Department of Chemistry, College of Engineering Trivandrum, Thiruvananthapuram, India.
Department of Chemistry, University College Thiruvananthapuram, Kerala University, Thiruvananthapuram, India.
Anticancer Agents Med Chem. 2021;21(15):2066-2074. doi: 10.2174/1871520621666201229115253.
Melanoma is one of the most common forms of skin cancer, and B-RAF is a mutated protein found in most melanomas. The important function of B-RAF is normal cell growth and survival. Most of the known B-RAF mutations are V600E mutations. Vemurafenib is the fluorine-based drug currently used for V600E mutations. However, this drug has side effects, therefore, more potent drugs with fewer side effects are required.
This study aims to develop a more effective lead compound as a B-RAF inhibitor from hydroxyquinone by structural modification of embelin, a naturally occurring hydroxybenzoquinone. It has the potency of detoxifying blood and is hence useful in a wide range of skin diseases. Thus, a fluorine substituted semisynthetic derivative of embelin, 5-(3-chloro-4-trifluoromethoxy phenyl amino)-2-hydroxy-3-undecyl- [1, 4] benzoquinone to fight against skin cancer was prepared.
Fluoro derivative of embelin was synthesized by the direct condensation of embelin with 3-chloro-4- trifluoromethoxy aniline. The structure of the product was characterized using various spectral data obtained from IR, H NMR, F NMR, C NMR, and mass spectrum. Various in vitro studies like antiproliferative study in A375 Cell Lines (B-RAF Elisa), western blotting analysis, gene expression study by reverse transcriptase PCR, caspase assay, flow cytometry analysis, clonogenic assay, and transwell migration assay were carried out to find its biological activity.
A semisynthetic derivative of Embelin 5-(3-Chloro-4-trifluoromethoxy phenyl amino)-2-hydroxy-3- undecyl- [1, 4] benzoquinone (EOCF) was prepared, and the structure of the derivative was confirmed by spectral analysis. The MTT assay proves that the fluoro derivative of embelin exhibited better anti-cancer activity in melanoma cell lines than the parent compound, embelin. Western blot analysis showed that B-RAF expression level was reduced by the addition of derivative than the parent compound embelin. The Caspase ELISA analysis indicated that the derivative was found to be a good apoptotic marker. From the flow cytometry analysis, it was observed that cell arrest occurs at the G/G phase. Its antimetastatic activity was determined using clonogenic assay. It indicated that the derivative EOCF inhibits the metastatic effects in melanoma cell lines. The migratory potential of melanoma cells was significantly reduced in the presence of EOCF when the transwell migration assay was conducted.
This work established that the potency of the synthesized compound was more than the parent compound, embelin, when it was structurally modified with 3-chloro-4-trifluoromethoxy aniline. The derivative can be used as a lead molecule for further drug discovery.
黑色素瘤是最常见的皮肤癌之一，而 B-RAF 是大多数黑色素瘤中发现的一种突变蛋白。B-RAF 的重要功能是正常细胞的生长和存活。大多数已知的 B-RAF 突变是 V600E 突变。目前，维莫非尼是用于 V600E 突变的氟基药物。然而，这种药物有副作用，因此需要更有效力且副作用更少的药物。
本研究旨在通过对天然存在的羟基苯醌恩布立尼的结构修饰，从羟基喹啉中开发出一种更有效的作为 B-RAF 抑制剂的先导化合物。它具有解毒血液的功效，因此在广泛的皮肤病中都有应用。因此，我们制备了一种用于对抗皮肤癌的氟取代半合成恩布立尼衍生物，5-(3-氯-4-三氟甲氧基苯基氨基)-2-羟基-3-十一烷基-[1,4]苯醌。
通过恩布立尼与 3-氯-4-三氟甲氧基苯胺的直接缩合合成了氟代恩布立尼衍生物。通过从 IR、H NMR、F NMR、C NMR 和质谱中获得的各种光谱数据对产物的结构进行了表征。进行了各种体外研究，如 A375 细胞系（B-RAF ELISA）中的增殖抑制研究、western blot 分析、逆转录 PCR 基因表达研究、caspase 测定、流式细胞术分析、集落形成试验和 Transwell 迁移试验，以发现其生物活性。
制备了恩布立尼的半合成衍生物 5-(3-氯-4-三氟甲氧基苯基氨基)-2-羟基-3-十一烷基-[1,4]苯醌（EOCF），并通过光谱分析确认了衍生物的结构。MTT 试验证明，与母体化合物恩布立尼相比，氟代恩布立尼衍生物在黑素瘤细胞系中表现出更好的抗癌活性。Western blot 分析表明，与母体化合物恩布立尼相比，添加衍生物后 B-RAF 的表达水平降低。Caspase ELISA 分析表明，该衍生物是一种良好的凋亡标志物。从流式细胞术分析可知，细胞在 G/G 期发生阻滞。通过集落形成试验确定了其抗转移活性。结果表明，衍生物 EOCF 抑制了黑素瘤细胞系中的转移作用。当进行 Transwell 迁移试验时，发现用 EOCF 处理后黑色素瘤细胞的迁移潜力显著降低。
本研究表明，与 3-氯-4-三氟甲氧基苯胺结构修饰后的合成化合物的效力强于母体化合物恩布立尼。该衍生物可作为进一步药物发现的先导分子。
