4886 Wang et al.
Asian J. Chem.
N
N
N
Wells containing no drugs were used as blanks. The IC50
values were defined as the concentration of compounds that
produced a 50 % reduction of surviving cells and calculated
using Logit-method. Tumor cell growth inhibitory rate was
calculated in the following eqn. 1:
Br
a
b
3H2O
N
1
N
2
N
3
OH
8
O
OH
1
OH
O
OH
4'
N
12 13
9
5'
6'
N
Br
+
7
6
2
c
3'
10
11 14
O
O
OH
3
N
1'
2'
N
3
5
4
5
Sample group OD
Control group OD)
O
O
O
Inhibition (%) = 1−
×100 %
4
Scheme-I:Synthesis route of compound 5. Conditions and regents: (a)
Benzene, reflux, 10 h, 100 %; (b) CCl4, NBS, hv, reflux, 10-12 h,
70 %; (c) DMF, K2CO3, 85 ºC, 4 h, 31.4 %
Angiogenesis assay: Fertilized White Leghorn chicken
eggs, provided by ChineseAcademy ofAgricultural Sciences,
were placed in an incubator as soon as embryogenesis started
and were kept under constant humidity of 65 % at 37 ºC. On
day 7, a square window was opened on the shell and physio-
logical saline (0.1 mL) was injected in to detach the shell
membrane. Then gelatin sponges carrying the compound 5
stimulators at 10 and 40 µg/egg were implanted, respectively.
The control group was treated with physiological saline. The
windows were sealed with medical adhesive tape and the
incubation went on till the experiment day. The above steps
were performed under sterile condition. On the 11th day, the
tapes were removed and the entire CAM was detached after
tissue fixation with methanol/acetone (1:1, v/v). Then we used
computer-assisted tracking of images to obtain absolute values
for the number of microvessels which were 1-100 µm in
diameter. Data were analyzed using t-test of statistics analysis
system, the values were expressed as mean s of 6 observations
and p < 0.05 was considered significant.
dissolved in benzene (17 mL). The mixture refluxed for 10 h
to evaporate the water of crystallization and compound 2 was
gained. Compound 3 was prepared from compound 2 (TMP,
4.000 g, 0.029 mol) and N-bromosuccinimide (4.201 g, 0.024
mol) in refluxing carbon tetrachloride. The reaction mixture
was illuminated by a 60W tungsten light bulb for 12 h. Then
the mixture was filtered and the filtrate evaporated under
vacuum and the crude oil-product was gained. Compound 3,
with 70 % purity, was not made further purification as it caused
a strong mucous membrane irritation.
9,10-Dioxo-4,5-dihydroxy-9,10-dioxo-2-anthra-
cenecarboxylic acid-3,5,6-trimethylpyrazin-2-methyl ester
(compound 5): Compound 3 (2.320 g, 10.791 mmol) and Rhe
(3.064 g, 10.791 mmol) were dissolved in dry DMF, then
K2CO3 (3.475 g, 25.195 mmol) was added, the mixture was
kept at 85 ºC for 4 h. The warm reaction mixture was poured
into ice-water and the crude product was extracted with ethyl
acetate.After drying the organic layer over anhydrous Na2SO4
and evaporating the solvent under vacuum, the crude product
was separated by flash chromatography with petroleum
ether:ethyl acetate (5:1) as eluent and recrystallized from
acetone. Compound 5: yellow crystals, m.p. 220.7-221.6 ºC,
Stability test:According to "Pharmacopeia of the People's
Republic of China" (2010 Edition), we prepared artificial
gastric juice and artificial intestinal juice by dissolving pepsin
and trypsase in pH = 1.2 and pH = 6.8 buffer, respectively.
Then two samples of compound 5 (1.60 and 1.68 mg) were
precisely weighed and mixed with 50 mL artificial gastric juice
and 50 mL artificial intestinal juice, respectively. We keep the
two artificial digestive solution in swing bed at 37 ºC and
sampled 1 mL preparation at hour 0, 1, 2, 4, 6, 8, 12 and 24 h,
respectively. Each sample was treated by the addition of 3 mL
acetonitrile as protein precipitation agent.After centrifugation
at 4500 rpm for 10 min, the supernatant was filtrated with
0.22 µm filtration film and then 10 µL filtrate was injected to
HPLC analysis at a UV wavelength of 430 nm.
1
yield 31.4 %. H NMR (CDCl3) δ (ppm): 12.042 (s, 1H,
8-OH), 11.975(s, 1H, 1-OH), 8.434(s, 1H, 4-H), 7.878 (d, 1H,
5-H), 7.352 (d, 1H, 7-H), 7.746 (t, H, 6-H), 5.521 (s, 2H,
O-CH2), 5.52 (s, 2H, CH2-2'), 2.64 (s, 3H, CH3-6'), 2.59 (s,
3H, CH3-5'), 2.56 (s, 3H, CH3-3'). 13C NMR (CDCl3) δ (ppm):
162.4 (C1), 120.4 (C2), 137.8 (C3), 124.9 (C4), 120.4 (C5),
137.5 (C6), 125.5 (C7), 162.9 (C8), 192.8 (C9), 180.9 (C10),
134.0 (C11), 115.8 (C12), 118.4 (C13), 133.5 (C14), 164.1
(C15). Pyrazine ring: 66.5 (2'-CH2), 151.7 (C2'), 144.3 (C3'),
149.0 (C5'), 149.5 (C6'), 21.6 (6'-CH3), 21.5 (5'-CH3), 20.5
(3'-CH3). HRMS (ESI) m/z: 419.3173 [M + H]+, calcd. (%)
for C23H19N2O6 419.1243.
Acute toxicity: Kunming mice (BeijingVital River Labo-
ratory Animal Technology Company Limited, China) of both
sexes, weighing 18-22 g, were divided into four groups of 10
animals matched in weight and size. The mice were placed in
cages and kept under standard environmental conditions with
a standard rodent diet and water ad libitum under a 12 h light-
dark cycle. They were deprived of food for 24 h but allowed
free access to tap water throughout the experiments. This
research was carried out in accordance with the "Regulation
for the Administration of Affairs Concerning Experimental
Animals" (State Council of China, 1988).
Bio-evaluation methods
Cytotoxicity evaluation: The cytotoxicity of 5 was tested
on human cancer cell line of Bel-7402 by the standard MTT
assay. The cancer cell Bel-7402 was provided by Chinese
Academy of Medical Sciences & Peking Union Medical
College. The growing tumor cells at a density 104 cells/mL
were exposed to various concentrations of the tested drugs
and incubated in a 96-well microtiter plate for 96 h (37 ºC,
5 % CO2). After MTT solution (20 µL, 5 mg/mL) was added
to each well, the plate was incubated for a further 4 h. Then
the media was removed. Formazan crystals were dissolved
with DMSO (150 µL). After mixing well, the absorbance was
quantified at 570 nm with a BIORAD 550 spectrophotometer.
The maximum suspended dose (80 mg/mL) of 5 was
prepared in 0.3 % Na-CMC water solution, then one group of
20 both sexes mice were administered the maximum tolerated
dose (0.4 mL/10 g) by oral administration. The other 20 mice,
control group, were gave 0.3 % Na-CMC (0.4 mL/10 g) via
gavage. The general behaviour of the mice was observed
Wang, Peng-Long
