C₆₀-（N,N-四氯邻苯二甲酰基）脱氢枞胺衍生物的合成及抗HIV活性研究

【作者】 周志；

【导师】 林中祥；

【作者基本信息】 南京林业大学 ， 林产化学加工工程， 2013， 博士

【摘要】 脱氢枞胺是松香的重要改性产品之一，是歧化松香胺的主要成分，广泛应用于手性拆分、造纸、胶粘剂、涂料、选矿、石油开采、医药、农药等领域。它是含有三个手性碳原子的天然手性化合物，具有许多天然药物都具有的芳香型三环二萜结构。富勒烯是碳的第三种同素异形体。自富勒烯发现以来，各种富勒烯衍生物的开发及在材料和生物医药领域的应用已被广泛的研究。研究发现，富勒烯衍生物具有一系列的生物学活性，如抗HIV病毒、抗癌、切割DNA、清除自由基等。近年来，连接有生物活性基团的富勒烯衍生物已引起人们极大的关注。许多生物活性分子如氨基酸、多肽、核苷酸、糖类、甾体化合物已被链接到富勒烯分子上，并已证明具有不同的生物学活性。本文将脱氢枞胺与四氯邻苯二甲酸酐反应生成N,N-四氯邻苯二甲酰基脱氢枞胺，并通过氧化反应在B环引入羰基，再在C环通过硝化、乙酰化、氧化、水解、取代等反应引入硝基、乙酰基、酯基、羟基及甲氧基等官能团，合成一系列的脱氢枞胺衍生物。脱氢枞胺衍生物的7位羰基与对甲苯磺酰肼经缩合反应生成脱氢枞胺对甲苯磺酰腙衍生物，然后与C₆₀通过[1+2]环加成反应生成C₆₀-N,N-四氯邻苯二甲酰基脱氢枞胺衍生物。将所得的C₆₀-N,N-四氯邻苯二甲酰基脱氢枞胺衍生物进行水性化改性，以增强其在生物环境中的溶解性，并对它们进行抗HIV-1逆转录酶、蛋白酶活性测试。本课题为探索和开发新的具有生物活性的松香胺衍生物提供了参考。论文的研究内容及结果如下：（1）以歧化松香胺为原料，通过与对甲苯磺酸的成盐反应，提纯制备了脱氢枞胺。通过脱氢枞胺与四氯苯酐的缩合反应，合成了N,N-四氯邻苯二甲酰基脱氢枞胺（2）。（2） N,N-四氯邻苯二甲酰基脱氢枞胺在氯化铝作用下进行傅-克酰基化反应生成N,N-四氯邻苯二甲酰基-12-乙酰基脱氢枞胺（8），然后与间氯过氧苯甲酸发生氧化反应生成N,N-四氯邻苯二甲酰基-12-乙酰氧基脱氢枞胺（9）。化合物9经7位烯苯基氧化、水解，再与碘甲烷作用分别得到N,N-四氯邻苯二甲酰基-7-氧代-12-乙酰氧基脱氢枞胺（10），N,N-四氯邻苯二甲酰基-7-氧代-12-羟基脱氢枞胺（11）和N,N-四氯邻苯二甲酰基-7-氧代-12-甲氧基脱氢枞胺（12）。（3）采用较温和的蒙脱土-硝酸铜（claycop）试剂对脱氢枞胺衍生物的C环进行硝化，得到N,N-四氯邻苯二甲酰基-12-硝基脱氢枞胺（3）和N,N-四氯邻苯二甲酰基-14-硝基脱氢枞胺（4）的混合物，N,N-四氯邻苯二甲酰基-7-氧代-13-硝基脱异丙基脱氢枞胺（7）。与传统的浓硫酸-浓硝酸的混酸体系相比，由于体系中释放出的硝酸根离子浓度较低，反应较为缓和，危险性小。（4）7位烯苯基氧化反应中，传统的方法是在Ac₂O/AcOH混合溶剂中使用CrO₃或Na₂CrO₄等六价铬试剂作氧化剂，该方法会产生大量铬的有毒废液，同时，由于反应物N,N-四氯邻苯二甲酰基脱氢枞胺（2）、N,N-四氯邻苯二甲酰基-12-乙酰氧基脱氢枞胺（9）及N,N-四氯邻苯二甲酰基-12-硝基脱氢枞胺（3）在Ac₂O/AcOH中溶解性差，所得产物产率很低（大约10¹5%）。因此，我们改用8倍量的叔丁基过氧化氢作氧化剂，以CrO₃/吡啶作催化剂，在CH₂Cl₂中室温搅拌反应25h，所得N,N-四氯邻苯二甲酰-7-氧代脱氢枞胺衍生物（6、10和5）产率分别为68.5%、65.5%、38.1%。相较于传统的CrO₃或Na₂CrO₄氧化剂，使用叔丁基过氧化氢氧化试剂，反应条件温和、产物后处理过程更为简单快捷、产率提高，同时不产生有毒废液，有利于环保。（5）使用对甲苯磺酰肼与N,N-四氯邻苯二甲酰脱氢枞胺衍生物的7位羰基或12位乙酰基发生缩合反应生成N,N-四氯邻苯二甲酰脱氢枞胺对甲苯磺酰腙衍生物（13¹8），反应溶剂的选择对产率影响很大，以苯/乙醇（4:1）作反应溶剂，0.09倍量的对甲苯磺酸作催化剂时，取得较好效果。（6）对甲苯磺酰腙衍生物（13¹8）在吡啶中与甲醇钠在室温下反应20min,然后加入C₆₀的氯苯溶液，70°C搅拌反应24h,生成单加成物、多加成物及未反应完的C₆₀的混合物，再经柱层析得N,N-四氯邻苯二甲酰基-7,7-C₆₀-脱氢枞胺（19），N,N-四氯邻苯二甲酰基-12-硝基（或乙酰氧基、甲氧基）-7,7-C₆₀-脱氢枞胺（20、21、22），N,N-四氯邻苯二甲酰基-13-硝基-7,7-C₆₀-脱异丙基脱氢枞胺（23）和N,N-四氯邻苯二甲酰基-12-乙基-23,23-C₆₀-脱氢枞胺（24）。N,N-四氯邻苯二甲酰基-12-乙酰氧基-7,7-C₆₀-脱氢枞胺（21）经水解生成N,N-四氯邻苯二甲酰基-12-羟基-7,7-C₆₀-脱氢枞胺（25）。根据文献，C₆₀与对甲苯磺酰腙衍生物反应生成[5,6]开环或[5,6]开环和[6,6]闭环异构体混合物，但在我们的实验中，没有发现[5,6]开环异构体生成，提纯产物只中有[6,6]闭环异构体。所以，该反应是通过卡宾机制实现的。产物经IR，UV-vis，¹H NMR，¹³C NMR，MALDI-TOF MS，元素分析等分析方法测试了结构，确认为目标产物。（7）乙二胺与N,N-四氯邻苯二甲酰基-C₆₀-脱氢枞胺衍生物（19²5）上的C₆₀发生加成反应，产物经酸化后得到七个水溶性的N,N-四氯邻苯二甲酰基-C₆₀-脱氢枞胺乙二胺二盐酸盐衍生物（26³2）。产物经MALDI-TOF MS检测确认为目标产物，且为乙二胺加成度为1²的混合物。（8）本文还探索了水溶性N,N-四氯邻苯二甲酰基-C₆₀-脱氢枞胺乙二胺二盐酸盐衍生物的生物活性。