Design,Synthesis And Antitumor Activity Evaluation Of ?-Lapachones

In order to improve drug efficacy and selectivity,antitumor drug design targeting specific cell signaling pathways,kinases and proteins in tumor pathogenesis is desirable.NQO1 is a class of flavoprotein enzymes commonly expressed in eukaryotic cells that actively participates in the metabolism of various quinones and their bioactivation in vivo through electron reduction reactions.The expression level of NQOl is highly upregulated in many solid tumor cells compared with normal cells.Due to its overexpression in many tumor cells and biological activation of quinones,NQO1 is considered a potential molecular target for the treatment of various tumors.NQO1-targeted bioreductive drugs,triggered prodrugs and inhibitors are promising candidates as cancer therapies.Natural o-quinone compound ?-lapachone(?-lap)is a typical NQO1-targeted bioreductive drugs.Under the mediation of NQO1,a large number of reactive oxygen species(ROS)are produced through the quinone-hydroquinone-quinone redox cycle.High concentration of ROS will seriously damage the redox microenvironment of tumor cells,induce apoptosis of tumor cells,and have strong antiproliferative activity against a variety of tumor cells.The binding sites of NQO1 are mainly consists of the plane active pocket composed of Trp105,Tyr126,Tyr128,Phe106,Gly150,Gly149 and coenzyme FAD,and a side hydrophobic pocket composed of Tyr128,His161,Phe 236 and Met131.Molecular docking studies reveals that although the ?-lap is effectively embedded into the active pocket of NQO1,it occupies only the bottom pocket parallel to the FAD without binding to the lateral pockets formed by residues of Tyr128,His161,Phe 236,and Met131.Additionally,the pyran ring of ?-lap is a rather unstable structure that is prone to ring-opening hydrolysis to form toxic metabolites,which also impede its further development as an antitumor drug.1.Design,synthesis and antitumor activity evaluation of 4-substituted o-naphthoquinoxalactams as NQO1 bioreductive drugs.Based on the structure of ?-lap,thirty 4-substituted-3,4-dihydrobenzo[h]quinoline-2,5,6-triones were designed by replacing the pyran ring of ?-lap with ?-lactam to increase the stability of ?-lap derivatives,and introducing various types of substituents in the 4 position of the ?-lactam to enhance the binding with the side pocket of NQO1 and the targeting of NQO1.The target compounds were synthesizing by using 1,2-naphthoquinone as the starting material and trimethylsilyl azide(TMSN3)to introduce an amino group at 4 position.Then the intermediate was refluxed with aldehyde and meldrum's acid in acetic acid in order to synthesize the target compounds BL1-BL30.Through NQO1 activity,molecular docking and tumor cell proliferation inhibition experiments,the structure-activity relationships(SAR)of these compounds were established,with 4-hydroxy-3-methoxyphenyl(BL14),3-hydroxy-4-methoxyphenyl(BL15)and 2-fluorophenyl(BL10)stand out as preferred candidates.The compound BL15,which showed strong NQO1-binding activity and anti-proliferation activity,could stop the mitotic division of HepG2 tumor cells in the S/G2 phase,inducing tumor cells apoptosis.Further investigation into the mechanism showed that BL15 caused intracellular redox imbalance through increased ROS production,resulting in a decrease in mitochondrial membrane potential that eventually leaded to tumor cell apoptosis.The HepG2 tumor xenograft animal model showed that intravenous administration of BL15 could effectively inhibit tumor growth,with the growth inhibition rate reaching 50.3%at 20 mg/kg.Compared with the vehicle group,all mice showed no significant change in body weight,a sign of low toxicity of this compound.2.Design,synthesis and antitumor activity evaluation of 3-(1-benzotriazole)-nor-?-lapachone as NQO1 bioreductive drugs.Nor-?-lapachone(nor-?-lap)is a demethylated analogue of ?-lap that exhibits selective strong anti-proliferative activity against a variety of tumor cells while has low inhibitory activity against normal cells.Nor-?-lap participates in NQO1-dependent redox cycles and can induce tumor cell apoptosis via ROS upregulation.Molecular docking shows that nor-?-lap is effectively embedded in the active pocket of NQO1 and forms efficient ?-? bonding with the isopyrazine of FAD.However,the lateral pockets formed by residuals of Met131,Met154,Phe236,Tyr128 and His161 are not occupied by nor-?-lap.In order to increase the NQO1 targeting efficiency and antitumor activity of nor-?-lap,we designed twelve 3-(1-benzotriazole)-nor-?-lapachones on the basis of nor-?-lap by introducing a benzotriazole substituent in its 3-position.Through NQO1 activity,molecular docking and tumor cell proliferation inhibition experiments,the SAR of these compounds were established,with the one substituting the 3rd position of nor-?-lap with 5-butylbenzotriazole(NB11)stands out as the preferred candidate.This compound could stagnate the mitosis of HepG2 tumor cells in the G0/G1 phase and induce tumor cell apoptosis.Further investigation into the mechanism showed that compound NB11 could lead to intracellular redox imbalance through increased ROS production,resulting in the drop of mitochondrial membrane potential that caused tumor cell apoptosis.The HepG2 tumor xenograft animal model showed that intravenous administration of NB11 could effectively inhibit tumor growth,with a growth inhibition rate reaching 52.3%at 20 mg/kg.Compared with the vehicle group,NB11 did not cause any significant change in the body weight of mice.3.Design,synthesis and antitumor activity evaluation of ?-lapachone-monastrol hybrids as NQO1 bioreductive drugs.Monastrol is the first small molecule Eg5 inhibitor,which can specifically bind to Eg5 and inhibit its function,induce the proliferation of tumor cells to arrest in mitosis,and promote apoptosis.This study adopted the concept of pharmacophore combination,and a series of novel ?-lapachone analogs BM1-BM15 was designed and synthesized by replacing pyran ring of ?-lap with tetrahydropyrimidinethione moiety of monastrol.These hybrids had potent antiproliferative activity against NQO1-rich cell lines(HepG2 and A549),while NQO1-defficient cell lines(H596 and LO2)were less sensitive to these hybrids.In the presence of DIC,an inhibitor of NQO1,the antiproliferative activity of these compounds against A549 decreased,indicating that the process of biological reduction and activation mediated by NQO1 is closely related to their antiproliferative activity NQO1 activity assay and docking study demonstrated that BM9 was a good substrate of NQO1.Furthermore,as suggested by cellular mechanistic research concerning antitumor activity,the representative compound BM9 resulted in ROS production depending on NQO1,then oxidative stress triggered apoptotic cell death.In HepG2 xenograft tumor animal model,intravenous administration of BM9 can effectively inhibit tumor growth.At 20 mg/kg,the growth inhibition rate was 58.7%without obvious effect on the weight and behavior of mice.In conclusion,we designed and synthesized 4-substituted o-naphthoquinoxalactams,3-(1-benzotriazole)-nor-?-lapachone and ?-lapachone-monastrol hybrids as NQO1 bioreductive drugs with ?-lap and nor-?-lap as the lead compounds.The NQO1 activity,molecular docking simulation and tumor cell proliferation inhibitory activity of the representative compounds were evaluated.Three NQO1 bioreductive drugs BL15,NB11 and BM9 with excellent antitumor activity in vitro and in vivo were found.The NQO1 activity of these three compounds was higher than that of ?-lap and nor-?-lap,and the targeting and cell selectivity of these compounds were improved to a certain extent,which laid a certain foundation for further structural optimization and the discovery of candidate compounds.