3.1. Chemistry
3.1.1. General
Reversine and all reagents were purchased from Sigma Aldrich (St. Louis, MO, USA) and used without further purification. The reaction yields were calculated for the products after chromatographic purification. Thin layer chromatography (TLC): Merck silica gel F254 or reversed phase Merck RP-18 F254 (Merck, Darmstadt, Germany), with visualization with UV light. Flash chromatography (FC): Merck Si 15–25 μm; preparative thin layer chromatography (PLC): 20 × 20 cm Merck Kieselgel 60 F254 0.5 mm plates. MW-assisted reactions were carried out using a mono-mode CEM Discover SP reactor (CEM, Matthews, NC, USA) in a sealed vessel (200 W max). NMR spectra were recorded on an Avance 400 spectrometer (Bruker, Billerica, MA, USA) using a 5 mm BBI probe 1H at 400 MHz and 13C at 100 MHz in CDCl3 (relative to δH 7.25 and δC 77.00 ppm), or in (CD3)2SO (relative to δH 2.50 and δC 39.50 ppm), δ values in ppm, J values in Hz; assignments are supported by heteronuclear single quantum correlation (HSQC) and heteronuclear multiple bond correlation (HMBC) experiments. Electrospray ionization (ESI)-MS mass spectra were recorded using a Bruker Esquire-LC spectrometer by direct infusion of a methanol solution (source temperature 300 °C, drying gas N2, 4 L/min, scan range m/z 100–1000). Electron ionization (EI) mass spectra (m/z; rel%) and high resolution (HR)-EI data were recorded with a Kratos-MS80 mass spectrometer (SIS, Ringoes, NJ, USA), heating at 213 °C for 2, at 276 °C for 1 and at 417 °C for 3.
3.1.2. Typical Reaction Procedure for Precursors 4 and 5
To a solution of 2,6-dichloropurine (260 mg, 1.38 mmol, 1.0 equiv) in EtOH (2 mL) cyclohexylamine or methylcyclohexylamine (1.52 mmol, 1.1 equiv) and Et3N (1.52 mmol, 1.1 equiv) were added. The reaction mixture was irradiated at 90 °C for 1.5 h in a microwave reactor. The obtained white precipitate was filtered and chromatographically purified under the indicated conditions.
2-Chloro-N-cyclohexyl-9H-purin-6-amine (4). TLC (CH2Cl2:MeOH = 94:6 v/v): Rf = 0.55. Purification by silica gel PLC (CH2Cl2:MeOH = 94:6 v/v) Yield: 65%. 1H-NMR (DMSO-d6) δ 13.05 (br s, 2H, NH2), 8.08 (s, 1H), 7.95 (brs, 1H, NH), 4.60 (br s), 3.96 (br s cyclohexyl CH), 1.89, 1.73 and 1.26 (series of m, 10H, cyclohexyl). 13C-NMR (DMSO-d6) δ 154.2, 152.9, 150.5, 139.2(N=CH), 117.8, 48.8 (cyclohexyl CH), 32.1 (2C), 25.1 (2C), 24.8. Significant HMBC correlation: 1.26 ppm with 48.8 ppm. ESI(−)-MS: m/z 250/252 in ca. 3:1 ratio, [M − H]−.
2-Chloro-N-cyclohexyl-N-methyl-9H-purin-6-amine (5). TLC (hexane: EtOAc = 4:6 v/v): Rf = 0.62. Purification by silica gel FC (hexane:EtOAc, from 7:3 to 4:6 v/v). Yield: 80%. 1H-NMR (CDCl3) δ 13.30 (br s, 1H, NH), 7.84 (s, 1H, H=CN), 4.88(br s, 1H, cyclohexyl CH), 3.18 (br s, 3H, NCH3), 1.86, 1.73, 1.17 (series of m, 10H, cyclohexyl). 13C-NMR (DMSO-d6, detectable signals) δ 154.5, 152.9, 138.7(HC=N), 118.3 (br), 25.7(2C), 25.4. ESI(−)-MS: m/z 264/266 in 3:1 ratio, [M − H]−.
