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A. Andreani et al. / European Journal of Medicinal Chemistry 46 (2011) 4311e4323
- aminoguanidine hydrochloride for compounds 17a,d,e,j,n,
(1H, m, ar), 8.43 (1H, d, th, J ¼ 1.5), 8.49 (1H, s, CH), 11.96 (1H, s, ex).
Anal. C14H12FN7O2S$HCl (C, H, N).
18a,b, 19d, 20eem, 21e,j, 22o, 23o, 30a,b, 34a,b.
- 2-hydrazino-2-imidazoline hydrobromide for compounds 24a,
25c, 31a and 35a.
22o. IR: 3462e3135,1696,1214, 851.1H NMR: 7.78 (4H, broad, ex),
7.98 (2H, d, ar, J ¼ 8.7), 8.33 (2H, d, ar, J ¼ 8.7), 8.56 (1H, s, th), 8.87 (1H,
s, CH), 12.02 (1H, broad, ex). Anal. C13H10FN7O2S$HCl (C, H, N).
23o. IR: 3467e3135, 1634, 1163, 861. 1H NMR: 7.81 (4H, broad,
ex), 7.95 (2H, d, ar, J ¼ 8.7), 8.29 (2H, d, ar, J ¼ 8.7), 8.56 (1H, s, th),
8.87 (1H, s, CH), 12.11 (1H, s, ex). Anal. C13H10BrN7O2S$HCl (C, H, N).
24a. IR: 3309e3170, 1675, 1240, 1030. 1H NMR: 3.77 (4H, s, 2ꢂ
CH2), 7.37 (1H, s, thio), 8.22 (1H, s, th), 8.41 (2H, broad, ex), 8.85 (1H,
s, CH), 12.23 (1H, broad, ex). Anal. C13H9Cl3N6S2$HBr (C, H, N).
25c. IR: 3580e3186, 1681, 1076, 897. 1H NMR: 2.82 (3H, s, CH3),
3.74 (4H, s, 2ꢂ CH2), 7.88 (1H, d, ar-5, J ¼ 8.4), 8.24 (1H, dd, ar-6,
J ¼ 8.4, J ¼ 2), 8.35 (3H, broad, ex), 8.57 (1H, s, CH), 8.68 (1H, d,
ar-2, J ¼ 2). Anal. C15H13ClN8O2S$HBr (C, H, N).
The reaction mixture was refluxed for 5e20 h according to a TLC
test, and the resulting precipitate, obtained from the reaction
mixture or after partial solvent evaporation, was collected by
filtration with a yield of 50e60% (17d,e,j, 19d, 20e,f,h,i,kem, 22o,
24a, 25c, 30b, 31a, 34a, 35a), 70e80% (17a,n, 18a, 20g,j, 21e,j, 23o,
34b), and 85e95% (18b, 30a). In the case of compound 34a diethyl
ether was added to the ethanol solution to induce crystallization.
17a. IR: 3305, 3140, 1686, 1231, 835. 1H NMR: 7.36 (1H, s, thio),
7.79 (4H, broad, ex), 8.22 (1H, s, th), 8.91 (1H, s, CH),11.89 (1H, s, ex).
Anal. C11H7Cl3N6S.2HCl (C, H, N).
17d. IR: 3283e3022, 1670, 1158, 974. 1H NMR: 7.47 (3H, m, ar),
7.67 (2H, m, ar), 8.48 (1H, s, th), 8.78 (4H, broad, ex), 8.90 (1H, s, CH),
12.13 (1H, s, ex). Anal. C13H11ClN6S$HCl (C, H, N).
30a. IR: 3469, 3105, 1682, 1637, 838. 1H NMR: 7.41 (1H, s, thio),
7.50 (1H, td, bzt, J ¼ 7.6, J ¼ 1.8), 7.57 (1H, td, bzt, J ¼ 7.6, J ¼ 1.8), 7.77
(4H, broad, ex), 8.13 (1H, dd, bzt, J ¼ 7.6, J ¼ 1.8), 8.41 (1H, s, CH),
8.68 (1H, dd, bzt, J ¼ 7.6, J ¼ 1.8), 12.03 (1H, s, ex). Anal.
C15H10Cl2N6S2$HCl (C, H, N).
