5alpha, 8alpha-peroxy ergosterol-quaternary ammonium salt derivative as well as preparation method and application thereof

Abstract

The invention provides a 5alpha, 8alpha-peroxy ergosterol-quaternary ammonium salt derivative as well as a preparation method and application thereof, and belongs to the technical field of medicinal chemistry. The preparation method comprises the following steps: firstly, synthesizing a peroxy-ergosterol bromate derivative by taking 5alpha, 8alpha-peroxy-ergosterol as a raw material; then, based on the molecular hybridization theory, a series of side chains with different amino substituent groups are introduced to the peroxy ergosterol bromate derivative, and a quaternary ammonium salt structure is constructed. A series of 5 alpha, 8 alpha-peroxy ergosterol-quaternary ammonium salt derivatives with clinical anti-tumor application potential are designed and synthesized. An anti-tumor activity test shows that the obtained derivative has different degrees of inhibiting effects on tumor cells of human lung cancer, liver cancer, breast cancer and the like and shows a remarkable inhibiting effect on human breast cancer MDA-MB-231 cells, and the anti-tumor activity of the derivative with the most excellent activity is improved by 69 times compared with that of a lead compound 5alpha, 8alpha-peroxy ergosterol.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical chemistry, and in particular to a 5α,8α-ergosterol peroxy-3-quaternary ammonium salt derivative, a preparation method and an application thereof. Background Art

[0002] Cancer is a major global threat to human health, with morbidity and mortality rates showing alarming trends. Current research indicates that chemotherapy remains an effective treatment for cancer, but adverse side effects severely limit the clinical application of many drugs. Therefore, the development of novel, highly effective, and low-toxic anticancer drugs is crucial for cancer treatment. In recent decades, natural products have played a crucial role in advancing cancer therapy. Their rich chemical diversity, unique biological activities, and low toxicity offer promising prospects for combating this complex disease. A literature review revealed that 113 of the 136 small-molecule anticancer drugs marketed between 1981 and 2014 were closely related to natural products. However, the majority of these small-molecule anticancer drugs are structurally modified versions of the active scaffolds of natural products. The development of directly druggable natural products, such as camptothecin, paclitaxel, and vincristine, is extremely rare. Therefore, identifying highly active natural lead compounds and modifying their structures is a key area of new drug development.

[0003] Ergosterol peroxide (EP) is a representative 5α,8α-peroxysterol extracted from the broken spores of the traditional Chinese medicinal herb Ganoderma lucidum. Literature research indicates that this compound has inhibitory effects on various cancers, including breast cancer, lung cancer, and liver cancer. However, its poor water solubility hinders its dissolution, absorption, and transport in the body, resulting in low bioavailability and limited potential for fully realizing its anti-tumor potential. Quaternary ammonium compounds, due to their unique structure and properties, hold great potential in drug development. The quaternary ammonium group exhibits excellent hydrophilicity, significantly improving the compound's water solubility and enhancing its dissolution and transport in the body. Therefore, it is of great significance to develop a 5α,8α-peroxyergosterol-3-quaternary ammonium salt derivative and its preparation method to improve the solubility of 5α,8α-peroxysterol, enhance its anti-tumor activity in vivo, and reduce its toxicity. Summary of the Invention

[0004] The purpose of the present invention is to provide a 5α,8α-peroxyergosterol-3-quaternary ammonium salt derivative and its preparation method and application, so as to solve the problems of poor water solubility, low bioavailability and poor anti-tumor activity of 5α,8α-peroxysterol in the prior art.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a 5α,8α-peroxyergosterol-3-quaternary ammonium salt derivative, wherein the 5α,8α-peroxyergosterol-3-quaternary ammonium salt derivative has a structure shown in formula (I):

[0007]

[0008] Wherein, n is selected from an integer of 3 to 5, and R is selected from

[0009] The present invention also provides a method for preparing the above-mentioned 5α,8α-peroxyergosterol-3-quaternary ammonium salt derivative, comprising the following steps:

[0010] (1) condensing 5α,8α-ergosterol peroxide and bromoalkylcarboxylic acid to obtain a 5α,8α-ergosterol peroxide-3-bromoate derivative;

[0011] (2) reacting a 5α,8α-peroxyergosterol-3-bromoate derivative with a nitrogen-containing compound to obtain a 5α,8α-peroxyergosterol-3-quaternary ammonium salt derivative.

[0012] The present invention also provides a use of the above-mentioned 5α,8α-ergosterol peroxy-3-quaternary ammonium salt derivative in the preparation of tumor prevention drugs or anti-tumor drugs.

[0013] Beneficial effects of the present invention:

[0014] This study, using 5α,8α-ergosterol peroxyperoxide as a starting material, first synthesized ergosterol peroxybromide derivatives. Based on molecular hybridization theory, a series of side chains with different amino substituents were introduced into the ergosterol peroxybromide derivatives to construct quaternary ammonium salt structures. A series of novel 5α,8α-ergosterol peroxy-3-quaternary ammonium salt derivatives with potential clinical anti-tumor applications were designed and synthesized. Antitumor activity tests showed that the resulting derivatives exhibited varying degrees of inhibitory activity against human lung, liver, and breast cancer cells, with significant inhibition against human breast cancer MDA-MB-231 cells. The most active derivative exhibited 69-fold increased anti-tumor activity compared to the lead compound, 5α,8α-ergosterol peroxyperoxide. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is a comparison chart of the in vivo toxicity evaluation of the experimental group and the blank control group in Example 4;

[0016] Figure 2These are the results of in vivo anti-tumor effect evaluation of the drug-treated group, cisplatin control group, and blank control group in Example 4, where A is a visual image of tumor tissue, B is a comparison image of tumor volume, C is a comparison image of tumor weight, and D is a comparison image of HE staining of tumor tissue. DETAILED DESCRIPTION

[0017] The present invention provides a 5α,8α-peroxyergosterol-3-quaternary ammonium salt derivative, wherein the 5α,8α-peroxyergosterol-3-quaternary ammonium salt derivative has a structure shown in formula (I):

[0018]

[0019] Wherein, n is selected from an integer of 3 to 5, and R is selected from

[0020] In the present invention, the 5α,8α-peroxyergosterol-3-quaternary ammonium salt derivative is selected from one of the following structural formulas:

[0021]

[0022]

[0023]

[0024] The present invention also provides a method for preparing the above-mentioned 5α,8α-peroxyergosterol-3-quaternary ammonium salt derivative, comprising the following steps:

[0025] (1) condensing 5α,8α-ergosterol peroxide and bromoalkylcarboxylic acid to obtain a 5α,8α-ergosterol peroxide-3-bromoate derivative;

[0026] (2) reacting a 5α,8α-peroxyergosterol-3-bromoate derivative with a nitrogen-containing compound to obtain a 5α,8α-peroxyergosterol-3-quaternary ammonium salt derivative.

[0027] In the present invention, in step (1), the bromoalkyl carboxylic acid comprises 4-bromobutyric acid, 5-bromovaleric acid or 6-bromohexanoic acid; and step (1) is carried out in the presence of a solvent, a catalyst and a dehydrating agent.

[0028] In the present invention, the solvent is preferably dichloromethane; the catalyst is 4-dimethylaminopyridine; and the dehydrating agent comprises one or more of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate, N,N-diisopropylethylamine, 1,3-diisopropylcarbodiimide and N,N-dicyclohexylcarbodiimide.

[0029] In the present invention, the mass volume ratio of the 5α,8α-ergosterol peroxyester and the solvent is 1-5 mmol:45-55 mL; the molar ratio of the 5α,8α-ergosterol peroxyester, the catalyst and the dehydrating agent is 1-5:0.1-0.15:1-3.

[0030] In the present invention, in step (1), the molar ratio of the 5α,8α-ergosterol peroxide to the bromoalkyl carboxylic acid is 1-5:3-5.

[0031] In the present invention, in step (1), the condensation reaction temperature is 15-25° C., and the reaction time is 6-8 h, preferably 6 h, 7 h, or 8 h.

[0032] In the present invention, in step (2), the nitrogen-containing compound comprises triethylamine, triallylamine, N,N-dimethylbutylamine, tripentylamine, N-methylpiperidine, N-methylmorpholine, isoquinoline, N,N-dimethylaniline, and N,N-dimethylbenzylamine; and the molar ratio of the 5α,8α-peroxyergosterol-3-bromate derivative to the nitrogen-containing compound is 0.1-1.0:6-15.

