J. Guo, B. Chen, Y. Yu et al.
European Journal of Medicinal Chemistry 207 (2020) 112848
but-2-en-1-yl)-3-hydroxy-2-(methylamino)butanamide (8g). White
(9e). White solid, 41%. 1H NMR (400 MHz, CDCl3)
d 8.27 (s, 1H), 7.98
solid, 85%. 1H NMR (400 MHz, MeOD)
d
8.26 (d, J ¼ 18.7 Hz, 2H), 7.81
(s, 1H), 7.76 (s, 1H), 7.17 (t, J ¼ 5.8 Hz, 1H), 5.77 (d, J ¼ 5.3 Hz, 2H),
(s,1H), 5.92e5.77 (m, 2H), 4.63 (d, J ¼ 4.8 Hz, 2H), 3.86 (d, J ¼ 4.0 Hz,
2H), 3.76 (p, J ¼ 6.4 Hz, 1H), 2.81 (d, J ¼ 6.6 Hz, 1H), 2.31 (s, 3H), 1.13
4.55 (d, J ¼ 4.5 Hz, 2H), 4.01e3.93 (m, 2H), 3.88 (s, 1H), 3.62 (t,
J ¼ 19.5 Hz, 2H), 3.22 (s, 1H), 3.10 (d, J ¼ 5.0 Hz, 1H), 1.23 (s, 3H). 13
C
(d, J ¼ 6.4 Hz, 3H). 13C NMR (101 MHz, MeOD)
d
173.29, 159.28,
NMR (101 MHz, CDCl3) d 170.27, 158.35, 146.43, 146.00, 138.07,
148.91, 147.12, 138.30, 131.06, 130.99, 128.39, 127.37, 125.13, 121.52,
70.79, 67.84, 47.39, 39.82, 33.81, 18.79. HR-ESI-MS: [MþH]þ m/
z ¼ 399.0985, calcd for C16H20N4O3, found 399.0937.
130.94, 130.18, 128.02, 126.91, 124.35, 120.40, 116.52, 67.10, 66.26,
46.73, 39.37, 35.75, 18.66. HR-ESI-MS: [MþH]þ m/z ¼ 424.0938,
calcd for C16H20N4O3, found 424.0892.
4.2.4. General synthetic procedure for the preparation of 9a-e (with
9a as an example)
4.3. Thermal shift assay
A
mixture of NaH (10 mg, 0.25 mmol) and
3
(30 mg,
The affinities of the compounds binding to SeThrRS were firstly
evaluated by using a fluorescence-based thermal shift assay (TSA)
as previously described. In brief, 100 mM HEPES pH 7.5, 150 mM
0.0781 mmol) in anhydrous DMF (1.2 mL) was stirred at room
temperature for 1 h. This white suspension was added iodoethane
(18.3 mg, 0.117 mmol), and the resulting reaction mixture was
stirred at room temperature for 6 h. The resulting mixture was
quenched with water, and the aqueous solution was extracted with
EtOAc (3 ꢂ 3.0 mL). The combined organic layers were washed with
brine (3 ꢂ 3.0 mL), dried with anhydrous Na2SO4, filtered, and
concentrated under vacuum to give the crude product. The crude
product was purified by flash silica gel column chromatography
(CH2Cl2/MeOH ¼ 5/1) to afford 9a (10.3 mg, 32%) as white solid.
4.2.4.1. (2S,3R)-N-((E)-4-(6,7-dichloro-4-oxoquinazolin-3(4H)-yl)
but-2-en-1-yl)-2-(ethylamino)-3-hydroxybutanamide (9a). White
NaCl, 4 ꢂ SYPRO orange fluorescent dye, 8
compounds (or 0.5% DMSO as blank control) were added into 96
well PCR plate to prepare 20 L reaction system. The PCR plate was
mM SeThrRS, 0.5 mM
m
placed in a StepOnePlus real-time fluorescent quantitative PCR
instrument (Life Technologies Inc.), and incubated at 25 ꢀC for
10 min. Then, the temperature was raised from 25 ꢀC to 95 ꢀC at a
rate of 1 ꢀC/min, and the fluorescent signal was recorded every 30 s
during this period. The protein melting temperature (Tm) values
were calculated by fitting the curves of fluorescence intensity
versus temperature using the StepOne™ software v2.3. The data
were the average of three independent experiments.
