開發具有治療和螢光特性的硝基還原酶響應性去氧核醣靶向和氮芥烷基化前藥

Abstract

本論文主要聚焦於設計並合成能透過腫瘤缺氧環境過度表達的硝基還原酶 (NTR) 作用之缺氧活化前藥。前藥p-CbmNM-CPT或o-CbmNM-CPT (圖1) 因水溶性極差，將其溶於70%二甲基亞碸/去離子水後，使用化學還原劑Na2S2O4進行釋放實驗，而其前驅物p-CbmNM則可溶於2%二甲基亞碸/PBS緩衝液中以酵素NTR反應。利用液相層析質譜儀 (LC-MS) 分析顯示p-CbmNM-CPT和o-CbmNM-CPT皆能釋放喜樹鹼和氮芥，但觀察到的是推論結構為兩分子AniDBOH加成到一分子氮芥上之NMDBOH荷質比訊號。而NTR作用p-CbmNM後觀測到的是氮芥水解的NMOH，以及推論結構為兩分子NMOH加成至一分子氮芥之TriNMOH荷質比訊號。並進一步量測氮芥之穩定性，證實其分子活性過高，會迅速水解或被親核基加成，而失去烷化去氧核醣核酸的預期效果，無法和喜樹鹼達到協同效應。 為了改善氮芥活性過高的問題，因此將氮芥修飾在強拉電子之硝基苯的對位來降低其烷化能力，鄰位則以酸脂基團連接抗腫瘤藥物5-氟尿嘧啶 (5-FU) 或能和去氧核醣小溝結合之螢光團VBTZAMe (圖2)。當前藥被NTR還原後成為推電子之苯胺才會呈現氮芥之烷化能力，同時可以進行1,4-脫去釋放活性藥物5-FU，或釋放VBTZAMe顯現螢光，達到雙藥協同性或具治療和顯影多功能的藥物分子，見圖2。實驗結果顯示，在沒有加入NTR前，PNM-FU在水溶液中就會自行水解釋放5-FU，無法達到期望前藥之治療偕同效果。反之，PNM-VBTZAMe則確實能經NTR作用後釋放VBTZAMe螢光分子，並與去氧核醣產生結合，使螢光增強1.6倍。並透過LC-MS分析釋放的產物，結果顯示苯胺氮芥仍有被水解的現象，同時也觀察到苯胺氮芥與輔酶NADH上之鹼基腺嘌呤所形成的加成產物訊號，間接證實苯胺氮芥能夠與去氧核醣之鹼基產生烷化，造成損傷。細胞存活度實驗顯示PNM-VBTZAMe確實能在具有NTR的HepG2癌細胞中轉變為活性烷化結構APNM達到毒殺細胞的效果；細胞顯影結果也指出在相同HepG2環境中，可觀測到原本不具螢光性質之前藥，只有在與NTR作用後經1,4-脫去反應之後能釋放出螢光發色團VBTZAMe，證實PNM-VBTZAMe是具有潛力的治療癌細胞與具有顯影功效之前藥有機小分子。

The thesis primarily focuses on the design and synthesis of hypoxia-activated prodrugs that can be activated by the overexpression of nitroreductase (NTR) in the hypoxic tumor microenvironment. The prodrugs p-CbmNM-CPT and o-CbmNM-CPT (Figure 1), due to their poor water solubility, were dissolved in a solution of 70% dimethyl sulfoxide (DMSO) and deionized water for release experiments utilizing the chemical reductant sodium disulfide (Na2S2O4). Meanwhile, the precursor p-CbmNM was soluble in a 2% DMSO/PBS buffer for enzymatic reactions with NTR. Liquid chromatography-mass spectrometry (LC-MS) analysis demonstrated that p-CbmNM-CPT and o-CbmNM-CPT could release camptothecin (CPT) and nitrogen mustard (NM). The observed signals were consistent with a proposed structure in which two molecules of AniDBOH were added to one molecule of NM, as indicated by a specific mass-to-charge ratio (m/z) signal. Following the action of NTR on p-CbmNM, NM hydrolysis produced NMOH, along with a proposed structure, TriNMOH, suggesting the addition of two molecules of NMOH to one molecule of NM, which was also represented by a specific m/z signal. Further measurements of NM stability confirmed that its molecular activity was excessively high, resulting in rapid hydrolysis or nucleophilic addition. This process diminished its intended alkylating effect on deoxyribonucleic acid (DNA) and hindered the achievement of a synergistic effect with CPT. To address the issue of the high reactivity of nitrogen mustard, modifications are made at the para position of a strongly electron-withdrawing nitrobenzene to reduce its alkylating ability. At the ortho position, a carbonate group is attached to the anticancer drug 5-fluorouracil (5-FU) or to a fluorescent moiety, VBTZAMe, which can bind to the minor groove of DNA (Figure 2). When prodrugs are reduced by NTR to form an electron-donating aniline, they exhibit the alkylating properties of nitrogen mustard. Concurrently, they can undergo 1,4-elimination to release either 5-FU or VBTZAMe, the latter of which exhibits fluorescence. This process achieves dual-drug synergy or results in a multifunctional drug molecule with both therapeutic and imaging capabilities. Experimental results demonstrated that, in the absence of NTR, PNM-FU would spontaneously hydrolyze in an aqueous solution, releasing 5-FU and failing to achieve the intended prodrug effect. In contrast, PNM-VBTZAMe successfully reacts with NTR to release the fluorescent molecule VBTZAMe, which binds to DNA and results in a 1.6-fold increase in fluorescence. Analysis of the released products using LC-MS confirmed that aniline mustard underwent hydrolysis. Furthermore, signals corresponding to the adduct formed between aniline nitrogen mustard and the adenine base of the NADH coenzyme were also detected, indirectly confirming that aniline nitrogen mustard has the potential to alkylate DNA bases, leading to damage. The results of the cell viability assay demonstrate that PNM-VBTZAMe can effectively release the active alkylating species APNM in HepG2 cells expressing NTR, leading to cell death. Additionally, the cell imaging results indicate that, in the same environment, PNM-VBTZAMe is initially non-fluorescent; however, a fluorescent dye (VBTZAMe) becomes observable after the prodrug interacts with NTR and undergoes a 1,4-elimination reaction. These findings confirm that PNM-VBTZAMe is a promising prodrug with both therapeutic potential against cancer cells and fluorescence imaging capabilities as an organic small molecule.