【作者】 肖川；

【导师】 刘在群； 宋志光；

【作者基本信息】 吉林大学 ， 有机化学， 2012， 博士

【摘要】 天然产物的结构修饰与改造是发现新药的重要手段和途径。香豆素和嘌呤核苷是两类非常重要的天然活性化合物。香豆素类化合物广泛存在于自然界中，具有多种生理活性。近年来对香豆素类化合物的修饰和改造已经成为国内外许多药学工作者的研究重点。嘌呤核苷及其衍生物也是一类具有显著生物活性的化合物，在众多领域尤其是在医药方面占有非常重要的地位。开发具有抗肿瘤、抗病毒活性的核苷及其衍生物己成为当今研究热点。以香豆素类和嘌呤核苷类化合物为基础，对其进行结构修饰和改造具有重要意义。因此，本文首先以香豆素类化合物为基础，通过引入不同的官能团，包括甲基、金刚烷胺、吡唑、烯丙基等对其进行修饰与改造，并对所合成的香豆素衍生物进行抗氧化、抗肿瘤活性测试。然后以嘌呤核苷类化合物为基础，合成系列6-位芳基（杂芳基）取代的7-脱氮杂嘌呤核苷，并评价其生物活性。取得的创新性研究结果如下：（1）以多种生理活性的呋喃香豆素花椒毒酚（xanthotoxol, Xan）为先导，从简单原料4-甲基-7-羟基香豆素出发，分别经多步反应得到Xan的4-位和4,9-位甲基取代物MXan和DMXan，并对合成路线做了探讨。通过采用不同的体外抗氧化模型，包括化学体系法中的二苯代苦味肼基自由基（DPPH）法、2,2-联氮-双-（3-乙基苯并噻唑啉-6-磺酸）二铵盐（ABTS+）法、galvinoxyl法、β-carotene-bleaching法和化学模拟的生物体系法中的Cu2+/GHS（谷胱甘肽）引发的DNA氧化损伤法和2,2-偶氮-二（2-脒基丙烷）盐酸盐（AAPH）引发的DNA氧化损伤法，来研究化合物Xan、MXan和DMXan的抗氧化活性。结果表明：将甲基引入的花椒毒酚的4-位和9-位没有影响在亚油酸的自然氧化和Gu2+/GSH引发的DNA氧化损伤中的抗氧化作用，但是却降低了清除ABTS+和DPPH自由基和抑制AAPH引发的DNA氧化损伤的能力。MXan和DMXan的4-位和9-位甲基在一定程度上起到了促氧化作用。（2）将金刚烷胺药效团引入到花椒毒酚中，经过三步反应简便、高效地合成了三种金刚烷胺修饰的补骨脂素衍生物AXan、AMXan和ADMXan。MTT法体外抗肿瘤检测表明：Xan、MXan、DMXan对人肺癌细胞A549和NCI-H460有一定的抑制能力；而金刚烷胺修饰物AXan、AMXan和ADMXan对这两种癌细胞的抑制能力有了较大提高，对A549有中度抑制作用，IC₅₀值在40μM左右。（3）将具有多种活性的吡唑环引入到香豆素骨架上，合成了一系列含有4,5-二氢吡唑结构的7-羟基香豆素衍生物，并对合成路线进行了探讨。通过将动力学方程引入到抗氧化活性的评价体系中，来比较合成的4,5-二氢吡唑香豆素的抗氧化活性。测试结果表明：当4,5-二氢吡唑香豆素中两个羟基处于相邻位置时清除DPPH自由基的能力和抑制AAPH引发的DNA氧化损伤的能力要强于两个羟基处于相间位置时的能力；4,5-二氢吡唑环1-位N-H的存在提高了4,5-二氢吡唑香豆素清除自由基和保护DNA的能力。所合成的7个4,5-二氢吡唑香豆素均能通过减缓硫代巴比妥酸活性物质（TBARS）的生成速率来抑制DNA的氧化反应并产生抑制期，并且保护DNA能力都强于阳性对照Xan。化合物3b和4a具有良好的抗氧化活性，有进一步研究的价值。（4）从简单原料2,4-二羟基苯甲醛和取代的苯乙酸出发，经过多步简单高效的反应合成了一系列7,8-位取代3-芳基香豆素。MTT法体外抗肿瘤检测表明：合成的3-芳基香豆素对HeLa细胞的抑制作用比较明显，其中化合物9a、9b、10a、10b对HeLa细胞有较强的抑制能力(IC₅₀=13.50～17.66μM)；将烯丙基引入到3-芳基香豆素的8-位提高了其抗肿瘤活性，而7-位甲氧基的存在不利于提高对SK-HEP-1、HepG2、SGC7901这三种癌细胞的抑制能力。（5）以嘌呤核苷类化合物xylocydine为先导，对其碱基进行修饰，开发了一条从简单原料四氰基乙烯和L-木糖出发经过多步反应合成一系列6-位芳基（杂芳基）-7-脱氮杂嘌呤核苷的路线，并对合成中关键的Suzuki反应的条件做了优化。目标化合物对CDK1/Cyclin B1和CDK2/Cyclin A2的体外激酶分析结果表明：三种嘌呤核苷衍生物3h、3i、3j对CDK2/CyclinA2表现出显著的抑制活性，其IC50分别为4.6μM、4.8μM和55μM。3h、3i、3j与xylocydine相比活性有了降低，但提高了对CDK2/CyclinA2抑制的选择性。进一步细胞周期分析表明3h、3i、3j在一定程度上能够将HeLa细胞周期阻滞在G1/S期，说明这三种化合物在细胞体内可能对CDK2的活性有抑制作用。为了解CDK2抑制剂的构效特点，我们对3h、3i、3j和xylocydine做了分子对接模拟，结果表明化合物3h-j有较高的对接值。另一方面，使用MTT法对上述目标化合物进行了抗肿瘤活性筛选，发现化合物3g对四种人源癌细胞（A549、HeLa、SK-HEP-1、MCF-7）有一定的抑制能力。并在3g的基础上对其进一步修饰得到目标化合物DMBPN和DPBPN，对其合成方法做了优化，开发了“一锅法”路线。MTT法体外抗肿瘤检测表明：DMBPN和DPBPN对上述四种癌细胞的抑制能力有较大的提高。DMBPN对这四种细胞均有显著的抑制能力，IC₅₀值都在10μM以下，对SK-HEP-1的抑制活性最好，IC₅₀为4.13μM。更多还原

【Abstract】 Structural modification and transformation of natural products is an important approachfor the discovery of new drugs. Coumarins and purine nucleosides are two very importanttypes of natural bioactive compounds. Coumarins, distributed in nature, possess a varietyof biological activities. Recently, widespread attention has been draw to modification andrenovation of the coumarins. Purine nucleosides and their analogues, an important class ofbiologically active compounds, play so important role in many fields, especially inmedicine, that current research has been increasingly focused on the discovery andsynthesis of novel compounds