Design, synthesis, and evaluation of 4-chromenone derivatives combined with N-acylhydrazone for aurora kinase A inhibitor

There is accumulating evidence that compounds containing N-acylhydrazone or 4-chromenone moieties can be active against multiple cancer cell types, yet the combined effect of these chemical groups is unclear. This study aimed to develop more effective anti-cancer compounds by combining 4-chromenone and N-acylhydrazone. Thirteen derivatives were designed, synthesized, and characterized, and their structures were identified using nuclear magnetic resonance spectroscopy and high-resolution mass spectrometry. Most of the derivatives exhibited moderate to high efficacy in inhibiting the clonogenicity of HCT116 colon cancer cells. In particular, derivative 12, (E)-N'-((6-methoxy-4-oxo-4H-chromen-3-yl)methylene)isonicotinohydrazide, strongly inhibited clonogenicity (GI50 = 34.8 μM) of HCT116 cells and aurora kinase A (aurA) activity in vitro (IC50 = 1.4 μM). In silico docking experiment predicted that derivative 12 interacts with aurA based on computational docking and calculations of binding free energy. When derivative 12 was exposed to HCT116 cells, the phosphorylation of aurA at Thr288 was dose-dependently decreased within 60 min. Further analysis showed that derivative 12 destroyed the mitotic spindle in HCT116 cells. These results suggest that derivatives of 4-chromenone combined with N-acylhydrazone are feasible in the development of selective aurA inhibitor and could be considered potential chemotherapeutic agents.

The aim of this study was to identify compounds displaying anti-cancer activities by designing derivatives of 4-chromenone combined with N-acylhydrazone. Unlike the phenyl group substitution in flavones, the 3-position of 4-chromenone was substituted with N-acylhydrazone in this study (Additional file 1: Fig. S4). Thirteen derivatives were synthesized, and their structures identified using nuclear magnetic resonance (NMR) spectroscopy and high-resolution mass spectrometry (HR/MS). The 13 derivatives synthesized here displayed similar structures, with halogen, methyl, or methoxy substituents at the 6-position of the 4-chromenone moiety, and a phenyl or pyrimidine group at the N-acylhydrazone moiety. The anti-cancer activities of the derivatives were measured using a long-term survival clonogenic assay for 7 days, which can distinguish differences in the survival of cancer cells caused by compounds with similar structures.
Aurora kinases are members of the mitotic serine/threonine kinase family and include aurora kinase A (aurA), aurora kinase B (aurB), and aurora kinase C (aurC). Among them, aurA is frequently overexpressed in various human cancer and has been shown to act as an oncogene, thereby developing aggressive tumors [17,18]. Inhibition of aurA arrests cells at the G2/M phase and suppresses tumor growth in vivo [19][20][21].
In our previous study, compounds containing an N-acylhydrazone moiety showed inhibitory effects on aurA [22]. Therefore, in vitro aurA kinase assays were performed for all 13 derivatives synthesized here. In order to elucidate the binding mode between aurA and the most promising 4-chromenone derivative at the molecular level, in silico docking was performed. aurA inhibitory effect of the lead compound was analyzed by biochemical experiments, including western blot analysis and immunofluorescence microscopy.

Chemical synthesis
The synthetic procedure for the preparation of derivatives of 4-chromenone combined with N-acylhydrazone 1-13 is shown in Scheme 1. First of all, the Vilsmeier-Haack reaction was performed between substituted 2-hydroxyacetophenone and N,N-dimethylformamide (DMF) in the presence of phosphorous oxychloride (POCl 3 )-furnished chromenone aldehyde (II). Excess quantities of DMF reacted with POCl 3 to form a reaction intermediate active chloroiminium ion of DMF, which rapidly reacted with 2-hydroxyacetophenone to form chromenone. The chromenone intermediate reacted with additional chloroiminium ions to produce substituted 3-chromenone aldehyde (II). The substituted 3-chromenone aldehyde (II) was condensed with various benzohydrazides (III) in the presence of catalytic quantities of glacial acetic acid to produce 4-chromenone-N-acylhydazone hybrid compounds 1-13.

