Xin et al.
9
168.5, 165.3 (2C), 164.7, 163.8, 159.0 (2C), 119.2, 66.5 (m, 1H), 4.59–4.25 (m, 2H), 4.00–3.78 (m, 8H), 3.19–3.15
(2C), 52.4, 49.1 (4C), 45.8, 43.7, 21.71 (2C). ESI-MS m/z: (m, 1H), 2.65–2.53 (m, 2H), 2.19–2.02 (m, 2H), 2.02–1.79
414.3 [M + H]+.
(m, 3H), 1.09–0.85 (m, 4H), 0.80–0.73 (m, 4H). 13C NMR
(4-((4-(2-Aminopyrimidin-5-yl)-6-morpholino-1,3,5- (CDCl3) δ 171.8, 167.1, 167.0, 164.6, 164.0, 159.1 (2C),
triazin-2-yl)(methyl)amino)piperidin-1-yl)(cyclopropyl) 121.31, 66.8 (2C), 57.4 (2C), 45.7, 43.6 (2C), 42.4, 11.0
methanone (B12): white solid, yield 79%. mp:>250 °C. (2C), 9.3, 7.3 (2C). ESI-MS m/z: 466.3 [M + H]+.
1H NMR (CDCl3) δ 9.19 (s, 2H), 5.52 (s, 2H), 5.20–5.05
(4-((4-(2-Aminopyrimidin-5-yl)-6-morpholino-1,3,5-
(m, 1H), 4.90–4.40 (m, 2H), 4.00–3.75 (m, 8H), 3.22– triazin-2-yl)(cyclopropyl)amino)piperidin-1-yl)(cyclo-
3.03 (m, 4H), 2.67–2.58 (m, 1H), 1.84–1.63 (m, 4H), pentyl)methanone (B18): white solid, yield 63%. mp
1.24 (t, J = 7.0 Hz, 1H), 1.00–0.90 (m, 2H), 0.80–0.75 233–236°C. Anal. calcd for C25H35N9O2: C, 60.83; H,
1
(m, 2H). 13C NMR (DMSO-d6): δ 171.3 (2C), 165.2 (2C), 7.15; N, 25.54; found: C, 60.86; H, 7.13; N, 25.57%; H
164.6, 159.1, 159.0, 119.1, 66.5 (2C), 56.5, 55.4, 52.6 NMR (CDCl3) δ 9.18 (s, 2H), 5.52 (s, 2H), 4.81 (d, J =
(2C), 44.9, 43.7, 28.7, 19.0, 10.8, 7.3 (2C). ESI-MS m/z: 8.7Hz, 1H), 4.43 (s, 1H), 4.09 (d, J = 8.7Hz, 1H), 3.80–
462.3 [M + Na]+.
3.73 (m, 8H), 3.11–2.85 (m, 2H), 2.60–2.50 (m, 2H),
(4-((4-(2-Aminopyrimidin-5-yl)-6-morpholino-1,3,5- 2.18–1.97 (m, 2H), 1.95–1.74 (m, 8H), 1.66–1.51 (m,
triazin-2-yl)(methyl)amino)piperidin-1-yl)(cyclobutyl) 2H), 0.92 (m, 2H), 0.79–0.66 (m, 2H). 13C NMR (CDCl3):
methanone (B13): white solid, yield 51%. mp >250 °C. δ 174.4, 167.1, 167.0, 164.6, 164.1, 159.1 (2C), 121.1,
1H NMR (CDCl3) δ 9.18 (s, 2H), 5.52 (s, 2H), 5.15–4.66 66.8 (2C), 58.3, 57.5, 45.6, 43.6, 42.2, 41.1 (2C), 31.2,
(m, 2H), 3.99–3.68 (m, 9H), 3.29–3.20 (m, 1H), 3.03– 30.3, 30.1, 26.1, 26.0, 18.4, 9.3, 9.2. ESI-MS m/z: 494.4
2.95 (m, 4H), 2.70–2.50 (m, 1H), 2.40–2.25 (m, 2H), [M + H]+.
2.25–2.17 (m, 2H), 2.09–1.81 (m, 2H), 1.80–1.52 (m,
4-((4-(2-Aminopyrimidin-5-yl)-6-morpholino-1,3,5-
4H). 13C NMR (DMSO-d6): δ 172 (2C), 165.2 (2C), triazin-2-yl)(cyclopropyl)amino)piperidin-1-yl)(tetrahy-
164.6, 159.1, 159.0, 119.1, 66.5 (2C), 52.5, 52.4, 44.4, dro-2H-pyran-4-yl)methanone (B19): white solid, yield
44.3, 43.7, 41.2, 36.8, 28.6, 25.1 (2C), 17.8 (2C). ESI-MS 58%. mp 149–151°C. Anal. calcd for C25H35N9O3: C,
m/z: 476.3 [M +Na]+.
