Detailed Description
In order to more clearly illustrate the technical scheme of the invention, the technical scheme of the invention is further described in detail as follows:
IC50 was 50% inhibition concentration, i.e., concentration corresponding to B/b0=50%, and half inhibition was used to measure the anti-tumor proliferation activity of the compound, with lower half inhibition indicating greater anti-proliferation activity.
Western Blot is a method of staining a gel-electrophoretically treated cell or biological tissue sample with a specific antibody, and obtaining information about the expression of a specific protein in the analyzed cell or tissue by analyzing the position and depth of staining.
1 The H-NMR spectrum is the response of hydrogen nucleus in magnetic field, and the position and number of hydrogen atom in organic molecule on carbon skeleton can be determined by utilizing nuclear magnetic resonance hydrogen spectrum, so as to deduce the carbon skeleton structure of organic substance.
The CCK-8 method is a method for cell proliferation and cytotoxicity determination, the principle of the method is based on the reduction reaction of WST-8 (water-soluble tetranitroimidazole salt), WST-8 is reduced into a water-soluble orange product, namely formazan (Formazan) dye, by mitochondrial dehydrogenase in cells in the presence of an electron coupling reagent, the yield of the product is proportional to the number of living cells, and therefore the number of cells can be indirectly reflected by measuring the absorbance of the product.
Example 1
The synthesis method of the glycyrrhetinic acid-gold (I) compound comprises the following steps:
Preparation of intermediate 2a Compound 1 (BMS-1) (1.00 g,2.10 mmol), K2CO3 (0.87 g,6.29 mmol), 1, 3-dibromopropane (0.63 g,3.15 mmol) and anhydrous DMF (20 mL) were added sequentially to a 50mL single-port flask, stirred at room temperature for 1-24h, monitored by TLC, diluted with ethyl acetate (100 mL) after completion of the reaction, rinsed sequentially with 1N HCl (10 mL) and saturated brine (10 mL. Times.3), dried over anhydrous sodium sulfate, the solvent was removed, purified by column chromatography (SiO2, EA/PE=1:10 (v: v)), concentrated, and dried to give a pale yellow oil, 1.04g, yield 84%.
Intermediate 2b was prepared in the same manner as 2a, wherein 1, 3-dibromopropane was exchanged for equimolar amounts of 1, 4-dibromobutane in 96% yield.
Intermediate 2c was prepared in the same manner as 2a, wherein 1, 3-dibromopropane was exchanged for equimolar amounts of 1, 5-dibromopentane, yield ：88％.1H NMR(500MHz,Chloroform-d)δ7.41(dt,J＝34.0,7.2Hz,5H),7.35–7.33(m,2H),7.31(d,J＝7.6Hz,1H),6.26(s,2H),5.11(s,2H),4.32–4.25(m,1H),4.16(dd,J＝11.8,5.3Hz,1H),3.94–3.85(m,2H),3.78(s,6H),3.44(dd,J＝11.4,4.8Hz,2H),3.17(s,1H),2.98(s,1H),2.91(s,1H),2.30(s,3H),2.29–2.21(m,1H),1.92(dd,J＝14.6,7.1Hz,2H),1.80–1.70(m,4H),1.68(s,2H),1.57(dt,J＝15.0,7.5Hz,4H).
Preparation of intermediate 3a 1-ethylimidazole (1.58 g,16.5 mmol) and anhydrous acetonitrile (100 mL) were added to a 100mL single neck round bottom flask, the mixture was heated to reflux with a reflux condenser, the reaction was carried out for 1-5 days, after TLC monitoring the reaction was complete, the oil bath was removed, the mixture was naturally cooled to room temperature, the solvent was removed by rotary evaporation under reduced pressure, and the column was purified (SiO2, the eluent polarity was raised from MeOH/DCM=1:100 (v: v) to MeOH/DCM=1:10 (v: v) gradient elution), concentrated, and dried to give a white solid, yield ：67.1％.1H NMR(500MHz,Chloroform-d)δ10.64(d,J＝1.8Hz,1H),8.56(s,1H),7.48–7.40(m,3H),7.40–7.35(m,1H),7.33(d,J＝1.7Hz,1H),7.32(d,J＝1.9Hz,1H),7.31–7.29(m,2H),7.25(d,J＝3.8Hz,1H),6.28(s,2H),5.14(s,2H),5.04–4.90(m,2H),4.56(s,1H),4.49(q,J＝13.8Hz,2H),4.39(ddd,J＝11.3,7.9,3.1Hz,1H),4.34(q,J＝7.4Hz,2H),3.81(s,6H),3.64(d,J＝72.4Hz,2H),2.68–2.42(m,4H),2.29(s,3H),2.24(d,J＝13.6Hz,1H),2.12(tt,J＝13.0,6.4Hz,1H),1.92(d,J＝13.6Hz,1H),1.80(d,J＝13.8Hz,1H),1.63(t,J＝7.4Hz,3H),1.39(s,1H).13C NMR(126MHz,Chloroform-d)δ162.88,160.85,143.24,141.73,136.82,134.53,134.34,130.62,129.33,128.35,128.15,127.00,125.75,124.45,120.39,91.37,69.58,55.75,46.81,45.19,30.11,21.77,16.28,15.43.