Spectroscopic analysis
The structures of the thirteen 4-chromenone/N-acylhydrazone derivatives synthesized here were determined using NMR spectroscopy and HR/MS. All compounds were dissolved in deuterated dimethyl sulfoxide (DMSOd 6 ), diluted to approximately 50 mM, and transferred into 2.5 mm NMR tubes. All NMR experiments were performed on a Bruker AVANCE 400 spectrometer system (9.4 T; Bruker, Karlsruhe, Germany) at room temperature, and the chemical shifts were referenced to tetramethylsilane (0 ppm). The detailed experimental procedure followed has been reported previously [23]. Ultra-performance liquid chromatography-hybrid quadrupole-time-of-flight mass spectrometry (UPLC-TOFMS) was carried out on a Waters ACQUITY UPLC system (Waters, Milford, MA) [24]. All HR/MS data were collected as positive modes. The FT-IR spectra of the 13 derivatives are provided as Additional file 2.

Clonogenic assay
HCT116 human colon cancer cell lines were counted and plated onto 24-well tissue culture plates (BD Falcon ™ ; Becton Dickson Immunocytometry System, San Jose, CA) at a density of 4 × 10 3 cells/well in Dulbecco's modified eagle's medium supplemented with 10% fetal bovine serum. The cells were treated with various concentrations of derivative compounds (0, 5, 10, and 20 μM) for 7 days, fixed with 6% glutaraldehyde, and stained with 0.1% crystal violet, as described previously [25]. The cell growth inhibitory concentration values were measured using densitometry, and the half-maximal cell growth inhibitory concentration (GI 50 ) values were calculated using the SigmaPlot program.

In vitro kinase assay
In vitro kinase assays were performed using the EMD Milliporesigma kinaseprofiler service assay protocol (MilliporeSigma Corp., St. Louis, MO). Aurora A kinase (aurA) was supplied by EMD Millipore Corp. The substrate for phosphorylation was 30 μM AKRRRLSS-LRA and the concentration of ATP was 10 μM [26]. All experiments were repeated thrice at five different concentrations (0, 5, 10, 20, and 80 μM). The half-maximal inhibitory concentrations (IC 50 ) were obtained on SigmaPlot software (SYSTAT, Chicago, IL, USA) using the sigmoid curve fit [27].

In silico docking
The 3D structure of aurA was available in the protein data bank (3uod.pdb) [28]. The 3D structure of derivative 12 was determined using the X-ray crystallographic 3D structure of (E)-4-hydroxy-N'-(3-methoxybenzylidene) benzohydrazide [29]. In silico docking experiments were performed as described previously [25] on an Intel Core 2 Quad Q6600 (2.4 GHz) Linux PC with SYBYL 7.3 (Tripos, St. Louis, MO) [30]. The binding site was determined using the LigPlot program [31], and 3D images were generated using the PyMOL program (The PyMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC. Portland, OR, USA). The logP values were obtained using the SYBYL 7.3 program.

Statistical analysis
The data are plotted as means with S.D. Statistical comparisons were performed using a one-way ANOVA followed by Dunnett's multiple comparisons test with the GraphPad Prism V8.3.1 software (GraphPad Software, San Diego, CA). A P-value < 0.05 was considered statistically significant.

The structures and names of the 4-chromenone/N-acylhydrazone derivatives
The structures and names of the thirteen 4-chromenone/N-acylhydrazone derivatives synthesized here are listed in Table 1.
Spectroscopic data of the 13 derivatives, including NMR and HR/MS, are as follows:   (6)

Inhibitory effect of 4-chromenone derivatives combined with N-acylhydrazone on clonogenicity of HCT116 cells
There are numerous methods to measure the anti-cancer activities of small compounds, including cytotoxicity assays in cancer cell lines. In this study, a long-term survival clonogenic assay was adopted (Fig. 1). The GI 50 values ranged between 34.76 and 85.22 μM (Table 1 and Fig. 2). Seven of the derivatives (3-9) contained common substituents; a 6-methoxy group in the 4-chromenone moiety and a phenyl group in N-acylhydrazone. The different substituents of N-acylhydrazone appeared to have different effects on the GI 50 values. More specifically, the 3-bromo (derivative 4), 3-fluoro (5), and 3-hydroxy (6) groups displayed the higher inhibitory activity of clonogenicity than the hydrogen (3), 3-methoxy (7), 4-fluoro (8), and 4-methoxy (9) groups. In compounds with a 4-methoxy group substituted to the phenyl ring of N-acylhydrazone, a 6-bromo group (derivative 2) on the 4-chromenone moiety displayed better activity than the 6-chloro (1) and 6-methoxy (9) groups. Derivatives 3 and 10 contained a phenyl group on the N-acylhydrazone moiety, yet derivative 10 displayed the higher inhibitory activity of clonogenicity than derivative 3. The three derivatives (11)(12)(13) that contained a pyridine group instead of a phenyl group on N-acylhydrazone appeared to cause relatively the higher inhibitory activity of clonogenicity.