合成的七个水溶性N,N-四氯邻苯二甲酰基-C₆₀-脱氢枞胺乙二胺二盐酸盐衍生物经上海国家新药筛选中心进行体外抗HIV-1逆转录酶和抗HIV-1蛋白酶的活性测试。测试结果表明：①7,7-C₆₀取代衍生物（26）、12-羟基-7,7-C₆₀取代衍生物（30）、23,23-C₆₀取代衍生物（32）抗HIV-1逆转录酶活性较好，半数抑制浓度(IC₅₀)分别为3.01μg/mL、5.24μg/mL、4.32μg/mL。但12-硝基-7,7-C₆₀取代衍生物（27）、12-乙酰氧基-7,7-C₆₀取代衍生物（28）、12-甲氧基-7,7-C₆₀取代衍生物（29）和C环脱异丙基-7,7-C₆₀取代衍生物（31）的活性相对较低，它们的IC₅₀值都大于8μg/mL。②七个水溶性N,N-（四氯邻苯二甲酰基）-C₆₀-脱氢枞胺衍生物都呈现出一定的抗HIV-1蛋白酶活性。在7,7-C₆₀取代衍生物（26³1）中，7,7-C₆₀取代衍生物（26）的抗HIV-1蛋白酶活性最强，其IC₅₀值为7.80μg/mL，12-乙酰氧基-7,7-C₆₀取代衍生物（28）次之，IC₅₀值为12.65μg/mL，12-羟基-7,7-C₆₀取代衍生物（30）活性最低，IC₅₀值为112.87μg/mL。另外三个衍生物，12-甲氧基-7,7-C₆₀取代衍生物（29）、12-硝基-7,7-C₆₀取代衍生物（27）和C环脱异丙基-7,7-C₆₀取代衍生物（31），它们的抗HIV-1蛋白酶半数抑制浓度(IC₅₀)分别为19.39μg/mL、29.50μg/mL、19.43μg/mL。与7,7-C₆₀取代衍生物（26³1）相比，23,23-C₆₀取代衍生物（32）活性更低，其IC₅₀值为633.94μg/mL。更多还原

【Abstract】 Dehydroabietylamine is one of important modified products of rosin and the maincomponent of disproportionated rosin amine. It is widely used in many fields such as chiralresolution，papermaking，adhesives，paint，beneficiation，oil exploitation，medicine，pesticide，etc. It is a natural chiral compound with three chiral carbon atoms and possesses an aromaticditerpene structure with three rings as many natural drugs. Fullerene is the third allotrope ofcarbon. Since the discovery of C₆₀, various fullerene derivatives have been developed and theirapplications in the felds of material and biomedical sciences have been studied extensively.The study found that C₆₀derivatives exhibit a range of interesting biological activities,including inhibiting HIV-1protease, antitumor, inducing DNA photocleavage, and scavengingfree radicals. Among these fullerene derivatives C₆₀hybrids containing bioactive groups havereceived considerable attention in recent years. Many bioactive molecules, such as amino acids,peptides, nucleotide, sugars and steroids have been linked to C₆₀, and these C₆₀derivatives hasbeen proved to have different biological activities.In this paper, dehydroabietylamine reacted with tetrachlorophthalic anhydride to giveN,N-（Tetrachlorophthaloyl）dehydroabietylamine, which was introduced carbonyl on B ringthrough oxidation, and then functional groups such as nitro group, acetyl group, ester group,hydroxyl group and methoxy group were introduced on C ring by nitration, acetylation,oxidation, hydrolysis and substitution reaction, and so a series of dehydroabietylamine deri-vatives were synthesized. Subsequently, the condensation reactions