- 學位論文
新且具活性protoflavone衍生物之合成
Synthesis of new, biologically active protoflavone derivatives
摘要
Abstract Protoflavones represent a unique and rare class of natural flavonoids with a non-aromatic B-ring and a hydroxyl group at C-1’. Antitumor properties of these compounds and some of their synthetic derivatives had previously been confirmed; their mechanism of action and in vivo efficacy make these compounds potentially valuable for related drug discovery initiatives. In our work, we aimed i) to prepare a variety of synthetic protoflavone analogs, ii) to investigate the formation of the protoflavone B-ring from the more common 4ʹ-hydroxyflavone moiety upon free radical scavenging, ii) to study the prepared compounds for their antitumor properties in vitro, with a particular emphasis on their potential to overcome multi-drug resistant cancer, and iv) to search for other potential bioactivities of these compounds, which are not related to the cytotoxic effect. By combining semi-synthesis from naturally occurring 4ʹ-hydroxyflavones and a 4-7 steps total synthetic approach, 50 new protoflavone derivatives were synthesized, whose chemical diversity involved various A-ring substitution patterns and 1ʹ-substituents. In silico DFT calculations and experimental data obtained by HPLC and CE analyses provided proof for the existence of an apigenin-protoapigenone-apigenin redox cycle, driven by relevant participants of the intracellular redox equilibrium, such as OH radicals and reduced glutathione. Cytotoxicity of the newly obtained compounds was tested on a panel of sensitive and multi-drug resistant cell lines. The ability of protoflavones to evade efflux-mediated MDR was confirmed both in ABCB1 and ABCG2 expressing cell lines, with the exception of protoapigenone, which was identified as an ABCG2 substrate. Moreover, MDR selective cytotoxicity was observed for most of the tested protoflavones in a breast cancer cell line adapted to doxorubicin (MCF-7Dox) and SAR revealed importance of the A-ring substitution, while in the uterine sarcoma MES-SA/Dx5, another doxorubicin-selected cell line, only the 1´-OH containing compounds showed relevant selectivity. Studies on the mechanism for the MDR selectivity suggested the involvement of changes in the antioxidant defense of the cancer cells during the evolution of resistance. Screening a sub-set of our compounds for xanthine oxidase (XO) inhibitory activity revealed protoapigenone 1-O-propargyl ether as the first non-planar flavonoid that can strongly and competitively inhibit this enzyme. In silico docking studies uncovered a hydrophobic pocket near the active center of XO, where the propargyl side chain was fitting perfectly, hence anchoring the compound, and leading to an efficient competitive inhibition. Our findings re-wrote the structure-activity relationships generally accepted for flavonoids concerning the need for a planar structure, and opened new possibilities for a rational design of flavonoid-type XO inhibitors. Altogether, our work provided several new protoflavone derivatives, new insights into their formation, and rich bioactivity and SAR data on these compounds. Our work also outlined promising new research directions for the future, among which the possible use of protoflavone analogs as new lead compounds against multi-drug resistant cancer might be of particular interest.
並列摘要
Abstract Protoflavones represent a unique and rare class of natural flavonoids with a non-aromatic B-ring and a hydroxyl group at C-1’. Antitumor properties of these compounds and some of their synthetic derivatives had previously been confirmed; their mechanism of action and in vivo efficacy make these compounds potentially valuable for related drug discovery initiatives. In our work, we aimed i) to prepare a variety of synthetic protoflavone analogs, ii) to investigate the formation of the protoflavone B-ring from the more common 4ʹ-hydroxyflavone moiety upon free radical scavenging, ii) to study the prepared compounds for their antitumor properties in vitro, with a particular emphasis on their potential to overcome multi-drug resistant cancer, and iv) to search for other potential bioactivities of these compounds, which are not related to the cytotoxic effect. By combining semi-synthesis from naturally occurring 4ʹ-hydroxyflavones and a 4-7 steps total synthetic approach, 50 new protoflavone derivatives were synthesized, whose chemical diversity involved various A-ring substitution patterns and 1ʹ-substituents. In silico DFT calculations and experimental data obtained by HPLC and CE analyses provided proof for the existence of an apigenin-protoapigenone-apigenin redox cycle, driven by relevant participants of the intracellular redox equilibrium, such as OH radicals and reduced glutathione. Cytotoxicity of the newly obtained compounds was tested on a panel of sensitive and multi-drug resistant cell lines. The ability of protoflavones to evade efflux-mediated MDR was confirmed both in ABCB1 and ABCG2 expressing cell lines, with the exception of protoapigenone, which was identified as an ABCG2 substrate. Moreover, MDR selective cytotoxicity was observed for most of the tested protoflavones in a breast cancer cell line adapted to doxorubicin (MCF-7Dox) and SAR revealed importance of the A-ring substitution, while in the uterine sarcoma MES-SA/Dx5, another doxorubicin-selected cell line, only the 1´-OH containing compounds showed relevant selectivity. Studies on the mechanism for the MDR selectivity suggested the involvement of changes in the antioxidant defense of the cancer cells during the evolution of resistance. Screening a sub-set of our compounds for xanthine oxidase (XO) inhibitory activity revealed protoapigenone 1-O-propargyl ether as the first non-planar flavonoid that can strongly and competitively inhibit this enzyme. In silico docking studies uncovered a hydrophobic pocket near the active center of XO, where the propargyl side chain was fitting perfectly, hence anchoring the compound, and leading to an efficient competitive inhibition. Our findings re-wrote the structure-activity relationships generally accepted for flavonoids concerning the need for a planar structure, and opened new possibilities for a rational design of flavonoid-type XO inhibitors. Altogether, our work provided several new protoflavone derivatives, new insights into their formation, and rich bioactivity and SAR data on these compounds. Our work also outlined promising new research directions for the future, among which the possible use of protoflavone analogs as new lead compounds against multi-drug resistant cancer might be of particular interest.
參考文獻
延伸閱讀
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