17e. IR: 3416e3022, 1675, 1168, 856. 1H NMR: 7.34 (2H, m, ar),
7.59 (1H, broad, ex), 7.73 (2H, m, ar), 8.10 (3H, broad, ex), 8.45 (1H, s,
th), 8.90 (1H, s, CH), 12.02 (1H, s, ex). Anal. C13H10ClFN6S$HCl (C, H,
N).
30b. IR: 3490e3148, 1670, 1220, 1034. 1H NMR: 3.82 (3H, s,
OCH3), 3.92 (3H, s, OCH3), 7.46 (1H, s, ar), 7.50 (1H, t, bzt, J ¼ 7.7),
7.56 (1H, t, bzt, J ¼ 7.7), 7.70 (4H, broad, ex), 7.72 (1H, s, ar), 8.12 (1H,
d, bzt, J ¼ 7.7), 8.34 (1H, s, CH), 8.69 (1H, d, bzt, J ¼ 7.7), 12.08 (1H, s,
ex). Anal. C19H17N7O4S$HCl (C, H, N).
17j. IR: 3478e3160, 1696, 1163, 825. 1H NMR: 7.70 (1H, m, ar),
7.90 (4H, broad, ex), 8.06 (1H, m, ar), 8.35 (1H, m, ar), 8.49 (1H, s,
th), 8.85 (1H, s, CH),12.01 (1H, s, ex). Anal. C13H9ClFN7O2S$HCl (C, H,
N).
31a. IR: 3580e3078, 1669, 1235, 933. 1H NMR: 3.76 (4H, s, 2ꢂ
CH2), 7.41 (1H, s, thio), 7.56 (2H, m, bzt), 8.14 (1H, dd, bzt, J ¼ 8.1,
J ¼ 1.2), 8.36 (1H, s, CH), 8.59 (2H, s, ex), 8.76 (1H, dd, bzt, J ¼ 8.1,
J ¼ 1.2), 12.41 (1H, broad, ex). Anal. C17H12Cl2N6S2$HBr (C, H, N).
34a. IR: 3298e3064, 1673, 1192, 888. 1H NMR: 7.30 (1H, t, py,
J ¼ 7.6), 7.45 (1H, s, thio), 7.71 (1H, t, py, J ¼ 7.6), 7.82 (4H, broad, ex),
7.88 (1H, d, py, J ¼ 7.6), 8.40 (1H, s, CH), 9.58 (1H, d, py, J ¼ 7.6),12.05
(1H, s, ex). Anal. C13H10Cl2N6S$HCl (C, H, N).
17n. IR: 3457e3129, 2233, 1680, 835. 1H NMR: 7.82 (4H, broad,
ex), 7.89 (2H, d, ar, J ¼ 8.1), 7.97 (2H, d, ar, J ¼ 8.1), 8.51 (1H, s, th),
8.90 (1H, s, CH), 11.94 (1H, s, ex). Anal. C14H10ClN7S$HCl (C, H, N).
18a. IR: 3428e3154, 1685, 1647, 1150. 1H NMR: 2.82 (3H, s, CH3),
7.39 (1H, s, thio), 7.63 (4H, broad, ex), 8.33 (1H, s, CH), 12.12 (1H, s,
ex). Anal. C11H9Cl2N7S2$HCl (C, H, N).
20e. IR: 3288, 3124, 1680, 1158, 840. 1H NMR: 7.34 (2H, m, ar),
7.57 (1H, d, th, J ¼ 4.4), 7.73 (2H, m, ar), 7.84 (4H, broad, ex), 8.47
(1H, s, CH), 8.74 (1H, d, th, J ¼ 4.4), 12.23 (1H, s, ex). Anal.
C13H11FN6S$HCl (C, H, N).
35a. IR: 3575e3032, 1677, 1256, 1035. 1H NMR: 3.77 (4H, s, 2ꢂ
CH2), 7.23 (1H, t, py, J ¼ 7.6), 7.43 (1H, s, thio), 7.62 (1H, t, py, J ¼ 7.6),
7.83 (1H, d, py, J ¼ 7.6), 8.39 (1H, s, CH), 8.56 (2H, broad, ex), 9.52
(1H, d, py, J ¼ 7.6), 12.20 (1H, broad, ex). Anal. C15H12Cl2N6S$HBr (C,
H, N).
20h. IR: 3324e3000, 1680, 1153, 820. 1H NMR: 7.56 (2H, m,
ar þ th), 7.75 (2H, m, ar), 7.80 (4H, broad, ex), 8.49 (1H, s, CH), 8.70
(1H, d, th, J ¼ 4.4), 12.13 (1H, s, ex). Anal. C13H10F2N6S$HCl (C, H, N).
