|Place of Conferral||北京|
|Keyword||白癜风 黑色素合成 查尔酮和呋喃香豆素衍生物 Wnt/β-catenin 信号 通路 Pka/p38 Mapk 信号通路|
白癜风是世界皮肤病三大顽症之一，易诊断难治疗，且无特效疗法，其发病机制涉及功能性黑素细胞凋亡和(或)丢失，开发具有促黑素合成功效的抗白癜风新药，将会产生巨大的应用价值和经济价值。据300年前的维吾尔医学经典著作《药用总库》记载，驱虫斑鸠菊(Vernonia anthelmintica (L.) willd)和补骨脂(Psoralen corylifolia L.)是治疗白癜风经典药材，后续研究表明，其有效成分分别为黄酮类化合物和呋喃香豆素类化合物，然而在实际应用过程中，此类化合物存在作用机制不清、作用部位和靶点不明确等缺点，严重限制了该类药物的二次开发。本论文以抗白癜风为导向从传统中药中锁定活性化合物，利用有机化学的方法，对活性成分母核进行衍生修饰，以达到提高活性和生物利用度，降低副作用等目的。本文对所合成的所有衍生物进行了结构表征和促黑素合成活性测试，对目标衍生物进行了细胞水平抗白癜风活性机制研究，为抗白癜风新药开发奠定了基础。1衍生物的合成本文以苯乙酮和苯甲醛为原料首次合成了18个全新的查尔酮衍生物(1-18)；以间二苯酚为原料合成了25个呋喃香豆素类衍生物(异补骨脂素5a-5i, 补骨脂素6a-6p)。其中23个衍生物未见文献报道。所有化合物均经过氢谱、碳谱和质谱鉴定。2 促黑色素生成活性的初步探讨在体外条件下，系统科学地研究了衍生物对酪氨酸酶活性、细胞毒性、相对黑素含量的影响。结果显示：大多数衍生物可促进小鼠B16黑素瘤细胞中酪氨酸酶活性，可刺激黑素生成，且对黑素瘤细胞没有明显毒性；。其中查尔酮衍生物：2、18两个化合物激动作用最强，50 μM的相同浓度下，相对黑素含量明显优于阳性对照8-MOP (8-甲氧基补骨脂)。呋喃香豆素衍生物：6m、6p两个化合物促黑素作用最强，50 μM的相同浓度下相对黑素含量优于阳性对照。本实验结果提示：这些衍生物可能是通过调节酪氨酸酶活性达到促进黑素合成或再生作用。3 两个目标衍生物促黑色素合成的机制研究为了深入探讨所合成衍生物对酪氨酸酶及黑素合成的作用机理，本实验综合化合物构效关系和生物活性检测结果，筛选出两个目标衍生物。在上述酶活性测定基础上，采用Western blotting技术，首次探讨了目标衍生物促黑色素合成的信号通路。结果显示，查尔酮目标衍生物BDFTP-D301012可以通过激活p-Akt308促进GSK3β的磷酸化，进而导致β-catenin入核，上调小眼畸形相关转录因子MITF的转录，最终增加酪氨酸酶活性和黑色素生成，信号通路的抑制剂实验证实了上述推论。而呋喃香豆素衍生物BDFTP-D201020促进黑素合成，可以通过调节PKA和p38 MAPK两条信号通路发挥作用,试剂盒检测细胞内cAMP水平呈浓度依赖性升高,同样，PKA和p38 MAPK两条信号通路的抑制剂对黑色素生成以及酪氨酸酶的呈现阻断效果。综合以上实验结果，本文共完成了43个衍生物的合成及活性测试，探究了其中两个目标衍生物在小鼠黑色素瘤B16细胞中的促黑素合成机制，结果表明两个目标衍生物对黑素合成均有显著的促进作用，而这一作用分别通过Wnt/β-catenin 信号通路和PKA/p38 MAPK信号通路途径发挥作用。本实验结果为动物实验提供数据支持，为临床用抗白癜风新药开发提供了理论基础和实验依据。
Vitiligo is one of three most difficult treatment dermatosis in the whole world. The features of the diseases are easy to diagnose, difficult to treat and have no special treatment. Its pathogenesis involves the self destruction of melanocytes. It is feasible to search for new agents which can enhance melanin synthesis and have enormous potential application value and economic value. Vernonia anthelmintica (L.) willd and Psoralen corylifolia L. are the most popular Chinese herbal medicines used for vitiligo and initially recorded in "Yao Yong Zong Ku" around 300 years ago. Some important flavonoid and furocoumarin compounds are isolated from these plant respectively, and it is suggested that they play an important role in vitiligo treatment. However, these compounds have some shortcomings in the clinical application, such as unclear functionary mechanism, unclear site and target of action, which is a strong disincentive to new drug development.This paper focuses on finding anti-vitiligo active compounds which come from the traditional Chinese medicine, and be modified the structure by using organic chemical technologies to improve activity and bioavailability of derivatives and reduce their toxic effects. In this paper, the structure characterization and activity test of the derivatives were carried out, all these work laid the foundation for the development of new anti- vitiligo drugs.1. The synthesis of derivatives 