58.92; H, 6.92; N, 24.74; found: C, 58.96; H, 6.89; N,
(4-((4-(2-Aminopyrimidin-5-yl)-6-morpholino-1,3,5- 24.78%; 1H NMR (CDCl3) δ 9.17 (s, 2H), 5.52 (s, 2H), 4.81
triazin-2-yl)(methyl)amino)piperidin-1-yl)(cyclopentyl) (d, J = 8.7Hz, 1H), 4.43–4.35 (m, 1H), 4.03–4.00 (m, 2H),
methanone (B14): white solid, yield 51%. mp 233–235°C. 3.80–3.75 (m, 8H), 3.48–3.44 (m, 2H), 3.15–3.11 (m, 1H),
1H NMR (CDCl3) δ 9.19 (s, 2H), 5.45 (s, 2H), 5.20–4.75 2.80–2.74 (m, 1H), 2.63–2.49 (m, 2H), 2.21–1.83 (m, 7H),
(m, 2H), 4.12–4.05 (m, 1H), 4.02–3.67 (m, 8H), 3.23–2.92 1.63–1.50 (m, 2H), 0.95–0.84 (m, 2H), 0.79–0.66 (m, 2H).
(m, 5H), 2.63–2.50 (m, 1H), 1.93–1.64 (m, 12H). 13C 13C NMR (CDCl3): δ 172.7, 167.1, 167.0, 164.6, 164.2,
NMR (DMSO-d6): δ 173.6 (2C), 165.2 (2C), 164.6, 159.1, 159.1 (2C), 121.1, 67.3 (2C), 66.8, 58.2, 57.4, 45.5, 43.6,
158.9, 119.1, 66.51 (2C), 52.6, 52.5, 44.8, 43.7, 41.3, 30.2 42.1, 37.7 (2C), 29.3, 29.1, 27.3, 18.4 (2C), 9.3 (2C).
(2C), 30.1 (2C), 26.2 (2C), 26.1 (2C). ESI-MS m/z: 468.3 ESI-MS m/z: 510.3 (M + H)+.
[M + H]+.
(4-((4-(2-Aminopyrimidin-5-yl)-6-morpholino-1,3,5-
triazin-2-yl)(methyl)amino)piperidin-1-yl)(cyclohexyl)
Antiproliferative assays by MTT method
methanone (B15): white solid, yield 53%. mp 181– VS-5584 was purchased from Shanghai Biochempartner
1
182 °C. H NMR (CDCl3) δ 9.19 (s, 2H), 5.55 (s, 2H), Company (Purity: 99%, high-performance liquid chroma-
5.05–4.75 (m, 2H), 4.07–4.00 (m, 1H), 3.82–3.70 (m, tography), and 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl-
8H), 3.29–2.99 (m, 4H), 2.62–2.51 (m, 2H), 1.80–1.75 2H-tetrazolium bromide (MTT) was purchased from Sigma
(m, 8H), 1.51–1.46 (m, 4H), 1.43–1.25 (m, 2H). 13C (St. Louis, MO, USA). The cellular chemosensitivity was
NMR (DMSO-d6): δ 173.7 (2C), 165.2 (2C), 164.6, determined by using a modified MTT method assay in
159.1, 159.0, 119.1, 66.51 (2C), 54.0, 52.5, 44.7, 43.7, human cell lines HCT-116 and MCF-7, which was similar
42.3, 41.1, 38.7, 29.7, 26.1, 25.7 (2C), 18.5, 17.2, 13.0. to the procedures described.16 In brief, HCT-116, MCF-7
ESI-MS m/z: 482.3 [M + H]+.
cells in 200μL culture medium were seeded into 96-well
(4-((4-(2-Aminopyrimidin-5-yl)-6-morpholino-1,3,5- microplates at 3000–5000 cells/well, respectively, and cul-
triazin-2-yl)(methyl)amino)piperidin-1-yl)(tetrahydro- tured in Dulbecco’s Modified Eagle Medium with 10%
2H-pyran-4-yl)methanone (B16): white solid, yield 67%. fetal bovine serum (FBS), or RPMI-1640 with 10% calf
mp 237–239°C. 1H NMR (CDCl3) δ 9.18 (s, 2H), 5.44 (s, serum, incubated at 37°C for 24h prior to drug exposure.
2H), 5.16–4.70 (m, 2H), 4.04–4.00 (m, 3H), 3.97–3.63 (m, Cell numbers were titrated to keep control cells growing in
8H), 3.48–3.44 (m, 2H), 3.18–3.01 (m, 4H), 2.80–2.64 (s, the exponential phase throughout the 72h incubation
2H), 2.06–1.79 (m, 4H), 1.70–1.45 (m, 4H, CH2). 13C NMR period. Cells were treated with final concentrations of 10.0,
(DMSO-d6): δ 172.6, 165.2 (2C), 164.6, 159.1, 159.0, 5.0, 1.0, and 0.5μM of tested compounds simultaneously
119.1, 66.8 (4C), 66.5 (2C), 52.6 (2C), 44.7, 43.7, 36.8, and incubated for 72h and then 20μL of MTT solution
29.5, 29.5. ESI-MS m/z: 506.3 [M +Na]+.
(5mg/mL in PBS) was added to each well at lucifugal con-
(4-((4-(2-Aminopyrimidin-5-yl)-6-morpholino-1,3,5- dition and incubated for 4h at 37°C. The formed purple
triazin-2-yl)(cyclopropyl)amino)piperidin-1-yl)(cyclopro- formazan crystals were pelleted at the bottom of the well,
pyl)methanone (B17): white solid, yield 46%. mp: >250°C. separated from the supernatant, and dissolved in 200μL of
1H NMR (CDCl3) δ 9.19 (s, 2H), 5.53 (s, 2H), 4.77–4.60 DMSO. The optical density at 570nm was determined by