Synthesis and biological evaluation of 2,4-diaminopyrimidines as selective Aurora A kinase inhibitors

Wen-Wen Qin a, 1, Chun-Yan Sang a, 1, Lin-Lin Zhang a, Wei Wei a, Heng-Zhi Tian a, Huan-Xiang Liu a, Shi-Wu Chen a, *, Ling Hui b, c, **

A B S T R A C T

The Aurora kinases are a family of serine/threonine kinases that interact with components of the mitotic apparatus and serve as potential therapeutic targets in oncology. Here we synthesized 15 2,4- diaminopyrimidines and evaluated their biological activities, including antiproliferation, inhibition against Aurora kinases and cell cycle effects. These compounds generally exhibited more potent cyto- toxicity against tumor cell lines compared with the VX-680 control, especially compound 11c, which showed the highest cytotoxicities, with IC50 values of 0.5e4.0 mM. Compound 11c had more than 35-fold more selectivity for Aurora A over Aurora B, and molecular docking analysis indicated that compound 11c form better interaction with Aurora A both from the perspective of structure and energy. Furthermore, compound 11c induced G2/M cell cycle arrest in HeLa cells. This series of compounds has the potential for further development as selective Aurora A inhibitors for anticancer activity.

Keywords: Aurora kinase Kinase inhibitors Pyrimidine Cell cycle

1. Introduction

The Aurora kinase family is a subfamily of serine/threonine ki- nases that is essential for the regulation of centrosome maturation, mitotic spindle formation, chromosome segregation and cytoki- nesis during mitosis [1,2]. The family includes three kinases designated as Aurora A, B, and C, which are very closely related in the kinase domain sequence. However, these kinases have quite different and nonoverlapping functions during mitosis [3]. Aurora A regulates the cell cycle and is associated with late S phase and entry into the M phase. It associates with the spindle poles and is involved in both centrosomal and acentrosomal spindle assembly [4,5]. Aurora B localizes to the centromeres in prometaphase, and then relocates to the spindle midzone at anaphase. It has functions associated with histone phosphorylation and chromatin conden- sation in prophase, chromosome alignment and segregation, regulation of a mitotic checkpoint at metaphase and a role in cytokinesis [6]. Aurora C has similar functions as Aurora B [7].
The expressions of Aurora A and Aurora B are elevated in a va- riety of human cancers and are associated with poor prognosis [8]. The potential roles of Aurora kinases in regulating cell mitosis and tumorigenesis make them attractive targets for anticancer therapy [9]. Many Aurora kinase inhibitors have been developed and introduced into clinical trials, including VX-680/MK-0457, ZM447439, Hesperadin, MLN8054, MLN8237, and AZD1152 (Fig. 1) [10e12].
ZM447439 [13], Hesperadin [14] and VX-680/MK-0457 [15] were the first generation of Aurora kinase inhibitors. These three small molecule chemical inhibitors occupy the ATP-binding site in Aurora kinases to inhibit catalytic activity. Unlike pan-Aurora ki- nase inhibitors, MLN8054 and MLN8237 are ATP-competitive and reversible Aurora A selective inhibitors, and are approximately 40- fold and 200-fold more sensitive towards Aurora A, respectively, compared with Aurora B [12]. MLN8237 is more potent than MLN8054 and causes less benzodiazepine-like side effects based on structure modulation by the addition of a methoxy group to either end of the MLN8054 molecule [16,17]. AZD1152 is an Aurora B se- lective inhibitor that showed 1000-fold selectivity for Aurora B over Aurora A and a panel of 50 additional kinases in enzymatic assays [18e21]. It thus still remains uncertain how exactly aurora A and B pan- or monospecific inhibitors induce tumor cell death and which type of inhibitor will be preferable from a therapeutic viewpoint. Recently, the research of novel selective Aurora inhibitors has become a new trend, and a lot of new active compounds have been developed [22e24].
Pyrimidine is the important pharmacology core in many Aurora inhibitors, such as VX-680, ENMD-2076, CYC-116 and ENMD-2076 [12]. To identify additional effective Aurora inhibitors, we designed a series of 2,4-diaminopyrimidine compounds, our modeling studies suggested that the pyrimidine core as well as the secondary aromatic amine of the compounds form hydrogen bonds with the hinge region of the kinase domain and show selectively inhibition to Aurora A over Aurora B. Introduction of cyclopentyl amine on the C-4 in pyrimidine can adopt a binding mode similar to VX-680 [25]. Furthermore, the differences of F, Cl, Br and NO2 at 5-C of pyrimidine was to investigate the effects of the electron- withdrawing on anti-proliferation and inhibition of Aurora ki- nase. Herein, we reported the synthesis, and evaluated their anti- proliferation activities, inhibition of Aurora kinase and effects on the cell cycle.

