4.55(s,2H,CH2),3.49(s,8H,4×CH3),2.41(s ,3H,CH3),2.19(dd,J=13.9,3.8Hz,1H),2.06-1.99(m,4H),1.95(d,J=6.6Hz,1 H),1.86-1.83(m,1H),1.74-1.68(m,2H),1.61-1.54(m,3H),1.52(d,J=5.1Hz,1 H),1.49-1.45(m,1H),1.43-1.39(m,1H),1.37(d,J=10.1Hz,1H),1.26-1.22(m, 4H), 1.00 (d, J=6.6Hz, 3H, CH3), 0.92-0.90 (m, 6H, CH3), 0.84-0.81 (m, 9H, CH3). 13 C NMR(150MHz,Chloroform-d)δ168.17,166.23,163.38,154.53,141.57,135.31,135.20, 134.79,132.41,131.04,130.54,130.47,130.14,120.14,81.94,79.51,71.17,56.26,51 .71,51.08,44.66,42.88,42.41,39.85,39.40,37.06,34.38,33.64,33.16,29.81,28.7 5,26.89,23.48,21.73,20.99,20.74,20.07,19.75,18.23,17.68,12.98.MS(ESI)[M+Na] + 822.4.

[0101] Example 6

[0102] 5α,8α-Peroxyergosterol-3-piperazine-(5-(4-methoxybenzyl)-2,4-thiazolidinedione) Preparation:

[0103] Let R in Example 1 be... The aromatic aldehyde (benzaldehyde) is replaced with R. The aromatic aldehydes were used in the same manner as in Example 1, and the final product was a pale yellow solid, 5α,8α-peroxyergosterol-3-piperazine-(5-(4-methoxybenzyl)-2,4-thiazolidinedione), with a yield of 72.8%.

[0104] 1 H NMR(600MHz,Chloroform-d)δ7.88(s,1H,CH2=CH2),7.48(d,J=8.3Hz,2H,Ar-H),7.00(d,J=8.3Hz,2H,Ar-H),6.51(d,J=8.5Hz,1H,CH2=CH2),6.24(d,J=8.5Hz,1 H,CH2=CH2),5.22(dd,J=15.2,7.7Hz,1H,CH2=CH2),5.14(dd,J=15.2,8.7Hz,1H, CH2=CH2),4.96-4.90(m,1H),4.55(s,2H,CH2),3.87(s,3H,OCH3),3.61-3.49(m, 8H,4×CH3),2.19(dd,J=13.5,5.1Hz,1H),2.05-2.00(m,4H),1.96(d,J=7.6Hz,1 H),1.87-1.83(m,1H),1.71(d,J=13.8Hz,2H),1.61-1.55(m,3H),1.52(d,J=4.7H z,1H),1.48-1.45(m,1H),1.43-1.39(m,1H),1.37(d,J=9.9Hz,1H),1.26-1.22(m ,4H),1.00(d,J=6.5Hz,3H,CH3),0.94-0.89(m,6H,CH3),0.85-0.80(m,9H,CH3). 13C NMR(150MHz,Chloroform-d)δ168.25,166.33,163.44,161.72,135.33,135.33,135. 23,134.61,131.06,125.95,118.34,114.95,81.96,79.53,71.20,56.29,55.64,51. 73,51.10,44.68,42.90,42.40,39.87,39.42,37.07,34.40,33.66,33.19,29.83,28 .77,26.91,23.50,21.01,20.76,20.08,19.77,18.25,17.69,13.00.MS(ESI)[M+Na] + 838.4.

[0105] Example 7

[0106] 5α,8α-Peroxyergosterol-3-piperazine-(5-(4-tert-butylbenzyl)-2,4-thiazolidinedione) Preparation:

[0107] Let R in Example 1 be... The aromatic aldehyde (benzaldehyde) is replaced with R. The aromatic aldehydes were obtained in the same manner as in Example 1, and the final product was a white solid, 5α,8α-peroxyergosterol-3-piperazine-(5-(4-tert-butylbenzyl)-2,4-thiazolidinedione), with a yield of 45.9%.

