Synthesis and radioprotective effects of novel benzyl naphthyl sulfoxide (sulfone) derivatives transformed from Ex-RAD

Article abstract of DOI:10.1039/c7md00573c

In this work, a series of novel benzyl naphthyl sulfoxides (sulfones) derived from Ex-RAD were designed and synthesized as potential radioprotective agents. Some of the compounds considerably protected HUVECs against ⁶⁰Co γ-irradiation, accompanied by the absence of cytotoxicity. Compared to Ex-RAD, compound 8n not only exhibited a significant protective effect on cell survival and radiation-induced DNA damage, but also remarkably enhanced the survival (100%) of mice in 30 days after being exposed to irradiation. The results suggested that some target compounds are valuable for further research as promising radioprotectors.

Full text of DOI:10.1039/c7md00573c

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RESEARCH ARTICLE
Synthesis and radioprotective effects of novel
benzyl naphthyl sulfoxide (sulfone) derivatives
Cite this: DOI: 10.1039/c7md00573c
transformed from Ex-RAD†
*
Lin Tang, Tao Peng, Gang Wang, Xiaoxue Wen, Yunbo Sun, Shouguo Zhang,
*
*
Shuchen Liu and Lin Wang
In this work, a series of novel benzyl naphthyl sulfoxides (sulfones) derived from Ex-RAD were designed
and synthesized as potential radioprotective agents. Some of the compounds considerably protected
HUVECs against 60Co γ-irradiation, accompanied by the absence of cytotoxicity. Compared to Ex-RAD,
compound 8n not only exhibited a significant protective effect on cell survival and radiation-induced DNA
damage, but also remarkably enhanced the survival (100%) of mice in 30 days after being exposed to irradi-
ation. The results suggested that some target compounds are valuable for further research as promising
radioprotectors.
Received 13th November 2017,
Accepted 9th February 2018
DOI: 10.1039/c7md00573c
rsc.li/medchemcomm
cells (HUVECs), lung fibroblast cells (HFL-1) and skin fibro-
1. Introduction
blast cells (AG1522).5 In in vivo studies, Ex-RAD has also
Providing protection from ionizing radiation injuries is not
only a matter of concern in therapeutic radiology, but also
has become an important issue due to the ever increasing
threats associated with the proliferation of nuclear materials,
terrorism and occupational risks associated with space
exploration.1,2 At present, amifostine (WR2721) is the only
radioprotector that has been clinically approved by the Food
and Drug Administration for reducing side effects in patients
undergoing radiotherapy.3 This drug has a good effect on ra-
diation injuries, but causes serious side effects, such as nau-
sea, vomiting and hypotension.4 Therefore, it is essential to
develop easily self-administered, less toxic, and more effective
radioprotectors.4
demonstrated significantly greater protection against 60Co
γ-irradiation than the vehicle when administered to mice
before radiation exposure (subcutaneous injection (s.c.) or
intragastric administration (i.g.)).5–8 Moreover, pre-clinical
pharmacokinetic studies in rats, dogs, rabbits and monkeys
demonstrate that Ex-RAD is well-absorbed following extravas-
cular administration, resulting in significant plasma expo-
sure.10,11 Mechanistically, the radioprotective effects of Ex-
RAD may involve the prevention of p53-dependent apopto-
sis.8 Attenuation of ataxia telangiectasia-mutated gene-p53-
mediated (ATM-p53-mediated) DNA damage response (DDR)
by Ex-RAD contributes to the mitigation of radiation-induced
hematopoietic toxicity.6 Further, Ex-RAD manifests its protec-
tive effects through the up-regulation of PI3-kinase/AKT path-
ways in cells exposed to radiation.9 Onconova Therapeutics
has completed four Phase I trials with Ex-RAD, three trials
with subcutaneous Ex-RAD in more than 50 healthy adults
and one trial with oral Ex-RAD in nine healthy adults; none
of these trials reported evidence of systemic side effects.12
The structure of Ex-RAD, associated with three types of
chemical structure, has been protected by a patent. The three
chemical structures (Fig. 1) are: styryl benzyl sulfoxides (sul-
fones) (II), diaryl acrylketone sulfoxides (sulfones) (III) and
diaryl vinyl sulfoxides (sulfones) (IV).