solid, 32%. 1H NMR (500 MHz, MeOD)
d 8.28 (s,1H), 8.23 (s,1H), 7.80
(s,1H), 5.87e5.79 (m, 2H), 4.63 (d, J ¼ 4.7 Hz, 2H), 3.85 (d, J ¼ 3.3 Hz,
2H), 3.77 (dd, J ¼ 12.4, 6.2 Hz,1H), 2.91 (d, J ¼ 6.4 Hz,1H), 2.59e2.50
(m, 2H), 1.13 (d, J ¼ 6.2 Hz, 3H), 1.06 (t, J ¼ 7.0 Hz, 3H). 13C NMR
4.4. Enzyme inhibition assay
(126 MHz, MeOD)
d
173.68, 159.24, 148.91, 147.12, 138.29, 131.06,
The aminoacylation activities of SeThrRS in the presence of
130.97, 128.39, 127.36, 125.09, 121.50, 68.80, 67.87, 47.37, 42.45,
different compounds were detected by measuring ATP consump-
39.80, 18.76, 13.86. HR-ESI-MS: [MþH]þ m/z ¼ 413.1142, calcd for
tion in the reaction [30]. The 10
SeThrRS, 4 M ATP, 10 -threonine, 500
30 mM HEPES pH 7.5, 150 mM NaCl, 30 mM KCl, 40 mM MgCl2,
1 mM dithiothreitol, 0.1% BSA and 100 M each compound. The
reactions were incubated at room temperature for 3 h, then
stopped by adding 10 L Kinase-Glo® Reagent (Promega Inc.). The
mL reaction consisted of 25 nM
C
16H20N4O3, found 413.1094.
m
mM
L
mg/mL E. coli total tRNA,
4.2.4.2. (2S,3R)-N-((E)-4-(6,7-dichloro-4-oxoquinazolin-3(4H)-yl)
but-2-en-1-yl)-2-((2-fluoroethyl)amino)-3-hydroxybutanamide (9b).
m
White solid, 25%. 1H NMR (400 MHz, CDCl3)
d
8.29 (s, 1H), 7.97 (s,
1H), 7.77 (s, 1H), 7.52 (s,1H), 5.76 (t, J ¼ 3.6 Hz, 2H), 4.56 (s, 2H), 4.51
(d, J ¼ 3.9 Hz, 1H), 4.40 (d, J ¼ 3.5 Hz, 1H), 4.06 (dd, J ¼ 6.2, 4.4 Hz,
1H), 3.90 (s, 2H), 3.00 (dtd, J ¼ 13.2, 8.8, 4.5 Hz, 2H), 2.85 (ddd,
J ¼ 15.2, 9.8, 3.6 Hz, 1H), 1.20 (d, J ¼ 6.3 Hz, 3H). 13C NMR (101 MHz,
m
luminescence was read on a FlexStation 3 multimode microplate
reader (Molecular Devices Inc.). The reactions consisted of three
groups: the blank group without compounds, the experimental
group with compounds, and the control group without enzyme.
The inhibitory rate for each compound was calculated as inhibition
rate (%) ¼ (RLUcompound e RLUblank)/(RLUcontrol eRLUblank) ꢂ 100%.
The inhibitory rate was measured in three independent assays, and
the average value was provided.
CDCl3)
d 172.00, 158.30, 146.33, 146.03, 138.03, 130.91, 130.51,
128.05, 126.96, 123.98, 120.45, 82.4 (d, J ¼ 166.7 Hz), 67.06, 66.44,
48.25 (d, J ¼ 1.9 Hz), 46.71, 28.67, 18.65. HR-ESI-MS: [MþH]þ m/
z ¼ 431.1048, calcd for C16H20N4O3, found 431.1006.