The intermediate 3b is prepared in the same way as 3a, only the raw material 2a is changed into 2b, and light yellow solid is obtained, and the yield is high ：65.2％.1HNMR(500MHz,Chloroform-d)δ10.43(d,J＝1.7Hz,1H),7.98(s,1H),7.45–7.40(m,3H),7.37(td,J＝3.4,1.7Hz,2H),7.33–7.30(m,2H),7.27(d,J＝1.8Hz,2H),6.26(s,2H),5.11(s,2H),4.53(td,J＝7.9,2.1Hz,2H),4.39(q,J＝7.4Hz,2H),4.31(dq,J＝11.1,6.0,5.5Hz,2H),4.22(d,J＝3.6Hz,2H),3.78(s,6H),3.48–3.40(m,2H),2.46(t,J＝11.7Hz,1H),2.28(s,5H),2.13–2.04(m,2H),1.89(d,J＝12.9Hz,2H),1.79–1.66(m,2H),1.61(t,J＝7.4Hz,3H),1.40–1.21(m,2H).13C NMR(126MHz,Chloroform-d)δ161.75,160.59,143.15,141.80,136.82,134.64,134.51,130.47,129.34,128.32,128.12,126.94,125.69,123.29,121.15,91.34,69.47,64.74,63.20,55.75,51.12,49.40,45.24,27.41,25.33,16.26,15.51.
The intermediate 3c is prepared in the same way as 3a, only the raw material 2a is changed into 2c, and the yellowish green solid is obtained, and the yield is ：63.4％.1H NMR(500MHz,Chloroform-d)δ10.47(s,1H),7.90(s,1H),7.44(d,J＝5.5Hz,3H),7.42(s,1H),7.36(t,J＝7.3Hz,1H),7.31(d,J＝7.2Hz,3H),7.27(d,J＝5.6Hz,1H),6.27(s,2H),5.13(s,2H),4.50(t,J＝7.3Hz,2H),4.45–4.40(m,3H),4.39–4.35(m,1H),4.31(dt,J＝11.1,5.7Hz,1H),3.80(s,6H),3.63(d,J＝12.1Hz,1H),3.56(s,1H),2.58(s,1H),2.35(s,1H),2.28(s,3H),2.25–2.18(m,1H),2.10(dd,J＝15.3,7.6Hz,2H),2.05–1.98(m,1H),1.82(ddd,J＝28.4,17.9,10.7Hz,4H),1.66(dd,J＝15.3,7.5Hz,2H),1.61(t,J＝7.4Hz,3H),1.35(dd,J＝27.3,8.5Hz,2H).13C NMR(126MHz,Chloroform-d)δ160.26,143.03,141.94,135.40,135.11,134.55,130.32,129.40,128.37,128.11,126.89,125.65,122.18,121.77,91.24,69.29,63.95,63.29,55.69,50.16,50.01,46.33,45.34,30.23,29.78,28.36,25.64,25.19,25.12,16.25,15.06.
The preparation method of the intermediates 6a-6c is the same as that of the intermediates 3a-3c, except that the raw material BMS-1 is replaced by GA;
Intermediate 6a, white solid, yield ：84.1％.1H NMR(500MHz,Chloroform-d)δ10.61(s,1H),7.61(t,J＝1.8Hz,1H),7.47(t,J＝1.7Hz,1H),5.58(s,1H),4.60–4.54(m,1H),4.46(dq,J＝18.5,7.3Hz,3H),4.24(dp,J＝23.8,5.9Hz,2H),3.24(dd,J＝11.0,5.3Hz,1H),2.77(dt,J＝13.5,3.6Hz,1H),2.40(p,J＝7.0Hz,2H),2.36(s,1H),2.03(ddt,J＝16.9,12.9,3.6Hz,3H),1.93–1.87(m,2H),1.82(dd,J＝13.6,4.6Hz,1H),1.70–1.62(m,7H),1.61(d,J＝3.5Hz,1H),1.46(t,J＝2.7Hz,1H),1.44(d,J＝2.5Hz,1H),1.42(d,J＝3.3Hz,1H),1.39(s,3H),1.34(dd,J＝13.3,3.0Hz,1H),1.31(d,J＝3.0Hz,1H),1.22(s,1H),1.19(s,3H),1.13(s,6H),1.07–1.03(m,1H),1.02(s,3H),0.98(dd,J＝13.0,4.3Hz,1H),0.82(d,J＝4.4Hz,6H),0.72(d,J＝12.4Hz,1H).13C NMR(126MHz,Chloroform-d)δ200.32,176.21,169.69,137.30,128.25,122.56,121.58,78.64,61.93,60.61,54.94,48.73,47.28,45.52,45.45,44.14,43.30,41.26,39.19,39.15,37.72,37.15,32.75,31.93,31.05,29.99,28.62,28.41,28.11,27.28,26.49,26.35,23.37,18.72,17.47,16.39,15.60,15.49.
