8
D. Fearon et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
CHCl3). mp: 291 °C. 1H NMR (500 MHz, DMSO d6) 5.16 (2H, s), 6.81
(1H, d, J = 2.4 Hz), 6.93 (1H, d, J = 2.4 Hz), 7.25 (1H, tt, J = 1.2 Hz,
7.3 Hz), 7.36–7.40 (2H, m), 7.44–7.48 (2H, m), 11.53 (1H, br
s).13C NMR (126 MHz, DMSO d6) 157.5 (C), 139.2 (C), 138.0 (C),
129.3 (CH), 126.9 (CH), 125.7 (CH), 119.6 (C), 117.8 (CH), 110.2
(CH). HRMS: Found 187.0877, calculated for C11H11N2O (M + H)+:
187.0866.
118.5 (CH), 113.1 (CH), 55.9 (CH3). HRMS: Found 322.1182, calcu-
lated for C18H16N3O3 (M + H)+: 322.1186.
4.4.3. 3,5-Dimethoxy-N-(2-oxo-5-(pyridin-4-yl)-1,2-dihydropyridin-
3-yl)benzamide (18)
Prepared using general procedure C to give a grey solid (32 mg,
34%): 3,5-dimethoxybenzoylchloride (53.6 mg, 0.27 mmol,
1
equivalent), 3-amino-5-(pyridin-4-yl)pyridin-2-one (50 mg, 0.27
mmol, 1 equivalent). Rf = 0.67 (DCM:EtOAc 1:1). mp: 280 °C. 1H
NMR (500 MHz, DMSO d6) 3.83 (6H, s), 6.75 (1H, t, J = 2.2 Hz),
7.06 (2H, d, J = 2.2 Hz), 7.61 (2H, dd, J = 4.6 Hz, 1.5 Hz), 7.83 (1H,
d, J = 2.5 Hz), 8.59 (2H, dd, J = 4.6 Hz, 1.5 Hz), 8.70 (1H, d, J = 2.5
Hz), 9.36 (1H, s), 12.58 (1H, s).13C NMR (126 MHz, DMSO d6)
165.4 (C), 161.1 (C), 157.9 (C), 150.7 (CH), 144.0 (C), 136.5 (C),
129.6 (C), 128.4 (CH), 123.3 (CH), 120.1 (CH), 115.5 (C), 105.6
(CH), 104.3 (CH), 56.0 (CH3). HRMS: Found 352.1291, calculated
for C19H18N3O4 (M + H)+: 352.1292.
4.3.2. 2-Methoxy-5-(pyrimidin-5-yl)pyridin-3-amine
Prepared using general procedure A to give a brown solid (142
mg, 72%): 2-dicyclohexylphosphino-2,4,6-triisopropylbiphenyl
(94 mg, 0.197 mmol, 0.2 equivalents), tris(dibenzylideneacetone)
dipalladium(0) (45 mg, 0.049 mmol, 0.05 equivalents), pyrim-
idinyl-5-boronic acid (124 mg, 1.182 mmol, 1.2 equivalents),
potassium phosphate (627 mg, 2.96 mmol,
3 equivalents), 5-
bromo-2-methoxypyridin-3-amine (200 mg, 0.985 mmol, 1 equiv-
alent) and n-butanol (7 mL). Rf = 0.3 (DCM: EtOAc 1:1). mp: 233 °C.
1H NMR (500 MHz, DMSO d6) 3.92 (3H, s), 5.19 (2H, s), 7.21 (1H, d,
J = 2.2 Hz), 7.78 (1H, d, J = 2.2 Hz), 9.02 (2H, s), 9.16 (1H, s).13C NMR
(126 MHz, DMSO d6) 157.4 (CH), 154.6 (CH), 152.9 (C), 133.4 (C),
132.0 (C), 131.0 (CH), 124.2 (C), 116.7 (CH), 53.6 (CH3). HRMS:
Found 203.0929, calculated for C10H11N4O (M + H)+: 203.0927.
4.4.4. 3-Fluoro-N-(2-oxo-5-(pyridin-4-yl)-1,2-dihydropyridin-3-yl)
benzamide (19)
Prepared using general procedure C to give a yellow solid (24
mg, 29%): 3-fluorobenzoylchloride (0.028 mL, 0.27 mmol, 1 equiv-
alent), 3-amino-5-(pyridin-4-yl)pyridin-2-one (50 mg, 0.27 mmol,
1 equivalent). Rf = 0.51 (DCM:EtOAc 1:1). mp: 292 °C. 1H NMR
(500 MHz, DMSO d6) 7.50 (1H, td, J = 2.1 Hz, 8.2 Hz), 7.62 (1H, t, J
= 8.0 Hz), 7.77 (1H, d, J = 2.0 Hz), 7.82 (1H, d, J = 7.8 Hz), 8.23 (2H,
d, J = 6.8 Hz), 8.26 (1H, brs), 8.82 (1H, d, J = 2.6 Hz), 8.83 (2H, d, J
= 6.8 Hz), 9.62 (1H, s), 13.01 (1H, s). 13C (126 MHz, DMSO d6)
164.8 (C), 162.5 (d, J = 244.8 Hz, CF), 158.1 (C), 152.2 (CH), 142.8
(C), 136.5 (d, J = 6.9 Hz, C), 132.7 (CH), 131.5 (d, J = 8.0 Hz, CH),
129.5 (C), 124.0 (d, J = 1.9 Hz, CH), 123.5 (CH), 121.9 (CH), 119.6
(d, J = 21.2 Hz, CH), 114.9 (d, J = 23.0 Hz, CH), 113.0 (C). HRMS:
Found 310.0984, calculated for C17H13N3O2F (M + H)+: 310.0986.
