Study On Antitumor Effect 8-position Modified Indolequinazolinedione Derivatives And The Metabolism In Rats

Objective:1.The molecular docking module in the computer virtual screening software was used to systematically carry out the virtual screening of anti-tumor active molecules of IQD ring derivatives.IQD,marketed drugs（fostamatinib,antroquinolol,palbociclib,vandetanib and Venetoclax,etc.）and original ligand（N-methyl-2-[[4-[（3,4,5-Trimethyl）Amino]-1,3,5-Triazin-2-yl]Amino]Benzamide,（2Z）-5’-Bromo-2,3’-Biindole-2’,3（1H,1’H）-Dione Amori Ate et al）.Based on the docking score by using the Libdock method,we initially and rapidly screened out the substitution sites with antitumor and synergistic effects from the 52 candidate molecules of IQD D-ring derivatives,and attempted to deduce the antitumor mechanism of IQD derivatives.2.Referring to the existing literature,five 8-substituted IQD derivatives（8-NO₂-IQD,8-Cl-IQD,8-F-IQD,8-CH₃-IQD and 8-OCH₃-IQD）were synthesized by heating and reflux reaction of 5-position isatin derivative and isatin anhydride in triethylamine and toluene.Aiming at the five 8-substituted IQD derivatives mentioned above,we carried out experiments of their anti-proliferation effects and tumor cell apoptosis signaling pathways on three common human tumor cells（MCF-7,Hep G2 and A549）in vitro:（1）Detection of the effect of the 8-substituted IQD derivatives on the proliferation activity of tumor cells by MTT method;（2）Crystal violet staining was used to observe the effect of the 8-substituted IQD derivative on the morphological changes of tumor cells.（3）Hoechst staining was used to observe the effect of the8-substituted IQD derivative on tumor cell apoptosis.（4）Annexin V-FITC/PI staining was used to detect the effect of the 8-substituted IQD derivative on the apoptosis of tumor cells.（5）Western-blotting was used to examine the effect of the 8-substituted IQD derivative on the expression of Akt,PI3K,p-Akt,p-PI3K,Fas,Fasl,Bad,Bcl-2and Bax proteins in tumor cells.3.Experimental study on metabolites and pharmacokinetics of 8-F-IQD in healthy SD rats was carried out.The rats were given8-F-IQD drug solution by gavage at the dose of 50mg/kg for three consecutive days.Tail vein blood was collected at set time points（0.5,1,1.5,2,3,4,8,12,and 24h）after the last dose and feces and urine were collected from each subject via a metabolism cage.Using LCMS-IT-TOF high-resolution mass spectrometer,the mobile phase was acetonitrile（A）-0.1%formic acid water buffer（B）,and gradient elution was conducted for 0～10 min and 20%（A）.10.1～20min,20%-50%（A）;20.1～35min,50%（A）-95%（A）;35.1～40min,95%-20%（A）;40.1～45min,20%（A）.Met ID 1.2 software performed metabolite profiling in blood,urine,and feces from test rats.A high performance liquid chromatography-triple quadrupole mass spectrometer（UPLC-QQQ）was used in positive ion mode and selective ion reaction monitoring（SRM）.A Dima Diamonsil C-18 column（100mm×2.1mm,1.8μm）was selected.The mobile phase consisted of acetonitrile（A）-water（B）with gradient elution of 0～7min,5%（A）-95%（B）;7.1-10min,95%（A）-5%（B）;10.1-20min,5%（A）-95%（B）.The prototype component 8-F-IQD in the blood of the test rats was analyzed.Results:1.The docking results of IQD derivatives were analyzed as follows:（1）The IQD derivative modified at position 8 was the substitution site with the strongest synergistic effect in the D ring.（2）To a large extent,the IQD ring derivatives may exert the antitumor effect by inhibiting the invasion and metastasis of tumor cells,but the scores of most of the IQD ring derivatives are lower than those of the marketed drugs and original ligands.2.Studies on the proliferative activity and apoptosis-inducing signaling pathways of five 8-substituted IQD derivatives on MCF-7,Hep G2 and A549 cells:Results:（1）8-NO₂-IQD and 8-F-IQD showed good cell proliferation inhibition effects on MCF-7,Hep G2 and A549 cells,with the most significant effect on A549 cells（***P<0.001）.（2）8-NO₂-IQD and 8-F-IQD can make A549 cells collapse or even rupture in a concentration-dependent manner.（3）Hoechst staining:Compared with the blank control group,8-NO₂-IQD and 8-F-IQD showed significant differences in the nuclear concentration rate of A549 in the administration group.（4）Annexin V-FITC/PI staining results of A549 cells after 8-NO₂-IQD induction of apoptosis showed that 8-NO₂-IQD could induce early and late apoptosis of A549 cells with concentration-dependent and significant differences（**P<0.01）;（5）Western-Blot experiments showed that after A549 cells were treated with 1.56,3.13and 6.25μmol/L 8-NO₂-IQD,the expressions of Fas,Fasl,Bax and Bad were significantly increased（*P<0.05 or**P<0.01）,the expressions of Akt,PI3K and Bcl-2 were down-regulated,Akt showed significant differences,and PI3K and Bcl-2showed no differences.p-Akt and p-PI3K expressions were not significantly changed.3.Study results of 8-F-IQD in rats:A total of 15 metabolites and protodrugs were detected in rat plasma,urine,and feces.Among them,the m/z information of267.0546,269.0760,333.0075,285.0652,445.1062 and others were the characteristic metabolic peaks of 8-F-IQD,m/z267.0546 was the prototype drug,m/z269.0760 was shown as the hydrogenated metabolic drug,m/z333.0075 was shown as the oxidized metabolic drug,m/z285.0652 was shown as the hydrolyzed metabolic drug and m/z445.1062 was shown as the hydrogenated+glucuronidation metabolic drug.Pharmacokinetic study method has good specificity and the linear relationship is good in the concentration range of 50.00-800.00 ng/m L（r=0.9951）.The accuracy of each index investigated by the method was less than 15.11%,and RSD values in the precision test were no more than 12.82%.The pharmacokinetic parameters of8-F-IQD from the 8-F-IQD blood concentration-time curve in rats were as follows:T_1/2=6.617?1.20h,C_max=703.548?119.45 ng/ml and T_max=2h.Conclusion:The D-ring modified IQD drug molecules may exert their tumor-inhibiting effects by inhibiting tumor cell invasion,metastasis and cycle arrest,and the8-position-modified IQD is the most important substitution site for antitumor efficacy in D-ring.The prepared 8-NO₂-IQD and 8-F-IQD have significant anti-tumor activity on three cell lines,and the 8-NO₂-IQD can induce apoptosis of human A549 cells through Fas/Fasl and mitochondrial pathway.8-F-IQD is mainly metabolized through oxidation,hydrolysis and glucuronidation in healthy SD rats,and it has the characteristics of rapid absorption and elimination.