3.1.3. Synthesis of 6-Morpholinopyridin-3-amine (6)
A mixture of 2-chloro-5-nitropyridine (303 mg, 1.91 mmol, 1.0 equiv), morpholine (0.5 mL, 5.74 mmol, 3.0 equiv) and Et3N (483 mg, 0.67 mL, 2.5 equiv) in CH2Cl2 (4 mL) was stirred at room temperature overnight. The reaction mixture was diluted with water (10 mL) and extracted with CH2Cl2 (30 mL × 3). The combined organic layers were washed with water (30 mL × 6) and brine (1 × 30 mL), dried over anhydrous Na2SO4 and concentrated in vacuo to give a yellow solid. 70 mg of the yellow solid (0.335 mmol, 1.0 eq) were diluted in EtOH (5 mL) and a spatula tip of catalyst Pd/C was added. The obtained mixture was hydrogenated for 2 h, using a Hypem XP hydrogen generator (h2planet, Milan, Italy), Pressure was set at 1.5 bar. The crude mixture was filtered on Celite, and the filtrate was evaporated to obtain a red solid. Yield: 93% over two steps. TLC (hexane:ethyl acetate = 4:6 v/v + Et3N): Rf = 0.15. 1H-NMR (CDCl3) δ 7.79 (d, J = 2.7 Hz, 1H), 7.01 (dd, J = 8.8, 2.7 Hz, 1H), 6.73 (brs, 2H, NH2), 6.56 (d, J = 8.8 Hz, 1H), 3.82 (m, 4H), 3.33 (m, 4H). 13C-NMR (CDCl3) δ 154.02, 135.09, 134.58, 126.42, 108.41, 66.80 (2C), 47.08 (2C). ESI(−)MS: m/z 178 [M − H]−.
3.1.4. Synthesis of N6-Cyclohexyl-N6-methyl-N2-(6-morpholinopyridin-3-yl)-7H-purine-2,6-diamine (1)
A mixture of 5 (22.5 mg, 0.085 mmol, 1 equiv), 6 (60 mg, 0,34 mmol, 4 equiv) and trifluoroacetic acid (0.1 equiv.), in ethanol (2 mL), was microwave irradiated at 120 °C, for 2.5 h. The reaction mixture was concentrated in vacuo and the residue was submitted to preparative HPLC (RP-18 LiChroSphere 7 µm, acetonitrile/water 1:1 + 0.1% TFA, flow 5 mL/min, detection at 268 nm, 3.1 min) obtaining 1 trifluoracetate salt as a white powder (10.7 mg, yield 25%). 1H-NMR (CDCl3) δ 12.40 (br s, 1H), 7.94 (brd, J = 7.8 Hz, 1H), 7.83 (s, 1H), 7.79 (brs, 1H), 7.03 (brd, J = 8.1 Hz, 1H), 6.57 (brd, J = 8.1 Hz, 1H), 3.82 (m, 4H), 3.35 (m, 4H), 3.20 (brs, NCH3), 2.16–1.28 (series of m, 10H). 13C-NMR (CDCl3) detectable signals by HSQC correlation δ 7.83 with δ 135.7 (C-8) and by HMBC correlations: δ 53.6, 151.9, 118.4, 133.6, 127.0, 108.6, 66.2, 46.6. ESI(+)-MS: m/z 409 [M + H]+; MS/MS (409): m/z 327. The solid, dissolved in methanol, was treated with Et3N, the mixture evaporated in vacuo and the residue eluted through RP-18 LiChrolut with water/methanol, gradient elution to obtain free 1. ESI(−)MS: m/z 407 [M − H]−; MS/MS(407): m/z 325. EI-MS: m/z 408 (M+, 5), 368 (4), 326 (1), 229 (2), 179 (23). HRMS(EI) calcd. for C21H28N8O, 408.23861, found 408.23683.
3.1.5. Typical Reaction Procedure for Products 2 and 3
Compound 4 or 5 (0.188 mmol, 1.0 equiv.) was dissolved in N-methyl-2-pyrrolidone and aniline (0.56 mmol, 3.0 equiv.) was added. The reaction mixture was heated at 150 °C for 14 h, then ice-water (15 mL) was added and the mixture was extracted with EtOAc (20 mL × 2). The combined organic layers were washed with ice-water mixture (15 mL × 5) and brine (20 mL), dried over anhydrous MgSO4, and concentrated under reduced pressure to give a brown syrup. The crude residue was purified by silica gel FC eluting with hexane/EtOAc (from 7:3 to 4:6 v/v). The purity of 2 and 3 was verified by analytical HPLC injection (Si60 LiChroSphere 15–25 µm, 254 nm) with hexane/2-propanol 9:1.
N6-Cyclohexyl-
N2-phenyl-7
H-purine-2,6-diamine (
2). TLC (EtOAc): R
f = 0.42. Yield: 70%.
1H-NMR (CDCl
3) δ 13.0 (br. s., 1H, NH), 7.51 (brd.,
J = 7.8 Hz, 2H), 7.31 (brt,
J = 7.8 Hz, 2H), 7.05 (brt,
J = 7.8 Hz, 1H), 7.03 (s. 1H, purine), 6.57 (s, 1H, NH, exchangeable by CD
3OD addition), 5.59 and 4.01 (1:1 two brs, 1H, NH), 2.05 (m, 1H), 1.74 (m, 2H), 1.64 (m, 2H), 1.33 (m, 6H), in agreement with reported data [
21].