2. Results and discussion

2.1. Chemistry

The general synthetic routes for intermediate anilines 4aeb and 7aeb are illustrated in Schemes 1 and 2, respectively. Treatment of p-aminobenzoic acid 1a with ditertbutyl dicarbonate ((BOC)2O) afforded 4-Boc-amino-benzoic acid 2a, and then condensation of compound 2a with N-methyl-4-amino-piperidine generated 3a under condensing agent tri(dimethylamino)benzotriazol-1- yloxyphos phonium hexafluorophosphate (BOP) in the presence of N,N-diisopropylethylamine (DIPEA). Finally, removal of the protecting group provided aniline 4a in the dichloromethane so- lution of trifluoroacetic acid [26]. To obtain compound 4b, 3- methoxy-4-amino-benzoic acid (1b), as the raw material, the re- action process was similar to preparing 4a. Another intermediate aniline 6aeb was prepared by substituted reaction of 4-chloro-1- nitrobenzene with morpholine or 4-methyl-piperazine, and the nitro was reduced by catalytic hydrogen under the catalysis of 10% Pd/C [27].
Our approach to the preparation of 2,4-diaminopyrimidines based on the double SN2 displacement of pyrimidine is shown in Scheme 3. The displacement of the 4-chloro group of 2,4-dichloro- 5-substituted pyrimidine by cyclopentyl amine, cyclopropyl amine, and n-propylamine provided 8aed, 9 and 10, which has already been widely reported in the literature [28]. Treatment of 8aed, 9 and 10 with the different anilines 4a,b or 7a,b in isopropanol in the presence of hydrochloride at 80 ◦C gave the target compounds 11aed, 12b,c and 13e19 [29]. The target compound 20 was synthesized by the nitro at C-5 of 16 and reduced with hydrogen gas under the catalysis of 10% Pd/C. The newly synthesized compounds were characterized by physicochemical and spectral means, and both analytical and spectral data of all the compounds were in full agreement with the proposed structures.

2.2. Biological activity

2.2.1. Cytotoxicities of compounds 11e20

The in vitro cytotoxicities of target compounds 11e20 were evaluated in a panel of four human tumor cell lines (cervical car- cinoma HeLa, lung carcinoma A-549, human colorectal adenocar- cinoma HCT-8 and hepatic carcinoma Hep-G2 cells), with VX-680 as a reference compound. The screening procedure was based on the standard MTT method [30], and the results are summarized in Table 1.
All the target compounds showed better or equivalent antiproliferation activity in the four human tumor cell lines compared with VX-680. Notably, compounds 11aed with different substitutions (F, Cl, Br and NO2) at C-5 of pyrimidine showed significantly different effects in regards to cytotoxicity. The anti- proliferation activity of compounds 11b and 11c substituted with chloride or bromide at C-5 of pyrimidine showed more potent antiproliferation effects compared with compounds 11a and 11d, in which C-5 in pyrimidine was substituted with fluorine and nitro, respectively. However, we did not find any obvious differences in cytotoxicities in compounds 12aec. The displacement of the ani- lines from 4a to 4b in the C-2 substitute on the pyrimidine ring led to increased or maintained cytotoxicity upon treatment of the four cell lines. However, substitution of 4a with 7a and 7b in the C-2 of pyrimidine resulted in decreased antiproliferation activity of the target compounds. We next investigated the replacement of the cyclopentyl group on the C-4 in pyrimidine by different groups, such as cyclopropyl or n-propylamine. Unfortunately, all the syn- thesized compounds showed equivalent or lower antiproliferation activities.
From the results of the in vitro cytotoxic assays, we found that compound 11c showed strong growtheinhibitory activities in the cervical carcinoma HeLa cell line. The IC50 value of 11c was 0.9 mM, which was 30-fold lower than VX-680. Next we selected com- pounds 11c and 12a to explore the effects on Aurora kinases and the cell cycle in HeLa cells.