[0108] 1H NMR(600MHz,Chloroform-d)δ7.91(s,1H,CH2=CH2),7.50(d,J=8.5Hz,2H,Ar-H),7.47(s,2H,Ar-H),6.51(d,J=8.5Hz,1H,Ar-H),6.24(d,J=8.5Hz,1H,Ar-H) ,5.22(dd,J=15.2,7.7Hz,1H,CH2=CH2),5.14(dd,J=15.2,8.4Hz,1H,CH2=CH2 ),4.96-4.89(m,1H),4.56(s,2H,CH2),3.55(d,J=61.1Hz,8H,4×CH3),2.19(dd ,J=13.6,3.5Hz,1H),2.06-1.99(m,4H),1.97-1.94(m,1H),1.86-1.83(m,1H) ,1.73-1.67(m,2H),1.62-1.56(m,1H),1.52(d,J=5.6Hz,1H),1.49-1.46(m,1H ),1.45-1.41(m,1H),1.40(d,J=5.4Hz,1H),1.34(s,9H,CH3),1.26-1.20(m,4 H),1.00(d,J=6.6Hz,3H,CH3),0.92-0.90(m,6H,CH3),0.84-0.81(m,9H,CH3). 13 C NMR(150MHz,Chloroform-d)δ168.18,166.22,163.39,154.57,154.52,135.30,135.20, 134.63,132.40,131.02,130.48,130.37,126.40,120.25,81.93,79.50,71.16,56.25,51 .70,51.07,44.65,42.86,42.41,39.84,39.39,37.05,35.18,34.37,33.64,33.15,31.1 7,28.75,26.88,23.47,20.98,20.73,20.06,19.75,18.22,17.68,12.97.MS(ESI)[M+Na] + 864.5.

[0109] Example 8

[0110] 5α,8α-Peroxyergosterol-3-piperazine-(5-(3-chloro-4-fluorobenzyl)-2,4-thiazolidinedione) Preparation:

[0111] Let R in Example 1 be... The aromatic aldehyde (benzaldehyde) is replaced with R. The aromatic aldehyde was obtained, and the rest were the same as in Example 1. The final product was a yellow solid, 5α,8α-peroxyergosterol-3-piperazine-(5-(3-chloro-4-fluorobenzyl)-2,4-thiazolidinedione), with a yield of 56.9%.

[0112] 1 H NMR(600MHz,Chloroform-d)δ7.81(s,1H,CH2=CH2),7.56(d,J=6.8Hz,1H,Ar-H), 7.41(d,J=7.6Hz,1H,Ar-H),7.31-7.24(m,1H),6.51(d,J=8.7Hz,1H,CH2=CH2),6 .24(d,J=8.5Hz,1H,CH2=CH2),5.21(dd,J=15.3,7.7Hz,1H,CH2=CH2),5.14(dd,J =15.3,8.4Hz,1H,CH2=CH2),4.98-4.88(m,1H),4.56(s,2H,CH2),3.55(d,J=63.6 Hz,8H,4×CH3),2.19(dd,J=13.7,5.4Hz,1H),2.05-2.00(m,4H),1.96(d,J=9.0Hz ,1H),1.87-1.83(m,1H),1.76-1.73(m,2H),1.61-1.56(m,3H),1.52(d,J=7.2Hz, 1H),1.48-1.45(m,1H),1.42-1.39(m,1H),1.37(d,J=9.9Hz,1H),1.26-1.21(m,4 H),1.00(d,J=7.1Hz,3H,CH3),0.91(d,J=4.2Hz,6H,CH3),0.84-0.81(m,9H,CH3). 13CNMR(150MHz,Chloroform-d)δ167.26,165.73,159.91,154.52,135.29,135.19,132.4 8,132.40,131.77,131.03,130.06,122.71,117.79,117.65,81.92,79.50,71.19,56.2 5,51.69,51.06,44.64,42.86,42.60,39.84,39.38,37.04,34.38,33.63,33.15,29.81 ,28.74,26.87,23.46,20.98,20.72,20.05,19.73,18.22,17.67,12.97.MS(ESI)[M+Na] + 860.3.