with antiviral and antitumor properties. Structuremodification and transformation based on coumarins and purine nucleosides has importantsignificance for the discovery of new drugs. Therefore, firstly, series of coumarins weresynthesized with the introduction of different functional groups, including methyl,amantadine, pyrazole, allyl etc., and then their antioxidant and antitumor activities weretested in vitro in this essay. Then series of6-（het） aryl-7-deazapurine nucleosides weresynthesized based on modification and their biological activity were also evaluated invitro. The innovative research results achieved are as follows:（1） A furanocoumarin xanthotoxol （Xan）, with variety of physiological activity, waschosen as lead compound. Its4-methyl and4,9-dimethyl analogues, MXan and DMXan,were synthesized by the multi-step reaction starting from simple starting materials4-methyl-7-hydroxycoumarin, and the synthetic route was also discussed. Different invitro antioxidant models, such as2,2′-diphenyl-1-picrylhydrazyl radical （DPPH）,2,2′-azinobis （3-ethylbenzothiazoline-6-sulfonate） cationic radical （ABTS+）, galvinoxylradical, β-carotene-bleaching in chemistry systems and Cu2+/glutathione （GSH）-mediatedand2,2′-azobis（2-amidinopropane hydrochloride）（AAPH）-induced oxidation of DNA inthe chemical simulation biological systems were employed to investigate the antioxidant capacity of Xan, MXan and DMXan. The results showed that methyl attaching toxanthotoxol did not affect its ability to protect linoleic acid against autoxidation and toinhibit Cu2+/GSH-induced oxidation DNA, but decreased its ability to scavenge ABTS+and DPPH, and to protect DNA against AAPH-induced oxidation. The hydroxyl group at8-position played a major role in the antioxidant of xanthotoxol, and the methyl at4-position and9-position exhibited prooxidant properties to a certain extent.（2） Amantadine pharmacophore was introduced into xanthotoxol. Three psoralenderivatives AXan, AMXan and ADMXan modified by amantadine were synthesizedthrough three simple and efficient reactions. The MTT test revealed that Xan, MXan andDMXan exhibited certain inhibition against human lung cancer cell line A549andNCI-H460. AXan, AMXan and ADMXan enhanced the inhibition capacity against the twocancer cell lines, having a moderate inhibitory activity on A549, with IC50value around40μM.（3） A series of7-hydroxycoumarin derivatives containing4,5-dihydropyrazole moietywere synthesized by introducing active pyrazole ring into coumarin skeleton, and thesynthesis was also discussed. Dynamics equations were introduced into the antioxidantsevaluation systems to quantitatively study the antioxidant activity of these4,5-dihydropyrazole coumarins. Test results showed that the two hydroxyl groups ortho toeach other in the benzene ring of4,5-dihydropyrazole coumarins possessed strongercapacity to trap DPPH and protect DNA against AAPH-induced oxidation than that of twohydroxyl groups meta to each other. The N-H in position1-N-unsubstituted4,5-dihydropyrazole ring could also enhance ability to scavenge radicals and to protectDNA. These seven4,5-dihydropyrazole coumarins all could inhibit the oxidation of DNAvia slowing down the rate of product of TBARS and give inhibition time in the process,which were all higher than positive control Xan.3b and4a were two novel and promisingantioxidants suitable for further development.