[0033] In the present invention, step (2) is carried out in the presence of a solvent, wherein the solvent comprises one or more of toluene, acetonitrile, n-butanol, tetrahydrofuran and N,N-dimethylformamide, preferably acetonitrile.

[0034] In the present invention, the reaction temperature in step (2) is 70-85° C., and the reaction time is 12-24 h.

[0035] The present invention also provides a use of the above-mentioned 5α,8α-ergosterol peroxy-3-quaternary ammonium salt derivative in the preparation of tumor prevention drugs or anti-tumor drugs.

[0036] The technical solutions provided by the present invention are 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.

[0037] Example 1

[0038] (1) 5α,8α-peroxyergosterol-3-pentylbromide Preparation:

[0039] 5α,8α-Peroxyergosterol (500 mg, 1.17 mmol) and 5-bromovaleric acid (847.22 mg, 4.68 mmol) were dissolved in dichloromethane (DCM, 50 mL), 4-dimethylaminopyridine (DMAP, 15 mg, 0.12 mmol) was added as a catalyst, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl, 449 mg, 2.34 mmol) was added as a dehydrating agent, and the mixture was reacted at 25°C for 6 h. The reaction endpoint was detected by thin layer chromatography, and the crude product was purified by silica gel column chromatography. The column developing solvent ratio was V 石油醚 :V 乙酸乙酯 =20:1 to obtain 5α,8α-peroxyergosterol-3-pentylbromide as a white solid with a yield of 94%.

[0040] 1 H NMR(600MHz,Chloroform-d)δ6.51(d,J=8.5Hz,1H),6.23(d,J=8.5Hz,1H),5.22(dd,J=15.2,7.6Hz,1H),5.14(dd,J=15.3,8.4Hz,1H),5.00( dt,J=11.7,6.1Hz,1H),3.41(t,J=6.6Hz,2H),2.30(t,J=7.3Hz,2H),2.14-2.10(m,1H),2.04-1.99(m,3H),1.95(dd,J=9.2,3.2Hz,2H),1.90- 1.87(m,2H),1.87-1.84(m,1H),1.77(d,J=7.6Hz,2H),1.72-1.68(m,1 H),1.63-1.59(m,1H),1.57(dd,J=11.4,8.2Hz,2H),1.53-1.49(m,2H), 1.48-1.45(m,1H),1.43-1.39(m,1H),1.35(d,J=10.6Hz,1H),1.29-1. 17(m,4H),1.00(d,J=6.6Hz,3H),0.92-0.89(m,6H),0.84-0.81(m,9H). 13C NMR(150MHz,Chloroform-d)δ172.15,135.21,135.07,132.32,130.95,81.75,79.41,69.52,56.18,51.61,51.01,44.56,42.78,39.75, 39.30,36.96,34.28,33.55,33.20,33.11,33.07,31.98,28.65,26.32,23.52,23.38,20.89,20.63,19.97,19.65,18.09,17.58,12.89.

[0041] (2) Preparation of 5α,8α-peroxyergosterol-3-((5-triethylamine-1)-pentanoic acid)-methyl ester (I-1):

[0042] 5α,8α-Peroxyergosterol-3-pentanobromide (80 mg, 0.14 mmol) was dissolved in acetonitrile, and 8.4 mmol of triethylamine was added. The mixture was heated to 80°C and refluxed for 24 hours. The reaction endpoint was monitored by thin-layer chromatography. After completion of the reaction, the mixture was distilled under reduced pressure and separated and purified by neutral alumina column chromatography (dichloromethane:methanol = 50-20:1) to obtain the white powder product, 5α,8α-peroxyergosterol-3-((5-triethylamine-1)-pentanoic acid)-methyl ester, in an 82% yield.

[0043] 1H NMR(600MHz,Chloroform-d)δ6.51(d,J=8.5Hz,1H),6.24(d,J=8.5Hz,1H),5.22(dd,J=15.2,7.7Hz,1H),5.14(dd,J=15.3,8.4Hz,1H),4.95(dt ,J=11.7,6.1Hz,1H),3.48(d,J=7.5Hz,6H),3.40-3.37(m,2H),2.41(t, J=7.0Hz,2H),2.11-2.08(m,1H),2.06-1.99(m,3H),1.97(dd,J=9.0,5.8 Hz,2H),1.87-1.84(m,1H),1.82-1.79(m,2H),1.77-1.75(m,1H),1.71(dd,J=10.0,5.6Hz,3H),1.60(d,J=3.2Hz,1H),1.56(d,J=8.8Hz,2H),1. 50-1.47(m,2H),1.46(s,1H),1.38(s,10H),1.23(td,J=13.4,11.6,7.1 Hz, 4H), 1.00 (d, J = 6.5Hz, 3H), 0.91 (d, J = 7.7Hz, 6H), 0.84-0.81 (m, 9H). 13 C NMR(150MHz,Chloroform-d)δ172.09,135.16,135.04,132.33,130.91,81. 82,79.49,69.68,57.21,56.17,53.53×3,51.61,51.06,44.57,42.76,39.71 ,39.26,36.95,34.33,33.29,33.20,33.04,28.62,26.27,23.35,21.63,21. 36,20.86,20.63,19.95,19.63,18.08,17.57,12.86,8.06×3.HRMS(ESI)m / z Calcd for C 39 H 66 NO4[M-Br] + :612.4986,found:612.5357.

[0044] Example 2

[0045] The difference from Example 1 is that triethylamine is replaced by triallylamine, and other conditions are the same to obtain a white powder product 5α,8α-peroxyergosterol-3-((5-triallylamine-1)-pentanoic acid)-methyl ester (Ⅰ-2) with a yield of 56%.

[0046] 1 1H NMR (600 MHz, Chloroform-d) δ 6.51 (d, J = 8.5 Hz, 1H), 6.24 (d, J = 8.5 Hz, 1H), 6.06 - 6.01 (m, 3H), 5.80 (d, J = 16.8 Hz, 3H), 5.75 (d, J = 10.1 Hz, 3H), 5.22 (dd, J = 15.2, 7.6 Hz, 1H), 5.14 (dd, J = 15.3, 8.5 Hz, 1H), 5.00 - 4.95 (m, 1H), 4.16 (d, J = 7.2 Hz, 6H), 3.37 (d, J = 9.4 Hz, 2H), 2.40 (t, J = 6.9 Hz, 2H), 2.09 (d, J = 5.2 Hz, 1H), 2.03 - 1.97 (m, 5H), 1.93 - 1.87 (m, 2H), 1.85 (d, J = 6.7 Hz, 1H), 1.73 - 1.70 (m, 1H), 1.66 (t, J = 7.4 Hz, 2H), 1.60 - 1.57 (m, 2H), 1.55 (d, J = 7.0 Hz, 1H), 1.51 (dd, J = 9.3, 6.1 Hz, 2H), 1.47 (d, J = 6.5 Hz, 1H), 1.41 - 1.38 (m, 1H), 1.36 (d, J = 11.4 Hz, 1H), 1.26 - 1.22 (m, 4H), 1.00 (d, J = 6.6 Hz, 3H), 0.91 (d, J = 6.9 Hz, 6H), 0.84 - 0.81 (m, 9H). 13 13C NMR (150 MHz, Chloroform-d) δ 172.13, 135.18, 135.06, 132.34, 130.93, 129.57 × 3, 124.14 × 3, 81.83, 79.49, 69.78, 61.71 × 3, 58.78, 56.19, 51.62, 51.06, 44.58, 42.78, 39.73, 39.29, 36.97, 34.32, 33.21, 33.17, 33.06, 28.65, 26.30, 23.37, 21.76, 21.57, 20.87, 20.63, 19.96, 19.65, 18.10, 17.58, 12.88. HRMS (ESI) m / z Calcd for C 42 H 66 NO4 [M - Br] + : 648.4986, found: 648.5406.

[0047] Example 3

[0048] The difference from Example 1 is that triethylamine is replaced by N,N-dimethylbutylamine. Other conditions are the same, and a light yellow powder product 5α,8α-peroxyergosterol-3-((5-N-butyldimethylamine-1)-pentanoic acid)-methyl ester (Ⅰ-3) is obtained with a yield of 86%.

