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MedChemComm
1
3
(
dt, J = 17.1, 6.0 Hz, 3H, 3 × CH, arom.). C-NMR (75 MHz,
into a solution of compound 6 (200 mg, 0.86 mmol) in anhy-
drous THF (30 ml). The mixture was stirred at room tempera-
ture until the reaction was completed as monitored by TLC.
CDCl ) δ: 20.47, 20.53, 20.67, 20.77, 21.57, 21.69, 35.86,
7.53, 72.37, 72.91, 118.89, 123.62, 124.74, 127.78, 133.48,
34.96, 141.22, 142.01, 146.78, 147.76, 149.29, 151.24,
3
3
1
1
The mixture was poured into saturated aqueous NH Cl
4
+
68.53, 168.63, 168.91, 170.16. MS (ESI): m/z [M + H] 499.0,
(30 ml) solution, and was then extracted with ethyl acetate
(3 × 20 ml). The combined organic layers were dried over
Na SO and the solvent was removed in vacuo. The crude
+
m/z [M + Na] 521.1. Anal. calcd for C24
6
H
28
N
2
O
8
: C, 62.64; H,
.07; N, 5.62. Found: C, 62.42; H, 6.14; N, 5.44.
Ethyl 3,5,6-trimethylpyrazine-2-carboxylate (5). A mixture
of TMP (5 g, 36 mmol) and KMnO (7 g, 44 mmol) in anhy-
drous ethanol (150 ml) was stirred at 50 °C for 10 h. The
solid was filtered, and the filter cake was washed with cold
water. The filtrate was washed with ethyl acetate (3 × 150 ml),
and acidified with 2 N HCl to adjust the pH of the solution to 3.
The water layer was washed with ethyl acetate (3 × 100 ml).
The combined organic layers were dried over Na
concentrated under reduced pressure to give a crude product
2.5 g). The crude product was then used in the next step
without further purification. To the crude (2 g) in ethanol
80 ml) was added 4-dimethylaminopyridine (DMAP) (1.8 g,
4 mmol) and 1-IJ3-dimethylaminopropyl)-3-ethylcarbodiimide
EDCI) (2.8 g, 14 mmol). The mixture was stirred at room
2
4
product was purified by column chromatography (petroleum
4
ether/ethyl acetate, 3/1) to afford compound 7 as a yellow
1
oil (310 mg, 67ꢀ yield). H-NMR (300 MHz, CDCl ) δ: 2.05
3
(s, 3H, CH ), 2.30 (s, 6H, 2 × CH ), 2.49 (s, 6H, 2 × CH ), 2.62
3
3
3
(s, 3H, CH
3
), 2.93–3.31 (m, 6H, 3 × CH
2
), 4.94–5.16 (m, 4H,
CH ), 5.28 (dd, J = 10.5, 3.3 Hz, 1H, CH), 5.44–5.73 (m, 2H, 2
2
1
3
× CH), 7.13 (t, J = 6.1 Hz, 3H, 3 × CH, arom.). C-NMR
(75MHz, CDCl ) δ: 20.51, 20.65 (2C), 21.33, 21.46, 21.78,
2
SO
4
and
3
36.87, 40.09, 40.33, 72.24, 77.22, 88.03, 119.12, 119.24,
123.37, 124.31, 127.25, 132.19, 132.28, 135.19, 141.03, 141.91,
147.10, 147.23, 149.21, 167.57, 168.18, 168.26, 170.02. MS
(
+
(
1
(
(ESI): m/z [M + H] 539.2. Anal. calcd for C H N O : C,
2
5
30 2 8
64.67; H, 6.36; N, 5.20. Found: C, 64.20; H, 6.32; N, 4.60.
temperature overnight. The solution was condensed and the
residue was dissolved in cold water (30 ml). The pH of the
solution was adjusted to 4–5 with 2 N HCl, followed by extrac-
tion with ethyl acetate (3 × 30 ml). The combined organic
layers were dried over Na SO , filtered and the solvent was
2 4
removed in vacuo. The resulting residue was purified by
Stability assay in vitro
The tested compounds were dissolved in DMSO (10 ml) to
make solution A (10.9 × 10 μM). To 600 μl of solution A was
added 100 μl of Tween 80, and the solution was then diluted
with physiological saline to make 10 ml of solution B (the final
concentration of each compound was 0.655 × 10 μM). Solu-
tion B (2 ml) was pre-warmed at 37 °C for 5 min, and was
then added 6.8 μl of carboxylesterase solution (equal to
3
3
column chromatography (petroleum ether/ethyl acetate, 3/1)
to afford compound 5 as a colorless oil (1.4 g, 60ꢀ yield).
