A Fluoro Derivative of Embelin, as Potent B-RAF Inhibitor in Melanoma.

Background: 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.

Objective: 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.

Methods: 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.

Results: 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.

Conclusion: 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.