[0049] 1 H NMR(600MHz,Chloroform-d)δ6.51(d,J=8.5Hz,1H),6.24(d,J=8.5Hz,1H),5.22(dd,J=15.2,7.7Hz,1H),5.14(dd,J=15.3,8.4Hz,1H),4.96(dt,J=11.7, 6.1Hz,1H),3.65-3.61(m,2H),3.49(d,J=8.7Hz,2H),3.37(s,6H),2.39(t,J =7.0Hz,2H),2.12-2.08(m,1H),2.02(dd,J=15.9,9.5Hz,3H),1.99-1.95(m, 2H),1.85(d,J=6.8Hz,2H),1.83(d,J=9.6Hz,2H),1.73-1.70(m,4H),1.60-1 .59(m,1H),1.57(d,J=4.2Hz,2H),1.50(d,J=10.7Hz,2H),1.47(d,J=6.6Hz, 1H),1.43(d,J=7.3Hz,2H),1.40-1.38(m,1H),1.36(d,J=11.6Hz,1H),1.25( s,4H),1.00(dd,J=7.0,3.0Hz,6H),0.91(d,J=7.9Hz,6H),0.84-0.81(m,9H). 13 C NMR(150MHz,Chloroform-d)δ171.98,135.17,135.05,132.34,130.92,81.81,79 .47,69.69,64.01,63.58,56.17,51.61,51.18×2,51.05,44.57,42.77,39.72,39 .27,36.96,34.32,33.37,33.20,33.05,29.69,28.62,26.29,24.63,23.36,22.0 6,21.52,20.87,20.63,19.95,19.64,18.09,17.57,13.73,12.87.HRMS(ESI)m / z Calcd forC 39 H 66 NO4[M-Br] +:612.4986,found:612.5631.

[0050] Example 4

[0051] The difference from Example 1 is that triethylamine is replaced by tripentylamine. Other conditions are the same, and a light yellow powder product 5α,8α-peroxyergosterol-3-((5-tripentylamine-1)-pentanoic acid)-methyl ester (Ⅰ-4) is obtained with a yield of 81%.

[0052] 1 H NMR(600MHz,Chloroform-d)δ6.51(d,J=8.5Hz,1H),6.24(d,J=8.5Hz,1H),5.22(dd,J=15.2,7.7Hz,1H),5.14(dd,J=15.3,8.4Hz,1H),4 .97(dt,J=11.7,6.0Hz,1H),3.51(s,2H),3.37-3.32(m,6H),2.42(d,J=4.0Hz,2H),2.09(dd,J=13.7,5.3Hz,1H),2.05-1.95(m,5H),1.89 (d,J=13.3Hz,2H),1.83(dt,J=15.9,7.4Hz,3H),1.73-1.68(m,8H),1.59(d,J=6.8Hz,1H),1.57(s,1H),1.55(d,J=7.0Hz,1H),1.52-1.4 8(m,2H),1.47(d,J=6.5Hz,2H),1.41-1.37(m,12H),1.25(d,J=8.1Hz,4H),1.00(d,J=6.6Hz,3H),0.94-0.90(m,15H),0.84-0.81(m,9H). 13 C NMR(150MHz,Chloroform-d)δ172.14,135.18,135.05,132.33,130.90,81.80,79.4 6,69.68,59.30×3,59.10,56.19,51.62,51.06,44.57,42.77,39.72,39.29,36.95, 34.31,33.24,33.18,33.05,28.63×3,28.45,26.30,23.36,22.25×3,22.01×3,21.6 3,21.61,20.87,20.62,19.95,19.63,18.08,17.57,13.85×3,12.87.HRMS(ESI)m / z Calcd for C 48 H 84NO4[M-Br] + :738.6395,found:738.7132.

[0053] Example 5

[0054] The difference from Example 1 is that triethylamine is replaced by N-methylpiperidine, and other conditions are the same to obtain a white powder product 5α,8α-peroxyergosterol-3-((5-N-methylpiperidine-1)-pentanoic acid)-methyl ester (Ⅰ-5) with a yield of 63%.

[0055] 1 H NMR(600MHz,Chloroform-d)δ6.51(d,J=8.5Hz,1H),6.24(d,J=8.5Hz,1H),5.22(dd,J=15.3,7.7Hz,1H),5.14(dd,J=15.3,8.4Hz,1H),4.95(dt,J=11.7 ,6.1Hz,1H),3.74-3.65(m,6H),3.32(s,3H),2.40(t,J=7.0Hz,2H),2.10(d, J=8.4Hz,1H),2.02(dd,J=16.4,9.0Hz,3H),1.98-1.96(m,1H),1.96(d,J=4. 7Hz,1H),1.92-1.89(m,4H),1.84(dt,J=10.6,6.9Hz,4H),1.77-1.75(m,1H ),1.74-1.70(m,3H),1.61(d,J=11.0Hz,1H),1.58-1.55(m,2H),1.53(d,J=1 0.6Hz,1H),1.51-1.47(m,2H),1.41-1.38(m,1H),1.37-1.34(m,1H),1.27-1 .22(m,4H),1.00(d,J=6.6Hz,3H),0.91(d,J=7.6Hz,6H),0.84-0.81(m,9H). 13C NMR(150MHz,Chloroform-d)δ172.08,135.16,135.06,132.33,130.91,81.83,79 .48,69.66,61.03,61.00,56.17,51.61,51.05,48.14,44.57,42.76,39.71,39.2 7,36.96,34.33,33.44,33.21,33.04,29.69,28.62,26.28,23.35,21.65,21.31, 20.87,20.79,20.62,20.18×2,19.95,19.63,18.09,17.57,12.87.HRMS(ESI)m / z Calcdfor C 39 H 64 NO4[M-Br] + :610.4830,found:610.5169.

[0056] Example 6

[0057] The difference from Example 1 is that triethylamine is replaced by N-methylmorpholine. Other conditions are the same to obtain a yellow powder product 5α,8α-peroxyergosterol-3-((5-N-methylmorpholine-1)-pentanoic acid)-methyl ester (Ⅰ-6) with a yield of 78%.

[0058] 1H NMR(600MHz,Chloroform-d)δ6.50(d,J=8.5Hz,1H),6.25(d,J=8.5Hz,1H),5.22(dd,J=15.3,7.7Hz,1H),5.14(dd,J=15.2,8.5Hz,1H),4.96-4.9 1(m,1H),4.08-4.03(m,4H),3.89(d,J=8.8Hz,2H),3.83-3.79(m,2H),3 .69-3.65(m,2H),3.51(s,3H),2.41(t,J=7.1Hz,2H),2.11(dd,J=13.9,5 .0Hz,1H),2.02(dd,J=16.0,9.7Hz,3H),1.97(d,J=3.6Hz,2H),1.87(dd,J=13.8,6.8Hz,4H),1.76-1.71(m,4H),1.60(d,J=11.2Hz,1H),1.55(d, J=7.3Hz,2H),1.53(s,1H),1.50-1.47(m,2H),1.40-1.33(m,2H),1.27-1 .19(m,4H),1.00(d,J=6.6Hz,3H),0.92-0.90(m,6H),0.84-0.81(m,9H). 13 C NMR(150MHz,Chloroform-d)δ172.21,135.16,135.09,132.36,130.91,81. 90,79.53,69.67,60.75×4,59.84,59.81,56.17,51.61,51.07,44.58,42.78 ,39.72,39.26,36.97,34.40,33.37,33.22,33.05,28.62,26.27,23.36,21 .55,21.15,20.87,20.65,19.96,19.65,18.10,17.59,12.88.HRMS(ESI)m / z Calcd for C 38 H 62 NO5[M-Br] + :612.4623,found:612.4981.

[0059] Example 7

[0060] The difference from Example 1 is that triethylamine is replaced by isoquinoline. Other conditions are the same, and a yellow product 5α,8α-peroxyergosterol-3-((5-isoquinoline-1)-pentanoic acid)-methyl ester (I-7) is obtained with a yield of 75%.

