【作者】 高艳清；

【导师】 商士斌；

【作者基本信息】 中国林业科学研究院 ， 林产化学加工工程， 2013， 博士

【摘要】 松节油是我国重要的天然可再生资源，其来源丰富，结构独特，具有广泛的生物活性。作为松节油的主要组分之一，β-蒎烯化学性质活泼，具有很多潜在的利用价值。本文以β-蒎烯为原料，经氧化、异构、Diels-Alder加成、酯化等化学反应，制备了二氢枯茗酸酰胺、二氢枯茗酸酯、二氢枯茗酸酰基硫脲、二氢枯茗酸肟酯、蒎烯–马来酸酯、蒎烯–马来酸酰胺等六类共45种新化合物；利用傅里叶变换红外光谱、核磁共振、质谱、元素分析等分析测试手段对合成的六类化合物进行了结构表征。对二氢枯茗酸衍生物进行了抑菌和体外抗肿瘤活性测试，根据测试结果，初步探讨了抑菌活性的构效关系和作用机制，研究结果为松节油的深加工利用提供基础。以β-蒎烯为原料，碱性高锰酸钾为氧化剂，水和叔丁醇的混合溶液为反应介质合成了诺蒎酸，并首次得到了诺蒎酸的晶体结构。诺蒎酸在硫酸的作用下，脱水，重排开环得到二氢枯茗酸，再经酰氯化、酯化、酰胺化、硫化等反应合成了酰胺、酯、酰基硫脲、肟酯等共36个新化合物，并首次得到了二氢枯茗酸苯酰胺的单晶。通过将β-蒎烯与马来酸酐进行Diels-Alder加成，在蒎烯母环上引入两个羧基结构，并合成了蒎烯-马来酸双酯加成物和蒎烯-马来酸酰胺衍生物。采用平板二倍稀释法，分别选取革兰氏阴性菌（大肠埃希氏杆菌Escherichia coli、绿脓假单胞菌Pseudomonas aeruginosa、肺炎克雷伯氏菌Klebsiella pneumoniae、产气肠杆菌Escherichia aerogenes）和革兰氏阳性菌（金黄色葡萄球菌Staphyloccocus aureu和表皮葡萄球菌Staphyloccocus epidermidi）六种菌株为受试菌种，对合成的二氢枯茗酸酰胺、二氢枯茗酸酯、二氢枯茗酸酰基硫脲、二氢枯茗酸肟酯、蒎烯-马来酸酯、蒎烯-马来酸酰胺等六类化合物进行了抑菌活性测试。测试结果表明，大部分的二氢枯茗酸衍生物具有一定的抑菌作用，其中二氢枯茗酸肟酯衍生物对六种菌种均具有较好的抑制性能，具有一定广谱抗菌性，化合物5a、5c、5d、5e、5g对产气肠杆菌的最小抑菌浓度均小于16μg/mL，低于三种对照品（新洁尔灭、氨苄青霉素钠、红霉素）；除化合物5g外，其他肟酯类化合物对绿脓假单胞菌的的最小抑菌浓度均小于2μg/mL；化合物5e、5d对金黄色葡萄球菌的最小抑菌浓度为8μg/mL，与对照品新洁尔灭效果相当；化合物5c、5d、5e对表皮葡萄球菌的最小抑菌浓度小于8μg/mL，与对照品新洁尔灭和氨苄青霉素钠效果相当。因此二氢枯茗酸肟酯衍生物具有进一步开发研究用以筛选抑菌剂的应用价值。二氢枯茗酸酯衍生物对金黄色葡萄球菌和表皮葡萄球菌的抑制效果较好，除化合物3f、3k、3m、3n外，酯类化合物对金黄色葡萄球菌的最小抑菌浓度小于16μg/mL。除化合物4f外，二氢枯茗酸酰基硫脲衍生物对产气肠杆菌的抑制效果较好。选取了四种肿瘤细胞（分别为人非小细胞肺癌细胞、人乳腺癌细胞、人肝癌细胞和人结肠癌细胞）为受试细胞，采用噻唑蓝（MTT）比色法对二氢枯茗酸酰胺、二氢枯茗酸酯、二氢枯茗酸酰基硫脲、二氢枯茗酸肟酯等四类衍生物进行了体外抗肿瘤活性活性测试。结果表明部分化合物对四种肿瘤细胞具有一定的抑制作用，其中，二氢枯茗酸酰基硫脲对体外培养的人乳腺癌MCF-7和人结肠癌HCT116肿瘤细胞有一定的生长抑制作用，表现在化合物4d对人乳腺癌MCF-7肿瘤细胞的半数抑制浓度IC50为26.58μg/mL，化合物4c对人结肠癌HCT116肿瘤细胞的半数抑制浓度IC50为56.67μg/mL，均小于100μg/mL。比较了蒎烯衍生物的结构与抑菌活性之间的关系，结果显示，二氢枯茗酸的基本结构是抑菌作用的关键基团。通过比较二氢枯茗酸衍生物上的不同的取代基对于抑菌效果的影响，初步探讨了二氢枯茗酸衍生物抑菌活性的构效关系和作用机制。在大多数情况下，对于相同类型的二氢枯茗酸衍生物，分子量越小，含支链取代基越少，抑菌效果越理想。比较了对氨基苯甲酸、二氢枯茗酸衍生物和磺胺类抗菌剂的结构，二氢枯茗酸与对氨基苯甲酸和磺胺类抗菌剂在分子结构上具有一定的类似性，因此，推断在细胞内，二氢枯茗酸衍生物可能会代替对氨基苯甲酸，从而形成细菌代谢物质的类似物，以细菌拮抗物质的方式发挥抑菌的作用。更多还原

