Design and Synthesis of Novel 2-Acetamido, 6-Carboxamide Substituted Benzothiazoles as Potential BRAFV600E Inhibitors - In vitro Evaluation of their Antiproliferative Activity.

The oncogenic BRAFV600E kinase leads to abnormal activation of the MAPK signaling pathway and thus, uncontrolled cellular proliferation and cancer development. Based on our previous virtual screening studies which issued 2-acetamido-1,3 benzothiazole-6-carboxamide scaffold as active pharmacophore displaying selectivity against the mutated BRAF, eleven new substituted benzothiazole derivatives were designed and synthesized by coupling of 2-acetamidobenzo[d]thiazole-6-carboxylic acid with the appropriate amines in an effort to provide even more efficient inhibitors and tackle drug resistance often developed during cancer treatment. All derived compounds bore the benzothiazole scaffold substituted at position-2 by an acetamido moiety and at position-6 by a carboxamide functionality, the NH moiety of which was further linked through an alkylene linker to a sulfonamido (or amino) aryl (or alkyl) functionality or a phenylene linker to a sulfonamido aromatic (or non-aromatic) terminal pharmacophore in the order -C6 H4 -NHSO2 -R or reversely -C6 H4 -SO2 N(H)-R. These analogs were subsequently biologically evaluated as potential BRAFV600E inhibitors and antiproliferative agents in several colorectal cancer and melanoma cell lines. In all assays applied, one analog, namely 2-acetamido-N-[3-(pyridin-2-ylamino)propyl]benzo[d]thiazole-6-carboxamide (22), provided promising results in view of its use in drug development.

BRAF paradox breakers PLX8394, PLX7904 are more effective against BRAFV600Ε CRC cells compared with the BRAF inhibitor PLX4720 and shown by detailed pathway analysis.

PLX7904 and PLX8394 are novel BRAFV600E inhibitors-BRAFi that are designed to evade the paradoxical MAPK activation, a trait for the name "paradox breakers"-PB. Current FDA approved inhibitors (Vemurafenib, Dabrafenib, Encorafenib) although improved progression-free survival of mtBRAF melanoma patients suffer from this treatment related side effect. mtBRAF Colorectal Cancer (CRC) is resistant to the approved BRAF inhibitors, although combinatorial treatment co-targeting BRAF and EGFR/MEK is offering a promising prospect. In an effort to explore the potential of the novel BRAF inhibitors-PB to impede CRC cell proliferation, they were tested on RKO, HT29 and Colo-205 cells, bearing the BRAFV600E mutation. This study shows that the BRAF paradox breakers PLX7904 and PLX8394 cause a more prolonged MAPK pathway inhibition and achieve a stronger blockage of proliferation and reduced viability than PLX4720, the sister compound of Vemurafenib. In some treatment conditions, cells can undergo apoptosis. Genomic analysis on the more resistant RKO cells treated with PLX7904, PLX8394 and PLX4720 showed similar gene expression pattern, but the alterations imposed by the PB were more intense. Bioinformatic analysis resulted in a short list of genes representing potential master regulators of the cellular response to BRAF inhibitors' treatments. From our results, it is clear that the BRAF paradox breakers present a notable differential regulation of major pathways, like MAPK signalling, apoptosis, cell cycle, or developmental signalling pathways. Combinatorial treatments of BRAFi with Mcl-1 and Notch modulators show a better effect than mono-treatments. Additional pathways could be further exploited in novel efficient combinatorial treatment protocols with BRAFi.