[0061] 1 1H NMR (600 MHz, Chloroform-d) δ 11.04 (s, 1H), 8.88 (d, J = 6.7 Hz, 1H), 8.71 (d, J = 8.3 Hz, 1H), 8.36 (d, J = 6.7 Hz, 1H), 8.13 (dt, J = 15.1, 8.1 Hz, 2H), 7.95 (t, J = 7.5 Hz, 1H), 6.51 - 6.48 (m, 1H), 6.22 (d, J = 8.5 Hz, 1H), 5.22 (dd, J = 15.3, 7.6 Hz, 1H), 5.16 (d, J = 8.4 Hz, 1H), 5.13 (d, J = 8.3 Hz, 2H), 4.90 (dt, J = 11.7, 6.1 Hz, 1H), 2.37 (t, J = 7.1 Hz, 2H), 2.21 (t, J = 7.9 Hz, 2H), 2.06 (dd, J = 13.9, 5.4 Hz, 1H), 2.03 - 1.93 (m, 5H), 1.85 (d, J = 6.8 Hz, 1H), 1.74 (d, J = 6.9 Hz, 2H), 1.70 - 1.67 (m, 1H), 1.57 (d, J = 6.2 Hz, 2H), 1.52 - 1.44 (m, 4H), 1.40 - 1.35 (m, 2H), 1.26 - 1.21 (m, 4H), 1.00 (d, J = 6.5 Hz, 3H), 0.91 (d, J = 6.9 Hz, 3H), 0.88 (s, 3H), 0.84 - 0.81 (m, 9H). 13 13C NMR (150 MHz, Chloroform-d) δ 172.21, 150.59, 137.31, 137.08, 135.17, 135.08, 134.57, 132.3, 131.38, 131.31, 130.84, 127.93, 127.09, 126.32, 81.84, 79.47, 69.57, 60.98, 56.17, 51.62, 51.06, 44.57, 42.76, 39.71, 39.27, 36.93, 34.32, 33.38, 33.15, 33.04, 30.95, 28.62, 26.23, 23.34, 21.21, 20.87, 20.63, 19.95, 19.63, 18.06, 17.57, 12.86. HRMS (ESI) m / z Calcd for C 42 H 58 NO4 [M - Br] + : 640.4360, found: 640.4369.

[0062] Example 8

[0063] The difference from Example 1 is that triethylamine is replaced by N,N-dimethylaniline. Other conditions are the same, and a light yellow powder product 5α,8α-peroxyergosterol-3-((5-N,N-dimethylaniline-1)-pentanoic acid)-methyl ester (Ⅰ-8) is obtained with a yield of 48%.

[0064] 1 H NMR(600MHz,Chloroform-d)δ7.96(d,J=8.3Hz,2H),7.62(t,J=7.8Hz,2H),7.52(t,J= 7.4Hz,1H),6.50(d,J=8.5Hz,1H),6.22(d,J=8.5Hz,1H),5.22(dd,J=15.2,7.7Hz,1H) ,5.14(dd,J=15.3,8.5Hz,1H),4.87(dt,J=11.6,6.1Hz,1H),4.48(dt,J=9.6,3.6Hz,2 H),3.96(s,6H),2.25(t,J=7.2Hz,2H),2.03-2.01(m,1H),1.97-1.92(m,3H),1.85(q,J =6.8Hz,1H),1.81-1.77(m,1H),1.75(d,J=9.7Hz,1H),1.68(s,1H),1.66(d,J=3.5Hz, 1H), 1.63 (t, J = 7.5Hz, 2H), 1.57 (d, J = 4.3Hz, 1H), 1.54 (d, J = 7.4Hz, 1H), 1.50 (d, J = 11. 2Hz,2H),1.48-1.44(m,3H),1.40-1.37(m,1H),1.37-1.34(m,1H),1.27-1.21(m,4H), 1.00(d,J=6.6Hz,3H),0.91(d,J=6.8Hz,3H),0.88(s,3H),0.82(dd,J=9.5,6.4Hz,9H). 13C NMR(150MHz,Chloroform-d)δ171.82,144.36,135.17,135.08,132.30,130.86×2,130 .84,130.47,120.83×2,81.78,79.42,69.51,68.24,56.15,55.08,55.05,51.60,51.0 2,44.54,42.75,39.71,39.26,36.92,34.28,33.44,33.10,33.03,28.62,26.18,23.3 4,22.91,21.29,20.86,20.61,19.94,19.62,18.07,17.57,12.86.HRMS(ESI)m / zCalcd for C 41 H 62 NO4[M-Br] + :632.4673,found:632.5098.

[0065] Example 9

[0066] The difference from Example 1 is that triethylamine is replaced by N,N-dimethylbenzylamine. Other conditions are the same, and a white powder product 5α,8α-peroxyergosterol-3-((5-N,N-dimethylbenzylamine-1)-pentanoic acid)-methyl ester (Ⅰ-9) is obtained with a yield of 88%.

[0067] 11H NMR (600 MHz, Chloroform-d) δ 7.66 (d, J = 7.3 Hz, 2H), 7.48 (s, 1H), 7.45 (d, J = 7.2 Hz, 2H), 6.50 (d, J = 8.5 Hz, 1H), 6.24 (d, J = 8.5 Hz, 1H), 5.22 (dd, J = 15.3, 7.7 Hz, 1H), 5.14 (dd, J = 15.3, 8.4 Hz, 1H), 4.99 (s, 2H), 4.98 - 4.94 (m, 1H), 3.65 - 3.61 (m, 2H), 3.28 (s, 6H), 2.38 (t, J = 7.0 Hz, 2H), 2.11 (dd, J = 13.9, 5.1 Hz, 1H), 2.02 (dd, J = 13.6, 11.8 Hz, 3H), 1.98 (d, J = 3.7 Hz, 1H), 1.97 - 1.94 (m, 2H), 1.91 (d, J = 8.3 Hz, 1H), 1.85 (d, J = 6.8 Hz, 1H), 1.72 (d, J = 3.2 Hz, 1H), 1.69 (d, J = 3.8 Hz, 1H), 1.67 (t, J = 7.6 Hz, 2H), 1.58 - 1.55 (m, 2H), 1.50 (dd, J = 10.1, 7.6 Hz, 2H), 1.47 - 1.45 (m, 1H), 1.38 (dd, J = 10.7, 7.1 Hz, 2H), 1.27 - 1.20 (m, 4H), 1.00 (d, J = 6.6 Hz, 3H), 0.92 - 0.90 (m, 6H), 0.84 - 0.81 (m, 9H). 13 13C NMR (150 MHz, Chloroform-d) δ 172.02, 135.18, 135.06, 133.24×2, 132.34, 130.92, 130.82, 129.30×2, 127.19, 81.83, 79.48, 69.73, 67.49, 63.38, 56.17, 51.61, 51.05, 49.76, 44.57, 42.77, 39.73, 39.28, 36.96, 34.33, 33.34, 33.20, 33.05, 28.63, 26.28, 23.36, 22.21, 21.52, 20.87, 20.63, 19.96, 19.64, 18.10, 17.58, 12.88. HRMS(ESI) m / z Calcd for C 42 1 64 4 + NO4[M - Br]

[0068] Example 10

[0069] (1) 5α,8α-peroxyergosterol-3-butylbromide Preparation:

[0070] 5α,8α-Peroxyergosterol (500 mg, 1.17 mmol) and 4-bromobutyric acid (781.56 mg, 4.68 mmol) were dissolved in dichloromethane (DCM, 50 mL). 4-Dimethylaminopyridine (DMAP, 15 mg, 0.12 mmol) was added as a catalyst and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl, 449 mg, 2.34 mmol) was added as a dehydrating agent. The reaction was carried out at 25°C for 6 h. The reaction endpoint was detected by thin layer chromatography. The crude product was purified by column chromatography. The column developing solvent ratio was V 石油醚 :V 乙酸乙酯 =20:1, and a white powder of 5α,8α-ergosterol-3-butylbromide was obtained with a yield of 80%.

[0071] 1 H NMR(600MHz,Chloroform-d)δ6.44(d,J=8.5Hz,1H),6.16(d,J=8.5Hz,1H),5.15(dd,J=15.3,7.6Hz,1H),5.08(dd,J=15.2,8.4Hz,1 H),4.97(m,1H),3.39(t,J=6.4Hz,2H),2.39(d,J=7.2Hz,2H),2.11(m,2H),2.05(d,J=3.5Hz,1H),1.95(s,2H),1.88(d,J=8.8Hz,2H ),1.78(s,1H),1.68(s,1H),1.63(d,J=13.6Hz,1H),1.54-1.48(m,3H),1.44(d,J=8.3Hz),1.40(m,1H),1.36(m,1H),1.30(s,1H),1 .20-1.15(m,3H),1.14(s,1H),0.93(d,J=6.7Hz,3H),0.84(d,J=8.4Hz,6H),0.77(s,3H),0.75(d,J=3.1Hz),0.74(d,J=3.1Hz,3H). 13C NMR(150MHz,Chloroform-d)δ170.6,134.2,134.0,131.3,129.9,80.7,78.4,68.7,55.2,50.6,50.0,43.5,41.8, 38.7,38.3,35.9,33.3,32.2,32.0,31.8,31.7,27.6,26.7,25.3,22.4,19.9,19.6,18.9,18.6,17.1,16.6,11.9.