（4）3-ArylCoumarins was chosen for study and series of7and8-substituted3-arylcoumarins were synthesized from simple raw materials2,4-dihydroxybenzaldehyde and substituted phenyl acetic acid through several efficient reactions. The MTT test revealed that the novel3-arylcoumarins could inhibits HeLa cells moreobviously, in which compounds9a,9b,10a and10b, exhibited good inhibitory activity onHeLa cells (IC₅₀=13.50¹7.66μM). Introducing allyl to8-position of3-arylcoumarinswas favorable to improve the activity for inhibiting human cancer cell and the methoxy at7-position may be adverse unfavorable for maintaining the antitumor activity againstSK-HEP-1, HepG2and SGC7901cell lines.（5） Xylocydine, a purine nucleosides CDKs inhibitor, was chosen as leading compoundand6-（het） aryl-7-deazapurine nucleosides were synthesized via base modification. Aroute of syntheses of6-（het） aryl-7-deazapurine nucleosides with tetracyanoethylene and L（-）-xylose as starting molecules though multistep reaction was developed and theconditions of key Suzuki reaction in the synthetic route was also optimized. Forty six newcompounds were obtained, including thirty target compounds and sixteen intermediateproducts. The results of CDK1/Cyclin B1and CDK2/Cyclin A2inhibitory assay of theobtained compounds indicated that three purine nucleoside derivatives,3h,3i and3jshowed significant activity on CDK2/Cyclin A2, with IC₅₀value of4.6μM,4.8μM and55μM respectively. Although the inhibition activity of3h,3i and3j decreased to comparewith the xylocydine, the selectivity to CDK2/Cyclin A2has been increased. Those threecompounds all induced G1/S phase arrest in Human epithelial carcinoma cell line （HeLa）,and the results suggested they may inhibit CDK2activity in vitro. Furthermore, molecularmodeling study was conduct to understand the structural features of CDK2inhibitors, thethree novel CDK2inhibitors3h-j showed high docking scores. On the other hand,compound3g was found exhibiting certain inhibitory activity against four kinds of humancancer cells （A549, HeLa, SK-HEP-1, and MCF-7） in the MTT method filtering on abovetarget compounds. The target compounds DMBPN and DPBPN were obtained by furthermodification based on3g and the synthetic route was optimized with a "one pot" method.The result of antitumor in vitro by MTT method showed that DMBPN and DPBPNimproved the inhibitory activity on the above four kinds of cancer cell dramatically.DMBPN had a significant inhibiton on these four types of cells with IC₅₀value under10 μM, among which the inhibitory activity was best for SK-HEP-1, with IC₅₀value of4.13μM.更多还原