| Psoriasis is an autoimmune skin disease characterized by excessive epidermal hyperplasia and chronic inflammation of the dermis,with a global incidence of about0.3%.The prevalence of psoriasis in China is about 0.120.47%.The total patients are up to 6.5 million.Currently,there is no effective therapeutic drug for psoriasis.It is of great significance to develop drugs for the treatment of psoriasis.The two new derivatives of glycyrrhetinic acid was used as the research object in this study.The results are as follow:1.The seletion of the optimal synthesis conditions of two new derivatives of glycyrrhetinic acid.Glycyrrhetinic acid was used as the parent compound and nitrogen heterocyclic groups were introduced into C-3 by esterification to prepare two new glycyrrhetic acid derivatives 3-O-?6-chloropyridine-3-formyl?-glycyrrhetic acid?GA1?and3-O-?5-methylpyrazine-2-formyl?-glycyrrhetic acid?GA2?.The yield of GA1 was 81.8%and the yield of GA2 was 86%.The best synthesis conditions were:the reaction solvent was tetrahydrofuran,the feed ratio was 1:2,and the reaction time was 4h.2.The confirmation of structure for the new glycyrrhetic acid derivatives.Compound GA1:IR(2930.09,2923.62,1736.65,1589.99,766.18,1647.34,1743.72,1125.59,1718.78,2930.09cm-1);MS?[M+H]+=610?;1HNMR?1HNMR?600MHz,CDCl3???pp m?:9.02?d,J=1.9Hz,1H?,8.26?dd,J=8.3,2.4Hz,1H?,7.44?d,J=8.3Hz,1H?,7.28?s,3H?,7.00?s,3H?,5.76?s,1H?,5.03?s,2H?,4.82?dd,J=11.8,4.7Hz,1H?,2.91?d,J=13.7Hz,1H?,2.44?s,2H?,2.30?s,5H?,1.42?s,4H?,1.18?s,5H?,1.05?s,6H?,1.02–0.95?m,5H?,0.91–0.86?m,6H??;13CNM R(13CNMR?151MHz,CDCl3???ppm?:200.20?C11?,181.24?C30?,169.53?C13?,164.12?C6'?,155.51?C5'?,151.16?C1'?,139.59?C3?,135.81?C12?,128.27?C2'?,125.52?C4'?,82.43?C3?,61.69?C9?,55.10?C5?,48.28?C14?,45.49?C18?,43.81?C20?,43.27?C8?,40.92?C19?,38.43?C10?,37.00?C4?,34.24?C1?,32.71?C22?,31.90?C17?,30.34?C7?,28.56?C16?,28.45?C21?,28.26?C15?,26.52?C29?,26.43?C27?,23.64?C2?,23.41?C23?,21.19?C24?,18.73?C28?,17.42?C26?,16.97?C6?,16.43?C25?),confirmed the structure for the 3-O-?6-chloro pyridine-3-formyl?-glycyrrhetinic acid.Compound GA2:IR(2986.24,2965.37,1720.05,1651.95,1720.05,1357.83,1720.05,3108.24,1450.82cm-1),MS?[M-H]-=589?,1HNMR??600MHz,CDCl3???p pm?:9.16?d,J=1.0Hz,1H?,8.67?s,1H?,5.75?s,1H?,4.93?dd,J=11.9,4.6Hz,1H?,2.90?dd,J=10.3,3.4Hz,1H?,2.71?s,3H?,2.43?s,1H?,2.23?dd,J=13.3,3.4Hz,1H?,2.08–2.02?m,2H?,2.00–1.90?m,2H?,1.88–1.79?m,2H?,1.70?dd,J=14.8,5.7Hz,1H?,1.65?d,J=13.6Hz,2H?,1.51?d,J=12.8Hz,1H?,1.47–1.43?m,3H?,1.41?s,3H?,1.25?t,J=12.2Hz,9H?,1.17?s,4H?,1.06?s,4H?,0.98?s,3H?,0.92?d,J=11.5Hz,1H?,0.86?s,3H?.?;13CNMR??151MHz,CDCl3???ppm?:200.23?C11?,181.20?C30?,169.55?C13?,163.58?C6'?,157.09?C3'?,144.80?C2'?,144.58?C4'?,141.44?C1'?,128.46?C12?,82.75?C3?,61.69?C9?,55.04?C5?,48.28?C14?,45.49?C18?,43.80?C20?,43.25?C8?,40.91?C19?,38.78?C10?,38.50?C4?,37.73?C1?,36.98?C22?,32.71?C17?,31.89?C7?,30.95?C16?,28.55?C21?,28.47?C15?,28.21?C29?,26.50?C27?,26.42?C2?,23.65?C23?,23.39?C24?,21.67?C28?,18.71?C5?,17.41?C26?,16.95?C6?,16.44?C25??,confirmed the structure for t he 3-O-?5-methyl pyrazine-2-formyl?-glycyrrhetinic acid.3.The establishment of the determination of glycyrrhetic acid derivatives.The HPLC chromatographic conditions for the determination of compound GA1were:the chromatographic column was taken as Welchrom?Ultimate xp-phenyl?4.6×250mm,5m?,acetonitrile?A?-water?B?as the mobile phase,gradient elution?015min gradient change from 75:25 to 90:10,1520min keep 90:10 elution conditions for 5min,2025min gradient change from 90:10 to 75:25?,flow rate was 1mL/min,detection wavelength was 229nm,injection volume was 10?Lcolumn,The column temperature was 25?.The linear relationship is good in the range of0.040.24mg/mL,the linear regression equation is A1=32816C1+200.5?r=0.9997,n=6?,and the content of GA1 measured is 99.13%.HPLC chromatographic conditions for the determination of compound GA2:the chromatographic column was Welchrom?Ultimate xp-phenyl?4.6×250mm,5?m?and acetonitrile?A?-water?B?as mobile phase gradient elution?gradient elution conditions were:The gradient of 06min varied from 60:40 to 82:18,612min from 82:18 to 80:20,1225min from 80:20 to 60:40?.The detection wavelength was 250nm,the flow rate was1mL/min,the injection volume was 10?L,and the column temperature was 25?.the linear relationship is good in the range of 0.040.24mg/mL,the linear regression equation is A2=18033C2-49.9?r=0.9992,n=6?,and the content of GA2 measured is 99.07%.4.The development of the preliminary stability test of glycyrrhetinic acid derivatives.The stability of glycyrrhetinic acid derivatives under high temperature,high humidity and strong light was studied by HPLC.It was discovered that the stability of GA1 decreased significantly after 10 days of high temperature,but was stable under high humidity and strong light,and GA2 was stable under high temperature,high humidity and strong light.5.GA1 and GA2 were discovered to inhibit the proliferation of HaCaT cells and reduce the expression of TNF-?,and were superior to glycyrrhetinic acid.Using tretinoin as a positive drug,the proliferation inhibitory effects of GA1 and GA2 on HaCaT cells were detected by CCK-8,clone formation test,EdU,flow cytometry and other methods.The results showed that when the concentration of GA1and GA2 were 25100?M and 22.590?M respectively,the cells became round and vacuolated with increasing concentration,and the inhibition was enhanced.HaCaT were blocked at G0/G1 phase,and GA2 was superior to GA1.The apoptosis of HaCaT cells caused by the expression of Caspase 3 may be one of the mechanisms of its proliferation inhibition.It was discovered that both GA1 and GA2 could decrease the expression of TNF-?,suggesting that GA1 and GA2 might also have anti-inflammatory effect.The results also showed that the above effects of GA1 and GA2 were superior to those of glycyrrhetinic acid. |
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