4.3.3. 3-Amino-5-(pyrimidin-5-yl)pyridin-2(1H)-one (10)
Prepared using general procedure B to give a brown solid (77
mg, 68%): 2-methoxy-5-(pyrimidin-5-yl)pyridin-3-amine (122
mg, 0.603 mmol, 1 equivalent), trimethylsilyl chloride (0.386 mL,
3.02 mmol, 5 equivalents) and sodium iodide (452 mg, 3.02 mmol,
5 equivalents) in acetonitrile (5 mL). Rf = 0.57 (DCM:EtOAc 1:1).
mp: 240 °C. 1H NMR (500 MHz, DMSO d6) 5.26 (2H, s), 6.82 (1H,
d, J = 2.4 Hz), 7.20 (1H, d, J = 2.4 Hz), 8.94 (2H, s), 9.06 (1H, s).13
C
NMR (126 MHz, DMSO d6) 157.9 (C), 156.7 (CH), 153.7 (CH),
139.6 (C), 131.6 (C), 119.5 (CH), 113.2 (C), 108.6 (CH). HRMS:
Found 189.0711, calculated for C9H9N4O (M + H)+: 189.0771.
4.4.5. Kinase profiling
IC50 values for MPS1, Aurora A and Aurora B inhibition were
determined as previously described,44 and Ki values were esti-
mated from the mean IC50 values using the Cheng-Prusoff equa-
tion.48 The protocol for screening against the 26-kinase panel
using a mobility shift assay is provided in the Supplementary
Material.
4.4. N-(2-Oxo-5-(pyridin-4-yl)-1,2-dihydropyridin-3-yl)benzamide
(16)
4.4.1. General procedure C
Benzoyl chloride (0.062 mL, 0.53 mmol, 1 equivalent) was
added dropwise to 3-amino-5-(pyridin-4-yl)pyridin-2-one (100
mg, 0.53 mmol, 1 equivalent) in pyridine (3 mL) and the mixture
was stirred for 16 h at room temperature. The solvent was
removed under reduced pressure and the residue taken up in
methanol and filtered to give the desired product as a light-yellow
solid (41 mg, 27%). Rf = 0.58 (1:1 DCM:EtOAc). mp: 291 °C. 1H NMR
(500 MHz, DMSO d6) 7.54–7.67 (5H, m), 7.82 (1H, d, J = 2.5 Hz),
7.90–8.00 (2H, m), 8.52–8.65 (2H, m), 8.77 (1H, d, J = 2.5 Hz),
9.39 (1H, s), 12.60 (1H, s).13C (126 MHz, DMSO d6) 165.6 (C),
157.8 (C), 150.7 (CH), 144.0 (C), 134.2 (C), 132.7 (CH), 129.5 (C),
129.3 (CH), 128.0 (CH), 127.7 (CH), 122.5 (CH), 120.2 (CH), 115.7
(C). HRMS: Found: 292.1075, calculated for C17H14N3O2 (M + H)+:
292.1086.
4.4.6. Crystallography
Co-crystals of 22 and 23 with MPS1 were produced by vapour
diffusion using the hanging-drop method. The well buffer con-
tained 0.2 M NaCO2H, 0.1 M BTP (pH 7.5) and 15% (w/v) PEG
3350. The protein solution contained 11.59 mg/mL MPS1, 50 mM
HEPES (pH 7.5), 150 mM NaCl and 5 mM DTT, supplemented with
1 mM compound (1% DMSO). Drops contained a 1:1 mixture of
well buffer and protein solution. Crystals grew over 3 days at 18
°C. Harvested crystals were briefly transferred to cryoprotectant
(22.5% ethylene glycol, 15.5% (w/v) PEG 3350 and 0.08 M Bis-Tris
propane pH 7.5) and flash cooled in liquid nitrogen. Crystals of
MPS1 in complex with 2 were obtained by back-soaking a crystal
of MPS1 with 23 in a new solution of 0.19 M NaCO2H, 0.095 M
BTP (pH 7.5), 14.25% PEG 3350 supplemented with 2 mM 2 for
48 h prior to harvesting. Structure solution and refinement data
are presented in supplementary material.
4.4.2. 3-Methoxy-N-(2-oxo-5-(pyridin-4-yl)-1,2-dihydropyridin-3-yl)
benzamide (17)
Prepared using general procedure C to give a yellow solid (23
mg, 27%): 3-methoxybenzoylchloride (0.038 mL, 0.27 mmol, 1
equivalent), 3-amino-5-(pyridin-4-yl)pyridin-2-one (50 mg, 0.27
mmol, 1 equivalent). Rf = 0.69 (DCM:EtOAc 1:1). mp: 287 °C. 1H
NMR (500 MHz, DMSO d6) 3.85 (3H, s), 7.21 (1H, dd, J = 1.7 Hz,
8.0 Hz), 7.43–7.60 (3H, m), 8.17–8.31 (3H, m), 8.79–8.88 (3H, m),
9.46 (1H, s), 13.01 (1H, s).13C NMR (126 MHz, DMSO d6) 165.6
(C), 159.9 (C), 158.1 (C), 152.3 (C), 142.8 (CH), 135.6 (C), 132.3
(CH), 130.5 (CH), 129.6 (C), 122.6 (CH), 122.0 (CH), 119.8 (CH),
Acknowledgements
This work was funded by The Wellcome Trust. We acknowledge
funding from Cancer Research UK (grant numbers C309/A8274
and C309/A11566) Institute of Cancer Research – United Kingdom.
We are grateful to Stefan Knapp, Structural Genomics Consortium,
Oxford, UK, for the generous gift of expression plasmids for the