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近日，蘭州大學(xué)藥學(xué)院劉映前教授團隊與華中科技大學(xué)同濟藥學(xué)院徐傳瑞教授團隊合作，在抗腫瘤新藥發(fā)現(xiàn)與腫瘤治療方面取得新進展。相關(guān)研究成果“Design and synthesis of Aza-boeravinone derivatives as potential novel topoisomerase I inhibitors”發(fā)表于Bioorganic Chemistry期刊（IF：5.275）上。蘭州大學(xué)藥學(xué)院2020級碩士研究生周勇和2019級碩士研究生白銀鵬為該論文共同第一作者，通訊作者為劉映前教授和徐傳瑞教授，蘭州大學(xué)藥學(xué)院為該論文的第一單位。

腫瘤是世界上大多數(shù)國家的主要死亡原因之一。根據(jù)世界衛(wèi)生組織（WHO）統(tǒng)計，2020年全球估計有1930萬新病例的產(chǎn)生和1000萬腫瘤病例的死亡，并且全球腫瘤的發(fā)病率和死亡率正在迅速增加。因此，研究和開發(fā)新的藥物和治療策略以控制腫瘤顯得尤為迫切和重要。其中，化療在腫瘤的治療中仍然扮演著舉足輕重的作用。DNA拓撲異構(gòu)酶I（Top I）抑制劑是抗腫瘤藥物的重要組成部分，在晚期實體瘤和女性癌癥中發(fā)揮著越來越重要的作用。Top I是真核生物中必需的酶，參與細胞DNA復(fù)制、轉(zhuǎn)錄、重組和染色體分離等眾多過程。多項研究表明，與正常細胞相比，Top I在腫瘤細胞中高度表達并且促進腫瘤生長。因此，Top I已經(jīng)被證明是抗腫瘤的重要靶點之一。

藏藥喜馬拉雅紫茉莉為紫茉莉科植物喜馬拉雅紫茉莉（Mirabilis himalaica （Edgew.） Heim.）的干燥根。Boeravinones是從喜馬拉雅紫茉莉中分離得到的一類天然產(chǎn)物，具有廣泛的生物學(xué)活性，如抗炎、抗菌、抗氧化、抗病毒和抗腫瘤等。但其自身抗腫瘤譜窄且活性較弱，需進一步進行結(jié)構(gòu)優(yōu)化或改造而增強活性。因此，基于天然產(chǎn)物Boeravinones的結(jié)構(gòu)骨架，該研究團隊采用生物電子等排和“Aza”結(jié)構(gòu)優(yōu)化策略設(shè)計合成了兩個系列的6H-色烯并[3,4-b]喹啉衍生物。

Figure 1. Structures of boeravinones from Boerhaavia diffusa and novel structures of 6H-chromeno[3,4-b]quinoline derivatives.

Figure 2. Reagents and conditions: (a) ethyl acetoacetate, AcOH, benzene,reflux; (b) Ph2O, 250 ℃; (c) NBS, AcOH/DCM(1:2), rt; (d) POCl3, reflux; (e)benzoyl peroxide, NBS, CCl4, reflux; (f) NaH, DMF, rt; (g) Pd(OAc)2, PPh3,K2CO3, DMF, 120 ℃; (h) N,N-Dimethylethylenediamine, pridine, 120℃.

作者首先評估了所得化合物對HepG2、A2780、Hela、HCT116、SW1990和MCF7等多種腫瘤細胞株的細胞毒活性。篩選結(jié)果發(fā)現(xiàn)，化合物ZML-8和ZML-14對HepG2具有顯著的抑制效果，IC50值分別為0.58μM和1.94 μM。此外，化合物ZML-8和 ZML-14對HepG2 和 L-02 細胞的選擇性高于對照藥Topotecan。因此，進一步研究了候選化合物ZML-8和ZML-14的抗腫瘤效果與作用機制。

Table 1. Antiproliferative Activities of Compounds ZML-8, ZML-14, ZML-22, and ZML-23 against Normal Human Liver Cell Line L-02.

通過流式細胞術(shù)、Western blotting等實驗研究表明，化合物ZML-8 和ZML-14劑量依賴性地誘導(dǎo)HepG2細胞周期G2/M期阻滯。此外，化合物ZML-8和 ZML-14誘導(dǎo)HepG2細胞凋亡并伴隨促凋亡蛋白Bax、Bad和Cleaved-caspase3的激活以及抗凋亡蛋白Bcl-2的降低。這些結(jié)果表明，化合物ZML-8和 ZML-14誘導(dǎo)Caspase依賴的細胞凋亡。

Figure 4. Compounds ZML-8 and ZML-14 caused G2/M cell cycle arrest of HepG2 cells. (A)Cell cycle distribution HepG2 cells treated with compound ZML-8 and ZML-14 for24 h, Topotecan was used as positive control. Treated cells were then stained with PI/RNase and analyzed using a flow cytometer. (B) Sums of percentages of each cycle were shown as mean ± S.D. for three independent experiments (*P <0.05, **P < 0.01, ***P < 0.001.vs. the control).

Figure 5. Compounds ZML-8 and ZML-14 induced apoptosis of HepG2 cells. (A) Apoptosis in HepG2 cells treated with compound ZML-8 or ZML-14. Cells were exposed to compound ZML-8, ZML-14, or Topotecan for 48 h. The apoptosis rates were then assessed by Annexin V/PI double staining determined by flow cytometry. (B) Apoptosis rates were shown onthe histogram for three independent experiments. Data were shown as mean ± SD.*P < 0.05 or **P < 0.01 or ***P < 0.001 vs. the control.

