Discovery And Mechanism Research Of Novel Pyrimidine-Based Derivatives As Reversal Agents Against ABCB1-Mediated MDR

In clinical antitumor therapy,the appearance of MDR(multidrug resistance)has become a major obstacle in successful cancer chemotherapy.Although it is reported that MDR is involved with multiple mechanisms,the overexpression of some members of the ABC(ATP-binding cassette)protein family is thought to be a major contributor to the development of MDR in cancer cells.ABCB1(P-glycoprotein),the important member of ABC transport proteins,has been found to be highly expressed in various tumor tissues,such as colon,liver,pancreas cancers.It could take advantage of the energy obtained by ATP hydrolysis and pump a variety of antitumor drugs out of the cancer cells,such as paclitaxel,camptothecin analogs,etc.,resulting the lower drug concentration in cancer cells and the development of drug resistance.Clinical studies have shown that the combination of ABCB1 modulators with chemotherapeutic agents could down-regulate the expression or activity of ABCB1 and up-regulate the concentration of chemotherapeutic agents to cytotoxic concentration in cancer cells,achieving the antitumor effect.However,although several ABCB1 inhibitors have entered the clinical trial stage,there still have been no satisfactory results so far because of some limitations such as insignificant clinical effects,pharmacokinetic interaction,or concerns about safety.Therefore,developing more selective and effective novel ABCB1 inhibitors to reverse MDR has become an urgent requirement.We identified three series of pyrimidine-based derivatives as novel reversal agents against ABCB1-mediated multidrug resistance and evaluated their reversal activity.The main work was listed as below:(1)Discovery of 1,2,3-triazole-pyrimidine hybrids as novel reversal agents against ABCB1-mediated multidrug resistance(series 1)We conducted the activity test based on our in-house structurally diverse molecular library(~500 compounds)and found that compound containing 1,2,3-triazole moiety and pyrimidine core could reverse the resistance of ABCB1-overexpressed colon resistant cancer cell line SW620/AD300 to PTX.As reported,various tyrosine kinase inhibitors(TKIs)containing a pyrimidine core,including gefitinib and erlotinib,have been identified as substrates or modulators of ABCB1 and other ABC transporters.In addition,triazole,as a crucial nitrogen-heterocycle,also attracted our attention due to its wide application as ABCB1 inhibitors to reverse MDR,as well as its easy accessibility.Based on the primary data and Molecular hybridization,we identified that a series of 1,2,3-triazole-pyrimidine hybrids as novel MDR reversal agents.Among them,compound 15,17,25,34 exhibited more potent reversal activity than positive compound Verapamil.SAR analysis suggested that 1,2,3-triazole moiety may play an important role in determining activity.We selected compound 25 for further mechanistic research and the results showed that compound 25 had no influence on the expression and cellular localization of ABCB1 in SW620/AD300 cells,but up-regulated the accumulation of PTX to cytotoxic concentration in SW620/AD300 cells by inhibiting the function of ABCB1 transporter,achieving the reversal of MDR.(2)Discovery of biacylamide-1,2,3-triazole-pyrimidine hybrids as novel reversal agents against ABCB1-mediated multidrug resistance(series 2)For further structurally optimization,on one hand,we retained the positive contributor to development of reversal activity,such as 1,2,3-triazole moiety,halogen group,aromatic ring center,positive-ionizable groups etc.;on the other hand,in order to develop the binding affinity to ABCB1 transporter,we introduced the aromatic ring and biacylamide moiety which have potential inhibitory activity against ABCB1.Finally,we obtained a series of biacylamide-1,2,3-triazole-pyrimidine hybrids(series 2).However,although docking data showed that biacylamide moiety indeed increased the hydrogen binding sites,the reversal activity of series 2 resulted in decreased activity compared with series 1.Furthermore,lipophilicity has been identified as an important factor correlated to potent inhibitory activity against ABCB1,since the inhibitors majorly bind to the big hydrophobic pocket located within the transmembrane protein of ABCB1 and lipophilicity helps them enter the cavity.To explore the reasons for the decreased reversal activity of series 2 compounds,the lipophilicity of series 1 compounds 34,35,36,38 and series 2 compounds 10,11,12,13 was tested and the results showed that the lipophilicity of series 2 compounds 10,11,12,13 with lower reversal activity was decreased.Based on the above results,we speculated that the weak reversal effect of series 2 may result from lower lipophilicity,more groups that solvate in water and lager molecular size.These results provided a direction for further structural optimization and activity improvement.(2)Discovery of biacylamide-pyrimidine hybrids as novel reversal agents against ABCB1-mediated multidrug resistance(series 3)On the basis of above two series compounds,we retained the biacylamide moiety which could increase the hydrogen binding sites but removed the 1,2,3-triazole moiety to reduce molecular size and improve lipophilicity.Finally,we obtained a series of biacylamide-pyrimidine hybrids(series 3)and evaluated their reversal activity.The results showed that series 3 displayed significantly increased reversal activity than series 1 and 2.Among them,series 3 compound 15,18,19,21 with higher reversal activity also showed higher lipophilicity than series 2 compound 27-29,34,which confirmed the importance of lipophilicity in developing reversal activity.We selected compound 19 with best reversal activity for further mechanistic research.Compound 19 had no influence on the expression and cellular localization of ABCB1 in SW620/AD300 cells but up-regulated the accumulation of PTX to cytotoxic concentration in SW620/AD300 cells by inhibiting the function of ABCB1 transporter,achieving the reversal of MDR.ATPase activity assay suggested that compound 19 was a stimulator of ABCB1 ATPase and may reverse ABCB1-mediated multidrug resistance by competitively displacing conventional anticancer drugs and itself being pumped out by ABCB1.In addition,compound 19 had no significant inhibitory activity on CYP3A4,suggesting that it had the potential a leading compound to further develop safe ABCB1 inhibitors.Finally,compound 19 also exhibited reversal activity without toxic side effects in vivo.In this study,our effort has yielded three series of pyrimidine-based derivatives as novel reversal agents against ABCB1-mediated multidrug resistance and evaluated their reversal activity,which enriches the structure type of ABCB1 modulators and gives strong support for the discovery of more safe and effective ABCB1 modulators.