[0113] Example 9

[0114] 5α,8α-Peroxyergosterol-3-piperazine-(5-(2-fluorobenzyl)-2,4-thiazolidinedione) Preparation:

[0115] Let R in Example 1 be... The aromatic aldehyde (benzaldehyde) is replaced with R. The aromatic aldehydes were obtained in the same manner as in Example 1, and the final product was a white solid, 5α,8α-peroxyergosterol-3-piperazine-(5-(2-fluorobenzyl)-2,4-thiazolidinedione), with a yield of 79.4%.

[0116] 1H NMR(600MHz,Chloroform-d)δ8.14(s,1H,CH2=CH2),7.53-7.51(m,1H,Ar-H),7.46-7 .42(m,1H,Ar-H),7.26(d,J=7.3Hz,1H,Ar-H),7.16(d,J=9.2Hz,1H,Ar-H),6.51(d,J =8.4Hz,1H,CH2=CH2),6.24(d,J=8.5Hz,1H,CH2=CH2),5.22(dd,J=15.3,8.4Hz,1H,C H2=CH2),5.14(dd,J=15.3,8.4Hz,1H,CH2=CH2),4.96-4.89(m,1H),4.57(s,2H,CH2) ,3.55(d,J=60.1Hz,8H,4×CH3),2.19(dd,J=12.8,4.9Hz,1H),2.07-2.00(m,4H),1.9 6(d,J=9.8Hz,1H),1.87-1.83(m,1H),1.74-1.70(m,2H),1.63-1.57(m,3H),1.52(d, J=6.4Hz,1H),1.48-1.45(m,1H),1.43-1.39(m,1H),1.37(d,J=10.1Hz,1H),1.26-1. 21(m,4H),1.00(d,J=6.6Hz,3H,CH3),0.92-0.89(m,6H,CH3),0.84-0.81(m,9H,CH3). 13 C NMR (150MHz, CDCl3) δ167.69,165.71,162.38,160.69,154.52,135.29,135.19,132. 40,131.02,129.04,124.86,123.47,116.90,116.30,81.93,79.50,71.17,56.24,51 .69,51.06,44.64,42.86,42.52,39.83,39.38,37.04,34.37,33.63,33.15,29.80,2 8.73,26.87,23.46,20.97,20.72,20.05,19.74,18.22,17.67,12.96.MS(ESI)[M+Na] + 826.4.

[0117] Example 10

[0118] 5α,8α-Peroxyergosterol-3-piperazine-(5-(2,4-dichlorobenzyl)-2,4-thiazolidinedione) Preparation:

[0119] Let R in Example 1 be... The aromatic aldehyde (benzaldehyde) is replaced with R. The aromatic aldehydes were obtained in the same manner as in Example 1, and the final product was a white solid, 5α,8α-peroxyergosterol-3-piperazine-(5-(2,4-dichlorobenzyl)-2,4-thiazolidinedione), with a yield of 56.4%.

[0120] 1 H NMR(600MHz,Chloroform-d)δ8.17(s,1H,CH2=CH2),7.52(s,1H,Ar-H),7.48(d,J=8.5Hz,1H,Ar-H),7.37(d,J=7.6Hz,1H,Ar-H),6.52(d,J=8.5Hz,1H,CH2=CH2), 6.24(d,J=8.5Hz,1H,CH2=CH2,5.21(dd,J=15.3,8.5Hz,1H,CH2=CH2),5.14(dd,J =15.3,8.5Hz,1H,CH2=CH2),4.96-4.90(m,1H),4.56(s,2H,CH2),3.55(d,J=62.6 Hz,8H,4×CH3),2.19(dd,J=13.5,5.5Hz,1H),2.06-1.99(m,4H),1.96(d,J=10.7H z,1H),1.88-1.82(m,1H),1.75-1.67(m,2H),1.60-1.57(m,3H),1.52(d,J=4.6Hz ,1H),1.48-1.45(m,1H),1.43-1.39(m,1H),1.37(d,J=10.1Hz,1H),1.27-1.22(m ,4H),1.00(d,J=6.5Hz,3H,CH3),0.92-0.90(m,6H,CH3),0.84-0.81(m,9H,CH3). 13C NMR (151MHz, CDCl3) δ167.38,165.43,137.05,136.80,135.33,135.21,132.45,131. 08,130.57,130.39,129.73,129.60,127.93,124.94,81.96,79.55,71.25,56.29,51 .73,51.10,44.68,42.90,42.58,39.87,39.42,37.08,34.40,33.66,33.19,29.83,2 8.77,26.91,23.50,21.01,20.76,20.08,19.77,18.25,17.70,13.00.MS(ESI)[M+Na] + 876.3.

