Alda-1類似物之半合成、全合成及其對ALDH2之活性促進研究

Abstract

粒線體醛去氫酶2 (mitochondrial aldehyde dehydrogenase 2, ALDH2)為人體乙醇代謝途徑上重要的酵素之一。約5.6億東亞人(佔世界人口8%)有不具活性之ALDH2*2對偶基因。近幾年的研究指出，此喪失活性之突變，除了使個體較容易被內生性或外源性醛類損害，也可能與心血管疾病、糖尿病、上呼吸消化道癌症等有關。粒線體醛去氫酶2活化劑最早於2008年以高通量篩選的方式篩選出來，其中以Alda-1具有最好的粒線體醛去氫酶2促進活性。 本實驗根據Alda-1之結構，製備出一系列類似物。以EDC偶合的方式，將piperonylamine與一系列的苯甲酸，或是piperonylic acid與一系列苯甲胺進行醯胺偶合反應，得到化合物3a–h及6a–e。考量到人為設計化合物時，常一味追求更強之結合親和力(binding affinity)而忽略其毒性，所以除了合成Alda-1類似物之外，本實驗以小檗鹼為起始物，製備得到存在於臺灣黃柏根部之微量成分：methyl anhydroberberilate，其具有與Alda-1相似之N-piperonylbenzamide結構。而後藉由野生型粒線體醛去氫酶2活性促進試驗，觀察結構上的改變對促進活性有何影響。 結構-活性關係之探討發現羰基之位置對於促進活性可能有重要影響：當羰基位於甲苯基團之苄基位時(即化合物3b–h)，促進活性較羰基位於3,4-亞甲二氧苄基團之苄基位，最多增強約51倍。由電腦分子模擬結果推測影響活性之關鍵因素為「羰基位於甲苯基團或3,4-亞甲二氧苄基團之苄基位時，化合物之醯胺基氫與Asp457主鏈羰基是否在可產生氫鍵之距離內」。其次，苯甲醯基2'或6'位置至少須存在一個取代基，使Alda-1類似物能與野生型粒線體醛去氫酶2以正確的方式作用，例如：化合物3a苯甲醯基2'或6'位置無任何取代基，不同於其它Alda-1類似物以3,4-亞甲二氧苄基團插入野生型粒線體醛去氫酶2受質出入口，而是以苯甲醯基芳香環插入酵素受質出入口，使其醯胺基氫與Asp457主鏈羰基之距離大於氫鍵可作用距離。此外，若2'或6'位置上之取代基使苯甲醯基之芳香環較為疏水性，其與酵素受質出入口許多疏水性胺基酸會有較佳之疏水性作用力，且其造成之立體障礙越小越好。最後，3,4-亞甲二氧苄基基團之苯環須與Phe459支鏈芳香環平行以得到較佳之π-πi交互作用。

ALDH2 is one of the most important enzymes in ethanol metabolism. There are around 560 million East Asians (nearly 8% of the world’s population) carrying the inactive ALDH2*2 allel. Recent studies indicated that ALDH2*2 would not protect individuals from the damages caused by endogenous or exogenous aldehydes, and it may relate to cardiovascular diseases and upper aerodigestive track cancers, etc. The earliest ALDH2 activators are discovered by high-throughput screening in 2008. Among these ALDH2 activators, Alda-1 has the best activating effects. A series of Alda-1 analogs were prepared in the study. Compounds 3a–h and 6a–e were prepared from coupling piperonylamine or piperonylic acid with a series of benzoic acid or benzylamine by EDC. Considering compounds are often designed to pursue higher binding affinity but ignoring the toxicity, a minor constituent (methyl anhydroberberilate) in the roots of Phellodendron amurense var. wilsonii, which is similar to the N-piperonylbenzamide structure of Alda-1, was prepared from berberine. Through the wild-type ALDH2 activation assay, the structural impact on the activating ability was disclosed. Investigation on the structure-activity relationship suggested that the position of carbonyl group in these compounds might play the most important role in activating wild-type ALDH2: when carbonyl groups were located at the benzylic position of benzyl group of these compounds (3b–h), the activating abilities were better than those which carbonyl groups were located at the benzylic position of piperonyl group up to 51-fold. The computational molecular modeling results point out the most important role in activating wild-type ALDH2 is the capability of forming hydrogen bonds between amide hydrogen of the compounds and the main chain carbonyl group of Asp457 of the wild-type ALDH2. Secondly, there must exist at least one substitution group on either 2' or 6' position on the benzoyl group to make the Alda-1 analogs interact with wild-type ALDH2 correctly; e.g., there are not any substitution group at the 2' or 6' position on the benzoyl group of compound 3a, and it inserts into the substrate exit of wild-type ALDH2 by the benzoyl group instead of piperonyl group which makes the distance between amide hydrogen of 3a and the main chain carbonyl group of Asp457 beyond the hydrogen-bondable distance. In addition, if the 2' or 6' substitution groups make the aromatic ring of benzoyl group more hydrophobic, it would have better hydrophobic interaction with the many hydrophobic amino acids around the substrate exit of the enzyme. Moreover, the smaller steric interactions caused by the 2' or 6' substitution groups, the better the activity achieved. Finally, the aromatic ring of piperonyl group should be parallel to the aromatic side chain of Phe459 to get a proper π-π interaction.