D
M. Tian et al.
Cytotoxicity Assay
Cytotoxicity was determined by MTT (3-(4,5-dimethylthiazol-
2
-yl)-2,5-diphenyltetrazolium bromide) assay. C6 cells were
cultured in DMEM/F12 containing 10 % fetal bovine serum and
grown to the logarithmic growth phase. After digestion with
0
.25 % trypsin, the cells were seeded into 96-well plates at a
3
ꢀ1
density of 5 ꢃ 10 cells well and 200 mL per well. The cells
were then incubated at 378C in a humidified atmosphere with
5
% CO for 24 h. The medium was removed, and 200 mL of
2
fresh DMEM containing 8 mL of different concentrations of L1
or D1 was added. L1 or D1 was dissolved in water at final
concentrations of 2000, 1000, 500, 250, 125, 62.5, 31.25, and
ꢀ1
1
5.625 mg L . After a 72 h incubation, MTT (20 mL per well)
was added, and the plates were incubated for another 4 h. The
optical density (OD) was measured at 490 nm in a spectro-
photometer. The cytotoxicity of each drug was determined to
select the appropriate concentration for further radioprotection
experiments.
3
350
3400
3450
3500
3550
3600
G
Fig. 2. ESR spectrum of L1 in DMF at room temperature.
Protective Effects of L1 and D1 Against Radiation Injury
to C6 Cells
activity of CAT was tested by the catalase assay method which
[29]
was defined by Aebi. The content of GSH was determined by
the GSH assay kit according to the manufacturer’s instructions.
In each group, six samples (n ¼ 6) were processed.
The protective effects of L1 and D1 against radiation injury to
C6 cells were determined by MTT assay. C6 cells were cultured
in DMEM/F12 containing 10 % fetal bovine serum to the log-
arithmic growth phase. After digestion with 0.25 % trypsin, cells
Statistical Analysis
3
were seeded into 96-well plates at a density of 5 ꢃ 10 cells
Statistical analysis of the data to determine significant variations
among groups was performed using SPSS statistical software.
One-way analysis of variance (ANOVA) was performed and
post hoc tests of multiple comparisons were performed using the
least significant difference (l.s.d.) test. The results are presented
as the mean ꢂ standard deviation (s.d.). In all cases, P , 0.05
indicated a statistically significant difference.
ꢀ1
well and cultivated for 24 h. The medium was then removed,
and 200 mL of fresh DMEM containing different concentrations
of L1 or D1 was added. The final concentrations of L1 and D1
ꢀ
1
were 125, 62.5, 31.25, or 15.625 mg mL . After treatment with
6
0
L1 or D1 for 15 min, the cells were subjected to Co gamma
irradiation (8, 10, or 12 Gy) for 30 min. After a 72 h incubation,
MTT (20 mL per well) was added, and the plates were incubated
for another 4 h. The OD was determined by a spectrophotometer
Results
ꢀ
1
at 490 nm. The dose rate used was ,4.64 Gy min . Phosphate
buffered saline (PBS) was used as a control. Four replicates were
used for each concentration of each drug.
Synthesis and Characterization of L1 and D1
Different structural aldehydes can be converted into nitronyl
nitroxyl radicals based on Ullman’s pioneering work. Thus, the
first step to obtain chiral nitroxy radicals L1 and D1 was to
synthesize chiral aldehydes. As shown in Scheme 1, the
precursor of L1 chloride was obtained by the acylation of
Survival Assays
Mice were randomly divided into nine groups: normal control
group, radiation exposure group, WR2721 pre-treatment group,
and three L1 or D1 pre-treatment groups (radiation þ
4
-fluorobenzoic acid, and the alcohol L-(2-(hydroxymethyl)
pyrrolidin-1-yl)(4-fluorophenyl)methanone was obtained by the
condensation of L-proline and 4-fluorobenzoyl chloride. The
chiral aldehydes were obtained by the rapid oxidation of L-(2-
ꢀ
1
ꢀ1
0
.1 mmol kg of L1 or D1, radiation þ 0.25 mmol kg of L1 or
ꢀ
1
D1, and radiation þ 0.5 mmol kg of L1 or D1). Each group
contained six mice. WR2721, L1, and D1 were dissolved in
.5 % dimethyl sulfoxide (DMSO) and administered through
(hydroxymethyl) pyrrolidin-1-yl)(4-fluorophenyl)methanone
0
with trichloroisocyanuric acid in the presence of catalytic
TEMPO. The enantiomer D-(2-(hydroxymethyl) pyrrolidin-1-
yl)(4-fluorophenyl)methanone was prepared using the same
method but instead using raw chiral D-proline. The chiral alde-
hydes reacted with 2,3-bis(hydroxyamino)-2,3-dimethylbutane
intraperitoneal injection to mice 30 min before radiation expo-
sure. This experimental process was repeated for 7 days. The
survival rate of mice was recorded by the survival percentage of
mice exposed to different doses of radiation (6.0, 6.5, or 7.0 Gy
gamma radiation). Experiments were performed in triplicate.
0
to form a stable white solid, N,N -dihydroxyimidazoline, which
could be oxidized to the target compounds L1 and D1 by NaIO4
at 08C, as shown in Scheme 1.
The UV-vis absorption spectra of L1 and D1 were measured
Biochemical Assays
The mice groups and drug administration method were the same
as described in the above section. At 24 h after radiation expo-
sure, blood samples (1.0 mL) were collected in test tubes and an
anticoagulant added. The blood samples were then centrifuged
immediately for 10 min (48C, 2000ꢃg). The activities of SOD,
MDA, CAT, and GSH were tested in the collected plasma
samples. The activity of SOD was measured by Kakkar
in CH Cl at room temperature. The spectra showed the absorp-
2 2
tion in the UV range at 368 nm arising from the p–p* transition
of the ONCNO units. In the IR spectra of L1 and D1, it is
noteworthy that the characteristic bands caused by the N–O
ꢀ1
stretching frequency are observed at 1350 cm . As shown in
Fig. 2, the ESR spectra of the two types of radicals dissolved into
[27]
ꢀ1
method. The MDA level was measured using a method based
[
DMF at a concentration of 0.001 mol L showed five major
lines in the ratio 1 : 2 : 3 : 2 : 1, which was expected for the
28]
on thiobarbituric acid (TBA) established by Jain et al.
The