94
Y.-q. Zeng et al. / European Journal of Medicinal Chemistry 119 (2016) 83e95
d
1
7.15e7.18 (m, 4H);
d
7.41e7.43 (m, 3H);
d
7.58e7.59 (d, 2H);
d
8.47 (s,
was shown in Table 6.
.2.1.32. N-(1-(3-cyclohexylthioureido)-2-methylpropyl)cinnama-
þ
H);
d9.20 (s, 1H)
d
9.95 (s, 1H, NeH). MS 381.5 [MþH] .
5
1
5
.2.1.25. N-(2,2-dimethyl-1-(3-o-tolylthioureido)propyl)cinnama-
mide (408). H NMR (400 MHz, DMSO-d
6
)
d
0.87e0.92 (m, 6H);
2.12 (s, 1H); 3.98 (s, 1H);
7.57e7.66 (d, 2H). MS 360.3
1
mide (396). H NMR (400 MHz, DMSO-d
3
4
6
)
d0.94 (m, 9H);
d2.18 (s,
d1.10e1.29 (m, 6H);
d1.56e1.87 (m, 5H);
d
d
H);
H);
d
d
6.15 (s, 1H);
7.57e7.59 (d, 2H);
d
6.71 (s, 1H);
d7.17e7.26 (m, 4H);
d
7.38e7.45 (m,
d
6.68e6.72 (d, 1H); 7.39e7.44 (q, 4H);
d
þ
þ
d8.19 (s, 1H). MS 382.1 [MþH] .
[MþH] .
5
.2.1.26. 5.2.26.
N-(cyclopropyl(3-o-tolylthioureido)methyl)cinna-
1.97e2.00 (m, 2H);
5.98e6.01 (t, 1H);
7.15e7.16 (m, 4H); 7.40e7.43 (m,
9.10 (s, 1H); 9.62 (s, 1H). CNMR
17.49; 21.94; 33.04; 51.70; 60.09; 62.50; 120.45;
25.73; 126.32; 127.68; 128.82; 128.92; 129.87; 129.97; 134.35;
5.2.1.33. N-(1-(3-cyclohexylthioureido)-2,2-dimethylpropyl)cinna-
1
1
mamide (397). H NMR (400 MHz, DMSO-d
6
)
d
d
mamide (409). H NMR (400 MHz, DMSO-d
1.57e2.10 (m, 5H); 4.10e4.12 (d, 1H); 5.19e5.24 (m, 1H);
(s, 1H); 6.03e6.05 (s, 1H); 6.47e6.59 (d, 1H); 7.35e7.38 (t, 2H);
7.50e7.54 (d, 2H); 7.65e7.70 (d, 2H). C NMR (DMSO, 100 MHz)
180.99; 164.67; 139.15; 134.85; 129.33; 128.82; 127.39; 122.03;
6
)
d0.98e1.35 (m, 14H);
d
d
3
(
2.08e2.32 (m, 4H);
6.71e6.75 (d, 1H, J ¼ 16 Hz);
H); 7.53e7.60 (m, 3H);
DMSO, 100 MHz)
d
3.72e3.81 (m, 2H);
d
d
d
d5.61
d
d
d
d
d
1
3
13
d
d
d
d
d
d
d
1
65.58; 52.10; 35.82; 32.14; 31.99; 25.37; 25.10; 24.24. MS 374.3
þ
þ
1
34.90; 139.01; 140.93; 166.20; 178.89. MS 366.2 [MþH] . The
[MþH] .
13
1
results of 2D C, H COSY(HMQC) spectra of compound 397 was
shown in Table 6.
6. Biological evaluations
5
.2.1.27. (E)-N-(1-(3-(4-methoxyphenyl)thioureido)-2-
6.1. Colorimetric determination of ATPase activity
1
methylpropyl)-3-(3,4,5-trimethoxyphenyl)acrylamide (402). H NMR
400 MHz, DMSO-d 0.91e0.98 (m, 6H); 2.18 (s, 1H); 3.68 (s,
H); 3.74 (s, 3H); 3.81 (s, 6H); 5.86 (s, 1H); 6.60e6.64 (d, 1H);
6.90e6.92 (d, 2H); 7.32e7.38 (m, 3H); 8.30 (s, 1H); 9.60 (s, 1H).