Intermediate 6b, white solid, yield ：90.5％.1H NMR(500MHz,Chloroform-d)δ10.56(d,J＝1.7Hz,1H),7.59(t,J＝1.8Hz,1H),7.46(t,J＝1.8Hz,1H),5.49(s,1H),4.51(t,J＝7.6Hz,2H),4.44(q,J＝7.3Hz,2H),4.23(dt,J＝12.0,6.2Hz,1H),4.10(dt,J＝11.5,6.0Hz,1H),3.25(dd,J＝11.0,5.2Hz,1H),2.75(dt,J＝13.4,3.5Hz,1H),2.35(s,1H),2.08(ddt,J＝16.0,8.0,4.8Hz,2H),2.01(dq,J＝11.9,3.5,2.5Hz,3H),1.90(dd,J＝4.3,2.6Hz,1H),1.89–1.85(m,2H),1.85–1.81(m,1H),1.79(dd,J＝9.0,5.6Hz,2H),1.67(d,J＝13.3Hz,2H),1.65–1.61(m,5H),1.50–1.45(m,1H),1.43(s,1H),1.41(s,1H),1.38(s,3H),1.32(d,J＝9.7Hz,2H),1.23–1.18(m,1H),1.16(s,3H),1.13(d,J＝3.8Hz,6H),1.08–1.03(m,1H),1.02(s,3H),0.98(dd,J＝12.9,4.3Hz,1H),0.82(d,J＝1.6Hz,6H),0.74–0.68(m,1H).13C NMR(126MHz,Chloroform-d)δ200.40,176.32,170.10,137.15,128.11,122.24,121.76,78.59,63.39,61.92,54.91,49.55,48.84,45.47,45.40,44.09,43.34,41.22,39.20,39.15,37.62,37.15,32.73,31.90,31.05,28.63,28.43,28.11,27.66,27.27,26.49,26.37,25.75,23.37,18.72,17.46,16.40,15.61,15.49.
Intermediate 6c, white solid, yield ：93.9％.1H NMR(500MHz,Chloroform-d)δ10.55(s,1H),7.63(s,1H),7.47(s,1H),5.56(s,1H),4.50–4.43(m,3H),4.42–4.35(m,1H),4.24–4.16(m,1H),4.06–3.98(m,1H),3.24(dd,J＝10.9,5.3Hz,1H),2.73(dd,J＝10.2,3.3Hz,1H),2.36(s,1H),2.07(d,J＝7.6Hz,1H),2.05(d,J＝8.3Hz,2H),2.02–1.97(m,2H),1.91(d,J＝13.3Hz,2H),1.87–1.80(m,2H),1.75(s,1H),1.72(s,1H),1.70(s,1H),1.67(s,1H),1.63(t,J＝7.4Hz,5H),1.54(d,J＝8.0Hz,1H),1.51(d,J＝7.4Hz,1H),1.45(s,1H),1.42(d,J＝3.3Hz,1H),1.41(s,1H),1.39(d,J＝5.1Hz,3H),1.34(dd,J＝8.8,5.0Hz,1H),1.31–1.29(m,1H),1.27(s,1H),1.21(d,J＝13.7Hz,2H),1.15(s,3H),1.13(s,6H),1.06(s,1H),1.01(s,3H),0.98(dd,J＝13.0,4.3Hz,1H),0.81(d,J＝2.8Hz,6H),0.72(d,J＝11.5Hz,1H).13C NMR(126MHz,Chloroform-d)δ200.60,176.26,170.34,137.00,128.08,122.37,121.49,78.56,63.85,61.92,54.90,49.95,48.89,45.51,45.34,44.06,43.35,41.25,39.21,39.14,37.58,37.14,32.74,31.91,31.06,30.28,28.62,28.40,28.11,27.24,26.52,26.36,23.35,23.30,18.73,17.46,16.41,15.62,15.54.
Preparation of final product 7 a. Under nitrogen protection, intermediate 6a (69.3 mg,0.10 mmol) and anhydrous DCM (10 mL) were added sequentially to a 50mL single-neck round bottom flask, ag2 O (13.9 mg,0.06 mmol), and dry DCM (10 mL) were added in sequence, after 12h of reaction at room temperature, me2 SAuCl (29.46 mg,0.10 mmol) and NaBr solid (10.3 mg,1.00 mmol) were added, reaction was continued for 12h, TLC monitoring was completed, after completion of the reaction, celite was removed by suction filtration to remove the solid, a small amount of anhydrous DCM (about 3 mL) was rinsed with the solid, the solvent was removed by rotary evaporation under reduced pressure, dissolved in a small amount of DCM (about 0.5 mL) and added dropwise to an appropriate amount of n-hexane (about 10 mL), white solid was precipitated, suction filtration was dried to obtain a white solid dissolved in anhydrous acetone (10 mL), intermediate 3a (70.53 mg,0.10 mmol), K2CO3 (41.46 mg,0.30 mmol) was added sequentially under nitrogen protection, and finally, stirring was carried out in a dark place, and dry film was added at room temperature, and 35 h, and dry film was added (35 mg, 48 mmol) was added, 48% of dry solvent was removed by rotary evaporation to a dry gradient (about 1:48 v) and a dry gradient was added, and the solvent was removed by dry gradient was removed, and the dry solvent was directly removed from the dry column was distilled 1.48% by dry chromatography (1: 48 mL) and distilled off (1: 5 mL).