13C-NMR (CDCl
3) δ 156.6, 154.4, 150.3 (v br), 139.9, 135.9, 129.2 (2C), 123.0, 121.2 (2C), 114.7 (br), 49.3 (v br), 33.3 (2C), 25.6, 24.9 (2C). ESI(+)-MS:
m/
z 309 [M + H]
+; MS/MS (309):
m/
z 227. EI-MS:
m/
z 308 (M
+, 100), 225 (67). HRMS(EI) calcd. for C
17H
20N
6, 308.17494, found 308.17510.
N6-Cyclohexyl-N6-methyl-N2-phenyl-7H-purine-2,6-diamine (3). TLC (EtOAc): Rf = 0.54. Yield: 74%. 1H-NMR (CDCl3) δ 12.61 (brs., 1H, NH), 7.54 (brd, J = 7.8 Hz, 2H), 7.31 (brt, J = 7.8 Hz, 2H), 7.04 (brt, J = 7.8 Hz, 1H), 6.92 (s., 1H, NH, exchangeable), 6.77 (s, 1H, purine), 5.17 (br s, 1H, NH), 3.39 (br s, 3H, NCH3), 1.90–1.12 (series of m, 10H). 13C-NMR (CDCl3) δ 154.8, 155.6, 152.3 (br), 140.2, 134.6, 129.1 (2C), 122.6, 120.6 (2C), 114.7 (br), 54.8 (very br), 30.1 (br, CH3), 25.7 and 25.8 (5C). ESI(+)-MS: m/z 323 [M + H]+; MS/MS (323): m/z 241 EI-MS: m/z 322 (M+, 95), 307 (50), 265 (56), 240 (72). HRMS(EI) calcd. for C18H22N6, 322.19059, found 322.19071.
3.2. Computational Analysis
DFT calculation was performed for the tautomers of
3 in the gas phase, in chloroform and in water by using Polarized Continuum Model (PCM). Calculations were carried out on a PC running at 3.4 GHz on an Intel i7 2600 quad core processor with 8 GB RAM and 1 TB hard disk with Windows 7 Home Premium 64-bit SP1 as an operating system. Ligands were build using PC Model version 6.0 (Serena Software, Bloomington, IN, USA). A Gaussian 03W revision E.01 program [
29] with graphical interface GaussView 4.0. was used in the geometry optimization at a density functional theory (DFT) level of theory and invoking gradient with 6-31G(d,p) basis set for all the atoms. The gradient-corrected DFT with the three-parameter hybrid functional (B3) [
30] for the exchange part and the Lee–Yang–Parr (LYP) correlation function [
31] were utilized. The optimized structural parameters were employed in the vibrational energy calculations at the DFT levels to characterize all stationary points as minima. No imaginary wave number modes were obtained for each optimized structure, proving that a local minimum on the potential energy surface was actually found. The minimized molecules were saved in pdb extension.
AutoDock Tools (ADT) package version 1.5.6rc3 was employed to generate the docking input files and to analyze docking results, whereas Autodock Vina 1.1.2 [
32] was taken for docking calculation. The structures of Aurora-B (PDB ID: 2VGO) and Mps1 (PDB ID: 3HF9) kinases were determined by X-ray crystallography with a resolution of 1.7 Å and 2.6 Å respectively [
12,
33]. The structures were modified as follows: the ligand and all the crystallization water molecules were removed saving the file in pdb extension. All the hydrogen atoms were added by AutoDock Tools (ADT) and Gasteiger–Marsili charges were calculated saving the resulting file in pdbqt extension. Rotatable bonds were defined for each minimized ligand molecule. For the docking calculation a grid box of 16 × 16 × 16 Å in
x,
y,
z directions was created, spacing of 1.00 Å and centered at
x = 10.52,
y = −0.323,
z = 3.0766 for 2VGO and a grid box of 14 × 14 × 14 Å in
x,
y,
z directions was created, spacing of 1.00 Å and centered at
x = 0.572,
y = 17.486,
z = 51.185 for 3H9F. Vina parameters were set as follows: exhaustiveness of the local search =100 and number of conformations to calculate =10. Results are expressed as energy associated to each ligand-enzyme complex in terms of Gibbs free energy values (
Table S1). The visual ligand-enzyme interactions were displayed using LigPlot [
34].
3.3. Biological Evaluation
3.3.1. General
The purity of the tested compounds is >99% as established by HPLC analysis. The compounds were dissolved in DMSO. All treatments were conducted 24 h after cell seeding in culture medium.