[0072] (2) Preparation of 5α,8α-peroxyergosterol-3-((4-triallylamine-1)-butyric acid)-methyl ester (I-10):

[0073] 5α,8α-Peroxyergosterol-3-butylbromide (80 mg, 0.14 mmol) was dissolved in acetonitrile, and 8.4 mmol of triallylamine was added. The mixture was heated to 80°C under reflux and allowed to react for 20 hours. The reaction endpoint was monitored by thin-layer chromatography. After completion of the reaction, the mixture was distilled under reduced pressure and separated and purified by neutral alumina column chromatography (dichloromethane:methanol = 50-20:1) to obtain the white powder product, 5α,8α-peroxyergosterol-3-((4-triallylamine-1)-butyric acid)-methyl ester (I-10), in a yield of 52%.

[0074] 11H NMR (600 MHz, Chloroform-d) δ 6.51 (d, J = 8.5 Hz, 1H), 6.24 (d, J = 8.5 Hz, 1H), 6.12 (td, J = 10.0, 7.2 Hz, 3H), 5.79 (d, J = 16.8 Hz, 3H), 5.74 (d, J = 10.2 Hz, 3H), 5.22 (dd, J = 15.2, 7.7 Hz, 1H), 5.14 (dd, J = 15.2, 8.5 Hz, 1H), 4.96 (dt, J = 11.7, 6.1 Hz, 1H), 4.14 (d, J = 7.2 Hz, 6H), 3.50 - 3.46 (m, 2H), 2.54 - 2.49 (m, 2H), 2.23 - 2.19 (m, 2H), 2.12 - 2.09 (m, 1H), 2.05 - 1.99 (m, 3H), 1.99 - 1.95 (m, 2H), 1.87 (d, J = 5.8 Hz, 1H), 1.85 (d, J = 6.7 Hz, 1H), 1.74 - 1.71 (m, 1H), 1.61 - 1.59 (m, 1H), 1.58 - 1.55 (m, 2H), 1.50 (d, J = 10.7 Hz, 2H), 1.41 - 1.38 (m, 1H), 1.37 (s, 1H), 1.26 - 1.22 (m, 4H), 1.00 (d, J = 6.6 Hz, 3H), 0.92 - 0.90 (m, 6H), 0.84 - 0.81 (m, 9H). 13 13C NMR (150 MHz, Chloroform-d) δ 171.54, 135.16, 134.99, 132.32, 130.92, 129.37 × 3, 124.24 × 3, 81.79, 79.48, 70.24, 61.61 × 3, 58.03, 56.17, 51.59, 51.03, 44.56, 42.75, 39.70, 39.26, 36.93, 34.29, 33.12, 33.03, 30.23, 28.62, 26.23, 23.34, 20.86, 20.61, 19.94, 19.62, 18.07, 17.88, 17.57, 12.86. HRMS (ESI) m / z Calcd for C 41 H 64 NO4 [M - Br] + : 634.4830, found: 634.4838.

[0075] Example 11

[0076] The difference from Example 10 is that triethylamine is replaced by N,N-dimethylbutylamine, and other conditions are the same to obtain a light yellow powder product 5α,8α-peroxyergosterol-3-((4-N-butyldimethylamine-1)-butyric acid)-methyl ester (Ⅰ-11) with a yield of 84%.

[0077] 1 H NMR(600MHz,Chloroform-d)δ6.51(d,J=8.5Hz,1H),6.24(d,J=8.5Hz,1H),5.22(dd,J=15.2,7.7Hz,1H),5.14(dd,J=15.3,8.5Hz,1H),4.9 7(dt,J=11.7,6.2Hz,1H),3.69(dd,J=9.9,4.7Hz,2H),3.50-3.47(m,2H),3.37(s,6H),2.50(t,J=6.6Hz,2H),2.13-2.11(m,1H),2.06-2.01 (m,5H),1.99-1.95(m,2H),1.89-1.86(m,1H),1.85(d,J=6.7Hz,1H),1.74(ddt,J=12.8,6.2,3.2Hz,4H),1.59-1.55(m,2H),1.51-1.48(m, 2H),1.44(d,J=7.3Hz,2H),1.40-1.38(m,1H),1.37(s,1H),1.26-1.22(m,4H),1.01(t,J=6.9Hz,6H),0.92-0.90(m,6H),0.84-0.81(m,9H). 13 C NMR(150MHz,Chloroform-d)δ171.39,135.16,135.00,132.34,130.94,81.82, 79.51,70.26,64.11,62.62,56.17,51.60,51.28×2,51.04,44.57,42.77,39.72 ,39.27,36.95,34.31,33.14,33.05,30.09,29.70,28.63,26.25,24.48,23.36 ,20.87,20.62,19.96,19.64,18.08,17.98,17.57,13.70,12.87.HRMS(ESI)m / z Calcd for C 38 H 64 NO4[M-Br] + :598.4830,found:598.4838.

[0078] Example 12

[0079] The difference from Example 10 is that triethylamine is replaced by tripentylamine. Other conditions are the same, and a white powder product 5α,8α-peroxyergosterol-3-((4-tripentylamine-1)-butyric acid)-methyl ester (Ⅰ-12) is obtained with a yield of 88%.

[0080] 1 H NMR(600MHz,Chloroform-d)δ6.47(d,J=8.5Hz,1H),6.19(d,J=8.5Hz,1H),5.18(dd,J=15.2,7.7Hz,1H),5.10(dd,J=15.3,8.4Hz,1H),4.9 3(dt,J=11.7,6.2Hz,1H),3.59-3.54(m,2H),3.33-3.25(m,8H),2.54(d,J=7.3Hz,2H),2.08-2.05(m,1H),1.99-1.94(m,5H),1.83(d,J=3.9 Hz,1H),1.81(d,J=6.8Hz,1H),1.69(q,J=6.9,4.9Hz,8H),1.56-1.53(m,2H),1.50(d,J=7.4Hz,1H),1.47-1.44(m,2H),1.42(d,J=6.5Hz,1 H),1.34(dq,J=7.7,4.0Hz,12H),1.21-1.17(m,4H),0.95(d,J=6.6Hz,3H),0.88(d,J=7.7Hz,9H),0.85(d,J=2.9Hz,6H),0.79-0.76(m,9H). 13 C NMR(150MHz,Chloroform-d)δ171.66,135.16,135.02,132.30,130.88,81.80,79 .47,70.13,59.27×3,58.28,56.17,51.61,51.05,44.55,42.75,39.70,39.26,36. 92,34.28,33.11,33.02,30.03,28.61,28.45×3,26.23,23.34,22.21×3,21.85×3 ,20.84,20.60,19.93,19.62,18.04,17.64,17.54,13.85×3,12.85.HRMS(ESI)m / z Calcd for C 47 H 82 NO2[M-Br] + :724.6238,found:724.6957.

[0081] Example 13

[0082] (1) 5α,8α-peroxyergosterol-3-hexylbromide Preparation:

[0083] 5α,8α-Peroxyergosterol (500 mg, 1.17 mmol) and 6-bromohexanoic acid (912.61 mg, 4.68 mmol) were dissolved in dichloromethane (DCM, 50 mL). 4-Dimethylaminopyridine (DMAP, 15 mg, 0.12 mmol) was added as a catalyst and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl, 449 mg, 2.34 mmol) was added as a dehydrating agent. The reaction was carried out at 25°C for 6 h. The reaction endpoint was detected by thin layer chromatography. The crude product was purified by column chromatography. The column developing solvent ratio was V 石油醚 :V 乙酸乙酯 =20:1, and a white powder 5α,8α-peroxyergosterol-3-hexylbromide was obtained with a yield of 94%.

