【作者】 吴敬德；

【导师】 徐文方；

【作者基本信息】 山东大学 ， 药物化学， 2012， 博士

【摘要】 1.立题背景长期以来,白血病一直是威胁人类生命健康的恶性血液疾病,并且随着工业化进程的发展、环境的恶化,诱发白血病的外因逐渐增多,使得白血病的发病率特别是儿童白血病的发病率呈上升趋势。随着分子生物学、细胞生物学的发展,白血病的发病机制研究不断深入,染色体易位、原癌基因及抑癌基因的变异是白血病发病的主要原因,信号通路等的改变对白血病的进程有直接的影响,但由于不同类型的白血病发病机制的差异,造成白血病治疗的复杂性及多变性。维甲酸类化合物是4000余个天然及合成化合物的总称,其中多种化合物已成为肿瘤治疗的一线用药,如全反式维甲酸(ATRA)通过诱导细胞分化作用而成为复发或难治性急性早幼粒细胞白血病(APL)治疗的首选及一线用药；他米巴罗汀(Tamibarotene, AM80),是特异性RARα激动剂,临床主要用于治疗各种类型的APL,由日本Nippon Shinyaku公司开发,2005年6月在日本首次上市(商品名Amnolake)。组蛋白去乙酰化酶(HDAC)通过参与调控基因的转录而与白血病及其它恶性肿瘤发生、发展密切相关,是肿瘤治疗中一类较新的作用靶点：通过核受体,HDAC可被募集至肿瘤细胞中,使肿瘤细胞中HDAC呈高表达,其后果包括：细胞中基因的正常转录因此受到抑制,最终导致肿瘤的发生；其次,白血病及其它恶性肿瘤中往往存在细胞的异常增殖及分化受阻,这与影响细胞生长及分化的相关蛋白的静默相关,而这些蛋白的静默是启动子区域DNA的异常甲基化,以及包含组蛋白去乙酰化在内的染色体结构发生改变的结果。这种HDAC与恶性肿瘤发生、发展间的相关性是寻找及研究组蛋白去乙酰化酶抑制剂(HDACIs)的理论基础。组蛋白去乙酰化酶抑制剂(HDACIs)按结构特点共分6大类,目前已有部分组蛋白去乙酰化酶抑制剂被批准上市,还有10多个处于临床试验阶段。组蛋白去乙酰化酶抑制剂能够缓解特定致癌基因产物引起的转录抑制,已证实在白血病及其它恶性肿瘤的治疗中具有确切的疗效,其对白血病的治疗机制主要包括：诱导细胞分化,特别是对ATRA产生耐药的病人尤其显著：HDACIs通过抑制HDAC活性,消除特定靶细胞组蛋白去乙酰化,使得白血病细胞恢复对ATRA的敏感性；其它作用机制还包括诱导白血病细胞的凋亡及抑制增殖；细胞周期阻滞等等。组蛋白去乙酰化酶抑制剂的优点在于,靶向性较好,相应地毒副作用较小缺点是生物利用度低、代谢快。目前已有多个HDACIs进入临床试验阶段,SAHA是FDA批准的第一个上市的HDACIs。2.目标化合物的设计对已有的HDACIs结构进行分析发现,HDACIs的结构一般由三部分组成：疏水性基团、连接基团及锌离子螯合基团。基于已知HDAC抑制剂(如SAHA)的三维结构及其与酶的结合模型,根据药物设计的拼合原理,同时借鉴前药及多靶点药物理论,本文以RARa选择性激动剂他米巴罗汀(AM80)为疏水性基团,以异羟肟酸、羧基等作为锌离子螯合基团,通过酯键或酰胺键(连接基团)将二者融合,设计合成了一系列结构全新的HDAC抑制剂。目标化合物进入到体内后,通过抑制HDAC的活性,从而抑制白血病细胞的增殖、促进其分化和诱导其凋亡,同时,其在体内酯酶或酰胺酶催化下可代谢释放出AM80, AM80作用于RARa受体而诱导白血病细胞的分化和抑制其增殖而发挥白血病治疗作用,实现与前体药物的协同调节作用。3.目标化合物的合成本研究以2,5-二甲基-2,5-己二醇为起始原料,经过卤代、付-克烷基化、硝化、催化氢化、酰化、缩合反应,然后经酯水解反应得到关键中间体他米巴罗汀(AM80)。最后他米巴罗汀与邻苯二胺,盐酸羟胺,羟基乙酸,甘氨酸等进行近一步的缩合得到目标化合物。目标化合物和具有全新结构的中间体化合物采用1H-NMR、ESI-MS谱进行了结构确证。4.目标化合物生物活性评价对所合成化合物进行了体外的初步活性评价。体外抑酶试验中,采用HDAC(HDAC试剂盒、Hela细胞提取物和HDAC-8)、氨肽酶-N和明胶酶三种酶分别进行活性筛选。验结果表明所有的目标化合物对HDAC有选择性抑制作用,其中化合物10b和12b对HDAC的抑酶活性与阳性对照SAHA相当。体外细胞实验通过MTT法测定了化合物10b和12b对人卵巢透明细胞癌ES-2,人结肠癌细胞株HCT116,人乳腺癌细胞株MDA-MB-231,人慢性粒细胞白血病细胞株K562,人急性早幼粒白血病细胞株HL-60和NB4以及人前列腺癌细胞株PC-3的增殖抑制活性。结果表明化合物10b和12b对ES-2细胞、PC-3细胞、K562细胞及HL-60细胞的抑制活性均优于阳性对照SAHA,对HCT116细胞、MDA-MB-231细胞及NB4细胞的抑制活性均略弱于阳性对照SAHA。10b对HL-60及NB4细胞的抑制活性明显优于对照组AM80,12b对NB4细胞的抑制活性亦明显优于AM80。5.药动学研究选取具有较好抑酶活性和增殖抑制活性的目标化合物12b和10b,以已批准上市的RARa受体选择性激动剂AM80为阳性对照药,采用口服和静脉注射两种给药途径研究了目标化合物在Wistar大鼠体内的药物动力学特性。结果显示,静注给药后,10b在血浆中以独立分子存在,5min至11h可持续释放出AM80,并且10b作为AM80的前体药物,与AM80单独给药相比,各药动学参数均有较大改变,如使AM80的MRT从4.44h延长到18.09h,t1/2从2.22h延长到3.88h,Tmax也从0.08h延长到4h,这些结果显示10b静注可以很好地和其前体药物发挥协同治疗作用。与静注给药相比,10b和12b口服给药均显示较低的生物利用度,血浆中仅检测到少量的前体药物及AM80。6.总结与展望本研究以他米巴罗汀为基本结构骨架设计合成了19个目标化合物,并对其进行了体外初步活性评价。其中,化合物10b和12b在体外表现出较好的抑酶活性及对多株肿瘤细胞很强的增殖抑制活性。体内药动学研究结果表明目标化合物可代谢释放出AM80,与其前体药物HDAC抑制剂形成协同治疗、调节作用,使其具有多靶点效应。这些化合物是很有研究前景的先导化合物,对于开发新一代HDAC抑制剂类抗肿瘤药物具有重要意义。更多还原

