【作者】 包凯；

【导师】 程卯生； 张为革；

【作者基本信息】 沈阳药科大学 ， 药物化学， 2008， 博士

【摘要】 红霉素是临床常用的一类大环内酯抗生素。在长期的临床实践和深入的药理研究中人们发现,红霉素等药物在作用于细菌的同时,对人体的生理机能也产生了一定的影响,除具有抗菌活性之外,还呈现出其它的生物活性,包括促进消化道运动活性、抗炎活性、拮抗黄体生成素释放激素活性以及抑制磷酸二酯酶-3活性等。另外,人们发现红霉素等抗生素在肿瘤治疗方面起到积极的作用。尽管曾有文献报道红霉素类大环内酯抗生素有辅助治疗肿瘤的作用,但迄今为止,红霉素衍生物具有抑制肿瘤细胞增殖活性的文献却未见报道。以发现并提高红霉素的抗肿瘤活性、探索大环内酯类化合物抗肿瘤活性构效关系为目标的红霉素衍生物的研究更是鲜有人涉足。本论文的研究工作就是在这一全新的领域进行了初步的探索。在红霉素衍生物的合成与活性研究中,我们偶然发现了一个结构新颖的红霉素衍生物——红霉素二聚物,并且随后的药理实验证实它具有一定的体外抗肿瘤活性。以此为先导化合物,通过改变二聚物的两个大环内酯单体的连接方式和位点,我们首先设计合成了两种类型的红霉素衍生物二聚物（“尾对尾”二聚物TTD和“头对头”二聚物HHD）;在此基础上,按照红霉素衍生物二聚物中去除一个红霉素衍生物母环的简化思路,设计合成了“尾部变化”单体（TM）和“头部变化”单体（HM）两类红霉素衍生物二聚物的结构简化物。共设计合成了73个结构新颖的红霉素衍生物作为抗肿瘤活性研究的目标化合物。在中间体的制备过程中,采用了稀盐酸水解法选择性地脱除红霉素衍生物母环的3位cladinose;用NaBH₄还原红霉素或克拉霉素制备了9位为羟基的红霉素衍生物;用碘氧化法制备了N-去甲基化红霉素衍生物;另外以四乙酸铅为氧化剂制备了侧链为乙酰基的十二元环红霉素衍生物及其N-去甲基化产物;超声作用下KBH₄/ZrCl₄还原9（E）-红霉素肟制备了9（S）-氨基红霉素衍生物。以N-去甲基化红霉素衍生物为原料,以1-溴-2-氯乙烷、反式1,4-二溴-2-丁烯和氯乙酰氯为烷基化或酰基化试剂,合成了“尾对尾”二聚物（TTD）BK-01～BK-39。9（E）-红霉素肟衍生物与反式1,4-二溴-2-丁烯通过O-烷基化等反应,合成了“头对头”二聚物（HHD）BK-40～BK-48。以N-去甲基化红霉素衍生物和含氮杂环为原料,以1-溴-3-氯丙烷、反式1,4-二溴-2-丁烯和氯乙酰氯为烷基化或酰基化试剂,制备了“尾部变化”单体BK-49～BK-60。以9（E）-红霉素肟类衍生物和含氮杂环为原料,以反式1,4-二溴-2-丁烯等为烷基化试剂,制备了“头部变化”单体（HM）BK-61～BK-73。利用MS、¹H NMR和¹³C NMR等仪器分析方法确定了所有目标化合物的化学结构,并对化合物的¹³C NMR数据进行了简单的归纳和分析。在体外活性测试过程中,我们选用了SGC-7901（人胃腺癌细胞）、KB（人口腔癌细胞）、HT-1080（人纤维肉瘤细胞）和人早幼粒白血病HL-60细胞株等肿瘤细胞株,以抗肿瘤药物顺铂为阳性对照药物,采用四甲基偶氮唑盐（MTT）比色法或者台盼蓝染色法完成了54个目标化合物的活性测试并且对构效关系进行了初步的总结。实验证实,“尾对尾”二聚物（TTD）和“头对头”二聚物（HHD）均具有确切的抗肿瘤活性。其中,4-HHD、4-TTD等类型的二聚物活性良好,部分化合物体外抑制肿瘤细胞增殖的IC₅₀可达到0.1μM数量级。另外我们首次发现由红霉素衍生物二聚物经结构简化而设计的“尾部变化”单体（TM）和“头部变化”单体（HM）等两种类型的二聚物结构简化物均有一定的抗肿瘤活性。其中HM呈现与4-HHD相近的良好活性,部分HM体外抑制肿瘤细胞增殖的IC₅₀可达到0.1μM数量级。与“头部变化”单体（HM）相比,“尾部变化”单体（TM）的体外抗肿瘤活性偏低。采用流式细胞仪PI染色法等方法,分别考察4-TTD类化合物BK-23与HM类化合物BK-63对人胃腺癌细胞株SGC-7901和人白血病细胞株HL-60的细胞凋亡和细胞周期影响。初步证明,红霉素衍生物二聚物BK-23和结构简化物BK-63均可诱导肿瘤细胞凋亡,两者在抗肿瘤作用机理方面存在一定的共性。不同结构类型的红霉素衍生物均具有诱导细胞凋亡活性,并呈现良好的剂量依赖关系,红霉素衍生物抗肿瘤活性的作用机理与诱导细胞凋亡密切相关。此外,我们对红霉素衍生物的合成方法及相关反应进行了一定的研究。将微波催化技术应用于红霉素衍生物的分子内酯交换反应中,发展了在有溶剂或无溶剂条件下由红霉素烯醇醚及其衍生物制备十二元环红霉素衍生物的新的简便方法。首次使用可溶性的NaOAc-CaCl₂作为微波催化无溶剂反应的载体,由红霉素及其衍生物出发,将脱水和分子内酯交换两步反应连续进行,建立了直接制备十二元环红霉素衍生物的“一瓶反应”（one-pot reaction）新方法。此外我们还首次发现了酯促进的由氰负离子极性反转引发的N-甲酰化及相关反应,并将此反应应用于其它结构类型的底物,建立了合成N-甲酰化产物、甲咪类化合物、苯并咪唑以及N-取代胺甲叉基丙二酸二甲酯衍生物等多种化合物的通用方法。更多还原

【Abstract】 Erythromycin,a typical macrolide antibiotic,had been extensively used in the clinic for more than 50 years.In addition to the anti-microbial effects,many clinical studies suggested that erythromycin and its derivatives had some other activities,such as prokinetic activity, anti-inflammatory activity,luteinizing hormone-releasing hormone antagonistic activity and phosphodiesterase-3 inhibitory activity.Moreover,they had been discovered to have a beneficial effect on the treatment of