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

Rong Jiaa, Pengju Suna, Yan Zhanga, Youjin Gea, Niefang Yua*

Institute of Molecular Design and Drug Discovery, School of Pharmaceutical Sciences, Central South University, Changsha 410013, Hunan, China

Correspondence
*Niefang Yu, Institute of Molecular Design and Drug Discovery, School of Pharmaceutical Sciences, Central South University, Changsha 410013, Hunan, China.
Email: [email protected]

Funding information
National Natural Science Foundation of China, Grant/Award Number: Nos. 81172927 and 81573297

ABSTRACT：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.

KEY WORDS
Pracinostat, histone deacetylases, HDAC inhibitors, hydroxamic acids, synthesis

This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/cbdd.13527

1 | 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 [1][2][4]. The hydroxamic acid-based compounds are among those HDAC inhibitors, which received the most widely studies[3]. 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[5][6]. Currently, Pracinostat is undergoing phase III clinical trials[5][6][7][8][9].
In pre-clinical animal models, Pracinostat is a potent pan-HDAC inhibitor with favourable pharmacokinetic properties[10][11]. As one of the inventors, the correpsonding author of this article has witnessed the broad and thoroughly structure-activity relationships based on the scaffold of Pracinostat[12][13]. 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, synthsize and compare their differences in biological activities in vitro.

2 | EXPERIMENTS AND METHODS
The aim of this study is to first access if similar compounds with only structural postion 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 knowlege, 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.

2.1 | General procedure for the synthesis of target compounds 2a-2p
The synthesis of target compounds 2 were shown in Scheme 2. 3-chloro-4-nitrobenzoic acid was selectively reduced by Borane-THF under room temperature and 4 thus obtained in high yield. Compound 6 could be obtained from 4 by managed oxidation and Horner–Wadsworth–Emmons reaction [14]. Amination of ethyl (E)-3-(3-chloro-4-nitrophenyl) acrylate 6 with amine R1NH2 under basic conditions gave the intermediates7a-7h. Heating a mixture of the 7, R2CHO and SnCl2·2H2O in AcOH/MeOH (1:9) at 45℃ overnight led to the desired compounds 8a-8p. Finaly, treatment of the intermediate 8 with excessive Hydroxylamine hydrochloride in sodium methoxide/methanol gave the objective hydroxamates 2 (scheme 1).
The synthetic route for the Pracinostat is shown in Scheme 2, which was prepared from ready available 4-Chloro-3-nitrocinnamic acid, 9.

2.2 | Molecular docking
The crystal structure of human HDAC homolog (PDB code: 1C3R) was retrieved from the Protein Data Bank. All target compounds were docked in the active sites by MOE. Both HDAC homolog and ligand were structurally optimized prior to the actual docking simulation. Prior to the docking, the receptor was pre-treated according to the standard procedure provided by MOE and energy minimized using the Amber-99 forcefield. The standard protocol implemented in MOE was performed in docking calculations. The binding modes were analyzed by 2D and 3D tool as well as SiteView of the MOE software.

2.3 | Biology

2.3.1 | HDAC inhibitory assay
HDAC inhibition were 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℃ 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 IC50 values. The IC50 values were calculated by Prism Graphpad Prism v.5 software.

2.3.2 | Cell proliferation assay
The antiproliferative activity of synthesized compounds 2a-2p was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay. Four human cancer cell lines (HCT-116, A549, MCF-7 and SW-1990) were treated for 72 h with various concentration of the isolated compounds. All cells were cultured in RPMI 1640 supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. Cells were kept at 37°C in a humidified incubator with 5% carbon dioxide. All compounds were dissolved in DMSO to make a stock solution at 10 mM and diluted with medium at different concentrations before use. Cells plated in 96-well plates were treated with Pracinostat or Pracinostat derivatives for 72 h at 37°C, and then incubated with MTT for 4 h. After MTT removal, 150 μL DMSO was added into each well and the absorption values were measured at 490 nm using a Cytation5 imaging reader (Bio Tek Instruments. Inc. USA). The IC50 values were obtained by Prism Graphpad Prism v.5 software.

2.3.3 | Western blotting
HCT-116 cells were exposed to different concentrations of compound 2a (0.33 μM 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 h. 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 (VRIPA buffer:Vprotease inhibitor=100:1) was used to lyse the cells on ice for 30 min, followed by centrifugation at 12000 rpm for 15 min at 4℃. The supernatants was collected and protein concentration was quantified by a BCA Protein Assay Kit (Solarbio). 25 μg 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 h. The membranes were incubated with the primary and secondary antibodies and detected using the ChemiDoc XRS+ Gel Imaging System (Bio-Rad, USA). Primary antibodies against human Acetyl-Histone H3 and Acetyl-Histone H4 were obtained from Cell Signaling Technology (Beverly, MA, USA). Acetyl α-tubulin antibodies were purchased from Abbkine, Inc. (California, USA).

3 RESULTS AND DISCUSSION

All the structures of 2a-2p and key intermediates were characterized by1H NMR, 13C NMR and ESI-MS and in full agreement with the proposed structures. The spectra were shown in supplemental material.Docking compound 2a into the HDAC homology model (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 the similar way as that of Pracinostat, while the rest of the substituents at postion-1 and postion-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 2.46Å, 2.68Å and 2.55Å, respectively, and the corresponding hydrogen bond energies are -1.4 kcal/mol, -1.1 kcal/mol and -0.9 kcal/mol. Similarly, the length of the hydrogen bonds formed between the N-hydroxyacrylamide group of 2a with the residues Tyr297, Gly140 and His132 are 2.81Å, 2.59Å and 2.69Å, respectively, and the corresponding hydrogen bond energies are -1.0 kcal/mol, -2.5 kcal/mol and -3.3 kcal/mol. Notably, the substituent in the 1-position of benzimidazole ring of Pracinostat forms a hydrogen bond with the residue Tyr91 (N−H separation = 2.62 Å, hydrogen bond energies = -0.7 kcal/mol ), whereas the compound 2a does not establish this interaction.

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 IC50 values of the prepared compounds tested against HDAC6 varied from 0.97 μM to 2.18 μM, higher than that of Pracinostat (IC50 = 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 anti-proliferative activities than Pracinostat, as listed in Table 2. In all cell lines tested, Pracinostat showed potent anti-proliferative activities with IC50 values ranging from 0.32 μM 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.

Table 2 Anti-proliferative activities of the synthetic compounds
Compound 2a were 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) were measured in HCT-116. The experiments (Figure 4) demonstrated that compound 2a did not increased the acetylation level of histone H3, histone H4, as well as α-tubulin. The results corresponded to the enzymatic and cell-based assays.

4| CONCLUSIONS
In summary, we have demonstated that the attached postion of N-hydroxyacrylamide of Pracinostat is vital important for the HDAC inhibitory actvities. The N-hydroxyacrylamide attached to the postion-5, related compound such as Pracinostat is a potent HDAC inhibitor with actylation levels increased significantly for H3, H4, and α-tubulin. However, all these acitivities dramatically decreased even lost when N-hydroxyacrylamide was attached to the position-6 of the benzamidazole 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)

CONFLICT OF INTEREST
The authors declare no conflict of interests.

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