The final product 7b was prepared in the same way as 7a, except that intermediate 6a and 3a were replaced with equimolar amounts of 6b and 3b, as a white solid with a yield ：53％.1H NMR(500MHz,Chloroform-d)δ7.43(d,J＝7.1Hz,3H),7.40–7.36(m,1H),7.33(d,J＝7.4Hz,2H),7.30(s,1H),7.28(s,2H),7.16(d,J＝16.0Hz,2H),7.09(dd,J＝10.1,1.8Hz,1H),6.28(d,J＝2.8Hz,2H),5.58(d,J＝7.9Hz,1H),5.13(d,J＝3.9Hz,2H),4.48–4.39(m,2H),4.28(q,J＝9.2,8.0Hz,8H),4.15(ddd,J＝20.7,9.8,5.6Hz,2H),3.86(d,J＝3.4Hz,6H),3.45(d,J＝11.8Hz,1H),3.25(dd,J＝11.2,5.3Hz,1H),2.91(s,1H),2.77(ddd,J＝12.6,7.6,3.8Hz,1H),2.36(s,1H),2.29(d,J＝2.9Hz,3H),2.19(d,J＝14.6Hz,1H),2.08–2.02(m,5H),2.01–1.96(m,4H),1.88(d,J＝14.6Hz,2H),1.82(dd,J＝10.4,5.3Hz,3H),1.72(t,J＝7.6Hz,3H),1.69–1.66(m,2H),1.65–1.62(m,3H),1.61(d,J＝3.9Hz,1H),1.56–1.52(m,6H),1.46(d,J＝3.1Hz,1H),1.44(s,1H),1.43–1.41(m,1H),1.39(s,1H),1.38(s,3H),1.35(s,1H),1.34(s,1H),1.31(s,2H),1.21(d,J＝14.1Hz,2H),1.14(t,J＝5.2Hz,9H),1.05(s,1H),1.02(s,3H),0.98(dd,J＝13.4,4.0Hz,1H),0.82(d,J＝2.5Hz,3H),0.80(s,3H),0.72(d,J＝11.6Hz,1H).13C NMR(126MHz,Chloroform-d)δ200.38,183.10,182.91,182.72,176.41,160.37,143.10,141.86,134.65,134.60,130.49,129.39,128.48,128.32,128.12,126.92,125.69,122.02,121.54,121.44,121.32,121.09,91.43,78.68,69.55,63.36,61.87,56.00,54.89,50.88,48.53,46.53,46.37,45.44,44.03,43.29,41.11,39.14,37.74,37.11,32.74,31.87,31.03,30.15,29.70,28.61,28.38,28.09,27.95,27.76,27.27,26.46,26.34,25.94,25.43,23.40,18.70,17.48,16.84,16.39,16.27,15.59.ESI-MS calcd.for C77H107AuN5O9[M–PF6]+1442.7729,found1442.84.HPLC% purity of 95.08%.
The final product 7c was prepared in the same way as 7a, except that intermediate 6a and 3a were replaced with equimolar amounts of 6c and 3c, as a white solid with a yield ：39％.1H NMR(500MHz,Chloroform-d)δ7.43(s,3H),7.37(d,J＝4.9Hz,1H),7.32(d,J＝6.4Hz,2H),7.25(s,2H),7.20(d,J＝10.2Hz,1H),7.15(d,J＝10.6Hz,2H),7.09(s,1H),6.29(s,2H),5.60(s,1H),5.14(s,2H),4.50(d,J＝15.2Hz,2H),4.27–4.21(m,8H),4.09(d,J＝20.6Hz,2H),3.87(s,6H),3.53(s,1H),3.25(d,J＝6.5Hz,1H),2.95(s,1H),2.76(s,1H),2.36(s,1H),2.28(s,3H),2.25–2.20(m,1H),2.06(s,1H),2.03(s,2H),1.97(s,6H),1.86(s,2H),1.82(s,1H),1.80(s,2H),1.70(s,2H),1.69(s,1H),1.67(s,1H),1.64(s,4H),1.55(s,1H),1.54(s,2H),1.53(s,2H),1.52–1.51(m,1H),1.46(s,2H),1.44(s,2H),1.41(s,1H),1.39(s,3H),1.35(s,1H),1.30(s,5H),1.23–1.20(m,3H),1.13(s,9H),1.04(s,1H),1.02(s,3H),0.99–0.95(m,1H),0.85(d,J＝5.9Hz,1H),0.81(d,J＝5.6Hz,6H),0.72(d,J＝10.7Hz,1H).13CNMR(126MHz,Chloroform-d)δ200.33,182.97,182.81,182.64,176.39,169.74,160.48,143.07,141.84,134.60(d,J＝11.5Hz),130.47,129.36,128.38(d,J＝22.7Hz),128.10,126.90,125.67,122.24,121.75(d,J＝20.2Hz),121.13(d,J＝7.0Hz),120.84,91.43,78.65,69.56,66.60,63.85(d,J＝6.2Hz),61.87,56.00,54.87,51.23(dd,J＝10.3,4.2Hz),50.97,48.59(d,J＝4.0Hz),46.42(d,J＝16.2Hz),45.45,44.02,43.30,41.12,39.13,37.71,37.11,32.74,31.93,31.51,31.44,31.37,31.23(d,J＝7.8Hz),31.04,30.53,30.15,29.70,29.36,28.61,28.35,28.09,27.27,26.41(d,J＝15.9Hz),23.41,23.19(d,J＝4.4Hz),22.82,18.71,17.48,16.92(dd,J＝7.6,3.4Hz),16.39,16.27,15.60.ESI-MS calcd.for C79H17auN5O9[M–PF6]+1470.8042,found 1470.88.HPLC% purity of 97.69%.