tumor.Although there were some reports in the past few years of macrolide antibiotics having helpful effects on antitumor treatment,to the best of our knowledge,the inhibitory activity of erythromycin derivatives on the proliferation of tumor cells had never been reported.Up to now, little attention had been paid searching for the novel erythromycin derivatives with antitumor activity.In this article,preliminary investigations are made in this field.In our study of "Synthesis & Bioactivities of Erythromycin Derivatives",a new class of erythromycin derivative,dimmer of erythromycin was discovered and after further investigation, it was found to show in vitro antitumor activity.Therefore it was picked as the lead compound and through the change of the connecting chain and the connecting nodes on erythromycin derivatives,two kinds of dimmers of erythromycin derivatives（"tail to tail" dimmers,TTD and "head to head" dimmers,HHD）were designed and synthesized at first;then by the removal of one of the macrolactone ring from the dimmers of erythromycin derivatives,two kinds of simplified analogues of macrolide dimmers,"tail variation" monomer（TM）and "head variation" monomer（HM）,were designed and synthesized.Totally,73 novel erythromycin derivatives were synthesized for the in vitro antitumor activity test.During the synthesis of intermediate,the cladinose moieties of erythromycin derivatives could be selectively removed upon the treatment with dilute hydrochloric acid in an aqueous solution;employing NaBH₄ as a reducing agent in methanol,9（S）-hydroxyl erythromycin derivatives could be prepared conveniently;N-demethylated erythromycin derivatives were obtained by N-demethylation with iodine and sodium acetate in methanol;The synthesis of 12-membered ring erythromycin derivatives（containing a acetyl side chain）and its N-monodemethyled product were accomplished by the use of lead tetraacetate as an oxygenant; 9（S）-Erythromycylamin was synthesized by selective reduction of erythromycin A 9（E）-oxime with NaBH₄/ZrCl₄ under ultrasonic irradiation.The synthesis of the "tail to tail" dimmers（BK-01～BK-39,TTD）was accomplished through the alkylation or acylation of the N-demethylated erythromycin derivatives."Head to head" dimmers（BK-40～BK-48,HHD）were prepared from the 9（E）-oxime erythromycin derivatives,by the use of（E）-1,4-dibromo-2-butene as the alkylating agents.Using N-demethylated erythromycin derivatives and nitrogen-containing heterocycles as the substrates, 1,3-bromochloropropane,（E）-1,4-dibromo-2-butene and chloroacetyl chloride as the alkylating or acylating agents,"tail variation" monomers（BK-49～BK-60,TM）were prepared."Head variation" monomers（BK-61～BK-73,HM）were