6
Journal of Chemical Research
J=6.46Hz, 3H). 13C NMR (150MHz, CD3OD): δ 173.76,
171.74, 152.69, 118.68, 75.81, 66.69, 64.41, 33.48, 31.62,
29.15, 28.99, 28.98, 28.77, 24.57, 22.30, 13.01. HRMS
(ESI): m/z [M+H]+ calcd for C16H27O7: 331.1835; found:
331.1753.
Table 4. Inhibition studies of AP derivatives against SphK1 at
10μM.
Compound
Inhibition (%)a
Compound
Inhibition (%)
VC
3b
3d
NT
34
44
3a
3c
28
32
(S)-2-((R)-3,4-dihydroxy-5-oxo-2,5-dihydrofuran-2-yl)-
2-hydroxyethyl nonanoate (3b). White solid; 90% yield.
1H NMR (600MHz, CD3OD): δ 4.73 (d, J=2.03Hz, 1H),
4.16-4.27 (m, 2H), 4.06-4.12 (m, 1H), 2.37 (t, J=7.4Hz,
2H), 1.59-1.68 (m, 2H), 1.25-1.39 (m, 10H), 0.90 (t,
J=6.43Hz, 3H). 13C NMR (150MHz, CD3OD): δ 174.02,
171.73, 152.65, 118.69, 75.80, 66.68, 64.43, 37.08, 32.72
(2C), 32.05, 31.10, 26.24, 25.94 (2C). HRMS (ESI): m/z
[M+H]+ calcd for C15H25O7: 317.1522; found: 317.1548.
VC: vitamin C; NT: no activity detected.
aValues are percent inhibitions against SphK1 at 10μM, averages of two
separate experiments, standard deviations were ±5%.
with an IC50 value of 40µM. Although AP displayed rela-
tively poor activity compared to cisplatin, it could still be
utilized as an excellent lead for the further development of
SphK1 inhibitors due to its outstanding effect on SphK1
and the fact that it is known to be non-toxic.
(S)-2-((R)-3,4-dihydroxy-5-oxo-2,5-dihydrofuran-2-yl)-
2-hydroxyethyl (1S,4r)-4’-pentyl-[1,1’-bi(cyclohexane)]-
4-carboxylate (3c). White solid; 94% yield. H NMR
1
Experimental
(600MHz, CD3OD): δ 4.73 (d, J=2.09Hz, 1H), 4.24-4.27
(m, 1H), 4.17-4.21 (m, 1H), 4.07-4.10 (m, 1H), 3.67 (t,
J=4.54Hz, 3H), 3.56 (t, J=4.95Hz, 3H), 2.37 (t,
J=7.43Hz, 2H), 1.60-1.65 (m, 2H), 1.24-1.39 (m, 18H),
0.89 (t, J=6.99Hz, 3H). 13C NMR (150MHz, CD3OD): δ
173.75, 171.73, 152.57, 118.69, 75.79, 72.12 (2C), 66.66,
64.40, 60.86 (2C), 33.47, 31.67, 29.33 (3C), 29.20, 29.06
(2C), 29.00, 28.78, 24.58, 22.33, 13.04. HRMS (ESI): m/z
[M+H]+ calcd for C24H39O7: 438.2618; found: 439.2686.
Materials
Vitamin C, thionyl chloride, N,N-dimethy-lacetamide,
dichloromethane, sodium bicarbonate, fetal bovine serum,
and other materials were obtained from commercial sources
and were used without further purification.
Apparatus
High-resolution mass spectrometry (HRMS) was per-
formed on an AB SCIEX X500R Accurate Mass Q-TOF
using electrospray ionization (ESI). The NMR spectra
were recorded on a Bruker AM-600 spectrometer
(Billerica, MA, USA) with tetramethylsilane as the inter-
nal standard.
(S)-2-((R)-3,4-dihydroxy-5-oxo-2,5-dihydrofuran-2-yl)-
2-hydroxyethyl octanoate (3d). White solid; 92% yield.
1H NMR (600MHz, CD3OD): δ 4.74 (d, J=2.02Hz, 1H),
4.17-4.28 (m, 2H), 4.07-4.11 (m, 1H), 2.37 (t, J=7.43Hz,
2H), 1.57-1.67 (m, 2H), 1.24-1.39 (m, 8H), 0.90 (t,
J=6.42Hz, 3H). 13C NMR (150MHz, CD3OD): δ 173.85,
171.76, 152.61, 118.72, 75.83, 66.74, 64.42, 33.57, 31.46,
28.78, 28.70, 24.61, 22.29, 13.13. HRMS (ESI): m/z
[M+H]+ calcd for C14H23O7: 303.1366; found: 303.1433.
The 1H and 13C NMR spectra for compounds 3a–d in detail
can be found in the Supporting Information.
Synthesis
The synthesis of AP derivatives was carried out using a
“one-pot” method. Scheme 1 shows the general synthetic
method for the compounds 3a–d. Carboxylic acid (10mmol)
with different substituents, DMAc (15mL), and CH2Cl2
(5mL) were added to a flask and the mixture stirred at room
temperature. Next, SOCl2 (13mmol) was slowly added drop-
wise at 0°C. The mixture was reacted at 10°C for 0.5h, and
then l-ascorbic acid (12mmol) was added. The resulting
mixture was stirred, heated, and maintained at 25°C for 1h.
A new spot appeared according to thin-layer chromatogra-
phy (TLC). The reaction mixture was extracted with ethyl
acetate, and the combined organic layers were washed with
brine and dried over Na2SO4. The organic layer was filtered
and concentrated under reduced pressure to dryness to pro-
vide the crude product, which was purified by silica gel chro-
matography to give the desired product. The Supporting
Information contains the NMR data of compounds 3a–d.
The kinase test in vitro
The SphKs assays were carried out as described previously.19
The assay was performed using Kinase-Glo Plus lumines-
cence kinase assay kit (purchased from Promega, Fitchburg,
USA). It measures kinase activity by quantitating the amount
of ATP remaining in solution following a kinase reaction.
Here, we used PF-543 (purchased from Selleckchem,
Houston, TX, USA) as a positive control. The reaction sys-
tem was 50μL. Compounds S2532 and S2118 were dis-
solved in pure DMSO to prepare 10mM stock solutions and
diluted with Kinase buffer (pH=7.4, composition: 40mML−1
Tris, 10mML−1 MgCl2, 0.1gL−1 BSA, 1mML−1 DTT, and
10µML−1 ATP). SphK1/2 was added to 96-well plates,
which were then treated with desired concentrations of
S2532 or S2118 (0.01, 0.1, 1, 10, and 100µM) for 40min at
30°C. The ATP test solution was then added, and the mix-
tures were incubated at room temperature for 5min. The
luminescence was immediately measured using a microplate
(S)-2-((R)-3,4-dihydroxy-5-oxo-2,5-dihydrofuran-2-yl)-
2-hydroxyethyl decanoate (3a). White solid; 95% yield.
1H NMR (600MHz, CD3OD): δ 4.73 (d, J=2.01Hz, 1H),
4.19-4.28 (m, 2H), 4.07-4.10 (m, 1H), 2.37 (t, J=7.41Hz,
2H), 1.57-1.67 (m, 2H), 1.24-1.38 (m, 12H), 0.89 (t,