合成人類胸苷酸激酶之抑制劑

Abstract

胸苷三磷酸(dTTP)的生成與供給在人體中是獨特且重要的。胸苷三磷酸(dTTP)的合成必須從胸苷酸(dTMP)經由胸苷酸激酶(thymidylate kinase, TMPK)進行磷酸化形成胸苷二磷酸(dTDP)，接著透過核苷酸激酶催化形成胸苷三磷酸(dTTP)。然而，其他的去氧核苷三磷酸(dNTP)是藉由核糖核苷酸還原酶(ribonucleotide reductase, RNR)將核苷三磷酸(NTP)還原而得到。因此在細胞進行DNA修復及合成時，需要胸苷酸激酶及核糖核苷酸還原酶共同作用來提供足夠的去氧核苷三磷酸(dNTPs)。在以往的研究發現，抑制胸苷酸激酶的表現，可有效降低胸苷三磷酸(dTTP)的含量並伴隨尿苷三磷酸(dUTP)大量生成到DNA，使得鹼基對不平衡，進而造成細胞凋亡。小分子化合物YMU1可以有效的抑制人類胸苷酸激酶(thymidylate kinase, TMPK)，並增加低劑量癌症藥物小紅莓素(doxorubicin)所誘導的細胞凋亡。 為了尋找更好的抑制劑，我根據中研院高通量藥物篩選的結果中挑選出化合物1及TDZD-8作為合成的目標。我們設計的新分子同時包含TDZD-8的五元環及YMU1的主結構，從電腦模擬來看，發現此共軛分子可分別和胸苷酸激酶的兩個位置有作用，因此我們認為它對於胸苷酸激酶的抑制活性會較YMU1來的好。本篇論文便是設計並嘗試合成此共軛分子及其衍生物，希望能找到更好的胸苷酸激酶抑制劑。 在此論文中，我首先利用自由基耦合反應建構雙苯環系統，再藉由兩步轉化得到化合物1。然而，雙苯環分子化合物1對於人類胸苷酸激酶並無任何抑制活性。接著我將重點放在YMU1–TDZD共軛化合物的合成。在成功建構TDZD衍生物後，發現它們對於人類胸苷酸激酶皆有很好的抑制效果。然而，當嘗試將TDZD衍生物接上YMU1衍生物時，發現在鹼性情況下TDZD五環會裂解成尿素衍生物。為了避免此情形，試著先將甘胺酸接上YMU1衍生物，最後一步再進行TDZD的合環反應。但利用硫醯氯在建構五環時，發現它會影響原本的YMU1主架構。儘管兩種合成途徑皆無法得到YMU1–TDZD共軛化合物，將YMU1–尿素共軛化合物進行生物活性的測試，發現它對於人類胸苷酸激酶皆有很好的抑制效果。因此再合成了四個YMU1–尿素共軛化合物，並發現它們對於人類胸苷酸激酶也都有不錯的抑制效果。

The biosynthesis of dTDP is unique in conversion of dTMP by thymidylate kinase (TMPK), though other dNDPs are directly produced from their corresponding NDPs by ribonucleotide reductase (RNR). DNA repair requires the coordination between RNR and TMPK to supply dNTPs. Blocking the function of TMPK would cause incorporation of dUTP during DNA repair in tumor cell. Furthermore, inhibition of TMPK is found to enhance tumor lethality on treatment with anticancer drugs. The combination treatment with YMU1 and doxorubicin is effective in chemotherapy against tumor cells. In addition to YMU1, the high-throughput screening in the Genomics Research Center, Academic Sinica revealed several small molecules with high human TMPK inhibition, including TDZD-8. By computer modeling, the designed conjugated molecule containing the moieties of YMU1 and TDZD-8 shows that these two heterocyclic cores can bind to TMPK and ATP active sites, respectively. We thus proceeded with the synthesis and bioassay of the YMU1―TDZD conjugated compound for bioactivity evaluation. First, I synthesized TDZD derivatives from condensation of isocyanate and isothiocyanate and found that they all showed good hTMPK inhibition. However, attempted amide bond coupling of TDZD derivatives and YMU1 derivatives failed because the TDZD ring will decompose to urea under basic conditions. Therefore, another synthetic strategy was designed to construct amide bond first, followed by the formation of TDZD ring to prevent TDZD ring from decomposition into urea. Nevertheless, formation of TDZD ring in the presence of sulfuryl chloride also affected the YMU1 moiety. Even though YMU1–TDZD conjugated compound cannot be synthesized by these two pathways, YMU1–urea conjugated derivatives did show higher inhibition than YMU1.