[0084] 1 H NMR(600MHz,Chloroform-d)δ6.47(d,J=8.5Hz,1H),6.20(d,J=8.5Hz,1H),5.19(dd,J=15.2,7.6Hz,1H),5.11(dd,J=15.3,8.4Hz,1H ),4.96(tt,J=11.3,5.2Hz,1H),3.37(td,J=6.8,2.4Hz,4H),2.30(t,J=7.5Hz,2H),2.25(t,J=7.4Hz,2H),2.11-2.07(m,1H),1.98(dd ,J=13.9,11.3Hz,3H),1.93-1.89(m,2H),1.85-1.83(m,2H),1.63-1.61(m,2H),1.55-1.51(m,2H),1.45(td,J=7.4,7.0,3.2Hz,4H),1 .40-1.36(m,1H),1.34(d,J=9.7Hz,1H),1.20(dt,J=9.8,3.9Hz,4H),0.97(d,J=6.6Hz,3H),0.87(d,J=7.5Hz,6H),0.81-0.77(m,9H). 13C NMR (150MHz, Chloroform-d) δ172.43,135.21,135.08,132.27,130.89,81.72,79.35,69.34,56.16,51.59,51.02,44.53,42.76,39.73,39. 29,36.94,34.29,33.80,33.56,33.19,33.05,32.38,28.64,26.30,2 4.08,24.05,23.36,20.88,20.61,19.96,19.64,18.08,17.58,12.87.

[0085] (2) Preparation of 5α,8α-peroxyergosterol-3-((6-triallylamine-1)-hexanoic acid)-methyl ester (I-13):

[0086] 5α,8α-Peroxyergosterol-3-butylbromide (80 mg, 0.14 mmol) was dissolved in acetonitrile, and 8.4 mmol of triallylamine was added. The mixture was heated to 80°C under reflux and allowed to react for 20 hours. The reaction endpoint was monitored by thin-layer chromatography. After completion of the reaction, the mixture was evaporated under reduced pressure and separated and purified by neutral alumina column chromatography (dichloromethane:methanol = 50-20:1) to obtain the yellow powder product, 5α,8α-peroxyergosterol-3-((6-triallylamine-1)-hexanoic acid)-methyl ester (I-13), in a yield of 78%.

[0087] 11H NMR (600 MHz, Chloroform-d) δ 6.43 (d, J = 8.5 Hz, 1H), 6.17 (d, J = 8.5 Hz, 1H), 5.99 (ddt, J = 17.2, 10.2, 7.2 Hz, 3H), 5.73 (d, J = 16.8 Hz, 3H), 5.65 (d, J = 10.2 Hz, 3H), 5.15 (dd, J = 15.2, 7.7 Hz, 1H), 5.07 (dd, J = 15.3, 8.5 Hz, 1H), 4.87 (tt, J = 11.2, 5.2 Hz, 1H), 4.08 (d, J = 7.2 Hz, 6H), 3.26 - 3.20 (m, 2H), 2.22 (t, J = 7.2 Hz, 2H), 2.03 - 2.00 (m, 1H), 1.97 - 1.92 (m, 2H), 1.89 (dd, J = 10.7, 3.2 Hz, 2H), 1.84 (td, J = 8.1, 4.1 Hz, 2H), 1.82 - 1.79 (m, 1H), 1.79 - 1.76 (m, 1H), 1.66 (ddd, J = 18.2, 9.4, 3.3 Hz, 2H), 1.60 - 1.57 (m, 2H), 1.52 (t, J = 2.8 Hz, 1H), 1.48 (d, J = 9.9 Hz, 2H), 1.44 - 1.40 (m, 2H), 1.31 (dt, J = 16.1, 10.4 Hz, 4H), 1.16 (td, J = 12.3, 10.1, 5.5 Hz, 4H), 0.93 (d, J = 6.6 Hz, 3H), 0.84 (d, J = 7.0 Hz, 6H), 0.77 - 0.73 (m, 9H). 13 13C NMR (150 MHz, Chloroform-d) δ 172.40, 135.13, 135.03, 132.28, 130.86, 129.21 × 3, 124.32 × 3, 81.79, 79.43, 69.39, 61.52 × 3, 58.76, 56.13, 51.59, 51.03, 44.53, 42.72, 39.68, 39.24, 36.92, 34.30, 33.94, 33.18, 33.00, 28.59, 26.26, 25.83, 24.15, 23.32, 22.07, 20.84, 20.60, 19.93, 19.60, 18.06, 17.55, 12.83. HRMS (ESI) m / z Calcd for C 43 1 68 14H + NO4[M - Br]

[0088] Example 14

[0089] The difference from Example 13 is that triallylamine is replaced by N,N-dimethylbutylamine. Other conditions are the same, and a light yellow powder product 5α,8α-peroxyergosterol-3-((6-N-butyldimethylamine-1)-hexanoic acid)-methyl ester (Ⅰ-14) is obtained with a yield of 73%.

[0090] 1 H NMR(600MHz,Chloroform-d)δ6.50(d,J=8.5Hz,1H),6.24(d,J=8.5Hz,1H),5.2 2(dd,J=15.2,7.7Hz,1H),5.14(dd,J=15.3,8.5Hz,1H),4.98-4.93(m,1H),3.55 (t,J=8.3Hz,2H),3.52-3.48(m,2H),3.35(s,6H),2.30(t,J=7.1Hz,2H),2.10(d d,J=13.8,5.1Hz,1H),2.02(dd,J=12.5,6.0Hz,3H),1.97(dd,J=14.1,5.3Hz,2H ),1.89(dd,J=9.0,4.6Hz,1H),1.87-1.84(m,1H),1.78-1.75(m,2H),1.70(ddd, J=16.5,11.5,5.5Hz,6H),1.57(d,J=3.8Hz,2H),1.51(d,J=9.1Hz,1H),1.48(d, J=7.2Hz,1H),1.43(dd,J=9.4,5.8Hz,4H),1.39-1.36(m,1H),1.34(d,J=10.2Hz ,1H),1.25(s,4H),1.02-0.99(m,6H),0.91(d,J=7.5Hz,6H),0.84-0.81(m,9H). 13C NMR(150MHz,Chloroform-d)δ172.48,135.17,135.07,132.33,130.90,81.83,79. 46,69.48,63.98,63.82,56.17,51.61,51.17×2,51.05,44.56,42.77,39.71,39.27 ,36.96,34.34,33.96,33.21,33.04,29.69,28.62,26.29,25.69,24.65,24.28,23 .35,22.57,20.87,20.63,19.95,19.64,18.09,17.57,13.74,12.86.HRMS(ESI)m / z Calcd for C 40 H 68 NO4[M-Br] + :626.5143,found:626.5153.

[0091] Example 15

[0092] The difference from Example 13 is that triallylamine is replaced by tripentylamine. Other conditions are the same, and a white product 5α,8α-peroxyergosterol-3-((6-tripentylamine-1)-hexanoic acid)-methyl ester (Ⅰ-15) is obtained with a yield of 80%.

[0093] 1H NMR(600MHz,Chloroform-d)δ6.44(d,J=8.5Hz,1H),6.17(d,J=8.5Hz,1H),5.15(dd,J=15.2,7.7Hz,1H),5.07(dd,J=15.2,8.5Hz,1H),4.92-4.87(m, 1H),3.32(d,J=8.0Hz,2H),3.27(t,J=8.5Hz,6H),2.24(t,J=7.2Hz,2H),2. 01(d,J=5.3Hz,1H),1.95(d,J=5.5Hz,3H),1.91-1.87(m,2H),1.83(d,J=11 .2Hz,1H),1.78(d,J=6.7Hz,1H),1.68-1.65(m,4H),1.61(d,J=8.5Hz,8H), 1.52(s,1H),1.51(d,J=5.7Hz,2H),1.48(d,J=7.0Hz,1H),1.44(s,1H),1.4 2(d,J=3.5Hz,1H),1.39(s,2H),1.38(s,2H),1.33-1.30(m,12H),1.18(s,4 H),0.93(d,J=6.6Hz,3H),0.86-0.83(m,15H),0.75(dd,J=6.5,3.5Hz,9H). 13 C NMR(150MHz,Chloroform-d)δ172.46,135.18,135.07,132.32,130.89,81.80,79.44 ,69.48,59.18×3,59.09,56.18,51.62,51.06,44.56,42.76,39.72,39.28,36.95,34. 32,33.93,33.20,33.04,28.62,28.43×3,26.30,25.83,24.23,23.35,22.24×3,22.1 0,21.94×3,20.86,20.62,19.95,19.63,18.08,17.57,13.85×3,12.86.HRMS(ESI)m / z Calcd for C 49 H 86 NO4[M-Br] + :752.6551,found:752.669.

