| Based on the review of research progress of lead compounds(PPD,PPT,Pyxinol,(24S)-Pyxinol,PD,PT,Ocotillol)and their derivatives,chemical synthesis,biological activity screening and preliminary pharmacokinetic evaluation were carried out.A series of new amino acid ester derivatives were directionally prepared;the anti-tumor activities of derivatives were then evaluated in vivo and in vitro;the pharmacokinetic parameters were obtained.As a result,an innovative candidate drug with good antihepatocarcinoma activity was successfully screened.The main innovative achievements are listed as follows:1.Synthesis of amino acid ester derivatives of ginsengeninsA total of 11 amino acids(alanine,methionine,glycine,leucine,proline,valine,glutamate,lysine,tyrosine,tryptophan and isoleucine),closely related to liver cancer,lung cancer,breast cancer,cervical cancer and colon cancer,were selected to react with the C3-OH of 7 ginsengenins {Pyxinol,(24S)-Pyxinol,PD,PT,Ocotillol,PPD and PPT} by esterification,and a total of 130 amino acid ester derivatives were prepared.The derivatives were separated and purified by silica gel column chromatography and recrystallization.The structures were identified by physical and chemical properties,HR-MS and NMR.Among the derivatives,there were 22 derivatives of Pyxinol,(24S)-Pyxinol,PD,PT and Ocotillol respectively.And there were 10 derivatives of PPD,PPT respectively.Except for derivatives 1b,1c,1f?1j,2b,2c,2f?2j,4c,8a?8d,8f?8j,9a?9d,9f?9j and 12 c,other 96 derivatives are all new compounds synthesized for the first time.2.Study on the anti-tumor activity of amino acid ester derivatives of ginsengenins(1)Study on the inhibition of cells proliferationCCK-8 method was used to evaluate the inhibitory effects of 7 lead compounds and 130 derivatives on the proliferation of Hep G2,A549,MCF-7,He La and HCT-116 cells in vitro.The results showed that most of the derivatives exhibited better anti-tumor activity than the lead compounds;9 active derivatives with IC50? 20 ?M were screened out;the IC50 value(12.3 ?M)of the compound 8e(3-L-Prolyl-Pyxinol)on Hep G2 cells was the lowest.The structure-activity relationship between ginsengenins amino acid ester derivatives and antitumor activity were as follows: i)The better hydrophilicity of amino acids,the stronger activity of derivatives;ii)the activities of the derivatives with Boc-removed was stronger than the derivatives with Boc group;iii)the activities of compounds with 24 R configuration were stronger than compounds with 24 S configuration;iv)the activities of compounds with five-membered ring connected to C-17 position was stronger than the compounds with six-membered ring or chain structure;v)the compounds without hydroxyl group at the C-6 position have stronger activity than the compounds with hydroxyl group at the C-6 position.(2)The effect of compound 8e on H22 tumor-bearing miceThe H22 tumor-bearing mouse model was established to evaluate the effect of compound 8e with body weight,tumor size and histomorphology,liver and kidney indexes,inflammatory factors and blood biochemical parameters as evaluation indexes.The results showed that compound 8e could inhibit tumor growth,increase the contents of TNF-? and IL-2,and reduce the content of VEGF in a dose-dependent manner.It is suggested that compound 8e played an anti-liver cancer role by improving the immune ability and inhibiting tumor angiogenesis.Moreover,the ALT,AST,BUN and CRE contents in tumor-bearing mice could significantly regulate by treated with compound 8e alone or combined with cyclophosphamide,suggesting that compound 8e could significantly alleviate and protect the damage of liver and kidney in tumor-bearing mice.(3)Metabolomics study of compound 8e against liver cancerThe serum and liver samples of hepatoma mice interfered by compound 8e were analyzed by using metabolomics method based on UPLC-Q/TOF-MS technology.The results showed that the levels of 21 endogenous substances in tumor-bearing mice treated with compound 8e were significantly re-regulated.The substances included L-tryptophan,4-hydroxyretinoic acid,all-trans-5,6-epoxyretinoic acid,prostaglandin I2,(5S)-hydroxy-docosatetraenoic acid,leukotriene B4,(15S)-HPETE,12,13-Ep OME,all-trans-retinoic acid,stearic acid,13-L-hydroxyoleic acid,sphingosine-1-phosphate,8,9-epoxyeicosatrienoic acid,Lyso PC(18:1(9Z)/0:0),PC(16:0/16:0),retinyl ester,?-linolenic acid,arachidonic acid,linoleic acid and bilirubin.It was deduced that compound 8e exerted anti-cancer effect by interfering with eight metabolic pathways such as arachidonic acid metabolism,linoleic acid metabolism,retinol metabolism,sphingolipid metabolism,?