Design, Synthesis And Activity Evaluation Of Methylated Polyphenol Derivatives As MDR Reversal Agents

Chemotherapy is one of the main methods of oncotherapy, however, the extensivemulti-drug resistance (MDR) in cancer cells has threatened the efficacy of anticancerdrugs including paclitaxel, vinblastine and doxorubicin, et al. The mechanism ofMDR is complicated, but one of most important mechanisms is the overexpression ofthree transmembrane transporters including P-glycoprotein (P-gp), multidrugresistance-associated protein (MRP) and breast cancer resistance-associated protein(BCRP). These three drug efflux proteins are belonged to the ATP-binding cassette(ABC) transporter superfamily. P-gp is the first discovered, most important and mostin-depth study among the three transmembrane transporters. Considerable efforts havebeen made to develop the MDR reversal agents targeting P-gp, and three generationsof P-gp inhibitors have been developed. The first generation reversal agents includecalcium channel inhibitors, such calcium channel blocker verapamil, and cyclosporine Aclinically used as the immunosuppressant. But the toxicity of this generationcompounds as a result of its high effective concentrations limits its use in clinicalpractice. The second generation MDR inhibitors are derivatives of the first generationreversal agents, including dexverapamil, PSC833, VX-710, S9788, et al. Althoughthese compounds showed high reversal activity and none toxicity, these compoundscombined with anticancer drugs would interfer with metabolism of the anticancerdrugs, which would lead to unpredictable side effects. And thus these MDR inhibitorsfailed to clinical application. The third-generation reversal agents includingzosuquidar, tariquidar, elacridar, which overcome the defects of the first twogenerations, are low-level toxic, efficient P-gp mediated MDR reversal agents. Thesecompounds have good development prospects. However, none of MDR inhibitors hasbeen used in clinic until now. So, looking for low toxicity, high efficiency andspecificity MDR reversal agents applicable to clinical oncotherapy is one of hotspotsin anticancer research field. Our group has been engaged in the searching for MDR reversal agents for many years, and has made some achivements. Based on themechanism of P-gp mediated tumor MDR and the previous research achivements ofour group, we designed and synthesized methylated EGCG and methylated quercetinderivatives respectively, and tested for the bioactivity of the target compounds. Thisthesis is divided into the following two parts:1. Design, synthesis and bioactivity analysis of methylated EGCG analogues as MDRreversal agents.It has been long reported that EGCG, the main ingredient of the tea polyphenol,could reverse P-gp mediated tumor MDR. The further study showed that the fullymethylated EGCG could reverse the P-gp mediated MDR more significantly. Ourgroup has synthesized several fully methylated EGCG analogues showing high MDRreversal activity.1μM compounds of1,2,3and4potentiated paclitaxel cytotoxicityon P-gp overexpressed LCC6MDR cell with the relative reversal factors of6.9,39.6,16.7and9.8respectively, much higher than the classic MDR reversal agent verapamil(RF=3.8) and EGCG (RF=1.2). Based on these research results, seven methylatedEGCG derivatives have been designed and synthesized, and four of them have beentested for their P-gp mediated MDR reversal activitives. All of these four compounds(13,14,16and21) inhibited P-gp modulating activities at1.0μM, with the RF being43.5,49,19.3and13.9respectively, and all these four tested compounds showedsignificantly higher activity than the positive control sample verapamil. Thecytotoxicity experiments showed that the four compounds had no cytotoxicity, whichprove that these four EGCG analogues are low toxicity and efficient MDR reversalagents.2. Design, synthesis and bioactivity analysis of methylated quercetin analogues asMDR reversal agents.Quercetin, one of the flavonoid compounds, has anti-inflammatory, antiviral,anticancer and antioxidant activities, and is widely distributed in nature. It has beenreported that quercetin showed good tumor MDR reversal activity, so using thequercetin as the leading compound to design and synthesize efficient MDR reversalagents becomes one of the focuses. Quercetin has the similar the polyphenol structure as EGCG. However, the main difference is that the presence of double bond in C-ringleads to the rings A and C in the same plane, but the ring C of EGCG has itsthree-dimensional spatial structure since it does not contain double bond. Methylatedphenolic compounds like permethyl epigallocatechin gallate (EGCG), permethylningalin B and its synthetic analogues also displayed potent P-gp-modulatingactivities. These results suggest that methylation of some natural phenolic compoundscould improve P-gp modulating activity, and multi-methoxy substituted phenyl ringsmay be an important pharmacophore for reversing MDR. It has been reported that themethylated marine alkaloid Ninglin B exhibited higher P-gp mediated MDR reversalactivity than Ninglin B. Here, we designed and synthesized14methylated quercetinderivatives (containing various side chains at O-3) and tested for their P-gp-, BCRP-and MRP1-modulating activities. Almost all quercetin derivatives exhibited nocytotoxicity towards LCC6, LCC6MDR or normal mouse fibroblast cells (L929) withIC50higher than100μM. The only slightly toxic compound is compound12, with IC50towards L929at17.6μM.1μM compound34displayed P-gp-reversal activity (RF of34=11.3), which is higher than the positive control sample verapamil (RF=3.3).Compounds22,29,31and34(at1.0μM) showed BCRP-reversal activities inHEK293/R2(RF of22=13.6, RF of29=11.8, RF of31=14.7and RF of34=12.6).Compounds22、23、28、31and34(at1.0μM) inhibited BCRP-mediated MDRinMCF7-MX100cell lines (RF of22=20.1, RF of23=8.3, RF of28=10.0, RF of31=9.5and RF of34=7.6).