合成及評估不同雜環化合物作為抗病毒及抗癌藥劑

Synthesis and Evaluation of Various Heterocyclic Compounds as Antiviral and Anticancer Agents

庫方昇(Vathan Kumar)

指導教授 ： 梁博煌

國立臺灣大學/生命科學院/生化科學研究所/博士(2014年)

https://doi.org/10.6342/NTU.2014.02958

摘要

雜環化合物廣泛分佈於自然界，且已成為不可或缺的上市藥物。在本論文中，我分別在第二、三章描述一些雜環化合物的合成及當作抗病毒藥劑來抑制神經胺酸酶及人類嚴重急性呼吸道症候群冠狀病毒(SARS-CoV)蛋白酶的活性，並在第四章描述一些藉破壞β-tubulin: CCT-β來抗癌的藥劑。
流感病毒抗藥性的發生讓新抗病毒藥劑的研究變得必要。在目前的研究我們使用高通量篩選從約6800個化合物找到KR-72039作為H1N1及H5N1神經胺酸酶(NA)的抑制劑。結構－活性研究得到系列中最佳的H5N1 NA抑制劑3e (IC50 = 2.8 μM)，具混合型酵素抑制模式(Kic = 2.9 μM及 Kiu = 5.6 μM)，並能抑制病毒複製 (EC50 = 27 μM)。電腦模擬顯示我們的化合物主要在結合NAs 活性區的loop-430。化合物3l 可同時抑制H7N9和H7N9-R292K NAs。這些化合物的CC50 >200 μM，因此可作為發展新一類抗流感藥物的起始。
雖然2005年後已沒有SARS病例，然而新一類型的冠狀病毒稍後被稱為中東急性呼吸道症候群冠狀病毒 (MERS-CoV) 在2012年初期發生，這是能感染人類的第六個冠狀病毒。雖然許多SARS-Co V蛋白酶的抑制劑已被發現，但尚未有抗冠狀病毒的藥物被研發上市。我們實驗室早先曾報導一些 pyrazole 化合物作為3CL蛋白酶的抑制劑，因此我繼續合成及試驗pyrazolone 類似物，並發現一些抑制劑。電腦模擬預測可能的結合部位可能在蛋白雙體交接位置導致影響雙體形成。最佳抑制劑3q，只需在7.9 μM 即可抑制酵素活性。有趣的是3l可藉結合到相似環境來抑制NAs及SARS蛋白酶的活性。
從我們已發表的論文(Lin et al., Cancer Res. 2009) I-Trp 和β-tubulin的Cys354形成共價性鍵結，因而破壞 β-tubulin: CCT-β複體。藉使用此熱點，陳世勳博士發現了一個可逆型破壞protein-protein interaction (PPI)的抑制劑，因此造成過度表現CCT-β的抗藥性癌細胞死亡。我們也從事SAR來發現必要的pharmacophore。發現在這個hit中 Nitro及其他芳香及疏水性基團對於活性是不能或缺的。

關鍵字

Neuraminidase ； SARS Co-V ； β-Tubulin

並列摘要

Heterocyclic compounds are widely distributed in nature and has become indispensable component of the marketed drugs. In this thesis, I describe the synthesis and structure-activity relationship (SAR) of some heterocyclic compounds as antiviral agents namely neuraminidase inhibitors, SARS-CoV protease inhibitors in chapter 2 and 3, respectively, and anti-cancer agents by disrupting β-tubulin: CCTβ complexes in chapter 4.

The emergence of resistance among flu viruses has made search for new antiviral agents essential. In the present study, we performed high-throughput screening on ~6800 compounds to identify KR-72039 as an inhibitor of H1N1 and H5N1 neuraminidases (NAs). Structure-activity relationship studies was conducted leading to the best inhibitor 3e which inhibited H5N1 NA (IC50 = 2.8 μM) with binding affinity (Kic = 2.9 μM and Kiu = 5.6 μM) as a mixed-mode inhibitor to block viral replication (EC50 = 27 μM). In silico analysis indicated that our compounds predominantly occupy loop-430 around NA active site. Compound 3l additionally inhibited both H7N9 and H7N9-R292K NAs. The CC50 for these compounds were >200 μM. These inhibitors serve as a starting point for the development of a novel class of antiflu agents.

Though there was no case of SARS after 2005, a new class of coronavirus which was later named as Middle East respiratory syndrome coronavirus (MERS-CoV) emerged in early 2012. This is the sixth coronavirus that could infect humans. Although many inhibitors of the SARS-CoV proteases have been discovered, no anti-coronaviral drug has been developed so far. We had earlier reported some pyrazole compounds as the 3CL protease inhibitors, so we continued to synthesize and test the pyrazolone analogs. A few compounds showed inhibitory activity. Docking experiments predicted the probable binding site of these compounds is at the dimer interface to block the active dimer formation. The best compound 3q inhibited the protease at 7.9 μM. Interestingly, 3l inhibited both neuraminidase and SARS protease by binding to the similar environments in both enzymes.

From our published results (Lin et al., Cancer Res. 2009) I-Trp forms covalent linkage with Cys354 of β-tubulin, thereby disrupting β-tubulin: CCT-β complexes. Using this as a hot spot, Dr. Shih-Hsun Chen identified a reversible inhibitor for disrupting this protein-protein interaction (PPI) and causing cell death of CCT-β overexpressed cancers, which are multidrug resistant. We performed SAR to identify the essential pharmacophore. Nitro substitution in the hit is indispensable for the activity along with aromatic or hydrophobic side chain.

並列關鍵字

Neuraminidase ； SARS Co-V ； β-Tubulin

參考文獻

Chapter 2 References

2. Watanabe, Y. et al. The changing nature of avian influenza A virus (H5N1). Trends Microbiol 20, 11-20 (2012).

3. Taubenberger, J.K. et al. 1918 Influenza: the mother of all pandemics. Emerg Infect Dis 12, 15-22 (2006).

4. CDC. Key Facts About Avian Influenza (Bird Flu) and Highly Pathogenic Avian Influenza A (H5N1) Virus. (2010 ).

6. Herfst, S. et al. Airborne transmission of influenza A/H5N1 virus between ferrets. Science 336, 1534-41 (2012).