Example 2
Glycyrrhetinic acid-gold (I) dominant compound selection:
(1) In vitro antiproliferative capacity assessment:
The cell lines related to the invention are all purchased from Shanghai cell banks of the national academy of sciences of China, and all cells are cultivated in a DMEM medium added with 10% fetal calf serum and 1% penicillin/streptomycin double antibody in an incubator with 5% CO2 at 37 ℃.
The specific experimental operation comprises digesting Hepa1-6, hepG2 and Hep3B cells in logarithmic phase with pancreatin, centrifuging, discarding supernatant, blowing with complete culture medium uniformly and counting, adjusting cell concentration to 2×104/mL, inoculating to 96-well plate (100 μL, 2×103 well), culturing overnight, dissolving compound to be tested in DMF (20 mM), gradient diluting with complete culture medium to make compound concentration 1.25,2.5,5,10,20 and 40 μM, adding 100 μL to original 96-well plate to make compound final concentration 0.625,1.25,2.5,5,10 and 20 μM, placing 96-well plate back into incubator for culturing for 72 hr, adding 20 μL MTT solution (5 mg/mL, PBS dissolving, pH=7.4) into 96-well plate, incubating for 4 hr, discarding supernatant, adding DMSO (200 μL), shaking at uniform speed for 10min, measuring absorbance at enzyme marker meter, setting 3 multiplex wells for each concentration (%), calculating cell viability (%), and analyzing data by using 34.0 software;
cell viability (%) = (ODd-OD0)/(ODC–OD0) ×100%
ODd is the absorbance value of the dosing group, OD0 is the zeroed hole absorbance value, and ODC is the blank group absorbance value;
As a result, the antiproliferative activity (IC50) of 7a,7b and 7c on HepG2 cells was 1.19.+ -. 0.15. Mu.M, 1.70.+ -. 0.20. Mu.M and 2.64.+ -. 0.36. Mu.M in this order, the antiproliferative activity of 7c on HepG2 cells was slightly better than that of auranofin (IC50 values of 72h and 24h of auranofin were 1.77.+ -. 0.29. Mu.M and 3.79.+ -. 0.49. Mu.M, respectively) and the IC50 values of 7c at 72h and 24h were 2.64.+ -. 0.36. Mu.M and 3.22.+ -. 0.31. Mu.M, respectively) decreased in value with the extension of the carbon chain of Linker, which shows that 7c was more effective than that of the positive medicine and the efficacy was maintained for a long period of time;
(2) Evaluation of the ability to inhibit the expression of PD-L1 protein:
Firstly, evaluating the expression condition of a compound on the PD-L1 protein of the HepG2 cell on the protein level by using Western Blot, using a confocal technology to intuitively evaluate the PD-L1 expression condition of the HepG2 cell treated by the compound, and selecting a dominant compound to perform the following stability and other pharmacodynamics evaluation by combining an antiproliferative activity result;
As a result, the expression of PD-L1 protein in HepG2 cells was evaluated by Western blotting, and the effect of PD-L1 was significantly reduced by 7b and 7c, and was poor, and the statistical graph of the relative expression level of PD-L1 protein also confirmed, and in addition, in order to more intuitively observe the expression of PD-L1 protein, the expression of PD-L1 in treated HepG2 cells was evaluated by using confocal techniques by 7a, 7b and 7c, and it was found that three compounds were able to inhibit the expression of PD-L1 protein on the surfaces of HepG2 cells, and the inhibition intensity was consistent with the result at the protein level, in comparison with DMF, and that 7c was selected as the advantageous compound of the present series for the subsequent stability and other pharmacodynamics evaluation in combination with the antiproliferative activity result of (1).