of7-carbonyl of dehydro-abietylamine derivatives with p-tosylhydrazide yielded the corresponding dehydroabietylaminep-tosylhydrazones, followed by [1+2] cycloaddition reactions with C₆₀to yield C₆₀-N,N-（tetr-achlorophthaloyl）dehydroabietylamine derivatives. These C₆₀-N,N-（tetrachlorophthaloyl）dehy-droabietylamine derivatives were waterborne modified to increase their solubility in biologicalenvironment and their biological activities of anti-HIV-1reverse transcriptase and anti-HIV-1protease were tested. The research project can be expected to provide a reference for exploringand developing new rosinamine derivatives with potential biological activity. Following are theworks and results of our study.（1） Dehydroabietylamine was purified by the salt-forming reaction of disproportionatedrosin amine and p-toluenesulphonic acid. N,N-（Tetrachlorophthaoyl）dehydroabietylamine（2）was synthesized by condensation reaction of dehydroabietylamine and tetrachlorophthalicanhydride.（2） N,N-（Tetrachlorophthaoyl）dehydroabietylamine was transformed into N,N-（tetrachlo-rophthaloyl）-12-acetyldehydroabietylamine（8） by Friedel-Crafts acylation in the presence ofAlCl3, followed by oxidation with m-chloroperbenzoic acid to generate N,N-（tetrachlorophthal-oyl）-12-acetoxydehydroabietylamine（9）. Compound9was transformed into N,N-（tetrachloro-phthaloyl）-7-oxo-12-acetoxydehydroabietylamine（10）, N,N-（tetrachlorophthaloyl）-7-oxo-12-h-ydroxydehydroabietylamine（11） and N,N-（tetrachlorophthaloyl）-7-oxo-12-methoxydehydroabi-etylamine（12）, respectively, by C-7benzylic oxidation, hydrolysis and the reaction with CH3I.（3） C ring of dehydroabietylamine derivatives（2） was mononitrated with the milder clay-supported copper（II） nitrate（claycop） to afford a mixture of N,N-（tetrachlorophthaloyl）-12-nitrodehydroabietylamine（3） and N,N-（tetrachlorophthaloyl）-14-nitrodehydroabiethylamine（4）, aswell as N,N-（tetrachlorophthaloyl）-7-oxo-13-nitrodehydroabiethylamine（4）. This reaction has the advantages of milder and high safety due to the lower concentration of nitate ion releasingin the claycop system, compared with the traditional H2SO4-HNO₃mixed acid system.