[0121] Example 11

[0122] 5α,8α-Peroxyergosterol-3-piperazine-(5-(2-methoxybenzyl)-2,4-thiazolidinedione) Preparation:

[0123] Let R in Example 1 be... The aromatic aldehyde (benzaldehyde) is replaced with R. The aromatic aldehydes were used in the same manner as in Example 1, and the final product was a yellow solid, 5α,8α-peroxyergosterol-3-piperazine-(5-(2-methoxybenzyl)-2,4-thiazolidinedione), with a yield of 88.2%.

[0124] 11H NMR (600 MHz, Chloroform-d) δ 8.28 (s, 1H, CH2=CH2), 7.45 (d, J = 7.7 Hz, 1H, Ar-H), 7.41 (t, J = 8.7, 8.7 Hz, 1H, Ar-H), 7.04 (t, J = 7.5, 7.5 Hz, 1H, Ar-H), 6.94 (d, J = 8.3 Hz, 1H, Ar-H), 6.51 (d, J = 8.5 Hz, 1H, CH2=CH2), 6.24 (d, J = 8.5 Hz, 1H, CH2=CH2), 5.22 (dd, J = 15.2, 7.7 Hz, 1H, CH2=CH2), 5.14 (dd, J = 15.2, 8.5 Hz, 1H, CH2=CH2), 4.95 - 4.90 (m, 1H), 4.55 (s, 2H, CH2), 3.89 (s, 3H, OCH3), 3.55 (d, J = 68.8 Hz, 8H, 4×CH3), 2.19 (dd, J = 13.6, 3.5 Hz, 1H), 2.06 - 1.99 (m, 4H), 1.95 (d, J = 6.5 Hz, 1H), 1.88 - 1.83 (m, 1H), 1.74 - 1.68 (m, 2H), 1.62 - 1.55 (m, 3H), 1.53 - 1.49 (m, 1H), 1.48 - 1.45 (m, 1H), 1.44 - 1.40 (m, 1H), 1.38 - 1.35 (m, 1H), 1.26 - 1.21 (m, 4H), 1.00 (d, J = 6.5 Hz, 3H, CH3), 0.92 - 0.90 (m, 6H, CH3), 0.84 - 0.81 (m, 9H, CH3). 13 13C NMR (150 MHz, CDCl3) δ 168.57, 166.19, 158.62, 135.30, 135.19, 132.49, 132.39, 131.01, 130.50, 129.54, 122.44, 121.30, 120.98, 111.25, 81.93, 79.49, 71.14, 56.24, 55.61, 51.69, 51.06, 44.64, 42.86, 42.29, 39.83, 39.38, 37.03, 34.37, 33.62, 33.15, 29.80, 28.73, 26.86, 23.46, 20.97, 20.72, 20.05, 19.74, 18.22, 17.67, 12.96. MS (ESI) [M+Na] + 838.4.

[0125] Example 12

[0126] 5α,8α-Peroxyergosterol-3-piperazine-(5-(3-methoxybenzyl)-2,4-thiazolidinedione) Preparation:

[0127] Let R in Example 1 be... The aromatic aldehyde (benzaldehyde) is replaced with R. The aromatic aldehydes were obtained in the same manner as in Example 1, and the final product was a pale yellow solid, 5α,8α-peroxyergosterol-3-piperazine-(5-(3-methoxybenzyl)-2,4-thiazolidinedione), with a yield of 78.5%.

