Q. Xiao, et al.
Bioorganic&MedicinalChemistry28(2020)115722
Table 3
Michaelis–Menten kinetics for phosphorylation of FTY720, IMMH001, 1, and 2 in whole blood.
Compound
R
Rat
Human
Vmax
Vmax
Km
CLint
Km
CLint
(pmol/min)
(μmol/L)
(μL/min)
(pmol/min)
(μmol/L)
(μL/min)
FTY720
–
61.3
10.2
64.6
11.2
0.950
0.437
0.334
0.175
0.31
0.53
2.63
2.15
0.03
23.7
3.9
0.013
0.012
0.049
0.025
IMMH001
CH2OH
H
37.24
16.15
4.13
6.9
3.9
85.25
48.37
23.60
12.6
0.06
0.78
0.43
42.62
54.12
86.55
5.7
1
2
7.6
4.2
10.7
11.2
CH3
0.38
Fig. 4. Substrate–velocity curves for phosphorylation of IMMH001, 1, and 2 in whole blood.
3.93–3.92 (m, 2H), 2.84–2.81 (m, 2H), 2.75–2.71 (m, 2H), 1.99–1.95
(m, 2H), 1.89–1.83 (m, 2H), 1.03 (t, 3H, J = 8.0 Hz); 13C NMR
(100 MHz, CDCl3): δ 165.5, 159.3, 140.1, 140.0, 139.2, 139.0, 133.1,
132.2, 132.1, 128.7, 128.6, 128.5, 127.3, 127.2, 126.0, 70.1, 52.1,
36.8, 31.5, 30.2, 20.7, 13.7. ESI-HRMS (m/z): calcd for C23H25N2O3 [M
+H]+ 377.1860, found 377.1861.
C22H27N2O2 [M + H]+ 351.2067, found 351.2061.
4.1.5.2. (R)-2-Amino-4-(4′-(2-propyloxazol-4-yl)-[1,1′-biphenyl]-4-yl)-2-
methylbutan-1-ol hydrochloride (2). White solid, yield 81%, mp: 214 °C;
20
1
[α]D –1.61 (c 0.2, CH3OH). H NMR (400 MHz, CD3OD): δ 8.27 (s,
1H), 7.80 (d, 2H, J = 8.0 Hz), 7.68 (d, 2H, J = 8.0 Hz), 7.62 (d, 2H,
J = 8.0 Hz), 7.35 (d, 2H, J = 8.0 Hz), 3.68–3.65 (m, 1H), 3.60–3.55
(m, 1H), 2.90–2.86 (m, 2H), 2.74–2.73 (m, 2H), 1.90–1.85 (m, 4H),
1.38 (s, 3H), 1.05 (t, 3H, J = 8.0 Hz); 13C NMR (150 MHz, CDCl3): δ
167.8, 142.1, 141.9, 140.7, 139.9, 136.0, 130.0, 128.3, 128.2, 127.2,
66.3, 58.8, 38.7, 30.8, 30.2, 21.6, 20.4, 14.0. ESI-HRMS (m/z): calcd for
4.1.4.2. (R)-4-(2-(4′-(2-propyloxazol-4-yl)-[1,1′-biphenyl]-4-yl)ethyl)-4-
methyloxazolidin-2-one (14b). White solid, yield 90%, mp: 185 °C; [α]D
20 14.4 (c 0.5, CHCl3); 1H NMR (400 MHz, CDCl3): δ 7.86 (s, 1H), 7.79
(d, 2H, J = 8.0 Hz), 7.61 (d, 2H, J = 8.0 Hz), 7.55 (d, 1H, J = 8.0 Hz),
7.25 (d, 1H, J = 8.0 Hz), 5.42 (bs, 1H), 4.22 (d, 1H, J = 8.0 Hz), 4.10
(d, 1 H, J = 8.0 Hz), 2.86–2.75 (m, 2H), 2.74–2.70 (m, 2H), 1.97–1.93
(m, 2H), 1.89–1.84 (m, 2H), 1.44 (s, 3H), 1.04 (t, 3H, J = 8.0 Hz);
13C NMR (100 MHz, CDCl3) δ 165.6, 158.7, 140.3, 139.9, 139.8, 138.8,
133.2, 129.8, 128.7, 127.3, 127.2, 125.9, 75.6, 57.6, 42.2, 30.1, 30.0,
26.0, 20.6, 13.7; ESI-HRMS (m/z): calcd for C24H27N2O3 [M+H]+
391.2016, found 391.2011.