（4） The conventional methods for C-7benzylic oxidations involve the use of chromium（VI）reagents, such as CrO₃and Na₂CrO₄, in a mixed solvent of Ac₂O/AcOH, which led toconsiderable amounts of toxic effuents and afforded low yields （approximately10¹5%） in theC-7benzylic oxidations of N,N-（Tetrachlorophthaoyl）dehydroabietylamine（2）, N,N-（tetrachlo-rophthaloyl）-12-acetoxydehydroabietylamine（9） and N,N-（tetrachlorophthaloyl）-12-nitrodehyd-roabietylamine（3） because of their poor solubility in Ac₂O/AcOH. So we used an excess oft-BuOOH （8equiv） as an oxidant and CrO₃/pyridine mixture as a catalyst, the reaction mixturewas stirred for25h at room temperature in CH₂Cl₂, the yields of N,N-（tetrachlorophthal-oyl）-7-oxodehydroabietylamine derivatives （6,10and5） was68.5%、65.5%、38.1%,respectively. Compared with the traditional CrO₃or Na₂CrO₄oxidant, using t-BuOOH as theoxidant, the reaction is milder，the post-treatment process of products is more simple and theyields is higher, meanwhile it produce few toxic effuents and has a great significance inenvironmental protection.（5） The condensation reactions of7-carbonyl or12-acetyl of N,N-（tetrachlorophthaloyl）dehydroabietylamine derivatives with p-tosylhydrazide yielded N,N-（tetrachlorophthaloyl）de-hydroabietylamine p-tosylhydrazone derivatives（13¹8）. The choice of reaction solvent had agreat infuence on the yields. A mixture of benzene/ethanol（4:1） was used as the reaction solventand p-tolu-enesulphonic acid（0.09equiv） was added as a catalyst, giving a good effect.（6） p-Tosylhydrazone derivatives（13¹8） were reacted with NaOMe in pyridine for20minat room temperature. A solution of C₆₀in chlorobenzene was then added, and the mixture wasstirred for24h at70°C. A mixture of the monoadduct, higher adducts and unreacted C₆₀wasobtained and then purifed by column chromatography to give N,N-（tetrachlorophthaloyl）-7,7-C₆₀-dehydroabietylamine（19）, N,N-（tetrachlorophthaloyl）-12-nitro（or acetoxy, methoxy）-7,7-C₆₀-dehydroabietylamine（20,21,22）, N,N-（tetrachlorophthaloyl）-13-nitro-7,7-C₆₀-deisopr-opyldehydroabietylamine（23） and N,N-（tetrachlorophthaloyl）-12-ethyl-23,23-C₆₀-dehydroabie-tylamine（24）. N,N-（tetrachlorophthaloyl）-12-acetoxy-7,7-C₆₀-dehydroabietylamine（21） was hy-drolysed to afford N,N-（tetrachlorophthaloyl）-12-hydroxy-7,7-C₆₀-dehydroabietylamine（25）.According to the literature, C₆₀reacted with p-tosylhydrazones to afford [5,6]-open isomers or amixture of [5,6]-open isomers and [6,6]-closed isomers. In our experiment, no traces of the[5,6]-open isomer were found and the only isolated product was the [6,6]-closed isomer. Thus,the carbene mechanism is realised in this reaction. The target compounds were characterized byIR, UV-vis,¹H NMR,¹³C NMR, MALDI-TOF MS and elemental analysis.