Figure 6. Western blotting analysis of Caspase-dependent apoptosis proteins in HepG2 cells after compounds ZML-8 and ZML-14 treatment. HepG2 cells were treated with compound ZML-8, ZML-14, or Topotecan for 48 h. The cells were harvested and lysed to detect cleaved Caspase-3, P-53 and Bcl-2 members (Bcl-2 and Bax) using Western blot. α-Tubulin was used as reference. (A) and (B) Representative western blots of proteins in compound ZML-8 or ZML-14 treated cells. (C) and (D) Quantification of western blot. Results are representative of three independent experiments. *p < 0.05,**p < 0.01, and ***p < 0.001 vs control.

根據(jù)上述實驗結(jié)果，化合物ZML-8和ZML-14通過誘導(dǎo)細胞周期阻滯和細胞凋亡來抑制細胞增殖，并且該結(jié)果與對照藥物Topotecan相似。為此，作者假設(shè)化合物ZML-8 和 ZML-14 可能具有與Topotecan相似的作用機制。DNA松弛實驗結(jié)果表明，化合物ZML-8 和 ZML-14顯著抑制Top I的酶活性。化合物ZML-8和ZML-14分別在2.5 μM 和5.0 μM濃度劑量下具有與1.0 μM的 Topotecan抑制效果。此外，Western blotting結(jié)果表明，化合物ZML-8 和 ZML-14誘導(dǎo)Top I蛋白水平降解。據(jù)報道，抑制 Top I 或捕獲 Top1cc 可導(dǎo)致 DNA 損傷。因此，作者進一步檢測了DNA損傷的生物標志物γ-H2AX的表達情況。研究發(fā)現(xiàn)，化合物ZML-18和ZMl-14處理后γ-H2AX的蛋白表達水平顯著增加，表明誘導(dǎo)了DNA損傷。分子對接結(jié)果進一步表明，化合物ZML-8和ZML-14可以與Top I-DNA 復(fù)合物相互作用，結(jié)合能分別為-9.4 kcal/mol和-9.7kcal/mol。

Figure 7. Compounds ZML-8 and ZML-14 down-regulated the Top1 activity and induced DNA damage. (A) The inhibitory activity of compounds ZML-8 and ZML-14 on Top1. Lane 1: relaxed DNA; Lane 2: supercoiled plasmid DNA (pBR322) only; Lane 3: DNA + Top1+DMSO; Lane 4-5: DNA + Top1+ ZML-8; Lane 6-7: DNA + Top1+ ZML-14; Lane 8: DNA + Top1+ Topotecan,were used as positive control. (B) and (C) HepG2 cells were treated with compounds ZML-8 and ZML-14 for 48 h, harvested and lysed for detection of Top1 and γ-H2AX. (D) and (E) Histograms of relative expression of Top1 and γ-H2AX. Data are shown as mean ± SD of three independent experiments. *p < 0.05, **p < 0.01, and ***p < 0.001 vs control.

Figure 8. Hypothetical binding mode of compounds ZML-8 and ZML-14 in the ternary Top1-DNA-drug complex (PDB ID: 1K4T). Protein is shown as a cartoon, and compounds ZML-8, ZML-14 and Topotecan were shown in stick. Key hydrogen-bonding interactions are indicated with yellow dashes. (A-C) Compounds ZML-8 (pink), ZML-14 (green) and Topotecan (blue) were shown in the ternary complex with a docking energy -9.40, -9.70 and -12.4 kcal/mol, respectively.(D) Overlapping of the binding pose of compounds ZML-8 (pink) and ZML-14(green) with Topotecan (blue).

綜上，基于天然產(chǎn)物Boeravinones的結(jié)構(gòu)骨架，團隊采用生物電子等排策略設(shè)計合成了兩個系列的6H-色烯并[3,4-b]喹啉衍生物，并評估了所得化合物對HepG2、A2780、Hela、HCT116、SW1990和MCF7等腫瘤細胞株的細胞毒活性。篩選結(jié)果表明，化合物ZML-8和ZML-14對HepG2具有顯著的抑制效果，IC50值分別為0.58μM和1.94 μM。此外，化合物ZML-8和ZML-14對HepG2和L-02細胞的選擇性高于對照藥Topotecan。細胞機制研究表明，化合物ZML-8和ZML-14誘導(dǎo)HepG2細胞周期G2/M期阻滯、細胞凋亡和DNA損傷。此外，化合物ZML-8和ZML-14顯著抑制Top I的酶活性，并且誘導(dǎo)Top I蛋白水平的降解。分子對接表明，化合物ZML-8和 ZML-14顯著的細胞毒活性是由于與Top1-DNA復(fù)合物的良好相互作用。

這項研究的重要進展為后續(xù)開發(fā)具有完整自主知識產(chǎn)權(quán)的抗腫瘤藥物奠定了基礎(chǔ)。課題組長期致力于天然源抗腫瘤藥物的設(shè)計合成與創(chuàng)制研究，為臨床抗腫瘤藥物的開發(fā)提供科學(xué)依據(jù)。研究工作得到了浙江省重點大學(xué)優(yōu)勢特色學(xué)科開放項目、甘肅省國際科技合作重點項目和國家自然科學(xué)基金共同資助。該項研究也是依托甘肅省中藏藥功效物質(zhì)開發(fā)與利用行業(yè)技術(shù)中心，開展源于天然源藏藥資源進行新藥開發(fā)研究取得的又一重要的科研成果，對于推動中藏藥功效物質(zhì)開發(fā)與利用具有重要意義。

原文鏈接：https://doi.org/10.1016/j.bioorg.2022.105747