1
H-NMR (300 MHz, CDCl
3
) δ: 1.44 (t, J = 7.1 Hz, 3H, CH
3
),
2
4
units per μmol substrate). At 2, 4, 8, 16, 32 and 64 min,
00 μl of methanol was added to 200 μl of this mixed solution.
2
.57 (d, J = 2.1 Hz, 6H, CH ), 2.75 (s, 3H, CH ), 4.47 (q,
3
3
+
J = 7.1 Hz, 2H, CH
2
). MS (ESI): m/z [M + H] 194.9. Anal.
Ĵ0.25 H O: C, 60.44; H, 7.35; N, 14.10.
Found: C, 60.14; H, 7.20; N, 14.28.
-IJ3,5,6-Trimethylpyrazin-2-yl)hepta-1,6-dien-4-ol (6). Com-
pound 5 (200 mg, 1 mmol) was dissolved in anhydrous THF
10 ml) and was stirred under nitrogen on an ice bath. Allyl-
The resulting solution was vortex-mixed for 30 s, followed by
centrifugation at a rate of 10000 rpm for 3 min. The superna-
tant (20 μl) was analyzed by HPLC.
calcd for C10
H
14
N
2
O
2
2
4
Biological evaluations
(
magnesium bromide (3.0 ml, 3.0 mmol) was added dropwise.
The reaction mixture was stirred overnight at room tempera-
ture. The reaction mixture was poured into aqueous saturated
Protective effect against t-BHP-induced injury in H9c2
cells. H9c2 cells were cultured in low glucose Dulbecco's
Modified Eagle's Medium (DMEM) (Hyclone) supplemented
with 10ꢀ fetal bovine serum (FBS, Gibco) and 100 units per
ml penicillin–streptomycin (Gibco). Cells were seeded in 96-
well plates and were incubated in a humidified atmosphere
NH
4
Cl (20 ml) solution and washed with ethyl acetate (3 × 20 ml).
SO , filtered
The combined organic layers were dried over Na
2
4
and the solvent was removed in vacuo. The resulting residue
was purified by column chromatography (petroleum ether/
(37 °C, 5ꢀ CO ) until grown to 90ꢀ confluence. To evaluate
2
ethyl acetate, 3/1) to afford compound 6 as a colorless oil
the protective effects of the new compounds, cells were
pretreated with drugs at various concentrations for 1 h,
followed by exposure to 150 μM t-BHP for 12 h. MTT was
added, and cells were then incubated for another 4 h. The
medium was removed and DMSO (150 μl) was added subse-
quently. After incubation for 30 min in the dark, the absor-
bance was read on a microplate reader (Bio-Rad Model 680,
Japan) at a wavelength of 490 nm. Cell viability was expressed
as percentage to the control cells.
1
(190 mg, 80ꢀ yield). H-NMR (300 MHz, DMSO) δ: 2.40 (d,
J = 3.7 Hz, 6H, 2 × CH
3
), 2.54–2.71 (m, 7H, CH
3 2
, 2 × CH ),
4
5
.92 (ddd, J = 10.4, 2.4, 1.3 Hz, 4H, 2 × CH ), 5.41 (s, 1H, OH),
.63 (ddt, J = 17.3, 10.2, 7.1 Hz, 2H, 2 × CH). C-NMR (75 MHz,
2
13
DMSO) δ: 21.25, 21.46, 23.46, 44.64 (2C), 76.63, 117.85 (2C),
34.79 (2C), 146.31, 148.10, 148.78, 152.74. MS (ESI): m/z
1
+
+
[M + H] 233.0, m/z [M + Na] 255.6. Anal. calcd for C H N O:
14 20 2
C, 72.38; H, 8.68; N, 12.06. Found: C, 72.40; H, 8.85; N, 11.86.
IJR)-4-IJ2-Acetoxy-3-oxo-3-IJ(4-IJ3,5,6-trimethylpyrazin-2-yl)-
hepta-1,6-dien-4-yl)oxy)propyl)-1,2-phenylene diacetate (7).
Compound 3 in 20 ml of anhydrous THF was added dropwise
Protective effect against Dox-induced injury in H9c2 cells.
Compound 7 was chosen to be further evaluated in Dox-induced
injury in H9c2 cells. Cells were cultured in low glucose
590 | Med. Chem. Commun., 2015, 6, 586–591
This journal is © The Royal Society of Chemistry 2015