Ex-RAD (I in Fig. 1), also known as ON 01210.Na
(4-carboxystyryl-4-chlorobenzyl sulfone, sodium salt), is
a
chlorobenzyl sulfone derivative developed by Onconova Ther-
apeutics (Newtown, PA, USA) as a radioprotector and mitiga-
tor.5 Unlike most radioprotectors, Ex-RAD is not a free-radical
scavenger or responsible for cell cycle arrest. Available data
suggest that Ex-RAD has a novel mechanism for radiation
protection involving DNA repair pathways.6 It is a water solu-
ble, non-toxic, synthetic molecule with potent radioprotective
properties both in vitro and in vivo.5,7–9 In in vitro studies, Ex-
RAD has shown potent radioprotective efficacy in several hu-
man cell lines, including human umbilical vein endothelial
The structure–activity relationship (SAR) analysis of the
above aryl sulfoxides (sulfones) indicated the following rules:
(1) in the part of the styryl segment, the bioactivity was
higher when 2-chloro, 4-chloro, 4-fluoro, 2-methoxy,
2,4-dimethoxy or 4-carboxyl was introduced to the styrene
side; alternatively, the benzene ring was replaced by a 3-furan
Beijing Institute of Radiation Medicine, Beijing 100850, P. R. China.
E-mail: wanglin@bmi.ac.cn, wanglin07@sina.com; Tel: +86 010 66932239
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
c7md00573c
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Fig. 1 Structure of Ex-RAD (I). Structures of aryl sulfoxides (sulfones) protected by a patent (II, III and IV).
ring. (2) In the part of the benzyl segment, introduction of
4-cyano, 4-chloro or 4-hydrogen could enhance the bioactiv-
ity. However, a decrease in activity was observed when the
para-position of the benzene ring was substituted with
methoxy or nitro. (3) The bioactivity of sulfoxides was similar
to the corresponding sulfones. (4) The trans-forms could exist
more stably and the synthetic yield was higher. But a similar
bioactivity was also retained in cis-forms.
Based on the results of SAR analysis, the ortho-position of
the styrene side could be chemically processed to get various
derivatives. In this article, to connect the ortho-position of
the styrene side with the 2-position of vinyl according to the
principle of isotones, a series of novel 2-naphthyl benzyl sulf-
oxide (sulfone) derivatives (Fig. 2) were designed rationally.
Subsequently, thirty-seven target compounds were prepared.
Their stereochemical and electronic structures were similar
to Ex-RAD while avoiding the extent of patent protection. Fur-
thermore, looking forward to obtaining more effective anti-
radiation agents, the radioprotective effects of the target com-
pounds were evaluated in vitro and in vivo.
positive control. Ex-RAD was synthesized by our lab. Unfortu-
nately, most of the compounds did not show significant ra-
dioprotective activity for the first screening at a concentration
of 40 μM (data are provided in the ESI†). However, 9 com-
pounds (8d, 8g, 8j, 8l, 9l, 8n, 9o, 8p and 9t) exhibited obvious
radioprotective effects at concentrations of 40 μM and 20 μM
(second screening). In particular, the survival of 8n-treated
cells was significantly (p < 0.05) higher than that of Ex-RAD-
treated cells. Results from cell survival after irradiation indi-
cated that 8n exhibited good radioprotective activity. The sur-
vival rates of cells treated with the 9 compounds (20 μM) are
summarized in Table 1. The cytotoxic activity of the 9 com-
pounds (100 μM) in HUVECs was also determined by the
MTS tetrazolium assay. The results demonstrated that they
were not toxic (data not shown).
2.2.2. Evaluation of the protective effect of 8n on
radiation-induced DNA damage. The alkaline comet assay was
employed to monitor the efficacy of 8n in protecting HUVECs
from radiation-induced DNA damage.5 The comet assay is a
common technique for measurement of DNA damage in indi-
vidual cells. Under an electrophoretic field, damaged cellular
DNA (containing fragments and strand breaks) is separated
from intact DNA and migrates further from the intact DNA,
yielding a classic “comet tail” shape under the microscope. The
extent of DNA damage is usually visually estimated by comet
tail measurement. Radiation-induced DNA damage results in
increased numbers of single-strand breaks (SSBs), double-
strand breaks (DSBs), and alkali-labile lesions. The degree of
DNA damage was measured by calculating the tail lengths with
Comet Assay Software Project Lab (CASP1.2.3 beta 2) after irra-
diation with 6.0 Gy. Fifty randomly selected comets were cap-
tured by fluorescence microscopy after staining with vista
green DNA dye. In control cells, there was a significant increase
in tail lengths. As shown in Fig. 3, 8n treated HUVECs had sig-
nificantly (p < 0.05) shorter tail lengths compared to the vehi-
cle control and Ex-RAD treated cells, which indicated that 8n
played an important role in the repair of DNA damage after ex-
posure to ionizing radiation.