-linolenic acid metabolism,glycerophospholipid metabolism,porphyrins and greenery metabolism,tryptophan metabolism.3.Study on the pharmacokinetics of compound 8e intragastrically administered in rats(1)Determination of lipid-water partition coefficient of compound 8eTo simulate vivo environment of the gastrointestinal,the conditions of p H 2.0,5.8 and 7.4 were used to determine the balanced distribution of compound 8e in the water-n-octanol buffer solution with shaking flask method.Then the concentrations of compound 8e in two phases were measured by HPLC-ELSD method.The lipid-water distribution coefficient(Log P)of compound 8e was determined as 2.07?2.19,which was lower than that of the lead compound Pyxinol,indicating that the introduction of amino acid ester group in the structure could increase the water solubility.(2)Study on the "blood concentration-time curve" of compound 8e intragastrically administered in ratsThe HPLC-ELSD quantitative analysis method for compound 8e and its main metabolite(Pyxinol)in rat plasma was established for the first time.The linear range was 5?250 ?g/ml,and the LLOQ was 5 ?g/ml.The specificity,accuracy,precision,extraction recovery,residual effect and stability were all met the analysis requirements.The plasma concentration-time curve of compound 8e intragastrically administered was measured with the established analysis method,and the basic pharmacokinetic parameters were obtained.The pharmacokinetic of compound 8e in vivo was a linear process.Compound 8e was slowly absorbed and eliminated in rats(CL: 0.02 L·h-1·kg-1;t1/2: 9.71 ?10.59 h;Tmax: 6.67?6.80 h),and the half-life was significantly longer than that of the lead compound Pyxinol reported in the literature.The apparent distribution volume Vd was 0.28?0.32 L/kg,which was less than the total volume of body fluid 0.667 L/kg in rat.It has a longer mean residence time in rats(MRT(0-t): 14.12?15.69 h),which could effectively prolong the half-life.The plasma concentration of the main metabolite(Pyxinol)was also quantitatively analyzed.The results showed that the concentration of Pyxinol in plasma was very low before prototype drug reached maximum concentration.While the concentration of Pyxinol in plasma gradually increased when the prototype drug was eliminated,and reached maximum at 16 h after intragastric administration compound 8e.(3)Study on the bioavailability of compound 8e intragastrically administered in ratsThe blood concentration-time curve of compound 8e after intravenous injection in rats was also studied.Taking the AUC(0-t)of intravenous administration as a reference,the absolute bioavailability of compound 8e intragastrically administrated was calculated to be 45.75%,which is slightly higher than the reported bioavailability of Pyxinol(43%).(4)Study on the biotransformation of compound 8e intragastrically administered in ratsUPLC-Q/TOF-MS method with high sensitivity and high resolution characteristics was used to determine the plasma,urine,feces and bile samples of rats administrated with compound 8e intragastrically.UNIFI metabolite analysis platform was then used to analyze and identify the main metabolites in above samples.A total of 5 phase I metabolites and 7 phase II metabolites were identified.The phase I metabolic reactions mainly included dehydration,dehydrogenation,water addition,oxidation and deprolylation.And phase II metabolic reactions included methylation,phosphorylation,sulfation and glucuronidation.In summary,the present study enriched the structural modifications of ginsengenins.An innovative candidate drug,compound 8e(3-L-Prolyl-Pyxinol),with good anticancer activity and ideal pharmacokinetic parameters in vivo was screened out.This study provided the theoretical basis and scientific data for the further research and development of natural products against cancer.The innovations of this paper are as follows:1.A total of 96 new ginsenoside amino acid ester derivatives were synthesized,which enriched the structural modification of ginsenoside.2.The derivatives with good inhibitory effect on HepG2,A549,MCF-7,HeLa and HCT-116 cells were screened out,which provided a theoretical basis for the follow-up research.3.The structure-activity relationship of amino acid ester derivatives of ginsengenins against tumor was discussed.4.The anti-hepatoma effect of the new compound 8e was studied,which proved that 8e had strong anti-hepatoma activity.5.Pharmacokinetic studies on oral absorption and biotransformation of the new compound 8e were carried out,which contributed to further research and development. |
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