【Abstract】 1. Research backgroundLeukemia, the malignant disease of hematopoietic system, has long been a threat to human life and health. With the development of the process of industrialization and environmental degradation, the incidence of leukemia, especially childhood leukemia incidence rate is rising.The pathogenesis of leukemia includes chromosomal translocations and hematopoiesis-related oncogenes or antioncogenes mutations. Signaling pathways affect the processs of leukemia. But due to the differences in pathogenesis of different types of leukemia, the cure methods become complexity and variability.Retinoids, the general term of more than4,000natural and synthetic compounds, include a variety of compounds which have enter the clinic and the first-line therapeutic agents of tumor treatment, such as all-trans retinoic acid (ATRA) and Tamibarotene(Am80). Am80is a selective RARa agonist developed by Japanese company Nippon Shinyaku in June2005(trade name:Amnolake).Histone deacetylase (HDAC), involved in the regulation of gene transcription with leukemia and other malignancies, is becoming the new target of cancer therapy: the discovery of the recruitment of HDAC enzyme by nuclear receptors in cancer provide a rationale for inhibition of HDAC activity to release transcriptional repression. Histone deacetylase inhibitors (HDACIs) is divided into six categories. HDACIs are amenable to the design of multi-targeted small molecules because their inherent chemical flexibility and synergy with various anticancer agents. HDACIs can release transcriptional repression caused by the specific oncogene product, and the leukemia treatment mechanisms include:causing cell arrest, differentiation and/or apoptosis of tumor cells, particularly those patients who were highly resistant to ATRA. The advantages of the histone deacetylase inhibitor is better targeted, accordingly toxicity smaller, the disadvantage is the low bioavailability and fast metabolism. Several HDACIs are now undergoing clinical trials and SAHA is the first FAD-approved pan-HDACi to enter the clinic.2. Rational drug design of target compoundsCurrently identified HDACIs shared the following structural character:Cap group, linker and ZBG (zinc binding group). Based on the3D structures of known HDACIs and binding modes of these compounds in complex with HDACs, according to the Principles of Hybridization and Prodrug, we designed and synthesized a series of novel HDAC inhibitors. The retinoic acid receptor RARα agonist Tamibarotene (AM80) was selected as a hydrophobic fragment of the new compounds; glycolic acid, glycine,6-aminocaproicacid and other connection chains of varying lengths as linkers; different hydroximic acid and carboxyl structure as Zn2+binding group. We hope that these new compounds play the role of combined treatment:at first the compounds could maintain the utility structure of HDACIs and have the activity of it, then the Am80can be disposed