Design, Synthesis And Mechanism Of In Vitro Antitumor Action Study Of Novel Chalcones Desmosdumotin C, FMC Analogues

Background: Malignant tumor is one of the larger public health problems in the word,a serious threat to human health and life. At present the main way in the treatment of malignant tumor diseases are surgery, chemotherapy, radiotherapy and so on, which chemotherapy has an irreplaceable role, but high toxicity and multidrug resistance lead to the failure of tumor chemotherapy and poor prognosis. Thus studying antitumor drug of high effection, low toxicity and reversing MDR have important significance in medicine and clinical research. Chalcone is natural product with antitumor activity, which structure medicated and then developed into new antitumor drug become a hot topic of scientific research workers.Objective: The aim of the present study is to design, synthesis and mechanism of in vitro antitumor action of a new skeleton Desmosdumotin C and a novel chalcone FMC,and then investigate antioxidant action of FMC analogues in vitro.Methods:1. The efficient three-step reaction is employed to prepare the analogues of desmosdumotin C. All compounds are firstly synthesized from 2,4,6-trihydroxyacetophenone by alkylation and 4-O-alkylation. Finally, substitutedaromatic aldehydes are condensed by the Claisen-Schmidt reaction.2. HL-60 tumor cells are treated with Desmosdumotin C and its analogues after 24 h and then the apoptosis rate are observed by APC-Annexin V/7-AAD double staining.The change of cell cycle is detected by flow cytometry. The apoptosis rate of HL-60 tumor cells treated with different concentrations of compound is observed and the mechanism of inducing tumor cell apoptosis is studied.3. The concise four-step reaction is employed to prepare the analogues of FMC. All compounds are synthesized from 2,4,6-trihydroxyacetophenone, formylation at3-position under Vilsmeier-Haack conditions is followed by introducing a methyl group at 5-position. The step of selective methylation at 2-position is achieved by TMSCHN2. Finally, substituted aromatic aldehydes are condensed by Claisen-Schmidt reaction.4. The cell proliferation inhibitions of FMC and its analogues in human cervical carcinoma cell Hela, human lung adenocarcinoma cell A549, human hepatocellular carcinoma cell SMMC-7721, human colon carcinoma cell HCT-8, human cervical lymph node metastasis of oral squamous cell carcinoma cancer cell GNM, prostate cancer cell DU145, acute myelogenous leukemia cell HL-60 and human umbilical vein endothelial cells HUVEC are assessed by SRB assay. The change of cell cycle is detected by flow cytometry. The apoptosis rate of HL-60 tumor cells treated with different concentrations of compound is observed by FITC-Annexin V/PI double staining.5. Preliminary evaluation of the FMC and its partial analogues antioxidant capacity in vitro by their DPPH radical scavenging and antioxidant capacity of iron(FRAP).Results:1. HL-60 tumor cells are treated with Desmosdumotin C and its 31 analogues after 24 h and then the cell apoptosis rate is detected by flow cytometry. The results showthat the inhibition rate of lead compound Desmosdumotin C is 9.73%, the inhibition rate of most analogues is higher than or equal to lead compound, the inhibition rate of 8 analogues are more than 40% and 4 compounds are between20%-40%(the inhibitory activity of TE-3 is best for 65.49%). G0/G1-phase cells are significantly increased while S-phase cells are decreased with the elevation of compound TE-10 concentration. Cell cycle progression is blocked in the G0/G1-phase. Study on the mechaniam of Inducing cell apoptosis show that compared with the control group, the expression rate of Fas and Fas L are significantly increased, the expression rate of Bax is increased while the expression rate of Bcl-2 is decreased and then make the ratio of Bcl-2/Bax decreased.2. The cell proliferation inhibitions of FMC and its 15 analogues are assessed by SRB assay. The results show that the analogues with 4-chloro, 4-bromo or 4-nitro substituted on ring-B exhibit the moderate against 8 cell lines, which are up to 10 times more potent than the lead compound, no selectivity. G0/G1-phase cells are significantly increased while S-phase cells are decreased with the elevation of compound FMC-6 concentration. Cell cycle progression is blocked in the G0/G1-phase. Apoptosis results show that the proportion of apoptosis increase obviously with the increase of concentration. When concentration is greater than3.13 μg·m L-1, appeare apoptosis and apoptosis rate is significantly higher than control group(6.95%).3. Preliminary pharmacological test show that FMC and its analogues have a good scavenging DPPH radical activity in vitro and antioxidant capacity of iron, including FMC-14 and FMC-15 were better than FMC, introducing O-dihydroxy on ring-B would significantly increase the antioxidant activity of the compounds.Conclusion:1. A long alkyl chain substituted on ring-A, sterically hindered subitituent(trifluoromethyl, nitro, isopropyl) or para substituted(halogen, methoxy) on ring-B have great contribution to improve the activity. The activity of heterocyclic substituted(naphthalene) on ring-B is decreased.2. Desmosdumotin C compounds induce the apoptosis of target cells through the Fas/Fas L, Bax/Bcl-2 signaling pathways, which play a role of antitumor.3. The analogues with para substituted on ring-B have great contribution to improve the activity, which are up to 10 times more potent than the lead compound FMC,no selectivity. Whether inducing tumor cell apoptosis play antitumor function remains to be further research.4. The analogues with 3,4-dihydroxy substituted or 3-methoxy-4hydroxy substituted on ring-B have great contribution to improve the antioxidant activity of the compounds, but the mechaniam of antioxidantion and structure-activity relationship analysis remains to be further research.