2.2.2. Compounds 11c and 12a selectively inhibit Aurora A over Aurora B kinase in HeLa cells

Many pyrimidine compounds, such as VX-680, ENMD-2076, CYC-116 and ENMD-2076, have entered into clinical trials as Aurora inhibitors [12]. To explore whether 11c and 12a had similar effects on inhibition of Aurora kinases in HeLa cells, we investigated the effects on Aurora A and B kinases by western blot [31] and enzyme- linked immunosorbent assay (ELISA) [32].
HeLa cells were treated with various concentrations of com- pound 11c (10 nM, 25 nM, and 50 nM) for 12 h. The expression levels of Aurora A and Aurora B were decreased in HeLa cells upon exposure to 11c in a dose-dependent manner. We also observed that the effect of 11c on Aurora A was more potent than on Aurora B, indicating that compound 11c showed selectivity of inhibition of Aurora A over Aurora B (Fig. 2A and C). Compound 12a showed the same effects of 11c, but its potency was weaker than 11c (Fig. 2B and D).
The selective effects of compounds 11c, 12a and VX-680 on Aurora A kinase inhibition were also confirmed by ELISA, as shown in Fig. 3. When HeLa cells were treated with less than 5 nM of compound 11c for 12 h, the expression levels of Aurora A and B showed no significant changes. When the concentration of 11c was increased to more than 10 nM, the expression levels of Aurora A and B protein rapidly decreased in HeLa cells, and Aurora A protein was reduced more than that of Aurora B. From Fig. 3A, we easily ob- tained the IC50 values of 11c for Aurora A and B as 0.012 mM and 0.430 mM, respectively. These results indicate that compound 11c was more than 35-fold more selective for Aurora A compared with Aurora B in HeLa cells. We also obtained IC50 values of 12a for Aurora A and B of 0.043 mM and 0.395 mM, respectively, and the selectivity of 12a for Aurora A was only 9-fold over Aurora B. However, the IC50 values of VX-680 for Aurora A and B was 0.261 mM and 0.453 mM, respectively, and no obvious selectivity of VX-680 for Aurora A over Aurora B. Obviously, the strong growtheinhibitory activities of compound 11c and 12a than VX- 680 were in accordance with their better selectivity of Aurora A over Aurora B.

2.2.3. Aurora kinase binding model of compound 11c

To gain insight into the interaction of compound 11c with Aurora A and Aurora B, docking simulation was performed using the Autodock 4.2 [33] with Lamarckian Genetic Algorithm [34]. All the figures displaying the docking results were obtained using the scientific software Pymol [35]. AutoGrid was used to produce grids based on the position of the ligand in the proteins (PDB code 3D14 for Aurora A and 4C2V for Aurora B). In the docking process, the protein was considered to be rigid, while the ligand was considered flexible. The ligand 11c was docked into the appropriate binding pocket of Aurora A and Aurora B using the autodock module and the calculated binding energy was —10.02 kcal/mol for Aurora A and —9.05 kcal/mol for Aurora B, respectively. The resulting dock- ing poses are shown in Fig. 4. According to the binding energy, 11c is more sensitive to Aurora A than Aurora B.
Overall, the binding pockets of Aurora A and Aurora B are highly hydrophobic. Thus, hydrophobic interaction is the main driving force for the binding of 11c to Aurora A and Aurora B. In addition to the hydrophobic interactions, 11c can also form several important H-bonds. For example, in Aurora A, 11c can form two H-bonds with Glu62 and Asp155. In Aurora B, the oxygen atom in the amide group and the nitrogen atom in the piperidine ring of 11c can form two H- bonds with Lys122. From the distance of the two atoms that form hydrogen bonds, Aurora A can form stronger hydrogen-bonding interactions compared with Aurora B. Additionally, from the shape of the binding pocket, 11c can fit much better with the binding pocket of Aurora A. These differences may explain why compound 11c binds better with Aurora A compared with Aurora B.