【Abstract】 As an important renewable natural resource in China, turpentine has the abundantavailability, a unique structure, and widely bioactivity. β-pinene, one important component ofturpentine, is valuable in some fields due to its high chemical reactivity.In this thesis,45derivatives of amide, ester, oxime ester, and acylthiourea fromdihydrocumic acid as well as amide and ester from pinene-maleic acid were synthesizedthrough oxidation, isomerization, Diels-Alder reaction, esterification, and so on. Structures ofthese compounds were characterized by FTIR, NMR, MS, and Elemental Analysis. Theantibacterial activity and antitumor activity of these compounds were studied. According to thedata obtained from antibacterial test, the mechanism and the structure-activity relationship ofantibacterial activity were preliminarily discussed. The research provided new approaches ofthe deep processing and the utilization of turpentine.Nopinic acid was synthesized using β-pinene as raw material, potassium permanganateof alkaline as oxidant, and water/tert-butanol (v/v=1:2) as reaction medium, through which thecrystal structure of nopinic acid was firstly obtained. Dihydrocumic acid was synthesizedthrough reaction of dehydration, rearrangement, and ring-opening in the presence of sulfuricacid.38compounds of amides, esters, acylthioureas, and oxime ester were synthesized throughacylation, esterification, vulcanization, and so on. The crystal structure of4-isopropyl-N-phenylcyclohexa-1,3-dienecarboxamide was obtained. Through Diels-Alder reaction of β-pinene and maleic anhydride, two carboxyl groups was introducted into pinene, then amide andester from pinene-maleic acid were synthesized.The antibacterial activity of the derivatives was estimated by the2-fold agar dilutionmethod. Gram-negative bacteria (Escherichia coli, Klebsiella pneumonia, Pseudomonasaeruginosa, Escherichia aerogenes) and Gram-positive bacteria (Staphyloccocus epidermidis,Staphyloccocus aureu) were selected as test species. The results showed that most derivatives from dihydrocumic acid displayed antibacterial activity. Oxime esters from dihydrocumic aciddisplayed excellent antibacterial activity against all test species. MIC of compounds5a,5c,5d,5e, and5g against Escherichia aerogenes were lower than16μg/mL (lower than threereferences: Bromogeramine, Ampicillin Sodium, and Erythromycin). Except compound5g,MIC of oxime esters against Pseudomonas aeruginosa were lower than2μg/mL. MIC ofcompounds5e and5d against Staphyloccocus aureu were8μg/mL (equivalent toBromogeramine). MIC of compounds5c,5d, and5e against Staphyloccocus epidermidis werelower than8μg/mL (equivalent to Bromogeramine and Ampicillin Sodium). Oxime estersderivatives of dehydrocumic acid have potential value for further research to explore excellentantibacterial agent. Esters from dihydrocumic acid showed good antibacterial activity againstStaphyloccocus epidermidis and Staphyloccocus aureu. Except compounds3f,3k,3m, and3n,MIC against Staphyloccocus aureu were lower than16μg/mL. Acylthiourea fromdihydrocumic acid showed good antibacterial activity against Escherichia coli.Four kinds of cancer cell (human non-small cell lung cells, human breast cancer cells,human hepatoma cells, and human colon cancer cells) were selected as test cancer cells. Theantitumor activity in vitro of the derivatives of amide, acylthiourea, ester, and oxime ester fromdihydrocumic acid were estimated by the MTT method. The results showed that a part ofderivatives from dihydrocumic acid displayed some antitumor activity in vitro against cancercells. Among them, acylthiourea from dihydrocumic acid showed some antitumor activity invitro against human breast cancer cell MCF-7and human hepatoma cancer cell HCT116. TheIC50of compound4d against human breast cancer cell MCF-7was26.58μg/mL, and the IC50of compound4c against human hepatoma cancer cell HCT116was56.67μg/mL, which werelower than100μg/mL.The structure of derivatives and antibacterial activity were analyzed, and the resultshowed that dehydrocumic acid was the key group for antibacterial activity. The effection ofdifferent substituent in derivatives of dehydrocumic acid was studied. The structure-activityrelationship of derivatives was investigated preliminarily. In most cases, for the same kind of derivatives, compounds with lower molecular weight and less brabched substituents showedbetter antibacterial activity. The structures of PABA, dehydrocumic acid, and sulfa antibacteiralagent were compared. There were some structural similarity between these three compounds.After penetration into cell membrane, it is possible that derivatives of dihydrocumic acid couldtake the place of PABA, and play antagonistic effects on the metabolism of bacterium.更多还原