C NMR (DMSO, 100 MHz) 18.12; 31.68; 55.23; 55.84; 60.11;
(
3
6
)
d
d
d
The assay procedure was adopted from previous reports with
some modifications [26]where indicated. Stock solutions of mala-
chite green (0.1% w/v), polyvinyl alcohol (2.3% w/v), and ammo-
nium molybdate tetrahydrate (1% w/v in 1 M HCl) were prepared
and mixed with water at the ratio of 2:1:1:2 to prepare the mala-
chite green reagent, which is stable at room temperature for at least
d
d
d
d
d
d
d
d
1
3
d
6
4.99; 104.96; 113.89; 125.50; 130.29; 130.59; 131.39; 138.68;
þ
139.38; 153.07; 156.45; 164.55; 180.34. MS 474.2 [MþH] .
2
h. After its colour changing from dark brown to yellow, the re-
5
.2.1.28. (E)-N-(1-(3-cyclohexylthioureido)-3-methylbutyl)-3-(3,4,5-
agent should be filtrated through a 0.22- M filtration membrane
m
1
trimethoxyphenyl)acrylamide (404). H NMR (400 MHz, CCl
3
D-d)
1.61e1.77 (m, 5H); 1.
5.44e5.48
prior to use. ATP, malachite green, polyvinyl alcohol and ammo-
nium molybdate tetrahydrate were purchased from SIGMA and
used without further purification.
d
9
(
0.90e0.93 (m, 6H);
2e2.15 (m, 2H); 3.89 (m, 9H);
m, 1H); 6.19 (s, 1H);
d
1.29e1.35 (m, 5H);
d
d
d
d4.09e4.11 (m, 1H); d
d
d6.47e6.51 (d, 1H);
d6.76e6.85 (m, 2H);
For compound screening, an aliquot of a master mixture of
d
7.27e7.34 (d, 1H);
d
7.57e7.61 (d, 1H); 8. 27 (s, 1H). MS 464.4
d
DnaK:DnaJ (2.0
X-100, 100 mM TriseHCl, 20 mM KCl, and 6 mM MgCl
added into each well of a 96-well plate (14 L per well). To this
solution, 1 L of either one of the test compounds (5 mM) or DMSO
m
M:3.5
m
M) prepared in assay buffer (0.017% Triton
þ
[MþH] .
2
, pH 7.4) was
m
5
.2.1.29. 5.2.29. (E)-N-(1-(3-cyclohexylthioureido)-2,2-
(405).
1.20e1.31 (m, 7H);
2.04 (s, 1H); 3.79 (s,
6.01e6.04 (s, 1H);
m
dimethylpropyl)-3-(3,4,5-trimethoxyphenyl)acrylamide
was added, and the plate was shaken for 10 min and incubated for
1
ꢁ
H NMR (400 MHz, DMSO-d6)
1.57e1.60 (d, 1H); 1.67 (s, 2H);
H); 3.86 (s, 1H); 5.05e4.09 (m, 1H);
6.67e6.74 (d, 1H); 6.95 (s, 2H);
H); 7.42e7.45 (d, 1H);
DMSO, 100 MHz) 181.00; 164.45; 152.99; 139.32; 138.72; 130.43;
21.30; 104.96; 65.50; 59.99; 59.61; 55.80; 52.16; 35.77; 32.15;
d0.89 (s, 9H);
d
30 min at 37 C.
d
3
d
1
(
1
d
d1.91 (s, 2H)
d
d
Ten microlitres of 2.5 mM ATP was added to start the reaction.
