W.W. Wong, et al.
BiochemicalPharmacology169(2019)113641
The DNA-PKcs (pdb code 5LUQ, 4.3 Å) [4] was prepared using the
into the crystal structure of DNA-PKcs using GOLD (v 5.6.1). The
binding site was defined by a centroid with the coordinates X: 31.8168,
Y: 10.5208, Z: −5.9434 and a radius of 8 Å. Six residues found within
the first shell of the proposed ligand binding site (Lys3753, Ile3803,
Trp3805, Thr3809, Met3929 and Ile3940) were treated as flexible
during docking. The side chains were sampled from the GOLD rotamer
library [48]. A H-bond interaction with the backbone NH of Leu3806
was specified as a constraint for docking. Images were created using
PyMOL (v1.7.0.1, Schrodinger, LLC).
5′), 6.98 (d, J = 2.3 Hz, 1H, H-8), 6.80 (s, 1H, H-3), 6.75 (d, J = 2.3 Hz,
1H, H-6); MS m/z 387.1 (MH+, 100%).
2.1.3. 5-Hydroxy-7-morpholino-2-phenyl-4H-chromen-4-one (IC87361)
Chromenone IC87361 was synthesised from triflate 2 [46] (1.00 g,
2.59 mmol), morpholine (0.27 mL, 3.1 mmol), Pd2dba3 (119 mg,
0.13 mmol), XPhos (247 mg, 0.52 mmol) and Cs2CO3 (1.86 g, 5.7 mmol)
in dry dioxane (30 mL) stirred at 110 °C for 16 h in a sealed tube. The
mixture was cooled and the solvent evaporated under reduced pressure.
The residue was partitioned between EtOAc (100 mL) and water
(100 mL). The organic fraction was sequentially washed with water
(50 mL) and brine (50 mL) and dried (MgSO4). The filtrate was eva-
porated and the residue purified by column chromatography, eluting
with 2% EtOAc/DCM, to give chromenone IC87361 (0.64 g, 77%) as a
yellow solid: mp 100–102 °C; 1H NMR (CDCl3) δ 12.60 (s, 1H, 5-OH),
7.89 (dd, J = 7.7, 1.6 Hz, 2H, H-2′, H-6′), 7.50–7.56 (m, 3H, H-3′, H-4′,
H-5′), 6.64 (s, 1H, H-3), 6.41 (d, J = 2.3 Hz, 1H, H-8), 6.31 (d,
J = 2.3 Hz, 1H, H-6), 3.88 (t, J = 4.9 Hz, 4H, H-2′′, H-6′′), 3.37 (t,
J = 5.0 Hz, 4H, H-3′′, H-5′′);13C NMR δ 182.0, 163.7, 161.9, 158.3,
156.3, 131.8 (2), 129.2 (2), 126.4 (2), 106.1, 104.2, 97.2, 91.7, 66.8
(2), 47.5 (2); MS m/z 342.2 (MH+, 100%); HPLC purity 99.1%.
2.3. Cell culture
HCT116 cells from ATCC were grown in AlphaMEM (ThermoFisher,
Waltham, MA) with 5% FBS (Moregate Biotech, Hamilton, NZ).
HCT116/POR-G cells expressing an N-terminally truncated POR,
copGFP and puromycin resistance genes, and HCT116/PORko-R cells
with biallelic knockout of POR and expressing RFP and neomycin re-
sistance markers, have been reported previously [49] and were pas-
characterised [50] head and neck squamous cell carcinoma (HNSCC)
cell line UT-SCC-54C was passaged in MEM with 4.5 g/L D-glucose,
20 mM HEPES and 10% FBS. A HAP1 line with a CRISPR-induced 11 bp
deletion in PRKDC at genomic location chr8:47912458 (clone
HZGHC024034c011), resulting in formation of a premature termina-
tion codon truncating DNA-PKcs from 469 to 107.8 kDa, and the par-
ental chronic myeloid leukaemia-derived HAP1 line (C631), were
purchased from Horizon Discovery and cultured in IMDM with 5% FBS.
The initially haploid lines were passaged for 4 weeks to allow sponta-
neous conversion to diploidy, which was confirmed by propidium io-
dide flow cytometry using HCT116 cells as a pseudodiploid reference.
All lines were passaged for < 3 months from mycoplasma-free (Plas-
moTest; InvivoGen, San Diego, CA) frozen stocks that were authenti-
cated by STR profiling.
2.1.4. 5-((1-Methyl-2-nitro-1H-imidazol-5-yl)methoxy)-7-morpholino-2-
phenyl-4H-chromen-4-one (SN38023)
A mixture of IC87361 (179 mg, 0.55 mmol), 5-(chloromethyl)-1-
methyl-2-nitro-1H-imidazole [47] (115 mg, 0.66 mmol) and Cs2CO3
(269 mg, 0.83 mmol) in dry DMF (10 mL) was stirred at 75 °C for 4 h.