[0094] Performance testing:

[0095] 1. The anti-tumor activity of the 5α,8α-peroxyergosterol-3-quaternary ammonium salt derivatives obtained in Examples 1 to 15 was tested. The test method and results are as follows:

[0096] (1) Main experimental instruments and reagents used in the test

[0097] Table 1 Main experimental instruments and reagents used in the test

[0098]

[0099]

[0100] (2) Selection of test cells and test compounds

[0101] In this test, 5α,8α-ergosterol peroxy (EP) and cisplatin were selected as compounds of the positive control group, and the 5α,8α-ergosterol peroxy-3-quaternary ammonium salt derivative obtained in Examples 1 to 15 was selected as the compound of the test group.

[0102] In this test, human liver cancer cells (HepG2), human breast cancer cells (MCF-7), human breast cancer (MDA-MB-231), human non-small cell lung cancer cells (A549) and human mammary epithelial cells (MCF-10A) were selected as test cells.

[0103] (3) Test method:

[0104] MTT method: The test cells in the logarithmic growth phase were collected and inoculated into 96-well culture plates, with 5.0×10 cells per well. 3 / 150 μL, placed in a 37 ° C, 5% CO2 incubator for culture, the next day the culture medium was removed, and 150 μL of different concentrations of compounds were added (the compound concentration was diluted in pairs, and each compound was set at 20 μmol / L, 10 μmol / L, 5 μmol / L, 2.5 μmol / L, 1.25 μmol / L, 0.625 μmol / L, 0.3125 μmol / L, 0.15625 μmol / L, a total of 8 concentrations, each test had 3 parallel wells, and repeated 3 times). No compound was added to the negative control group. After 48 h, 20 μL of 5% MTT working solution was added to each well. After further culture for 4 h, 150 μL of dimethyl sulfoxide (DMSO) was added to each well to terminate the reaction. The absorbance (OD) value of each well at 492 nm was detected by a microplate reader, and the cell growth inhibition rate was calculated.

[0105] The IC value of the drug was calculated based on the cell growth inhibition rate (%) of the drug at different drug concentrations. 50 IC 50 : The concentration of the test compound that inhibits 50% of cell growth.

[0106] (4) The anti-tumor activity test results of the 5α,8α-peroxyergosterol-3-quaternary ammonium salt derivatives obtained in Examples 1 to 15 are shown in Table 2.

[0107] Table 2 In vitro antitumor activities (IC 50 , μmol / L)

[0108] Compound A549 MCF-7 HepG2 MDA-MB-231 MCF-10A Example 1 6.82 12.86 6.99 8.26 18.47 Example 2 6.40 5.12 5.03 0.41 10.54 Example 3 7.56 3.65 7.80 2.94 9.93 Example 4 2.76 0.72 3.04 0.25 11.61 Example 5 6.74 5.41 10.05 5.29 16.19 Example 6 6.68 6.58 9.89 12.46 24.58 Example 7 4.43 5.46 5.10 2.87 10.05 Example 8 6.65 7.89 7.82 5.82 13.45 Example 9 4.68 7.72 7.42 8.97 15.27 Example 10 0.86 8.74 10.32 1.92 10.56 Example 11 6.54 13.39 16.85 5.28 12.08 Example 12 0.61 0.95 6.31 0.62 9.47 Example 13 0.63 3.59 11.86 0.83 8.95 Example 14 7.94 10.16 19.70 8.54 13.08 Example 15 0.51 3.68 8.22 1.46 11.24 Cisplatin 11.64 4.05 6.29 10.37 8.01 EP 14.95 19.42 18.05 17.26 23.45

[0109] As shown in Table 2, the inhibitory effects of the 5α,8α-peroxyergosterol-3-quaternary ammonium salt derivatives obtained in Examples 1 to 15 on the human breast cancer MDA-MB-231 cell line are generally stronger than those on other cell lines. For the MDA-MB-231 cell line, the 5α,8α-peroxyergosterol-3-quaternary ammonium salt derivatives obtained in Examples 2, 4 and 12 showed a strong inhibitory effect (IC 50 <1 μmol / L), among which the 5α,8α-peroxyergosterol-3-quaternary ammonium salt derivative obtained in Example 4 had the strongest antitumor activity, IC 50 The value was increased by 69 times compared with EP. For the MCF-10A cell line, the 5α,8α-peroxyergosterol-3-quaternary ammonium salt derivative obtained in Example 4 showed an IC 50 =11.61 μmol / L cytotoxicity, indicating that the 5α,8α-peroxyergosterol-3-quaternary ammonium salt derivative obtained in Example 4 has good selectivity (SI=46.44). Thus, the 5α,8α-peroxyergosterol-3-quaternary ammonium salt derivative obtained in the present invention has good selectivity and antitumor activity against human MDA-MB-231 cells and can be used as a lead compound for the preparation of new antitumor drugs.

[0110] 2. The water solubility of the 5α,8α-peroxyergosterol-3-quaternary ammonium salt derivatives and EP obtained in Examples 1 to 15 was tested. The test method and results are as follows:

[0111] (1) Test method: The obtained 5α,8α-peroxyergosterol-3-quaternary ammonium salt derivative and EP were determined by ultraviolet spectrophotometry. 5 mg of 5α,8α-peroxyergosterol-3-quaternary ammonium salt derivative or EP were weighed and prepared into an aqueous solution with a concentration of 1 mg / mL. The solution was ultrasonically treated at room temperature for 30 min and then filtered through a microporous filter membrane. The absorbance of the 5α,8α-peroxyergosterol-3-quaternary ammonium salt derivative and EP was measured at 206 nm using a 200-900 nm ultraviolet spectrophotometer. The water solubility of the 5α,8α-peroxyergosterol-3-quaternary ammonium salt derivative was evaluated by comparing the absorbance values at the same wavelength.

[0112] (2) The results of the water solubility test of the 5α,8α-peroxyergosterol-3-quaternary ammonium salt derivatives obtained in Examples 1 to 15 are shown in Table 3.

[0113] Table 3 Water solubility of 5α,8α-peroxyergosterol-3-quaternary ammonium salt derivatives and EP of Examples 1 to 15

[0114]

[0115]

[0116] The solubility test results show that the water solubility of the 5α,8α-peroxyergosterol-3-quaternary ammonium salt derivatives obtained in Examples 1 to 15 is significantly improved compared to the parent compound. Under the same experimental conditions, the solubility of the parent EP is set to 0.0100 mg / mL. With this as a reference, the water solubility of the compounds modified with quaternary ammonium salts generally reaches 0.3000 mg / mL, which is more than 29 times higher than that of the parent EP. Among them, the water solubility of Example 4 is increased by 87 times. This significant change is due to the positively charged nitrogen atoms in the quaternary ammonium salt structure. Its strong hydrophilicity can form stable hydration with water molecules, greatly promoting the dissolution process of the compound in the solvent, thereby improving the bioavailability of the drug and laying the foundation for the further development of new drugs with high efficiency and low toxicity.

[0117] 3. The in vivo toxicity evaluation of the 5α,8α-peroxyergosterol-3-quaternary ammonium salt derivative obtained in Example 4 was performed. The test method and results are as follows:

[0118] (1) Test method: Healthy BALB / c female mice of similar weight were selected. After adaptive feeding for one week, they were randomly divided into different experimental groups with 6 mice in each group. A blank control group and experimental groups of Example 4 with different doses were set up to ensure that the experimental results were statistically significant and repeatable. Example 4 was administered to mice by intraperitoneal injection. According to the results of the preliminary experiment, two low-dose and high-dose groups were determined, and different doses of Example 4 were administered respectively. The control group was given an equal amount of normal saline. The drug was administered once every two days for 14 days, and the general condition of the mice was closely observed during this period. After 14 days, the main organs of the mice were fixed, dehydrated, embedded, and sliced, and HE staining was performed. The tissue morphological changes were observed under an optical microscope to check whether there were pathological changes such as cell damage, inflammatory response, and necrosis, and the toxicity of Example 4 was evaluated from the histological level.