d
d
d
Thus, the final reaction volume was 25
the tested compounds was 200
80
the plates were shaken gently. Immediately following this step,
10 L of 34% sodium citrate was added to terminate the nonenzy-
m
L, and the concentration of
ꢁ
d
d
7.21e7.25 (d, 1H);
d
7.24e7.26 (d,
m
M. After 3 h of incubation at 37 C,
13
d
d
7.68e7.70 (d, 1H);
d8.16 (s, 1H). C NMR
mL of the malachite green reagent was added into each well, and
d
m
þ
3
2
1.98; 25.38; 25.11; 24.27; 13.98. MS 464.4 [MþH] . The results of
matic hydrolysis of ATP. After the samples were mixed thoroughly
13
1
ꢁ
D
C, H COSY(HMQC) spectra of compound 405 was shown in
and incubated at 37 C for 15 min, the OD620 was measured using a
Table 6.
SpectraMax M5 (Molecular Devices, Sunnyvale, CA, USA).
5
.2.1.30. (E)-N-(1-(3-cyclohexylthioureido)-2-methylpropyl)-3-
6.2. Screening of resistance reversal agents
1
(
3,4,5-trimethoxyphenyl)acrylamide (406). H NMR (400 MHz, -d)
d
d
1.02e1.34 (m, 11H);
4.09e4.11 (m, 1H);
d
1.63e2.05 (m, 6H);
5.14e5.18 (m, 1H); 6.17 (s, 1H);
7.57e7.61 (d, 1H); 8.02 (s, 1H). MS
d
3.87e3.89 (m, 9H);
The breast cancer cell lines BT474 and BT/LapR1.0 were selected
for the in vitro evaluation of the cell viability and coefficient of drug
interaction (CDI) of the thiourea derivatives. The epidermal growth
factor (EGFR: ErbB-1, ErbB-2) tyrosine kinase inhibitor lapatinib
d
d
d6.55e6.59
(
d, 1H);
d
6.74e6.78 (m, 2H);
d
d
þ
4
50.2 [MþH] .
(10 mM in DMSO, BioVision, Cat: 1624-100, Lot: 50324) was used as
5
.2.1.31. N-(1-(3-cyclohexylthioureido)-3-methylbutyl)cinnamamide
the positive control. The cell viability was measured using the
CellTiter-Glo® kit (Promega, Part: G755B, Lot: 32513501, EXP: 2014-
05). The cells were digested in 1 mL of 0.25% trypsin (Gibco) sup-
1
(
d
(
d
d
407).
H
NMR (400 MHz, DMSO-d
1.03e1.33 (m, 6H); 1.53e1.66 (m, 7H);
6.63e6.67 (d, 1H, J ¼ 15.68 Hz);
7.48e7.52 (d, 1H, J ¼ 15.68 Hz); 7.56e7.62 (d, 2H, J ¼ 6.76 Hz);
8.69 (s, 1H). CNMR (DMSO, 100 MHz) 21.80; 22.22;
4.24; 24.28; 25.10; 31.82; 32.20; 42.19; 52.48; 57.25; 121.17;
6
)
d
d
0.88e0.89 (m, 6H);
1.82e1.91 (m, 2H); 4.01
7.37e7.45 (m, 5H);
d
d
s, 1H);
d
d
plemented with 2 mL of medium (containing 10% FBS, Gibco) and
5
d
seeded (1 ꢀ 10 cells/mL, 50
m
L/well, 5000 cells/well) into 96-well
13
ꢁ
7.76 (s, 1H);
d
d
plates. The plates were incubated for 24 h at 37 C. The serially
diluted compounds and control were added into each well, and the
cells were incubated for 72 h at 37 C. A volume of the CellTiter-Glo
2
þ
ꢁ
1
27.52; 128.84; 134.58; 139.40; 165.14; 179.33. MS 374.3 [MþH] .
13
1
The results of 2D C, H COSY(HMQC) spectra of compound 407
Reagent (50 mL) equal to the volume of the cell culture medium