The mixture was cooled and diluted with EtOAc (80 mL). The organic
fraction was washed sequentially with water (3 × 50 mL), brine
(50 mL), and then dried (MgSO4). The solvent was evaporated and the
residue was purified by column chromatography, eluting with a gra-
dient (0–5%) of MeOH/DCM, to give SN38023 (146 mg, 57%) as a
yellow solid: mp 200–203 °C; 1H NMR [(CD3)2SO] δ 7.99–8.04 (m, 2H,
H-2′, H-6′), 7.52–7.59 (m, 3H, H-3′, H-4′, H-5′), 7.41 (s, 1H, H-4‴), 6.78
(d, J = 2.2 Hz 1H, H-8), 6.71 (d, J = 2.2 Hz, 1H, H-6), 6.68 (s, 1H, H-3),
5.35 (s, 2H, CH2O), 3.89 (s, 3H, NCH3), 3.77 (t, J = 4.7 Hz, 4H, H-2′′,
H-6′′), 3.41 (t, J = 4.9 Hz, 4H, H-3′′, H-5′′); 13C NMR δ 175.3, 159.4,
159.2, 157.8, 154.3, 146.2, 133.8, 131.3, 131.1, 129.0 (2), 128.3, 125.9
(2), 106.5, 108.0, 96.8, 94.2, 65.8 (2), 60.7, 46.8 (2), 34.5; MS m/z
2.4. DNA-PK enzyme inhibition
Inhibition of DNA-PK kinase activity was evaluated by Reaction
Biology Corporation (Malvern, PA) with the HotSpot assay platform
[51], quantifying [γ-33P]-ATP phosphorylation of a peptide substrate
10 μg/ml DNA and 10 μM ATP.
463.1 (MH+
,
100%); HRMS calcd for C24H23N4O6 (MH+
) m/z
463.1612, found 463.1616 (0.9 ppm); HPLC purity 98.8%, with <
0.01% of residual IC87361. Stock solutions were prepared in DMSO at
30 mM and stored frozen at −80 °C for in vitro studies.
2.5. Radiolytic reduction
2.1.5. 5-Hydroxy-7-(morpholino-d8)-2-phenyl-4H-chromen-4-one
IC87361)
(d8-
Solutions of SN38023 (10 μM) in deoxygenated 0.1 M sodium for-
mate/5 mM sodium phosphate buffer (pH 7.0), with a final DMSO
concentration of 0.1%, were prepared in a Bactron Pd/H2-scrubbed
anaerobic chamber (Sheldon Manufacturing, Cornelius, OR), trans-
ferred to HPLC vials, sealed, gamma irradiated (cobalt-60 Eldorado 78,
2.5 Gy/min) and stored at −80 °C until analysis by LC-MS.
Pd(OAc)2 (Chem-Impex International, Wood Dale, IL; 40 mg,
0.18 mmol) and
BINAP (Chem-Impex International, Wood Dale, IL;
0.12 g, 0.20 mmol) were added to toluene (20 mL) and the mixture was
stirred under nitrogen at 40 °C for 30 mins. Cs2CO3 (0.84 g, 2.58 mmol),
triflate 2 (0.70 g, 1.81 mmol) and d8-morpholine (0.20 mL, 2.17 mmol)
were added and the mixture was stirred at reflux for 24 h. The mixture
was cooled to 20 °C and solvent removed in vacuo. The resulting residue
was purified by column chromatography, eluting with a gradient
(0–4%) of EtOAc/DCM, to give chromenone d8-IC87361 (0.35 g, 58%)
as a yellow solid: mp 245–248 °C; 1H NMR (CDCl3) δ 12.58 (s, 1H, 5-
OH), 7.89 (m, 2H, H-2′, H-6′), 7.47–7.57 (m, 3H, H-3′, H-4′, H-5′), 6.61
(s, 1H, H-3), 6.37 (d, J = 2.3 Hz, 1H, H-8), 6.27 (d, J = 2.3 Hz, 1H, H-
6); 13C NMR (CDCl3) δ 182.0, 163.7, 161.9, 158.3, 156.3, 131.8 (2),
129.2 (2), 126.4 (2), 106.0, 104.1, 97.1, 91.6, 65.7 (quintet, 2), 46.5
(quintet, 2); HRMS calcd for C19H10D8NO4 (MH+) m/z 332.1732, found
332.1732 (0.13 ppm); HPLC purity 98.7%.
2.6. Metabolism and cellular uptake
Trypsinised cells (5 × 105 cells/well in 24-well plates) were in-
cubated to allow attachment for 2 h before addition of compounds
(final volume 0.5 mL, ≤1% DMSO). For anoxic exposures, trypsinsed
cell pellets were transferred to the anaerobic chamber, resuspended in
medium previously equilibrated in the chamber for 3 d and seeded into
similarly equilibrated plates. At various times, 14C-mannitol (370 GBq/
mmol, American Radiolabelled Chemicals, Inc) was added (740 Bq in
10 μL/well), mixed gently, extracellular medium was removed and
1 mL ice-cold MeOH was added to extract compounds from the cells.
For determination of overall metabolism, the MeOH extract was added
to the extracellular sample. For analysis of intracellular and
2.2. Molecular modelling studies
Ligands for docking were prepared using LigPrep (Maestro v11.4).
3