20i. IR: 3488e3114, 1680, 1061, 799. 1H NMR: 7.57 (1H, d, th,
J ¼ 4.2), 7.78 (6H, m, 2ar þ 4ex), 8.23 (1H, s, CH), 8.70 (1H, d, th,
J ¼ 4.2), 12.00 (1H, s, ex). Anal. C13H9F3N6S$HCl (C, H, N).
20j. IR: 3442e3124, 1685, 1168, 851. 1H NMR: 7.55 (1H, d, th,
J ¼ 4.3), 7.72 (1H, m, ar), 7.80 (4H, broad, ex), 8.10 (1H, m, ar), 8.39
(1H, m, ar), 8.54 (1H, s, CH), 8.68 (1H, d, th, J ¼ 4.3), 12.05 (1H, s, ex).
Anal. C13H10FN7O2S$HCl (C, H, N).
5.2. Biology
5.2.1. Cell-based screening assay
The NCI screening is a two stage process [23], beginning with
the evaluation of all compounds against the 60 cell lines at a single
concentration of 10ꢀ5 M. Compounds exhibiting significant growth
inhibition were evaluated against the 60 cell panel at five concen-
tration levels by the NCI according to standard procedures (http://
sure time was 48 h.
20k. IR: 3473e3094, 1680, 1061, 840. 1H NMR: 7.59 (1H, d, th,
J ¼ 4.6), 7.71 (1H, m, ar), 7.78 (4H, broad, ex), 8.29 (1H, s, CH), 8.38
(1H, m, ar), 8.48 (1H, m, ar), 8.70 (1H, d, th, J ¼ 4.6), 12.05 (1H, s, ex).
Anal. C13H10FN7O2S$HCl (C, H, N).
20l. IR: 3450e3109, 1737, 1173, 861. 1H NMR: 6.84 (4H, broad,
ex), 7.56 (1H, d, th, J ¼ 4.4), 7.73 (3H, m, ar), 7.99 (1H, m, ar), 8.52
(1H, s, CH), 8.73 (1H, d, th, J ¼ 4.4), 12.17 (1H, s, ex). Anal.
C14H11F3N6S$HCl (C, H, N).
5.2.2. High-throughput kinase assay methodology
High-throughput assays for kinase inhibition were conducted in
384-well plates and utilized IMAP technology (Molecular Devices,
Inc.). In brief, recombinant kinase (RSK1, RSK2, CHK1, CHK2, or
Aurora A) was incubated with fluorescent peptide substrate in
20m. IR: 3565e3119, 1624, 1030, 825. 1H NMR: 7.54 (1H, d, th,
J ¼ 4.5), 7.80 (4H, broad, ex), 7.90 (1H, dd, ar-6, J ¼ 8.4, J ¼ 2.1), 7.99
(1H, d, ar-5, J ¼ 8.4), 8.29 (1H, d, ar-2, J ¼ 2.1), 8.54 (1H, s, CH), 8.67
(1H, d, th, J ¼ 4.5), 11.96 (1H, s, ex). Anal. C13H10BrN7O2S$HCl (C, H,
N).
buffer containing 10
mM ATP. Phosphorylated peptide was then
bound to IMAP beads which resulted in an increase in the polari-
zation signal which was read on a Tecan Ultra microplate reader.
Data were normalized to control values and concentrations effec-
tive in inhibiting kinase activity by 50% (IC50) were read from
concentration response curves by linear interpolation.
21e. IR: 3252, 1675, 1235, 1168, 846. 1H NMR: 2.52 (3H, d, CH3,
J ¼ 1.3), 7.34 (2H, m, ar), 7.72 (2H, m, ar), 7.80 (4H, broad, ex), 8.44
(1H, s, CH), 8.47 (1H, d, th, J ¼ 1.3), 12.12 (1H, s, ex). Anal.
C14H13FN6S$HCl (C, H, N).
5.2.3. Cell culture and immunoblotting
MCF-7, MCF-10A, T47D and HEK 293 cells were obtained from
ATCC and cultured and maintained in RPMI medium supplemented
21j. IR: 3500e3000, 1680, 1168, 912. 1H NMR: 2.52 (3H, d, CH3,
J ¼ 1.5), 7.71 (1H, m, ar), 7.74 (4H, broad, ex), 8.09 (1H, m, ar), 8.37