Example 3
Stability evaluation of glycyrrhetinic acid-gold (I) compound:
Nuclear magnetic method (hydrogen spectrum) by reacting compound 7c (5 mM) in 10% D2 O/90% DMSO-d6 mixed deuteration reagent containing GSH (5 mM) at room temperature, collecting1 H-NMR spectra at different time points, and analyzing1 H-NMR spectrum change;
As a result, 7c was co-incubated in aqueous DMSO-d6 at room temperature for 7 days, no distinct new peak and change in chemical shift were seen in the hydrogen spectrum, which suggests that 4c was stable in aqueous solution, 7c and GSH were mixed in a molar amount of 1:1, and co-incubated in a DMSO-d6 mixed solution containing 10% D2 O at room temperature for 72 hours, at 6 hours, a triplet at 3.5ppm and a quartet at 4.5ppm were seen, and at 12 hours, but it was presumed that only about 10% of 7c reacted at 24 hours, which suggested that compound 7c was able to react slowly with GSH, and that 7c was stable in total.
Example 4
Pharmacodynamic evaluation of glycyrrhetinic acid-gold (I) compounds in HepG 2:
(1) Advantageous compounds in vitro inhibitor antiproliferative assay:
To explore the specific mechanism of anti-proliferation of the dominant compounds initially, four inhibitors (NAC, sal, fer-1, 3-MA) were used to co-culture with the dominant compounds, respectively, in HepG2 cell lines, and the CCK-8 method was used to detect cell viability;
The results were that NAC was co-cultured with 7c for 24h, and was able to dose-dependently reverse the inhibition of cell proliferation by 7c, and similarly ERS inhibitor Sal (Salubrinal) was able to achieve a similar reversal effect, whereas the iron death inhibitor, fer-1 (Ferrostatin-1) and autophagy inhibitor 3-MA (3-METHYLADENINE), were not observed to have a significant reversal effect, in conclusion, the antiproliferative capacity of 7c in HepG2 cells might be associated with oxidative stress, endoplasmic reticulum stress;
(2) The inhibition ability of the dominant compounds on HepG2 TrxR was evaluated:
① TrxR pure enzyme activity assay:
preparing TrxR pure enzyme, diluting dominant compound to proper concentration, adding 25 mu L enzyme solution and 25 mu L compound diluent into 96-well plate, incubating at room temperature, adding 225 mu L reaction solution into each well of 96-well plate immediately after mixing, adding 25 mu L DTNB (5 mg/200 mu L,95% ethanol dissolution) into each well, and continuously detecting absorbance change at 405nm for 300 seconds immediately by enzyme label instrument;
② Intracellular TrxR activity assay:
Inoculating HepG2 cells in a logarithmic growth phase to 6cm, when the cell fusion degree is 70%, administering, setting a DMF solvent control group and an administration group with different concentrations, collecting cells after 24 hours, operating according to a kit instruction, and detecting the protein concentration;
③ Intracellular TrxR probe detection:
inoculating HepG2 cells in a logarithmic growth phase into a 6-pore plate, after attaching the cells, dosing, adding TRFS-green staining working solution after 24 hours, staining for 30 minutes at 37 ℃, discarding the supernatant, washing for 2 times by using PBS, and observing under a fluorescent microscope;
④ Immunofluorescence (confocal) detection:
Placing the sterilized cell climbing sheet in a 24-well plate, inoculating HepG2 cells in a logarithmic growth phase into the well for overnight culture, adhering the cells, dosing, taking out the 24-well plate after 24 hours, discarding a culture medium, washing 3 times with PBS, adding 4% paraformaldehyde, fixing for 30 minutes at 37 ℃, taking out the cell climbing sheet, washing 3 times with PBS, adding 0.1% Triton to permeate for 10 minutes, washing 3 times with PBS, adding 1% BSA prepared with PBS for sealing for 1 hour, washing three times with PBS, adding primary anti-working solution TrxR, GRP78, CHOP, CRT, HMGB and PD-L1 at 4 ℃ overnight, taking out the 24-well plate, washing 3 times with PBS, wrapping with tin foil, slowly shaking 2 hours (room temperature) with a rabbit secondary antibody shaker containing Fluorescent (FITC) marks, washing 3 times with PBS, incubating 10 minutes at room temperature in a dark place, washing 3 times with PBS, sealing the cell climbing sheet, and then placing the cell climbing sheet in a Leica DMi8 fluorescent microscope or TCSSP laser confocal microscope for shooting;
The results show that the green fluorescence of the TrxR is remarkably reduced after 7c (4 mu M) treatment, which shows that 7c is used for remarkably inhibiting the expression of TrxR in HepG2 cells, in addition, 7c can be used for inhibiting the expression of TrxR protein in HepG2 cells in a dose-dependent manner, the relative expression amount of the TrxR is respectively reduced by 24%, 55% and 75% compared with that of a DMF group in 7c with the concentration of 1 mu M,2 mu M and 4 mu M, the difference between the TrxR and experimental data of only about 10% of 7c reacted with GSH in example 3 is large, the results show that 7c is mainly not used for inhibiting the activity of thiol on the TrxR, but is mainly used for carrying out coordination reaction with strong nucleophilic selenol anions in the active center of the TrxR, in addition, after the HepG2 cells are treated for 24 hours, the TrxR enzyme activity of the HepG2 cells is detected by using a TrxR enzyme activity kit, and the results show that 7c has the activity of inhibiting effect on the TrxR in the cells is improved, and meanwhile, the stability of the TrxR activity is improved;