[0128] 1 H NMR(600MHz,Chloroform-d)δ7.89(s,1H,CH2=CH2),7.39(t,J=7.4,7.4Hz,1H,Ar- H),7.11(d,J=7.9Hz,1H,Ar-H),7.03(s,1H,Ar-H),6.99(d,J=8.3Hz,1H,Ar-H),6.5 1(d,J=8.4Hz,1H,CH2=CH2),6.24(d,J=8.5Hz,1H,CH2=CH2),5.22(dd,J=15.1,7.8H z,1H,CH2=CH2),5.14(dd,J=15.3,8.5Hz,1H,CH2=CH2),4.95-4.91(m,1H),4.56(s, 2H,CH2),3.85(s,3H,OCH3),3.50(s,8H,4×CH3),2.19(dd,J=13.6,3.5Hz,1H),2.0 6-1.99(m,4H),1.96(d,J=9.5Hz,1H),1.88-1.83(m,1H),1.74-1.68(m,2H),1.60-1 .56(m,3H),1.52-1.49(m,1H),1.44-1.41(m,1H),1.38-1.35(m,1H),1.29-1.23(m, 4H), 1.00 (d, J=7.1Hz, 3H, CH3), 0.91 (d, J=4.4Hz, 6H, CH3), 0.85-0.81 (m, 9H, CH3). 13C NMR (150MHz, CDCl3) δ168.34,166.27,163.32,160.34,135.32,135.22,134.62,132.4 3,131.05,130.39,122.85,121.78,116.85,115.17,81.94,79.53,71.20,56.28,55.50 ,51.72,51.10,44.67,42.88,42.45,39.85,39.42,37.07,34.40,33.65,33.18,29.82, 28.75,26.98,23.49,20.99,20.75,20.07,19.76,18.24,17.68,12.99.MS(ESI)[M+Na] + 838.4.

[0129] Example 13

[0130] 5α,8α-Peroxyergosterol-3-piperazine-(5-(3,4-dimethoxybenzylidene)-2,4-thiazolidinedione) Preparation:

[0131] Let R in Example 1 be... The aromatic aldehyde (benzaldehyde) is replaced with R. The aromatic aldehydes were used in the same manner as in Example 1, and the final product was a yellow solid, 5α,8α-peroxyergosterol-3-piperazine-(5-(3,4-methoxybenzyl)-2,4-thiazolidinedione), with a yield of 76.8%.

[0132] 1H NMR(600MHz,Chloroform-d)δ7.87(s,1H,CH2=CH2),7.15(d,J=8.4Hz,1H,Ar-H),7.02(s,1H,Ar-H),6.96(d,J=8.4Hz,1H,Ar-H),6.51(d,J=8.5Hz,1H,CH2=C H2),6.24(d,J=8.5Hz,1H,CH2=CH2),5.22(dd,J=15.2,7.7Hz,1H),5.14(dd,J =15.2,8.5Hz,1H),4.97-4.87(m,1H),4.55(s,2H,CH2),3.94(d,J=6.3Hz,6H,O CH),3.49(s,8H,4×CH3),2.19(dd,J=13.5,5.0Hz,1H),2.06-2.00(m,4H),1.9 6(d,J=7.9Hz,1H),1.88-1.82(m,1H),1.74-1.68(m,2H),1.61-1.55(m,3H),1. 53-1.50(m,1H),1.49-1.44(m,1H),1.44-1.41(m,1H),1.39-1.36(m,1H),1.2 5(s,4H),1.00(d,J=6.6Hz,3H,CH3),0.91(s,6H,CH3),0.84-0.81(m,9H,CH3). 13 C NMR (150MHz, CDCl3) δ168.10,166.19,151.46,149.48,135.31,135.20,134.77,132.43, 131.05,126.22,124.91,118.65,112.53,111.54,81.94,79.51,71.18,56.28,56.18,56 .07,51.71,51.09,44.67,42.88,42.41,39.84,39.41,37.06,34.39,33.65,33.17,29.8 1,28.75,26.89,23.49,20.99,20.74,20.06,19.75,18.23,17.68,12.98.MS(ESI)[M+Na] + 868.4.

[0133] Example 14

[0134] 5α,8α-Peroxyergosterol-3-piperazine-(5-(2,3,4-trimethoxybenzyl)-2,4-thiazolidinedione) Preparation:

[0135] Let R in Example 1 be... The aromatic aldehyde (benzaldehyde) is replaced with R. The aromatic aldehydes were used in the same manner as in Example 1, and the final product was a pale yellow solid, 5α,8α-peroxyergosterol-3-piperazine-(5-(2,3,4-trimethoxybenzyl)-2,4-thiazolidinedione), with a yield of 78.8%.

