EXPERIMENTAL
Melting point (mp) was determined using an X-4 micro melting point apparatus (INESA, Shanghai, China). NMR
spectra were recorded in CDCl on a Bruker Biospin GmbH spectrometer with TMS as the internal standard. Mass spectra
3
were obtained using an Agilent 6330 ion trap mass spectrometer (ESI-MS). Flash column chromatography was performed on
silica gel (200–300 mesh, Qingdao, China). Thin-layer chromatography (TLC) was performed on precoated silica gel plates
(Merck GF254). All other reagents and solvents were commercially available and used without further purification.
3,5,6-Trimethylpyrazin-2-yl)methanol (1a). TMP (1, 10.8 g, 80 mmol) in acetic acid (16 mL) was quickly added to
H O (30%, 9 mL), and the mixture was stirred for 3 h at 75ꢆC. TLC was performed to detect the product. Sodium hydroxide
2
2
solution (50%) was added and the pH was adjusted to 10. Next, the solution was extracted with CHCl (50 mL ꢇ 3), washed
3
and dried with anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a white solid (TMP N-oxide, m).
TMP N-oxide (used directly without further purification) was dissolved in acetic anhydride (20 mL), stirred for 3 h at
150ꢆC, concentrated, and added to a sodium hydroxide solution (20%, 75 mL), and the mixture was stirred overnight at
ambient temperature. The mixture was extracted with CHCl , dried with anhydrous sodium sulfate, and concentrated under
3
reduced pressure to obtain the crude product. This was recrystallized with petroleum ether to yield 1a as a canary yellow
+
1
acicular crystals (7.17 g, 59% for two steps), mp 89ꢆC. ESI-MS m/z 153 [M + 1] , C H N O. H NMR (400 MHz, CDCl ,
8
12
2
3
13
ꢈ, ppm): 2.41–2.53 (9H, m, CH ), 4.22–4.25 (1H, t, OH), 4.68 (2H, d, CH ). C NMR (100 MHz, CDCl , ꢈ, ppm): 19.21,
3
2
3
21.17, 21.28, 60.83, 146.65, 147.39, 147.57, 149.50.
3,5,6-Trimethylpyrazine-2-carbaldehyde (1b). Compound 1a (6.08 g, 40 mmol) was dissolved in CHCl (60 mL),
3
MnO (24 g, 280 mmol) was added, and the mixture was refluxed for 6 h, cooled, filtered, and concentrated. The crude product
2
was purified by column chromatography and eluted with ethyl acetate–petroleum ether (1:6) to yield 1b as bright yellow
+
1
crystals (4.2 g, 70%), mp 86ꢆC. ESI-MS m/z 151 [M + 1] , C H N O. H NMR (500 MHz, CDCl , ꢈ, ppm): 10.10 (1H, s,
8
10
2
3
13
CHO), 2.75 (3H, s, CH ), 2.56 (6H, s, CH ꢇ 2). C NMR (125 MHz, CDCl , ꢈ, ppm): 21.39, 22.18, 141.91, 149.98, 151.54,
3
3
3
155.45, 194.33.
(2E)-3-(3,5,6-Trimethylpyrazin-2-yl)acrylic Acid (1c). Compound 1b (3.0 g, 20 mmol), together with malonic
acid (4.16 g, 40 mmol), was dissolved in pyridine (6 mL). Piperazine (0.344 g, 4 mmol) was added and the mixture was
refluxed for 2 h at 120ꢆC. When the reaction cooled, the mixture was diluted with CH Cl and placed in an ice bath, and
2
2
concentrated hydrochloric acid was added. Solids were removed by filtration, and the filtrate was concentrated under reduced
pressure to obtain the crude product, which was recrystallized with EtOH to yield a canary yellow powder (2.3 g, 60%),
+ 1
mp 273ꢆC, C H N O . ESI-MS m/z 193 [M + 1] . H NMR (500 MHz, CDCl , ꢈ, ppm, J/Hz): 7.90 (1H, d, J = 15.3, CH),
10 12
2
2
3
13
7.04 (1H, d, J = 15.3, CH), 2.62 (3H, s, CH ), 2.53 (6H, d, J = 6.3, CH ꢇ 2). C NMR (125 MHz, CDCl , ꢈ, ppm): 20.32
3
3
3
(CH ), 21.67 (CH ꢇ 2), 122.99, 140.16, 142.66, 148.94, 150.34, 152.77.
3
3
®
Assessment of Antitumor Activity by MTS Assay. Cell proliferation was measured using the CellTiter 96
A
AQ
One Solution cell proliferation assay (Promega, Madison, WI, USA). Cells were harvested in the logarithmic growth
ueous
4
phase, seeded into 96-well plates at a density of 3 ꢇ 10 cells/mL, and cultured in RPMI 1640 medium containing 5% FBS at
37ꢆC in a humidified incubator (5% CO ) for 24 h before exposure to various concentrations of compounds for a further 24 h.
2
Next, 10 ꢀL of MTS (Progema, Madison, WI) was added to each well, and the cells were incubated for an additional 1 h at
37ꢆC to convert the MTS into formazan. Three independent experiments were performed, and cell growth inhibition was
calculated according to the following equation:
Growth inhibition = (1 – OD of treated cells/OD of control cells) ꢇ 100%.
Half maximal inhibitory concentrations (IC ) were obtained from linear regression analysis of the concentration–
50
response curves plotted for each tested compound.
5
3
Dual-Luciferase Reporter Gene Assay. HEK293 cells were plated at a density of 10 cells/cm in 96-well plates in
100 ꢀL of DMEM basic medium containing 10% fetal bovine serum, 100 U/mL penicillin, and 0.1 mg/mL streptomycin.
Following overnight incubation, humanADRA-expression vectorsADRA1A, ADRA1B, orADRA1D or anADRB2-expression
vector were co-transfected with reporter vectors (CRE) and pGL4.74 [hRluc/TK] (as a control for transfection efficiency) into
cells using Lipofectamine 2000 transfection reagent (Invitrogen Life Technologies, CA, USA). The medium was removed
after 4 h and replaced with high-glucose DMEM for 18 h. Cells were then treated with agonist (PE, at concentrations of 10 ꢀM)
and antagonists (1, 3, and 10 ꢀM) and incubated for 8 h.
Firefly and Renilla luciferase activities, expressed as relative light units (RLUs), were determined using a Dual-Glo
luciferase assay kit (Promega) according to the manufacturer’s instructions. RLUs were measured using a luminometer
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