（7） Ethylenediamine reacted with C₆₀of N,N-（tetrachlorophthaloyl）-C₆₀-dehydroabiety-lamine derivatives（19²5） by addtion reaction, then the products were acidified to giveN,N-（tetrachlorophthaloyl）-C₆₀-dehydroabietylamine ethylenediamine dihydrochloride derivat-tives（26³2）. The target compounds were characterized by MALDI-TOF MS, and were themixture of1²different addition degrees of ethylenediamine.（8） In this paper, the bioactivities of water-soluble N,N-（tetrachlorophthaloyl）-C₆₀-dehydroabietylamine ethylenediamine dihydrochloride derivatives were also investigated, andthe anti-HIV-1reverse transcriptase and anti-HIV-1protease activities of the sythesized sevenwater-soluble N,N-（tetrachlorophthaloyl）-C₆₀-dehydroabietylamine ethylenediamine dihydro-chloride derivatives were tested by The National Center for Drug Screening. The test results showed that:①7,7-C₆₀-substituted derivative（26）,12-hydroxy-7,7-C₆₀-substituted derivative（30）and23,23-C₆₀-substituted derivative（32） showed relatively high anti-HIV-1reverse transcr-iptase activities, and their median inhibition concentration(IC₅₀) were3.01μg/mL,5.24μg/mLand4.32μg/mL, respectively. But the anti-HIV-1reverse transcriptase activities of12-nitro-7,7-C₆₀-substituted derivative（27）,12-acetoxy-7,7-C₆₀-substituted derivative（28）,12-methoxy-7,7-C₆₀-substituted derivative（29） and C ring deisopropyl-7,7-C₆₀-substituted derivative（31）were relatively low, their IC₅₀values were greater than8μg/mL.②All seven water-solubleN,N-（tetrachlorophthaloyl）-C₆₀-dehydroabietylamine derivatives presented a certain anti-HIV-1protease activities. In the7,7-C₆₀-substituted derivatives（26³1）, the anti-HIV-1proteaseactivity of7,7-C₆₀-substituted derivative （26） was highest, its IC₅₀value was7.80μg/mL,closely followed by12-acetoxy-7,7-C₆₀-substituted derivative（28）, its IC₅₀value was12.65μg/mL, and that of12-hydroxy-7,7-C₆₀-substituted derivative（30） was lowest, its IC₅₀value was112.87μg/mL. Three other derivatives,12-methoxy-7,7-C₆₀-substituted derivative（29）,12-nitro-7,7-C₆₀-substituted derivateive（27） and C ring deisopropyl-7,7-C₆₀-substituted derivative（31）, their IC₅₀value were respectively19.39μg/mL、29.50μg/mL and19.43μg/mL.Compared with that of7,7-C₆₀-substituted derivatives（26³1）, the anti-HIV-1protease activityof23,23-C₆₀-substituted derivative（32） is lower, its IC₅₀value was633.94μg/mL.更多还原