[0136] 1 H NMR (600MHz, Chloroform-d) δ8.18 (s, 1H, CH2=CH2), 7.21 (d, J=8.8Hz, 1H, Ar-H), 6. 78(d,J=8.9Hz,1H,Ar-H),6.52(d,J=8.5Hz,1H,CH2=CH2),6.24(d,J=8.5Hz,1H,CH2 =CH2),5.22(dd,J=15.2,7.6Hz,1H,CH2=CH2),5.14(dd,J=15.3,8.4Hz,1H,CH2=CH2 ),4.96-4.89(m,1H),4.55(s,2H,CH2),3.93(d,J=8.5Hz,6H,OCH3),3.88(s,3H,OCH 3),3.62-3.49(m,8H,4×CH3),2.19(dd,J=13.5,5.0Hz,1H),2.06-2.01(m,4H),1.96 (dd,J=9.4,2.8Hz,1H),1.86-1.83(m,1H),1.75-1.69(m,2H),1.61-1.55(m,3H),1. 52-1.50(m,1H),1.48-1.45(m,1H),1.43-1.39(m,1H),1.39-1.36(m,1H),1.26-1.2 3(m,4H),1.00(d,J=6.6Hz,3H,CH3),0.92-0.90(m,6H,CH3),0.84-0.81(m,9H,CH3). 13C NMR (150MHz, CDCl3) δ168.59,166.27,156.44,154.07,142.50,135.33,135.22,132.44,1 31.06,130.04,124.78,120.49,119.55,107.74,81.95,79.53,71.19,62.05,61.09,56.28 ,51.73,51.10,44.68,42.90,42.31,39.86,39.42,37.08,34.40,33.67,33.18,29.82,29. 45,28.76,26.91,23.50,21.00,20.76,20.07,19.76,18.25,17.69,13.00.MS(ESI)[M+Na] + 898.4.

[0137] The antitumor activity of the 5α,8α-peroxyergosterol-3-thiazolidinedione derivatives obtained in Examples 1-14 was tested. The test methods and results are as follows:

[0138] (1) Main experimental instruments and reagents used in the test

[0139] Table 1. Main experimental instruments and reagents used in the test.

[0140]

[0141]

[0142] (2) Selection of test cells and compounds used in the test

[0143] In this test, 5α,8α-peroxyergosterol (EP) and cisplatin were selected as compounds in the positive control group, and the 5α,8α-peroxyergosterol-3-thiazolidinedione derivatives obtained in Examples 1-14 were selected as compounds in the test group.

[0144] This test selected human liver cancer cells (HepG2), human breast cancer cells (MCF-7), and human non-small cell lung cancer cells (A549) as test cells.

[0145] (3) Test method:

[0146] MTT assay: Test cells in logarithmic growth phase were collected and seeded in 96-well plates at a density of 1.0 × 10⁶ cells per well. 5 / 100μL, incubated at 37℃ in a 5% CO2 incubator. The next day, the culture medium was removed, and 100μL of different concentrations of the compound were added (the compound concentration was double-diluted, and each compound was set with 80μmol / L, 40μmol / L, 20μmol / L, 10μmol / L, 5μmol / L, and 2.5μmol / L for a total of 6 concentrations, with 3 parallel wells for each test and 3 replicates). No compound was added to the negative control group. After 48h, 10μL of diphenyltetrazolium bromide (MTT) was added to each well, and the culture was continued for 4h. Then, 100μL of dimethyl sulfoxide (DMSO) was added to each well to terminate the reaction. The cells were incubated at room temperature for 1h, and the absorbance (OD) value of each well at 492nm was detected using a microplate reader to calculate the cell growth inhibition rate.

[0147] Cell growth inhibition rate (%) = (mean OD value of control group - mean OD value of drug group) / mean OD value of control group. The IC50 of the drug is calculated based on the cell growth inhibition rate (%) of different drug concentrations. 50 IC 50 : The concentration of the test compound that inhibits cell growth by 50%.

[0148] (4) The results of the antitumor activity test of the 5α,8α-peroxyergosterol-3-thiazolidinedione derivatives obtained in Examples 1 to 14 are shown in Table 2.