[0119] (2) The in vivo toxicity evaluation results of the 5α,8α-peroxyergosterol-3-quaternary ammonium salt derivative obtained in Example 4 are as follows: Figure 1As shown, HE staining of the heart, liver, spleen, lungs, and kidneys of the control mice showed normal tissue structure and cell morphology. The myocardial fibers of the heart were neatly arranged, and the myocardial cells were normal in morphology. The hepatocyte cords of the liver were orderly arranged, and the hepatic lobule structure was clear. The splenic corpuscles of the spleen were intact, and the lymphocytes were evenly distributed. The alveoli of the lungs were intact, and the alveolar walls and bronchial epithelial cells were morphologically normal. The glomeruli and renal tubules of the kidneys were normal in structure, and the lumen was unobstructed. HE staining of the major organs in the groups treated with different doses of compound 3d also showed no obvious abnormalities. Cell morphology, tissue structure, and cell arrangement were similar to those in the control group, with no signs of cell degeneration, necrosis, inflammatory infiltration, or tissue structural damage. This indicates that within the experimental dose range, Example 4 had no significant adverse effects on the normal structure and function of these important organs, demonstrating good in vivo safety. Therefore, in subsequent experimental protocols, the concentration gradient was set at 10 mg / kg and 20 mg / kg.

[0120] IV. The in vivo anti-tumor effect of the 5α,8α-peroxyergosterol-3-quaternary ammonium salt derivative obtained in Example 4 was evaluated. The test method and results are as follows:

[0121] (1) Test method: 4T1 cells in the logarithmic growth phase were collected and counted, and 3×10 6 Cells were inoculated into the right forelimb of BALB / c mice and the tumor volume was 100 mm. 3 BALB / c mice were randomly divided into a blank control group (Control), a treatment group according to Example 4 (treatment doses of 10 mg / kg and 20 mg / kg, respectively), and a cisplatin control group (treatment dose of 10 mg / kg), with 6 mice in each group. The treatment was intraperitoneally injected for 14 consecutive days. The body weight of the mice was recorded every 2 days, and the length and width of the tumor were measured with a vernier caliper to calculate the tumor volume. The tumor volume formula is: volume = (long diameter × short diameter) 2) / 2. After the experiment, mice were sacrificed, and subcutaneous tumors were removed, weighed, and photographed. Tumor volume inhibition rate was calculated as (1 - tumor volume of the treated tumor / tumor volume of the blank control) × 100%. Tumor tissue removed from mice was fixed with 4% paraformaldehyde for 48 hours, dehydrated, and embedded in paraffin blocks. 4μm sections were sliced using a microtome. After spreading in warm water, the sections were removed and baked in a 60°C incubator for 1 hour to ensure firm adhesion to the slides. The baked sections were dewaxed and hydrated in xylene, 100% ethanol, 95% ethanol, 80% ethanol, 70% ethanol, and distilled water, sequentially soaking for 3-5 minutes each step to restore the sections to a hydrated state. The hydrated sections were stained in hematoxylin solution for 5-10 minutes to stain the cell nuclei blue. The sections were then rinsed with tap water to remove excess dye. The sections were then stained with eosin solution for 2 minutes. Immerse the slides in 70% ethanol, 80% ethanol, 90% ethanol and anhydrous ethanol for 10 seconds each, make them transparent in xylene for 5 minutes, seal the slides with neutral resin, and observe under a microscope after the resin glue is dry.

[0122] (2) The results of the in vivo antitumor effect evaluation of the 5α,8α-peroxyergosterol-3-quaternary ammonium salt derivative obtained in Example 4 are as follows: Figure 2 As shown, tumor changes Figure 2 As shown in A, on the 14th day of the experiment, the average tumor volume of mice in the blank control group reached (1946.18±94.31) mm 3 In comparison, the tumor growth of the group treated with Example 4 was significantly inhibited. The average tumor volume of the group treated with the low concentration (10 mg / kg) was (1238.47±52.49) mm on day 14. 3 Compared with the blank control group, the volume was significantly reduced, with an inhibition rate of 36.38%. On the 14th day, the inhibitory effect of the high concentration (20 mg / kg) administration group was equivalent to that of the positive control group, with an average tumor volume of only (728.12±43.28) mm 3 , the inhibition rate was 62.59%. In addition, the tumor volume ( Figure 2 Middle B), weight change curve ( Figure 2 In Figure C), it can be seen that the tumor volume and weight of the Example 4 treatment group were statistically significantly different from those of the blank control group (p < 0.01 or p < 0.001), indicating that Example 4 can effectively inhibit the growth of tumors in breast cancer-bearing mice. Figure 2Middle D) shows that the breast cancer tissue sections of the blank control group showed that the cancer cells were tightly arranged and irregular in shape, the nuclei were large and dark in color, the nuclear-cytoplasmic ratio was significantly increased, and the cells showed obvious atypia. The changes in the medication group became more obvious as the dosage increased. In the Example 4 medication group, some cancer cells showed morphological changes, the cell volume decreased, and the cell nuclei shrank. In the high-dose group, the looseness of the cancer cells increased, the cancer cells decreased significantly, more necrotic areas were visible in the field of view, and the cell nuclei were fragmented and dissolved. This further shows that Example 4 can have a significant effect on the morphology and tissue structure of breast cancer cells and inhibit the development of tumors.

[0123] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A 5α,8α-peroxyergosterol-3-quaternary ammonium salt derivative, characterized in that The 5α,8α-peroxyergosterol-3-quaternary ammonium salt derivative has a structure shown in formula (I): Wherein, n is selected from an integer of 3 to 5, and R is selected from 2. The 5α,8α-peroxyergosterol-3-quaternary ammonium salt derivative according to claim 1, characterized in that The 5α,8α-peroxyergosterol-3-quaternary ammonium salt derivative is selected from one of the following structural formulas:

3. The method for preparing the 5α,8α-peroxyergosterol-3-quaternary ammonium salt derivative according to claim 1 or 2, characterized in that: The steps include: (1) condensing 5α,8α-ergosterol peroxide and bromoalkylcarboxylic acid to obtain a 5α,8α-ergosterol peroxide-3-bromoate derivative; (2) reacting a 5α,8α-peroxyergosterol-3-bromoate derivative with a nitrogen-containing compound to obtain a 5α,8α-peroxyergosterol-3-quaternary ammonium salt derivative.

4. The method for preparing the 5α,8α-peroxyergosterol-3-quaternary ammonium salt derivative according to claim 3, characterized in that: In step (1), the bromoalkyl carboxylic acid comprises 4-bromobutyric acid, 5-bromovaleric acid or 6-bromohexanoic acid; and step (1) is carried out in the presence of a solvent, a catalyst and a dehydrating agent.

5. The method for preparing the 5α,8α-peroxyergosterol-3-quaternary ammonium salt derivative according to claim 3 or 4, characterized in that: In step (1), the molar ratio of the 5α,8α-ergosterol peroxide to the bromoalkyl carboxylic acid is 1-5:3-5.

6. The method for preparing the 5α,8α-peroxyergosterol-3-quaternary ammonium salt derivative according to claim 5, characterized in that: In step (1), the condensation reaction temperature is 15-25° C. and the reaction time is 6-8 h.

7. The method for preparing the 5α,8α-peroxyergosterol-3-quaternary ammonium salt derivative according to claim 3, 4 or 6, characterized in that: In step (2), the nitrogen-containing compound comprises triethylamine, triallylamine, N,N-dimethylbutylamine, tripentylamine, N-methylpiperidine, N-methylmorpholine, isoquinoline, N,N-dimethylaniline, and N,N-dimethylbenzylamine; and the molar ratio of the 5α,8α-peroxyergosterol-3-bromate derivative to the nitrogen-containing compound is 0.1-1.0:6-15.

8. The method for preparing the 5α,8α-peroxyergosterol-3-quaternary ammonium salt derivative according to claim 7, characterized in that: The step (2) is carried out in the presence of a solvent, wherein the solvent comprises one or more of toluene, acetonitrile, n-butanol, tetrahydrofuran and N,N-dimethylformamide.

9. The method for preparing the 5α,8α-peroxyergosterol-3-quaternary ammonium salt derivative according to claim 6 or 8, characterized in that: The reaction temperature in step (2) is 70-85° C., and the reaction time is 12-24 h.

10. Use of the 5α,8α-peroxyergosterol-3-quaternary ammonium salt derivative according to claim 1 or 2 in the preparation of tumor prevention drugs or anti-tumor drugs.

Images