In silico molecular docking
To predict whether derivative 12 can directly bind to aurA, we analyzed the binding mode between aurA and derivative 12 using an in silico docking experiment. Of over 100 three-dimensional (3D) structures of aurA deposited in the protein data bank (PDB), the X-ray crystallographic structure 3uod.pdb [28] was selected as the 3D structure of aurA for in silico docking. This structure originated from Homo sapiens aurA expressed in Escherichia coli BL21(DE3), with a resolution of 2.50 Å. Its 3D structure contains the ligand 4-[(4-{[2-(trifluoromethyl)phenyl]amino}pyrimidin-2-yl)amino]benzoic acid (named as TPAP) (Additional file 1: Fig. S5), which is composed of two benzene rings and pyrimidine and has a molecular weight of 374 Da. Similarly, the thirteen derivatives synthesized here consist of three rings, and their molecular weights range between 322 and 401 Da. Human aurA consists of 403 amino acids and 3uod. pdb includes the majority of the protein, from Lys123 to Lys401. Importantly, it contains the protein kinase  The 3D structure of derivative 12 was determined based on the previously reported X-ray crystallographic 3D structure of (E)-4-hydroxy-N'-(3-methoxybenzylidene)benzohydrazide (Additional file 1: Fig. S6) [29]. The apo-protein of aurA without a ligand was obtained using the Sybyl program and, after energy minimization, compared with the crystallographic structure revealing a root mean squared deviation of 0.65 Å. The binding site was determined based on published data and LigPlot program analysis as Glu260, Gly140, Leu139, Thr217, Val147, Leu263, Ala160, Glu211, Tyr212, Arg137, Gly216, Ala213, and Arg220. To confirm the validity of our in silico docking process, TPAP (the ligand contained in 3uod. pdb) was docked into the apo-protein of aurA, revealing that it docked inside the same binding site as in the crystal structure of aurA (3uod.pdb).
The Sybyl program provides the flexible docking method FlexX, and performs 30 iterations of the docking procedure so that 30 protein-ligand complexes were generated. The binding energy ranged between -21.2 and -26.9 kcal/mol. Because the first complex showed both the lowest binding energy and best docking pose, its complex was selected. As shown in Fig. 3a, derivative 12 was effectively docked into aurA in this complex. Analysis by the LigPlot program revealed the residues participating in the interaction (Additional file 1: Fig. S7). The three residues Tyr219, Leu263, and Ala213 showed hydrophobic interactions with derivative 12. The hydrogen atom of the amine group of Arg220 forms a hydrogen bond (H-bond) with the oxygen atom in the ketone group of the 4-chromenone moiety with a distance of 2.86 Å. The oxygen atom of Leu139 forms an H-bond with the hydrogen atom of the secondary amine of N-acylhydrazone (3.20 Å). In addition, the oxygen atom of the ketone group of N-acylhydrazone forms two H-bonds with two amine groups in Arg137 (2.69 Å and 2.96 Å). An image of the binding site of the derivative 12-aurA complex is shown in Fig. 3b, where three residues forming H-bonds with derivative 12 are marked. While the ligand TPAP interacts with 13 residues of aurA, derivative 12 appears to interact with six residues. The volumes of derivative 12 and TPAP were calculated using the Sybyl/MOLCAD module as 234.1 Å 3 and 246.2 Å 3 , respectively. The larger volume of TPAP may, therefore, explain the difference in individual residue interactions mentioned above. However, while TPAP forms H-bonds with two residues of aurA, derivative 12 does so with three residues. Based on these data, it can be predicted that derivative 12 could directly bind to and inhibit the aurA kinase activity.