2.2.4. Compound 11c induces cell cycle arrest in G2/M phase

The Aurora A inhibitor induces common phenotypic effects such as G2/M accumulation, spindle defects and chromosome misalignment, and has enabled the identification of previously unknown Aurora A-regulated cellular functions [36]. Many studies have shown that VX-680 induces cell cycle arrest in the G2/M phase. To determine whether compound 11c has similar effects on tumor cells, we investigated its effects on cell cycle progression using fluorescence-activated cell sorting analysis of HeLa cells stained with propidium iodide [37] (Fig. 5). Treatment of HeLa cells with 11c resulted in a dose-dependent accumulation of cells in the G2/M phase with a concomitant decrease in the population of G1 phase cells. After 12 h of treatment with 2 mM or 5 mM of 11c, the percentages of cells in G2/M phase arrest were 33.2% and 43.5%, respectively, compared with 10.2% in untreated cultures. These results demonstrate that 11c interfered with cell proliferation by arresting the cell cycle in G2/M.

2.2.5. Compound 11c activates cyclin B1

Cyclin B is a member of the cyclin family and regulates the progression of cells into and out of M phase [38]. Thus, we exam- ined the effect of 11c on the expression of cyclin B in HeLa cells [39]. As shown in Fig. 6, treatment of cells with 11c resulted in an apparent increase of cyclin B, demonstrating that compound 11c can interfere with cell cycle progression. This conclusion is consistent with the cell cycle analysis results.

3. Conclusions

Aurora kinases have been of interest as potential therapeutic targets in oncology. Here we describe a series of 2,4- diaminopyrimidine small molecule inhibitors that exert their cytotoxic activities in human tumor cell lines through inhibition of Aurora kinases. We specifically demonstrate that compound 11c was selective for inhibition of Aurora A over Aurora B in HeLa cells, and the molecular docking analysis revealed that 11c form better interaction with Aurora A than that with Aurora B. Treatment of HeLa cells with compound 11c also results in G2/M accumulation. These results suggest that these compounds have potential for further development in vivo as anticancer agents.

4. Experiment

4.1. Chemistry

All starting materials and regents were purchased commercially and used without further purified, unless otherwise stated. All re- actions were monitored by thin layer chromatograph (TLC) on silica gel GF254 (0.25 mm thick). Column chromatography (CC) was performed on Silica Gel 60 (230e400 mesh, Qingdao Ocean Chemical Ltd., China). Melting points were determined in Kofler apparatus and were uncorrected. IR spectra were measured on a NicoLET iS5 spectrometer on neat samples placed between KBr plates. 1H NMR and 13C NMR spectra were recorded with a Varian Mecury-400BB or Mecury-600BB spectrometer with TMS as an internal standard, all chemical shift values are reported as ppm. Mass spectra were recorded on a Bruker Dalton APEXII49e and Esquire6000 (ESI-ION TRAP) spectrometer with ESI source as ionization, respectively.