to play the activity of the Am80in vivo.3. Synthesis of designed target compoundsThe important intermediate AM80was synthesized using2,5-Dimethyl-2,5-hexanediol as starting material through a reaction sequence including halogenation reaction, Friedel-Crafts alkylation, nitration, catalytic hydrogenation, acalytion, condensation and hydrolysis. The condensation of Am80with o-phenylenediamine, hydroxylamine hydrochloride, hydroxy acetic acid and glycin led to all the target compounds. The structures of target compounds and important intermediate were identified by’H-NMR, ESI-MS spectra4. Biological evaluation of the synthesized target compoundsPreliminary activity assay was carried out in vitro. Three kinds of enzymes were selected, which are HDAC, APN and Gelatinase. The results showed that most target compounds exhibited highly selective inhibition against HDAC as compared with APN and MMP-2. The compounds10b and12b were as potent as the positive control SAHA against HDAC. Compounds10b and12b were also assayed for their anti-proliferation activity towards ES-2, HCT116, MDA-MB-231, K562, HL-60, NB4and PC-3cell lines. MTT method was employed. The result showed that10b and12b exhibited higher antiproliferation activity against ES-2, PC-3, K562and HL-60cell lines than positive drug SAHA, and as compared with the AM80,10b have stronger antiproliferative activity against HL-60cell line and12b have stonger antiproliferative activity against NB4cell line.5. Pharmacokinetic studyThis study was performed to describe the compounds (10b,12b) concentration-time courses administrated either orally or intravenously to rats, to select an optimized administration scheme.12b and10b which have interesting bioactivity were administered (oral and i.v.) to rats to investigate whether it is actually metabolized to tamibarotene(AM80). The results show that after i.v. administration,10b kept the utility at first then disposed AM80after5minutes. Comparison of tamibarotene concentration profile after i.v. administration of10b, the MRT prolonged from4.44h to18.09h, t1/2from2.22h to3.88h, Tmax from0.08h to4h. These results indicate10b is possibly useful as a prodrug of AM80. Compared with iv administration,10b and12b all had low bioavailability after oral administration.6. Conclusion and prospectA series of novel HDAC inhibitors based on the scaffold of AM80were designed and synthesized. Preliminary enzyme activity assay and anti-proliferation assays of seven cell lines showed10b and12b possess potential inhibitory activity. Pharmacokinetic study showed that10b and12b, especially10b disposed AM80after i.v. administration and play the role of synergistic effects. These compounds are promising lead compounds for developing new generation of HDAC inhibitors as antitumor agents.更多还原