4.2.4.3. (2S,3R)-N-((E)-4-(6,7-dichloro-4-oxoquinazolin-3(4H)-yl)
but-2-en-1-yl)-3-hydroxy-2-(isopropylamino)butanamide
(9c).
8.28 (d, J ¼ 11.4 Hz, 2H),
The compounds with an inhibitory rate higher than 50% at
100 mM were further measured for their IC50 values. The inhibitory
rates of the compounds at various concentrations were measured,
and IC50 values were calculated by fitting the curves with GraphPad
White oil, 28%. 1H NMR (500 MHz, MeOD)
d
7.83 (s, 1H), 5.83 (dt, J ¼ 10.6, 7.6 Hz, 2H), 4.64 (d, J ¼ 5.0 Hz, 2H),
3.85 (s, 2H), 3.79 (dd, J ¼ 12.6, 6.5 Hz, 1H), 3.02 (d, J ¼ 5.9 Hz, 1H),
2.75e2.68 (m, 1H), 1.14 (d, J ¼ 6.1 Hz, 3H), 1.04 (d, J ¼ 6.0 Hz, 3H),
Prism 7 using the function of inhibition rate (%) ¼ 100/(1 þ [I]/IC50
)
1.00 (d, J ¼ 6.1 Hz, 3H). 13C NMR (126 MHz, MeOD)
d
173.86, 159.26,
B, where B corresponds to the slope factor and [I] corresponds to
148.93, 147.15, 138.32, 131.00, 130.92, 128.41, 127.39, 125.17, 121.53,
67.84, 66.26, 47.35, 39.83, 26.71, 22.12, 20.82, 18.76. HR-ESI-MS:
[MþH]þ m/z ¼ 427.1298, calcd for C16H20N4O3, found 427.1255.
the compound concentration.
4.5. Isothermal titration calorimetry
4.2.4.4.
(2S,3R)-2-(allylamino)-N-((E)-4-(6,7-dichloro-4-
oxoquinazolin-3(4H)-yl)but-2-en-1-yl)-3-hydroxybutanamide (9d).
The thermodynamic binding constant of compound 8g with
SeThrRS was determined by isothermal titration calorimetry (ITC).
White solid, 78%. 1H NMR (500 MHz, CDCl3)
d
8.29 (s, 1H), 7.96 (s,
1H), 7.77 (s, 1H), 7.44 (s, 1H), 5.82e5.73 (m, 3H), 5.14 (d, J ¼ 17.1 Hz,
1H), 5.06 (d, J ¼ 10.1 Hz, 1H), 4.56 (s, 2H), 4.00 (d, J ¼ 4.8 Hz, 1H),
3.94e3.85 (m, 2H), 3.22 (d, J ¼ 4.7 Hz, 2H), 2.97 (s, 1H), 1.17 (d,
20 mM SeThrRS and 200 mM compound 8g were prepared with a
buffer containing 20 mM HEPES pH 7.5 and 150 mM NaCl, and
loaded onto the sample cell and injection syringe of MicroCal VP-
ITC microcalorimeter (MicroCal Inc.), respectively. The injection
J ¼ 6.0 Hz, 3H). 13C NMR (126 MHz, CDCl3)
d 172.23, 158.28, 146.30,
146.05, 138.04, 134.92, 130.92, 130.61, 128.07, 126.97, 123.97, 120.46,
115.81, 67.10, 65.61, 50.69, 46.71, 39.08, 18.62.HR-ESI-MS: [MþH]þ
m/z ¼ 425.1142, calcd for C16H20N4O3, found 425.1099.
volume of the first titration was 5
10 L with a 180 s interval between each injection. The dissociation
constant (Kd), thermodynamics of binding ( H and S) and stoi-
mL, and other 24 injections were
m
D
D
4.2.4.5. (2S,3R)-2-((cyanomethyl)amino)-N-((E)-4-(6,7-dichloro-
4-oxoquinazolin-3(4H)-yl)but-2-en-1-yl)-3-hydroxybutanamide
chiometry of binding (N) were determined by fitting the calori-
metric data using ORIGIN software (MicroCal Inc.).
10