Minor structural modifications to Pracinostat produce big changes in its biological responses

A series of compounds similar to Pracinostat that contained benzimidazole ring and N‐hydroxyacrylamide attached at 5‐ or 6‐position were designed, synthesized, and evaluated as HDAC inhibitors. It was interesting to find that the corresponding derivative 1 with N‐hydroxyacrylamide attached at 5‐position was a potent HDAC inhibitor while the others at 6‐position were not. This is the first time to demonstrate the position difference plays important role in the HDAC inhibitory activities of the cinnamic hydroxamates.


| INTRODUCTION
HDACs have emerged as typical effective therapeutic targets for anticancer agents. To date, five HDAC inhibitors have been approved for cancer therapy and many more in different phases of clinical trials for diverse indications (Chen et al., 2018;Rahman et al., 2016;Zang et al., 2018). The hydroxamic acid-based compounds are among those HDAC inhibitors, which received the widely studies (Mottamal, Zheng, Huang, & Wang, 2015). Among them, Pracinostat 1 is a promising compound, which has been granted breakthrough therapy designation by FDA for the treatment of patients with newly diagnosed acute myelocytic leukemia (AML) who are ≥75 years of age or unfit for intensive chemotherapy Garcia-Manero et al., 2017). Currently, Pracinostat is undergoing phase III clinical trials Ganai, 2016;Garcia-Manero et al., 2017;Kang et al., 2017;Novotny-Diermayr et al., 2012).
In preclinical animal models, Pracinostat is a potent pan-HDAC inhibitor with favorable pharmacokinetic properties (Ning et al., 2013;Razak et al., 2011). As one of the inventors, the corresponding author of this article has witnessed the broad and thoroughly structure-activity relationships based on the scaffold of Pracinostat (Wang et al., 2009(Wang et al., , 2011. However, no examples were reported in the literature since about the synthesis and bioevaluation of Pracinostat derivatives with N-hydroxyacrylamide attached to the 6-position of benzimidazole ring, from our hands or from any other groups ( Figure 1). Thus, we put ourselves to design, synthesize, and compare their differences in biological activities in vitro.

METHODS
The aim of this study is to first access whether similar compounds with only structural position changes could induce any differences in their enzymatic and cell-based inhibitory activities. Secondly, we would investigate their corresponding signaling pathways ( Figure 2). To our knowledge, a typical HDAC inhibitor should be able to increase the expression of Ac-H3, Ac-H4, and Ac-α-tubulin, in addition to their corresponding enzymatic and cell-based inhibitory activities.
The synthetic route for the Pracinostat is shown in Scheme 2, which was prepared from ready available 4chloro-3-nitrocinnamic acid, 9.

| HDAC inhibitory assay
HDAC inhibition was detected using the Amplite ™ Fluorimetric HDAC Activity Assay Kit (Green Fluorescence, AAT Bioquest ® , Inc), Hela nuclear extract (BioVision), and HDAC6 (BPS Bioscience) following the manufacturer's protocol. The assay was performed in a volume of 25 μl at 37°C in 384-well white plates. The final components of the assay ingredients were 10 μl enzyme solution, 2.5 μl test compounds, and 12.5 μl HDAC Green ™ Substrate. The compounds were dissolved in dimethyl sulphoxide (DMSO) at a concentration of 10 mM and diluted in assay buffer before use. The highest concentration of Pracinostat is 10 μM, and the highest concentration of others targeted compounds is 20 μM. The experiment was carried out in triplicate for all investigated IC 50 values. The IC 50 values were calculated by Prism Graphpad prism v.5 software.

| Western blotting
HCT-116 cells were exposed to different concentrations of compound 2a (0.33 and 10 μM), and the control group was treated with Pracinostat at a concentration of 0.33 μM, both of them were treated for 24 hr. Total protein was extracted, and Western blotting was performed as normally described.
The treated cells were collected, RIPA buffer (Solarbio) contained a protease inhibitor (Selleck) cocktail (V RIPA buffer : V protease inhibitor = 100:1) was used to lyse the cells on ice for 30 min, followed by centrifugation at 225 g for 15 min at 4°C. The supernatants were collected, and protein concentration was quantified by a BCA Protein Assay Kit (Solarbio). Twenty-five micrograms of proteins were fractionated by SDS-PAGE (8%-14% gradient gels), electrophoresed, and transferred on to a PVDF membrane and blocked with 5% non-fat milk in TBS for at least 1 hr. The membranes were incubated with the primary and secondary antibodies and detected using the ChemiDoc XRS+ Gel Imaging System (Bio-Rad). Primary antibodies against human acetyl-histone H3 and acetyl-histone H4 were obtained from Cell Signaling Technology. Acetyl α-tubulin antibodies were purchased from Abbkine, Inc.