C
23H29N2O2 [M+H]+ 365.2224, found 365.2212.
4.2. Phosphorylation level evaluation
The LC–MS/MS system consisted of a Surveyor auto-sampler, a
Surveyor LC pump, a TSQ Quantum Access™ triple quadrupole mass
spectrometer with an electrospray ionization (ESI) source and Xcalibur
2.0 software for data acquisition and analysis spectrometer (Thermo
Finnigan, Santa Clara County, CA, USA). The analytes were chroma-
tographed by injection of a 10 µL sample into a ZorbaxSB-C18 column
(3.5 µm, 2.1 mm × 100 mm). The mobile phase consisted of solvent A
(0.1% formic acid in water) and solvent B (0.1% formic acid in me-
thanol). The analytes were eluted, at a flow rate of 0.2 mL/min, with
the following gradient elution: from 0 to 1.5 min, 5% B; linear increase
to 95% B in 0.2 min; 95% B during 3 min, decrease to 5% B in 0.2 min;
and stabilization at initial conditions for 3 min. The mass spectrometer
was set for multiple-reaction monitoring and was operated in a positive-
ion mode with ESI source. The spray voltage was set at 3700 V, and the
sheath gas and auxiliary gas at 30 and 15 psi. The capillary temperature
was set at 350 °C. The transition ion pairs were at m/z 308.3/255.3 for
FTY720, m/z 388.3/255.3 for FTY720-P, m/z 351.3/334.3 for
IMMH001, m/z 431.3/316.3 for IMMH001-P, m/z 351.3/334.3 for 1,
m/z 431.3/304.3 for 1-P, m/z 365.3/348.3 for 2, m/z 445.3/318.4 for
2-P.
4.1.5. General procedure for the synthesis of compounds 1 and 2
Compounds
14a/b
was
diluted
in
the
solvent
of
methanol:H2O = 10:1 (v:v), then potassium hydroxide (10 equiv) was
added, and the reaction was refluxed for 20 h. After cooling to room
temperature, water was added into the reaction mixture and extracted
with CH2Cl2. The combined organic layer was washed with water and
brine, dried over Na2SO4, filtered, and evaporated. The crude product
was chromatographed (silica gel, dichloromethane/methanol = 10:1),
and then acidification with 1 mol/L HCl in Et2O (2 mL) to afford the
desired products 1 and 2.
4.1.5.1. (R)-2-Amino-4-(4′-(2-propyloxazol-4-yl)-[1,1′-biphenyl]-4-yl)
20
butan-1-ol hydrochloride (1). White solid, yield 80%, mp: 210 °C; [α]D
–6.76 (c 0.3, CH3OH). 1H NMR (400 MHz, CD3OD): δ 8.19 (s, 1H), 7.79
(d, 2H, J = 8.0 Hz), 7.65 (d, 2H, J = 8.0 Hz), 7.60 (d, 2H, J = 8.0 Hz),
7.35 (d, 2H, J = 8.0 Hz), 3.83–3.81 (m, 1H), 3.65–3.62 (m, 2H),
3.31–3.24 (m, 2H), 2.84–2.80 (m, 4H), 1.99–1.82 (m, 4H), 1.03 (t, 3H,
J = 8.0 Hz); 13C NMR (100 MHz, CD3OD): δ 165.9, 140.2, 139.8,
139.7, 138.5, 134.0, 129.8, 128.5, 128.5, 128.2, 126.7, 126.6, 125.5,
60.5, 52.7, 30.8, 30.7, 29.3, 20.2, 12.5. ESI-HRMS (m/z): calcd for
Declaration of Competing Interest
The authors declare that they have no known competing financial
7