Introduction Cancer has been a significant public health

Introduction Cancer has been a significant public health problem in the world with more patients being diagnosed every year. However, chemotherapy from non-specific cytotoxic agents that as an effective means of cancer treatment is limited by severe side effects and poor selectivity. Thus, the development of mechanism-based targeted antitumor drugs has made significant progress in the past few years. Unfortunately, their clinical effectiveness is generally unsustainable in long-term treatment and the drug resistance always limits the use of these agents based on complex signaling networks of cancers. Nowadays, new treatment strategies, such as combined, multi-targeted therapies or recently, immunotherapy have been advocated.4, 5, 6, 7 Protein kinases in different cell signaling pathways play key roles in the processes of governing cellular proliferation, differentiation and evasion from apoptosis.8, 9, 10 Targeting kinases like Raf and PI3K have become one of the most pursued stavudine mg of anticancer agents.11, 12, 13 The approval of Raf kinase inhibitor such as sorafenib (1), PI3K inhibitor such as pictilisib has been adopted to produce higher potency and selectivity.14, 15 In addition, targeting epigenetic modifiers particularly histone deacetylases (HDACs) have recently set the foundation for a new generation of anti-cancer drugs.16, 17 Several small molecule HDAC inhibitors have been approved by FDA or NMPA for the treatment of various haematological and solid tumors, such as vorinostat and chidamide (2).18, 19 The therapeutic combination of an HDAC inhibitor and a kinase inhibitor, such as epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors, Raf or PI3K inhibitors, can overcome kinase inhibitor resistance and induce apoptosis in human solid cancers in a synergistic manner. However, combination therapy may be associated with unwanted drug-drug interactions, inappropriate pharmacokinetics, toxicity and scarce patient compliance. In order to overcome the problems related to the polypharmacological approach, the development of dual inhibition of HDAC and the tyrosine kinase signaling pathways has been recently proposed as a promising strategy which could provide novel drug candidates for the treatment of multifactorial diseases, such as CUDC-907, FK228.21, 22, 23, 24 Our group has also explored HDAC inhibitors based on a high-throughput screening campaign and found a lead compound 10a with IC50 of HDAC1 5.23 μM. Interestingly, compound 10a possessed the o-aminobenzamide structure similar to chidamide and acylaminophenoxybenzamide scaffold similar to sorafenib (Fig. 1). Based on the screening of kinase inhibitory activity assay in vitro, compound 10a also showed an modest inhibition against BRafV600E with IC50 = 1.78 nM. In this report, we describe the identification of a novel series of phenoxybenzamide dual Raf/HDAC inhibitors, which were obtained by lead optimization of 10a through a structure–activity relationship (SAR) study. These studies led to the discovery of a novel scaffold and potential dual Raf/HDAC inhibitors. Our initial efforts on the lead compound 10a focused on converting the pyridyl moiety of 10a to various aryl group (Table 1). Compound 10a and its derivatives (10b-j) were prepared as outlined in Scheme 1. Acid 3 was esterified to give methyl ester 4, which was then reacted with methylamine to afford N-methyl amide 5. Then compound 5 was converted to the ether 6 via nucleophilic substitution by using p-aminophenol. Furthermore, substituted aromatic carboxylic acids 7a-j were treated with SOCl2 to generate acyl chlorides 8a-j, which were reacted with 6 to produce amides 9a-j. Finally, these amides were reduced with iron powder in water/ethanol to afford final amines 10a–j. On the other hand, we probed the effect of different substituents on amide (Table 1). Similar chemistry was applied to the synthesis of compounds 16a-c in Scheme 2. To study the biological activity, compounds 10a–j and 16a-c were evaluated against BRafV600E activity assays with sorafenib as a positive control and HDAC1 enzyme assay with chidamide as a positive control. As shown in Table 1, most compounds exhibited moderate inhibitory activity against both BRafV600E and HDAC1. Instead of pyridine with benzene and naphthalene, compound 10b and 10j showed about 8-fold and 6-fold increase against BRafV600E activity. Hydrophilic groups at terminal phenyl couldn’t enhance the enzyme activity but could enhance HDAC1 activity, such as compounds 10g, 10h and 10i. Interestingly, compound 10e with Cl and CF3 still had the higher activity against HDAC1. Different types of amides had relatively significant effect on both BRafV600E and HDAC1 inhibition. The potency at enzymatic level of compound 16a with primary amine was significantly decreased compared to 10e. One tertiary amine was introduced at the carbonyl group (16c), which resulted in loss of HDAC1 and reduction of BRafV600E activity. However, another secondary amine 16b could sustain the HDAC1 activity compared to 10e. Among the compounds tested, compound 10e showed powerful inhibitory activity against HDAC1 (IC50 = 1.17 μM) and BRafV600E (IC50 = 0.073 μM).