prepared by utilizing 9（E）-oxime erythromycin derivatives and nitrogen-containing heterocycles as the substrates,（E）-1,4-dibromo-2-butene as the alkylating agents.All the target compounds and intermediates were characterized by the applications of MS, ¹H NMR and ¹³C NMR.The data of ¹³C-NMR spectra were discussed briefly.54 of the target compounds were evaluated for their in vitro antitumor activities against human SGC-7901,KB,HT-1080 and HL-60 cell lines by MTT assay or Trypan blue staining. And the structure-activity relationships study of them was also performed initially.The in vitro antitumor activity of "tail to tail" dimmers（TTD）and "head to head" dimmers （HHD）was firmly established by our assay.4-HHD and 4-TTD showed potent antitumor activity and the IC₅₀values of some erythromycin derivatives were between 0.1 and 1.0μM.In addition,both "tail variation" monomers（TM）and "head variation" monomers（HM）were shown to be active in the assay.Some of the HM were found to retain much of the activity of their parent compounds（4-HHD）,with an IC₅₀of 0.1-1.0μM.Compared to HM,TM 1ed to the decrease of antitumor activity.BK-23（4-TTD）and BK-63（HM）induced apoptosis in human SGC- 7901 or HL-60 cell lines and their influence on cell cycle were detected by flow cytometry performed by PI staining. Flow cytometry suggested that the dimmers of erythromycin derivatives BK-23 and the simplified analogue BK-63 could induce apoptosis which revealed that they may have a similar mechanism.Erythromycin derivatives containing different structures could induce apoptosis in a dose-dependent fashion and the mechanism of the antitumor activity of erythromycin derivatives highly correlated with the induction of apoptosis.We also focused our attention on the synthesis of erythromycin derivatives and related reactions.For the first time,we developed two methods to prepare the ring-contracted erythromycin derivatives via microwave-assisted intramolecular transesterification under either solvent-containing or solvent-free conditions.In addition,we found a novel water soluble sodium acetate-doped calcium chloride system to be used as the solid support in the microwave-assisted reactions and developed a new,straightforward synthesis of ring-contracted erythromycin derivatives by one-pot reaction of erythromycin and its derivatives via intramolecular dehydration and transesterification.Furthermore,we found a novel N-formylation and related reactions via umpolung of cyanide ion promoted by esters and developed a new,general method for the synthesis of formamides,N,N’-disubstituted formamidines,benzoimidazole,N-（aminomethylene）malonate and its derivatives.更多还原