[0149] Table 2. In vitro antitumor activity (IC50) of the 5α,8α-peroxyergosterol-3-thiazolidinedione derivatives obtained in Examples 1-14. 50 (μM)

[0150]

[0151]

[0152] Table 2 shows that the 5α,8α-peroxyergosterol-3-thiazolidinedione derivatives obtained in Examples 1-14 generally exhibited stronger inhibitory effects on the human lung cancer A549 cell line than on other cell lines. Specifically, the 5α,8α-peroxyergosterol-3-thiazolidinedione derivatives obtained in Examples 1, 3, 4, 6, 9, and 14 showed potent inhibitory effects on the A549 cell line (IC50). 50 <7μM), among which the 5α,8α-peroxyergosterol-3-thiazolidinedione derivative obtained in Example 14 showed the strongest antitumor activity, IC50. 50The value was 2.9 times that of EP. For the MCF-7 cell line, the 5α,8α-peroxyergosterol-3-thiazolidinedione derivatives obtained in Examples 1, 3, 5, and 14 showed potent inhibitory activity (IC50 < 10 μM), with the 5α,8α-peroxyergosterol-3-thiazolidinedione derivatives obtained in Examples 1 and 14 exhibiting the strongest antitumor activity, IC50 < 10 μM. 50 The values ​​were 8.48 μM and 8.96 μM, respectively. For the HepG2 cell line, the 5α,8α-peroxyergosterol-3-thiazolidinedione derivatives obtained in Examples 1, 3, 6, and 14 exhibited strong antitumor activity. It is evident that the 5α,8α-peroxyergosterol-3-thiazolidinedione derivatives obtained in this invention possess good selectivity and antitumor activity against human A549 cells, and can be used as lead compounds for further development of novel antitumor drugs.

Claims

1. A 5α,8α-peroxyergosterol-3-thiazolidinedione derivative, characterized in that, The structural formula of the 5α,8α-peroxyergosterol-3-thiazolidinedione derivative is as follows: ； R1 is selected from one of -H, -F, -Cl, and -OCH3; R2 is selected from one of -H, -Cl, and -OCH3; and R3 is selected from one of -H, -F, -Cl, -Br, -CH3, -OCH3, and -C(CH3)3.

2. The method for preparing the 5α,8α-peroxyergosterol-3-thiazolidinedione derivative of claim 1, characterized in that, Includes the following steps: (1) Preparation of thiazolidinedione derivatives: A. Thiazolidinedione, aromatic aldehyde, piperidine, and ethanol were mixed and refluxed, followed by post-treatment to obtain 5-substituted benzyl-2,4-thiazolidinedione; B. 5-substituted benzyl-2,4-thiazolidinedione, potassium carbonate, potassium iodide, N,N-dimethylformamide, and ethyl bromoacetate were mixed and reacted, followed by post-treatment to obtain 5-substituted benzyl-2,4-thiazolidinedione-3-ethyl acetate; C. 5-substituted benzyl-2,4-thiazolidinedione-3-ethyl acetate, hydrochloric acid, and glacial acetic acid were mixed and refluxed, followed by post-treatment to obtain the thiazolidinedione derivative. (2) Preparation of 5α,8α-peroxyergosterol-3-piperazine carboxylate: a) 5α,8α-peroxyergosterol, p-nitrophenyl chloroformate, dichloromethane, and pyridine were mixed and reacted, followed by post-treatment to obtain 5α,8α-peroxyergosterol-3-p-nitrobenzoate; b) 5α,8α-peroxyergosterol-3-p-nitrobenzoate, piperazine, dichloromethane, and triethylamine were mixed and reacted, followed by post-treatment to obtain 5α,8α-peroxyergosterol-3-piperazine benzoate; (3) The thiazolidinedione derivative, 5α,8α-peroxyergosterol-3-piperazine carboxylate, solvent and catalyst were mixed and reacted to obtain the 5α,8α-peroxyergosterol-3-thiazolidinedione derivative. The structural formula of the aromatic aldehyde is: ; where R is selected from , , , , , , , , , , , , , One of them; The steps (1) and (2) are not in any particular order.