18 chalone derivatives (1-18) were synthesized from Acetophenone and Benzaldehyde for the first time, and 25 furancoumarin derivatives ( isopsoralen 5a-5i, psoralen 6a-6p) were synthesized with resorcinol as raw material. A total of 43 derivatives were synthesized, and 23 derivatives were new. The structure of the derivatives was identified by mass spectrometry, hydrogen spectrum and carbon spectrum of NMR. 2. Study on the activity of promoting melanogenesis The effect of these compounds on tyrosinase activity, cell cytotoxicity and relative melanin content were studied systematically for the first time. The results show that several derivatives can directly activate tyrosinase activity and have no obvious toxicity to melanoma cells. For the Chalcone derivatives: compounds 2 and 18 had the strongest agonistic effect, and the relative melanin content was much better than 8-MOP in the same concentration of 50 μM. For the Furancoumarin derivatives: compounds 6m and 6p had the strongest agonistic effect, and the relative melanin content was better than 8-MOP in the same concentration of 50 μM. The results suggest that these derivatives may promote the synthesis and regeneration of melanin by regulating tyrosinase activity. 3. Study on the melanin synthesis mechanism of two target derivatives In order to explore the mechanism of tyrosinase and melanin synthesis by the derivatives, two target derivatives were screened by comprehensive structure-activity relationship and biological activity detection results. Western Blotting was used for explaining the signal pathway of melanin synthesis by target derivatives. The results showed that the Chalcone derivative BDFTP-D301012 could promote the phosphorylation of GSK3β by activating p-Akt308, thus leading to the nucleation of β-catenin, up regulating the transcription of MITF and eventually increasing the activity of tyrosinase activity and melanin synthesis. The Furancoumarin derivative BDFTP-D201020 promoted melanin synthesis by regulating PKA and p38 MAPK signaling pathways, the intracellular cAMP content was increased dose–dependently. Similarly, the relative inhibitors blocked BDFTP-D201020 induced-melanin formation and tyrosinase activity increasing. Based on the above results, the synthesis and activity test of 43 derivatives were completed, and the melanin synthesis mechanism of two target derivatives in murine melanoma B16 cells was explored. The results showed that the two target derivatives had significant promoting effect on melanin synthesis, and this effect was regulated by Wnt/β-catenin and PKA/p38 MAPK signaling pathways respectively. The results of this paper can provide data supports for animal experiments, and supply theoretical and experimental bases for the development of new anti-vitiligo drugs.
|尹丽. 查尔酮衍生物和呋喃香豆素衍生物的设计、合成及促黑素合成机制研究[D]. 北京. 中国科学院大学,2018.|
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