| RESULTS AND DISCUSSION
All the structures of 2a-2p and key intermediates were characterized by 1 H NMR, 13 C NMR, and ESI-MS and in full agreement with the proposed structures. All above spectra are available in Supplemental Material. Docking compound 2a into the histone deacetylaselike proteine (HDLP, homology of HDAC. PDB code: 1C3R) by MOE software (Figure 3), which revealed that the structural moiety of N-hydroxyacrylamide of the target compound 2a interacted with the residues in the active sites of HDAC homology model in a similar way as that of Pracinostat, while the rest of the substituents at position-1 and position-2 are quite different. As illustrated in Figure 2a, the N-hydroxyacrylamide group of the Pracinostat and 2a has three H-bond interactions with the residues Tyr297, Gly140, and His132 in the ligand-binding pocket, respectively. The length of the hydrogen bonds formed between the N-hydroxyacrylamide group of the Pracinostat with the residues Tyr297, Gly140, and His132 are 2.46, 2.68, and 2.55 Å, respectively, and the corresponding hydrogen bond energies are −1.  The enzyme inhibition assay results showed that all the prepared compounds (2a-2p) exhibited significantly weaker Pan-HDAC inhibitory activities than Pracinostat (Table 1). Specifically, the IC 50 values of the prepared compounds tested against HDAC6 varied from 0.97 to 2.18 μM, higher than that of Pracinostat (IC 50 = 0.10 μM).
The compounds 2a-2p were evaluated for their cellular potency against HCT-116, MCF-7, A549, and SW1990 cell lines in comparison with Pracinostat. Consistent with the results of the HDAC enzyme activity, all the target compounds showed poorer antiproliferative activities than Pracinostat, as listed in Table 2. In all cell lines tested, Pracinostat showed potent antiproliferative activities with IC 50 values ranging from 0.32 to 0.52 μM. Compounds 2a-2p showed weak inhibitory activity against SW1990 and MCF-7 while seemed almost lost activity against A549 and HCT-116 cells.
Compound 2a was chosen for further investigation in comparison with Pracinostat. The acetylation of histone 3 (Ac-H3), histone 4 (Ac-H4), and α-tubulin (Ac-α-tubulin) was measured in HCT-116. The experiments (Figure 4) demonstrated that compound 2a did not increase the acetylation level of histone H3, histone H4, as well as α-tubulin. The results corresponded to the enzymatic and cell-based assays.

| CONCLUSIONS
In summary, we have demonstrated that the attached position of N-hydroxyacrylamide of Pracinostat is vital important for the HDAC inhibitory activities. The N-hydroxyacrylamide F I G U R E 4 Western blot analysis of biochemical markers for apoptosis induction and inhibition by 2a and Pracinostat in HCT-116 cell line. Cells were treated with 2a at a concentration of 0.33 and 10 μM, respectively, and the control group treated with Pracinostat at a concentration of 0.33 μM. Levels of Ac-α-tubulin, Ac-H3, and Ac-H4 were probed by specific antibodies. GAPDH was used as the loading control attached to the position-5 related compounds such as Pracinostat is a potent HDAC inhibitor with acetylation levels increased significantly for H3, H4, and α-tubulin. However, all these activities dramatically decreased even lost when Nhydroxyacrylamide was attached to the position-6 of the benzimidazole ring. These results were also consisted with the cell-based inhibitor activities. Docking results reveals that the hydrogen bond interaction between amino unit of the side chain at position 1 (Figure 1) and the hydroxyl group of the tyrosine (Tyr91) plays an important role in HDAC inhibition by Pracinostat. Thus, minor structural modifications might produce big changes in its biological responses and we hope this observation might be useful in the arena of drug hunting.

ACKNOWLEDGMENTS
This work was supported by the National Natural Science Foundation of China (Nos. 81172927 and 81573297).