3. The method for preparing the 5α,8α-peroxyergosterol-3-thiazolidinedione derivative according to claim 2, characterized in that, In step A of step (1), the molar volume ratio of thiazolidinedione, aromatic aldehyde, piperidine and ethanol is 0.0005~0.002mol: 0.0005~0.002mol: 0.0005~0.002mol: 2~3mL; the reflux reaction temperature is 70~80℃, and the reflux reaction time is 8~10h; the post-processing includes sequentially extracting, washing, combining organic phases, drying, filtering and reducing pressure on the product obtained from the reflux reaction.

4. The method for preparing the 5α,8α-peroxyergosterol-3-thiazolidinedione derivative according to claim 2 or 3, characterized in that, In step B of step (1), the molar volume ratio of 5-substituted benzyl-2,4-thiazolidinedione, potassium carbonate, potassium iodide, N,N-dimethylformamide and ethyl bromoacetate is 0.002~0.003 mol: 0.0024~0.0036 mol: 0.002~0.003 mol: 9~11 mL: 0.002~0.003 mol; the reaction temperature is 40~50℃, and the reaction time is 5~7 h; the post-treatment includes sequentially extracting, washing, combining organic phases, drying, filtering, reducing pressure and purifying the product obtained from the reaction.

5. The method for preparing the 5α,8α-peroxyergosterol-3-thiazolidinedione derivative according to claim 4, characterized in that, In step C of step (1), the molar volume ratio of 5-substituted benzyl-2,4-thiazolidinedione-3-ethyl acetate, hydrochloric acid and glacial acetic acid is 0.001~0.0021 mol: 1~3 mL: 2~4 mL; the reflux reaction temperature is 70~80℃ and the reflux reaction time is 5~8 h; the post-treatment includes sequentially extracting, washing, combining organic phases, drying, filtering and reducing pressure on the product obtained from the reflux reaction.

6. The method for preparing the 5α,8α-peroxyergosterol-3-thiazolidinedione derivative according to claim 2 or 5, characterized in that, In step (2) a, the molar volume ratio of 5α,8α-peroxyergosterol, p-nitrophenyl chloroformate, dichloromethane, and pyridine is 0.003~0.005mol: 0.007~0.009mol: 12~18mL: 0.01~0.015mol; the reaction includes a first reaction and a second reaction; the temperature of the first reaction is 0~4℃, and the time of the first reaction is 4~7min; the temperature of the second reaction is 20~30℃, and the time of the second reaction is 1~2h; the post-processing includes sequentially extracting, washing, combining organic phases, drying, filtering, reducing pressure, and purifying the product obtained from the reaction.

7. The method for preparing the 5α,8α-peroxyergosterol-3-thiazolidinedione derivative according to claim 6, characterized in that, In step b of step (2), the molar volume ratio of 5α,8α-peroxyergosterol-3-p-nitrobenzoate, piperazine, dichloromethane, and triethylamine is 0.003~0.005 mol: 0.007~0.009 mol: 12~18 mL: 1~1.5 mL; the reaction temperature is 20~30℃, and the reaction time is 1~2 h; the post-treatment includes sequentially subjecting the reaction product to reduced pressure and purification.

8. The method for preparing the 5α,8α-peroxyergosterol-3-thiazolidinedione derivative according to claim 2, characterized in that, In step (3), the solvent is dichloromethane or N,N-dimethylformamide; the catalyst is one or more of 1-ethyl-(3-dimethylaminopropyl)carbamate hydrochloride, 4-dimethylaminopyridine, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, and N,N-diisopropylethylamine; the molar volume ratio of thiazolidinedione derivative, 5α,8α-peroxyergosterol-3-piperazine carbamate, solvent and catalyst is 1~5 mmol:1 mmol:50~60 mL:2~6 mmol.

9. The method for preparing the 5α,8α-peroxyergosterol-3-thiazolidinedione derivative according to claim 8, characterized in that, In step (3), the reaction temperature is 20~30℃ and the reaction time is 3~5h.

10. The use of the 5α,8α-peroxyergosterol-3-thiazolidinedione derivative of claim 1 in the preparation of antitumor drugs or tumor drugs, characterized in that, The tumors described are human non-small cell lung cancer cells, human liver cancer cells, and human breast cancer cells.

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