(3) The ability of the dominant compounds to induce apoptosis and cycle arrest in HepG2 cells was evaluated:
Inoculating HepG2 cells in logarithmic growth phase into 6-well plates (2×105 cells/well) for culturing overnight, and collecting cells after different administration groups are treated for 24 hours; detecting apoptosis or cell cycle by using an Annexin V-FITC apoptosis kit or a cell cycle detection kit according to the operation of a kit instruction;
as a result, the ability of 7c to induce apoptosis of HepG2 cells at 0.5. Mu.M was comparable to that of auranofin (4. Mu.M), and the ability of 7c to induce apoptosis exhibited a certain dose-dependence, but after 2. Mu.M, the apoptotic cells were no longer further improved, on the other hand, after 7c treatment, the HepG2 cell cycle was mainly blocked in G0/G1 phase, this cycle blocking pattern was consistent with that of (4. Mu.M) auranofin, except that 7c also blocked the cell cycle in G2/M phase, and auranofin was mainly in S phase, so 7c could induce apoptosis and cycle blocking of HepG2 cells;
(4) Evaluation of the damaging ability of the dominant compounds to HepG2 mitochondria:
Inoculating and dosing HepG2 cells in logarithmic growth phase for 24 hours, discarding culture medium, washing by PBS, adding JC-1 probe working solution, dyeing for 30 minutes at 37 ℃, discarding working solution, washing by PBS, observing under a fluorescence microscope, discarding culture medium after 24 hours, washing by PBS, digesting and collecting cells, adding JC-1 dyeing working solution, centrifuging (800 rpm,5 min), discarding working solution after PBS washing (800 rpm,3 min), re-suspending cells by using cell dyeing buffer, analyzing by using a flow cytometer, and processing data by using FlowJo software;
The results show that after the HepG2 cell is treated by the 7c, compared with the DMF control group, the red fluorescence is weakened, the green fluorescence is obviously enhanced, the mitochondrial membrane potential is reduced, and the flow cytometry analysis results also prove that the result proves that the 7c can induce mitochondrial dysfunction, but the 7c has less strong MMP reducing capacity than that of the auranofin;
(5) Evaluation of the ability of the dominant compounds to induce oxidative stress, endoplasmic reticulum stress and ICD effect on HepG 2:
① Evaluation of the ability of the dominant compounds to induce oxidative stress in HepG 2:
inoculating and dosing HepG2 cells in the logarithmic growth phase for 6 hours, discarding a culture medium, washing with PBS, adding DCFH-DA staining working solution, standing and staining at 37 ℃ for 30 minutes, discarding the working solution, washing with PBS, observing under a fluorescence microscope, and photographing;
As a result, DHE (Dihydroethidium) staining and flow cytometry results show that the amount of active oxygen in HepG2 cells caused by 7c is dose-dependent, the capacity of 7c to cause active oxygen increase at a concentration of 1 mu M or even 0.5 mu M is similar to that of auranofin (4 mu M), and compared with the result that the IC50 value of 7c is equivalent to that of auranofin for 24 hours, the fact that the auranofin is probably not a specific TrxR inhibitor is shown to a certain extent, the IC50 value of 24 hours and 72 hours of 7c is similar to that of 7c, the fact that 7c can enter cells rapidly to play a role, and the main target point of the 7c is TrxR, namely, the 7c is a TrxR inhibitor which is more specific than the auranofin;
② Evaluation of the ability of the dominant compounds to induce endoplasmic reticulum stress in HepG 2:
Inoculating HepG2 cells in logarithmic growth phase into 12-well plates (5×104/well) for overnight culture, and after treatment for 24h in different administration groups, staining with Fluo 4-AM probe, photographing under a fluorescence microscope, configuring and incubating the probe according to the instruction;
The results of evaluating the ability of 7c and oxaliplatin to promote the increase in the expression levels of calnectin and CHOP at the same concentration (4 μm), showed that 7c and oxaliplatin can both greatly increase the expression levels of these two proteins, but this ability 7c is stronger than oxaliplatin (3.45-fold and 3.42-fold increase in the expression levels of Calnexin and CHOP, respectively, compared to DMF groups, and 2.34-fold and 2.56-fold increase in oxaliplatin, respectively, after 7c treatment), the results of the confocal experiment also again confirm the above conclusion;
The Ca2+ level change in the cells is detected by a calcium fluorescent probe Fluo4-AM, and the result shows that after being treated by 7c (4 mu M) and oxaliplatin (4 mu M) for 24 hours, compared with a DMF group, obvious punctiform or blocky green fluorescence appears in cytoplasm of HepG2 cells, which indicates that a large amount of Ca < 2+ > is released, and the strength of the fluorescent probe is analyzed by flow cytometry, so that the capability of the 7c to cause Ca2+ release is stronger than that of the oxaliplatin with the same concentration;
③ The ability of the dominant compounds to induce ICD effects of HepG2 was evaluated:
immunofluorescence from the same (4) was used to assess the ability of dominant compounds to elicit ICD biochemical Signatures (DAMPs) and to detect the presence of HepG2 efflux HMGB1 as follows:
inoculating HepG2 cells in logarithmic growth phase into six-well plates, adding 24h after dosing, discarding culture medium, washing with PBS for 2 times, adding 200 μl of lysate into each well, collecting after full blowing, centrifuging at 4deg.C under 12000g for 5min, collecting supernatant, and placing in 96-well plates, wherein HMGB1 level is determined according to ELISA manufacturer standard operation;
The results showed that the characteristic green surface plaques were observed on confocal pictures of HepG2 cells treated with 7c (4 μm) or oxaliplatin (4 μm), indicating that CRT was transluminally transferred from endoplasmic reticulum vesicles to the tumor cell surface and that the green surface plaques of 7c were more pronounced than those of oxaliplatin group, indicating that 7c resulted in CRT efflux of HepG2 cells stronger than oxaliplatin, in addition, the flow cytometry results showed that the curves were significantly right shifted after 7c and oxaliplatin treatments and that 7c group was more pronounced than oxaliplatin group movements, and finally, western blot analysis results remained consistent with confocal and flow results, with 7c treatment resulting in CRT protein expression levels 2.91 times that of DMF group, and 2.21 times that of oxaliplatin group;
Evaluating the excretion of HMGB1 from HepG2 cells after 7C and oxaliplatin treatment, wherein the expression level of HMGB1 (S-HMGB 1) and HMGB1 (C-HMGB 1) in the culture medium is increased after 7C or oxaliplatin treatment, the expression level of HMGB1 (C-HMGB 1) in the cells is reduced, and the excretion capacity of HMGB1 protein caused by 7C is stronger than that of oxaliplatin;
The effect of 7C (4. Mu.M) treated Hepa1-6 cells on DCs in C57BL/6 mice was examined by flow cytometry, and the expression of both markers CD80 and CD86 for DCs maturation was increased, and the proportion of DCs in group 7C was doubled (31.6% vs 15.6%) compared to the DMF control group;
In conclusion, 7c can promote the release DMAPs of HepG2 cells and the maturation of murine DCs cells, and thus 7c can elicit ICD effects.
Example 5
The ability of glycyrrhetinic acid-gold (I) compounds to inhibit angiogenesis was evaluated using a zebra fish model:
zebra fish embryos are purchased from a biotechnology company, the embryos are placed in a standard zebra fish culture system, the temperature is kept at 28.0+/-1.0 ℃ through illumination circulation at day and night, all experiments are carried out on the 2 nd day after embryo transplantation is successful, a control group is treated by 0.1% DMF, the treatment groups are 7C (2 mu M) and auranofin (2 mu M) respectively, three days are continuously observed, fluorescent images of the embryos of each group are photographed, and the embryos are anesthetized by 0.03mg/mL of tricaine during photographing;
As a result, angiogenesis of the zebra fish embryo treated with Compound 7c was significantly inhibited, the area of blood vessels under the zebra fish intestine was significantly reduced after treatment with 7c (4. Mu.M) compared with the Control group (Control) (and there were many breaks indicating that 7c inhibits angiogenesis of the zebra fish embryo; angiogenesis inhibition of 7c was stronger compared with auranofin; example 6)
Study of antitumor immune mechanisms of glycyrrhetinic acid-gold (I) compounds in animals:
Male C57BL/6 mice (18-22 g) are purchased from a medical college, all animal experiments are carried out according to guidelines approved by a university institution and local ethical committee (the use license number of experimental animals is SYXK (Su) 2023-0077), food and water can be freely obtained under standard environmental conditions (the temperature is 22-24 ℃ and the light/dark period is 12 hours, the humidity is 50+/-10%), the mice start experiments after one week of adaptive feeding, the Hepa1-6 cells inoculated on a 10cm dish are collected after digestion, resuspended and counted by blowing with a serum-free cell culture medium, the cell density is adjusted to 1X 106/100 mu L/mouse, the left side underarm of the mice is inoculated, the intraperitoneal injection is carried out 1 time every 2 days, the weight of the tumor-bearing mice is recorded every day, the tumor volume is measured by a vernier caliper, the calculation formula of the tumor volume is V (cm3)＝a2 Xb/2, (a: short diameter; V: long diameter; tumor volume) two weeks later, the mice are taken out, the tumor and the major organs are taken out, and a subsequent tumor tissue flow analyzer is used for carrying out after tumor tissue tumor cell analysis and immune cell lysis is carried out;
The results show that the tumor volume and the weight of the mice treated by the compound are obviously reduced compared with the control group, the flow analysis results show that the proportion of mature DC cells and activated T cells is obviously increased, which indicates that the antigen presentation and lymphocyte killing effect are enhanced after the compound is treated, the proportion of myeloid-lineage suppressor cells (MDSCs) and regulatory T cells (Tregs) is reduced, which indicates that the immunosuppressive ability of tumors is weakened, the increase of M1 type macrophages is reduced, and the decrease of M2 type macrophages is accompanied, which indicates that the tumor immune microenvironment is changed from immunosuppressive type to immunopotentiator type;
Thus, compound treatment not only reduces tumor growth and development from the chemotherapeutic route, but also activates immune responses in mice, reversing immune escape of tumors.