Design, Synthesis And Biological Activity Of Novel Bifunctional Anticancer Nucleoside Conjugates

With increasing incidence and mortality,cancer has become the leading cause of death in China.It has been estimated that new cancer cases and deaths were 4.3 million and 2.8 million in 2015,respectively.It is imperative to develop new anticancer drugs for our country and even the world.Traditional anticancer drugs lacks selectivity for tumor,which is the leading reason of side effects.In our investigation,OHCT derivatives were designed and synthesized from the terpenone(OHCT)which is the bioactive molecule.We discovered a bifunctional OHCT conjugate named dT-QX,that has selective killing effect on tumor cells.The chemical structure of dT-QX is composed of two parts:the deoxythymidine which is from antiviral nucleoside drugs Zidovudine,and the quinoxaline moiety of OHCT.The two parts are linked together by a carbon linker through amide bond and a thiazole ring.More importantly,dT-QX is not only an active molecule with selective killing effect on tumor cells but also an effective photosensitizer in photodynamic therapy(PDT).In order to clarify the structure-activity relationship of dT-QX conjugates,a variety of dT-QX analogues and contrasts were designed and synthesized.Three types of dT-QX analogues were designed as follows:1)increasing length of linker between the two functional groups with two carbon atoms increments,from C2 to C8;2)modification the quinoxaline structure,such as F or Cl substitution;3)cis and trans isomers of the OHCT functional group.The control compounds of dT-QX analogues are:the two functional groups were substituted respectively,such as the replacement of deoxythymidine group with galactopyranoside structure,or replacement of the quinoxaline structure with pyrenyl group.The in vitro cytotoxicity indicates the deoxythymidine group and quinoxaline structure were the essential structures of dT-QX analogues for anticancer activity.Through the comparison of the activities of dT-QX analogues,the structure-activity relationship of these compounds was found as follows:1)with the increased linker length,the activity of the compounds increased;the activity of C6 is similar to that of C8,so the 'activity is C8=C6>C4>C2;2)the position of the substituted halogen atom leads to a different activity,Fa>Fb;3)the activity of cis dT-QX isomer is higher than that of trans isomer.In addition,the activity of halogen substituted analogues was slightly lower than that of dT-QX without UVA irradiation.However,the result is reversed under UV irradiation,and the activity of 2Cl is the highest among all dT-QX analogues.We also compared the invitro anticancer activity of 2C1 compound with commercially available anticancer drugs gemcitabine and sunitinib.The EC50 of gemcitabine on hepatocellular carcinoma cell Bel-7402 and pancreatic cancer cell PANC-1 are between 50-100 ?mol/L,and the EC50 of sunitinib on these two kinds of cells are all 11-13 ?mol/L.Without UV irradiation,the EC50 of 2C1 conjugates on the two kinds of cells are 20-50 ?mol/L,but the EC50 on Bel-7402 and PANC-1 cells of 2C1 decreases to 0.67 ?mol/L and 1.3 ?mol/L,respectively with UVA irradiation of the intensity 3.6 J/cm.It is showed that the anticancer activity of 2C1 conjugates is ten times than that of sunitinib.We also studied the mechanism of the significant increase of anticancer activity of dT-QX analogues under UVA irradiation.Flow cytometry analysis showed that,under UVA irradiation,2C1 conjugates accelerated the process of cell apoptosis,and there is a different mechanism of the cytotoxicity of 2C1 conjugates under UVA irradiation.HPLC analysis of cell lysates showed that 2C1 conjugates induced a significant increase of some species in cells under UVA irradiation,but 2C1 did not change itself.By using the reactive oxygen species(ROS)probe DCFH-DA,the cells showed significant production of ROS in the 2C1 conjugates treatment group under UVA irradiation.Therefore,the 2C1 conjugates and UVA irradiation are the necessary conditions for the production of ROS.We also analyzed the intensity of UVA irradiation,and found that 2C1 conjugates under the intensity of 3.0 mW/cm2 for 20 min of UVA irradiation(3.6 J/cm)is enough to induce the production of reactive oxygen species in cells.Further study on the rate of ROS production by dT-QX analogues with UVA irradiation showed that the ROS production rate of 2C1 conjugates group is much faster than that of C6 conjugates group,which is consistant with the cancer cell cytotoxicity results.We also found that the G-cis as the control compound,has the low ability to induce the production of ROS.Subsequent experiment found that G-cis has the ability of quenching ROS that associated with galactopyranoside structure.