3.3.2. Cell Culture
p53 wild-type (MCF-7 Vector) and silenced version of MCF-7 (MCF-7 shp53) were received from Reuven Agami [
17]. HCT116 p53
+/+ and the derivative p53
−/− cell lines were obtained from Bert Vogelstein [
19]. All cell lines were cultured in RPMI pH 7.4 (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% Fetal Bovine Serum (Mill Creek, Rochester, MN, USA), 1X L-Glutamine and Pen/Strep (Lonza, Basel, Switzerland) at 37 °C with 5% CO
2 in a humidified atmosphere. For MCF-7, 0.5 µg/mL Puromycin (Sigma Aldrich) was added to the culture medium to maintain selection of the vectors.
3.3.3. Cell Viability Assay (MTT Assay)
For the MTT assay, 5 × 103 cells/100 µL were plated into 96-well tissue culture plates. The day after, cells were treated with medium containing 0.1% DMSO alone or medium containing several concentrations of reversine or analogues (0.01 µM, 0.1 µM, 0.5 µM, 1 µM, 5 µM, 10 µM, 50 µM, 100 µM and 300 µM). After 24 h incubation, cell viability was determined by tetrazolium dye 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) (5 mg/mL in phosphate buffer saline (PBS); Sigma Aldrich) which was added to fresh medium (final concentration: 0.5 mg/mL). The yellow redox indicator MTT is reduced to a dark blue final product, MTT-formazan, by the mitochondrial dehydrogenases of living cells. Following 1 h-incubation at 37 °C, the medium was discarded and cells were lysed by adding 100 µL DMSO. Finally absorbance was examined at 590 nm wavelength using an Infinite M200 microplate reader (TECAN, Mannedorf, Switzerland). Each experiment has been run in triplicate and results are the merge of three experiments.
3.3.4. Cell Cycle Assay
Cell cycle analysis was performed on at least 30,000 events for each sample with FACSCantoTM A using BD Diva Software 6.1.3 (BD Bioscience, San Jose, CA, USA) and the DNA profile was analyzed by ModFit LT 4.1 (Verity Software House, Topsham, ME, USA). Cells were treated with 0.1% DMSO, 1 µM Reversine or different concentrations of each 1–3 (1 µM, 2.5 µM, 5 µM and 10 µM) for 24 h, detached with trypsin-EDTA, collected by centrifugation and washed three times with PBS. Cells were stained with propidium iodide using BD CycletestTM Plus DNA Kit (BD Bioscience) and then analyzed by flow cytometry. Results obtained are the merge of three experiments.
3.3.5. Membrane and Nuclei Staining
After treatments, cultured cells were washed twice with PBS and fixed with 4% formaldehyde/PBS for 20 min at room temperature. After three washes in PBS, cells were stained with Hoechst (Thermo Fisher Scientific, Waltham, MA, USA; 1:10,000) and Cell Mask (Thermo Fisher Scientific, Waltham, MA, USA; 1:10,000) and then visualized with a Zeiss Axio Observer Z1 instrument (Carl Zaiss, Oberkochen, Germany) using Zeiss AxioVision v.4.8.1.
3.3.6. Western Blot
Cells were cultured in 10 cm2/well and treated with 1 µM reversine or different concentrations of analogues (1 µM, 2.5 µM, 5 µM and 10 µM) for 24 and 96 h. 0.1% DMSO was used as negative control. Cells were detached with trypsin-EDTA, collected by centrifugation and washed with PBS. Samples were then lysed in 150 µL RIPA buffer (Sigma Aldrich) and the proteins were quantified by BCA assay (Thermo Scientific). 15 µg of extracted proteins were loaded on NuPAGETM 4–12% Bis-Tris acrylamide Gel (Thermo Fisher Scientific) and then transferred onto a nitrocellulose membrane using a Tris-Glycine buffer. Blocking was performed overnight with 5% not-fat dry milk, 0.1% TWEENTM and PBS1X. Immunodetection was obtained using the following primary antibodies: p53 (DO1, 1:4000; Santa Cruz Biotechnologies, Dallas, TX, USA) and GAPDH (1:10,000; Santa Cruz Biotechnologies). The secondary antibody used was horse alpha-mouse Human Recombinant Peroxidase (1:10,000; Vector Laboratories, Burlingame, CA, USA). Finally, western blots were analyzed by ECL and detected with ChemiDocTM XRS+ (Bio-Rad, Hercules, CA, USA) using ImageLab software (Bio-Rad).
3.3.7. Statistical Analysis
Data are presented as mean ± SEM of three independent experiments. GraphPad Prism 6 software (GraphPad software, La Jolla, CA, USA) was used and ANOVA test was performed for comparisons. Statistical significance was defined as a p value less than 0.05 in all tests.