For further understanding of the interaction between these i

For further understanding of the interaction between these inhibitors and proteins (BRafV600E and HDAC1) for guiding the SAR, compound 10e, the most potent compound of this series, was chosen as a representative for docking into the BRafV600E (PDB code: 1UWJ) and HDAC1 (PDB code: 1C3R), revealing excellent shape complementarity between ligand and the binding pocket. As shown in Fig 2., 10e binds to the BRaf ATP binding pocket, interacting with three aromatic residues: Trp530 of the hinge region, Phe582 at the end of the catalytic loop, and Phe594 of the DFG motif, which also contacts the central phenyl ring of the inhibitor. At one end of the inhibitor, the lipophilic trifluoromethyl phenyl forms hydrophobic interactions with the surrounding amino osmi synthesis residues (Val503, Ile571, Ile591). The aliphatic side chains of Lys482, Leu513, and Thr528 contact the central phenyl ring of the inhibitor. In addition to the van der Waals interactions, the phenylamine of compound 10e forms one essential hydrogen bond with the hinge region Cys531. The amide group forms two hydrogen bonds with Asp593 and Lys482. In addition, the methyl amide side group directs to the solvent accessible region of the enzyme. On the other hand, the docking mode of 10e in complex with HDAC1 reveals that this compound binds in the HDAC1 pocket with the similar manner to chidamide. The 4-chloro-3-(trifluoromethyl)phenyl moiety of 10e projects outside into the solvent, weakly contacting hydrophobic residues on the protein surface. Phenoxyphenyl moiety exits the reaction center along the long tubular channel and is stacked between Phe198 and Phe141. The o-aminobenzamide group entered into the active site by chelating the essential catalytic zinc ion, and forms three hydrogen bonds with His131, Tyr297 and Gly140, respectively. However, the only one chelating bond derived from the amino group may be an important cause of its reduced activity. In order to further ascertain the pan-inhibition of Raf kinases and HDACs, two representative compounds 10e and 10f with better HDAC1 and BRafV600E inhibition were selected and evaluated against recombinant human HDAC1, HDAC6, HDAC8 enzymes and ARaf, BRafV600E and CRaf, using chidamide and sorafenib as the positive control compounds. As shown on Table 2, it is noteworthy that this set of structures show promising pan-inhibition for Raf and selective activity against HDAC1. To test the anticancer activities of the synthesized compounds, we evaluated antiproliferative activities of representative compounds 10c, 10d, 10e, 10f and 10j against human chronic myelogenous leukemia cell line K562, human acute myelogenous leukemia cell line MV4-11, human hepatocellular carcinoma cell line Hepg2 and breast cancer cell line MDA-MB-468 by applying the MTT colorimetric assay. The IC50 values were summarized on Table 3. According to the inhibition data, all tested compounds showed obvious antiproliferative activities. Consistent with this observation of HDAC1 and BRafV600E inhibition, we found that 10e and 10f potently inhibited the growth of those cancer cells derived from both hematologic and solid tumors. Especially, compound 10e exhibited the better anti-proliferative activity in Hepg2 and MDA-MB-468 cell lines than sorafenib and chidamide. In conclusion, a series of phenoxybenzamide analogues have been prepared and assessed for their Raf and HDAC inhibitory activity and cytotoxicity against the K562, MV4-11, Hepg2 and MDA-MB-468 cell lines. Moreover, the selectivity assay of the respective compounds showed that they were not only excellent selective inhibition of HDAC1, but also pan-Raf inhibitors. Preliminary SAR data and docking mode revealed the o-aminobenzamide moieties were essential for binding to the proteins BRafV600E and HDAC1. In addition, compound 10e exhibited higher potency against Hepg2 and MDA-MB-468 cell lines than sorafenib and chidamide. In summary, we have demonstrated the example of dual-action inhibitors targeting the Raf and HDAC as a promising approach to search for efficient anticancer multi-target agents.