Inhibitory effect of derivative 12 on phosphorylation of aurora kinases A
Phosphorylation at Thr288 in the activation loop of aurA is crucial for its activation [34,35]. To validate whether derivative 12 could inhibit the phosphorylation of aurA at the cellular level, we treated HCT116 cells with Fig. 3 Images of the complex of derivative 12 and the apo-protein of aurora A kinase generated by the PyMol program (a) and its binding site (b), where Arg137, Leu139, and Arg220 of aurora A kinase form hydrogen bonds with derivative 12 various concentrations (0, 50, and 100 μM) of derivative 12. Phosphorylation of aurA on Thr288 was significantly reduced (p < 0.001, n = 3) in a dose-dependent manner (Fig. 4a). In contrast, phosphorylation of aurB on Thr232 and aurC on Thr198 were only slightly decreased at 100 μM. A time-course experiment confirmed that the addition of 50 μM derivative 12 significantly inhibited phosphorylation of aurA within 60 min, while the phosphorylation of aurB and aurC were relatively unaffected (Fig. 4b). These data suggest that derivative 12 preferentially inhibited aurA over aurB and aurC.

Effect of derivative 12 on the mitotic microtubule network
AurA is a mitotic kinase localized to the mitotic pole of spindle microtubules and has important functions in centrosome maturation, chromosome alignment, and mitotic spindle assembly [36,37]. To investigate whether derivative 12 affects mitotic spindle assembly during mitosis, we analyzed the morphological features of mitotic spindles using immunofluorescent staining of α/β tubulin in mitotic cells. Phosphorylation of histone H3 on Ser10 plays a role in chromatin condensation during mitosis and is therefore used as a marker for mitotic cells [38]. Thus, we analyzed the mitotic spindle network in phospho-histone H3 (Ser10)-positive mitotic cells. In untreated cells, we observed that bipolar spindle microtubules were attached to chromosomes arranged along with the metaphase plate in phospho-H3 (Ser 10)-positive cells, exhibiting typical features of the metaphase stage of mitosis (Fig. 5, top panels). In contrast, treatment with derivative 12 induced a disorganized arrangement of microtubules and abnormal chromosome alignment in phospho-H3 (Ser 10)-positive cells (Fig. 5, bottom panels). These data suggest that inhibition of aurA by Fig. 4 Effect of derivative 12 on the inhibition of aurora kinases. HCT116 cells were treated either with various concentrations of derivative 12 (0, 50, or 100 μM) for 60 min (a) or 50 μM derivative 12 over a time-course (0, 60, or 120 min) (b). Western blot analysis was performed using phospho-specific antibodies against aurA (Thr288)/aurB (Thr232)/aurC (Thr198). The anti-GAPDH antibody was used as an internal control. The band intensities of phosphorylated (p)-aurA, (p)-aurB, and (p)-aurC relative to GAPDH level were measured using ImageJ software. The data are presented as means ± SD (n = 3). NS, not significant; ***, P < 0.001 according to Dunnettt's multiple comparisons test derivative 12 is functionally linked to aberrant mitotic progression.
Here, we report that most of the derivatives exhibited moderate to high efficacy in inhibiting the clonogenicity of HCT116 colon cancer cells and in vitro aurA kinase activity. In particular, derivative 12, (E)-N'-((6-methoxy-4-oxo-4H-chromen-3-yl)methylene)isonicotinohydrazide, exhibited the suppression of clonogenicity of HCT116 cells with GI 50 value of 34.8 μM and the inhibition of in vitro aurA kinase activity with IC 50 value of 1.4 μM. In silico docking experiment predicted the interaction of derivative 12 with aurA. Furthermore, treatment of HCT116 cells with derivative 12 dosedependently prevented the phosphorylation of aurA at Thr288 within 60 min. In addition, derivative 12 disrupted the mitotic spindle dynamics in HCT116 cells.
Lipinski's rule of five is a method to evaluate the likelihood of a chemical compound being an orally active drug in humans based on its chemical and physical properties [39]. As listed in Table 1, all of the 13 derivatives studied here displayed logP values less than 5 and molecular weights less than 500 Da (ranging from 322 to 401 Da). In addition, the numbers of hydrogen bond donors and acceptors of the 13 derivatives were less than 5 and 10, respectively. Hence, all derivatives synthesized here satisfy Lipinski's rule.
Collectively, these results suggest that derivatives of 4-chromenone combined with N-acylhydrazone could be considered as potential chemotherapeutic agents.
However, there are limitations to this study, as we have focused on the design, characterization, and in vitro effects of these compounds. Future research should be directed towards evaluating the effectiveness of the 4-chromenone/N-acylhydrazone compounds in vivo, and ultimately clinical trials for the future benefit of cancer patients.
Additional file 2. The FT-IR spectra of the 13 derivatives.