4.4. General synthetic procedure of compounds 8e10

To a solution of 2,4-dichloro-5-fluoropyrimidine 7a (0.83 g, 5 mmol) in THF (10 mL) at 0 ◦C was slowly added THF solution of cyclopentylamine (0.74 mL, 7.5 mmol). The resulting mixture was stirred at 0 ◦C for 3 h (monitored by TLC). The solvent was removed in vacuo and water (20 mL) was added and extracted with CH2Cl2 (3 × 10 mL). Combined organic phase was washed with brine, dried over anhydrous Na2SO4, filtered and concentrated. The crude product was purified by column chromatography on silica gel (dichloromethane/methanol 50:1) to give 8a (0.83 g, 77%) as a solid. Similar procedure as that described for 8a gave pure 8bed, 9 and 10 as a solid.

4.5. Biology

4.5.1. Cytotoxicity assays

Cells were incubated at 37 ◦C in a 5% CO2 atmosphere. The MTT assay were used to determined growth inhibition. The synthetic compounds and reference compound VX-680 were dissolved in saline for five concentrations (0.001e100 mM). The A-549, HepG2, HCT-8 and HeLa cells were plated in 96-well plates and allowed to attach for 4e6 h, then exposed in quadplex well for 48 h. The media was aspirated, and 10 mL of 5 mg/mL MTT solution (dilute in sterile PBS) diluted in serum-free media was added to each well. After 4 h of incubation, the solution was centrifuged for 10 min under 2000 rpm. The supernatant was mixed with 150 mL DMSO, and shaken on an oscillator. The absorbance at l490 was determined on a plate reader. IC50 values were determined from a log plot of percent of control versus concentration [36].

4.5.2. Western blot analysis

HeLa cells (1 × 10 6 cells) exposed to compound 11c were collected into tubes and then washed with PBS. Cell pellets were lysed with lyses buffer (50 mM TriseHCl, pH 7.5, 10 mM EDTA, 0.2 M NaCl, 1.5 mM PMSF and 1% SDS). Cell lysates were boiled for 10 min, centrifuged and stored at —20 ◦C. Cell lysates containing 10e20 mm g protein were separated and transferred to nitro cellulosefilters. The blots were incubated with the corresponding antibodies and developed [36].

4.5.3. Analysis of cell cycle by flow cytometry

For cell cycle analysis, we used the cervical carcinoma HeLa cell line grown in RPMI-1640 supplemented with 10% (v/v) heati- nactivated fetal calf serum, 2 mM L-glutamine, 100 units/mL penicillin, and 24 mg/mL gentamicin and incubated at 37 ◦C in a humidified atmosphere of 5% CO2 and 95% air. Untreated and drugtreated cells ((3e5) × 105) were harvested and fixed overnight in 70% ethanol at 4 ◦C. Cells were then washed three times with PBS, incubated for 1 h with 1 mg/mL RNase A and 20 mg/mL propidium iodide at room temperature, and analyzed with a flow cy- tometer (COULTER EPICS XL, USA) as described previously [35].

4.5.4. ELISA experiment

Use Purified Human Aurora A or B antibody to coat microtiter plate wells, make solid-phase antibody, then add Aurora A or B to wells, Combined Aurora A which With HRP labeled, become antibody-antigen-enzyme-antibody complex, after washing Completely, Add TMB substrate solution, TMB substrate becomes blue color At HRP enzyme-catalyzed, reaction is terminated by the addition of a sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450 nm [32].

4.5.5. Molecular docking study

The docking simulation was performed using the Aurodock 4.2 software. Before docking, the protein structure was minimized firstly. The crystal waters were removed and the Kollman united atom charges and polar hydrogen was added to the two proteins. The ligand in the crystal structure was used to determine the location of a docking grid box and was then removed prior to grid generation in next step. Gasteiger charges were assigned to the new constructed structures in Autodock. At the same time, the Non- polar hydrogen atoms were merged and the rotatable bonds were defined. Based on the ligand in the crystal structure, the grid maps of the protein were produced using AutoGrid module embedded in Autodock software. As result, a grid size of 60 × 60 × 66 Å points and 0.375 Å spacing were generated. Each docking process was performed in 250,000 energy evaluation with 10 conformations kept and the most favorable pose of each compound was displayed.

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