2. Results and discussion
2.1. Chemistry
We first synthesized compound 5b by using 2-naphthol as a
starting material, as described in the literature.13–15 Com-
pounds 7a–7t were synthesized from the reaction of 5a–5b
with 6a–6j in the presence of NaOH.16 Finally, the sulfides
7a–7t were oxidized to obtain the corresponding sulfoxides
(8a–8r) and sulfones (9a–9t) with H2O2 in acetic acid.17 All the
synthesized compounds were purified by recrystallization or
silica gel column chromatography, and the structures of some
target compounds with better biological activity were character-
ized by 1H NMR, 13C NMR and HR-MS spectra analyses.
2.2. Biological activity
2.2.1. Evaluation of the radioprotective activity and cyto-
toxicity of target compounds. The in vitro radioprotective ac-
tivity of the target compounds was first evaluated by a radia-
tion assay using HUVECs as a model of normal cells.5 Cell
survival after irradiation was measured by the MTS tetrazo-
lium assay. Cells were pretreated with the target compounds
24 h before irradiation (8.0 Gy), and Ex-RAD was used as a
2.2.3. Radioprotective efficacy of 8n in mice. In in vitro
studies, compound 8n exhibited a significant radioprotective
effect. In order to complete the study of this compound, we
tested the radioprotective effectiveness of 8n in vivo on C57/
BL male mice. The survival was monitored for 30 days post-ir-
radiation.5,8 Ex-RAD and nylestriol were used as positive con-
trols. As shown in Fig. 4, 8n was given via intraperitoneal in-
jection (i.p.) at a dose of 300 mg kg−1 24 h and 15 min (two
doses) before irradiation at 8.0 Gy, resulting in a net survival
of 100% (p < 0.05, Fisher's extract test). The survival in the
group given nylestriol i.g. 24 h (one dose) before irradiation
Fig. 2 The structures of 2-naphthyl benzyl sulfoxide (sulfone) derivatives.
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Table 1 The survival rates in some compound-pretreated cells after irradiation
Compound (20 μM)
Structure
Survival rate (%)
Vehicle control
8d
—
43.7 3.4
52.0 1.9
8g
8j
57.2 0.9
59.3 2.0
51.3 3.5
52.4 1.2
77.2 2.6
8l
9l
8n
9o
8p
9t
56.7 2.4
55.1 1.6
53.1 4.6
Ex-RAD
—
57.5 4.9
Fig. 3 DNA damage as determined by the alkaline comet assay in HUVECs. The untreated group was composed of normal cultured cells without
irradiation. A. The fluorescence microscopy images of HUVECs 24 h post-irradiation. B. DNA damage was measured by calculating the tail lengths
24 h post-irradiation (6.0 Gy). 8n-treated HUVECs had significantly (p < 0.05) shorter tail lengths compared to the vehicle control and Ex-RAD
treated cells.
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Fig. 4 A. Kaplan–Meier 30 day survival rates were observed in male C57/BL mice (n = 10 per group). Mice that received 8n exhibited a significant
increase in survival (100%) as compared with the vehicle control group and the Ex-RAD group. Note: There was a significant difference among the
survival curves (p = 0.0104, df = 3, chi squared value = 11.25, log-rank test). B. The body weights of male C57/BL mice (n = 10 per group) were
measured 30 days post-irradiation. The body weights of mice in group 8n were statistically significant (p < 0.05) and heavier than those of the
control and Ex-RAD groups between 10 d and 30 d post-irradiation.
at 8.0 Gy also showed a net survival of 100%. However, the
survival rate in the group that was given Ex-RAD s.c. (300 mg
kg−1) 24 h and 15 min (two doses) before irradiation was only
60%. The survival in the vehicle control group was 50%.
Moreover, the body weights of mice in group 8n (Fig. 4B)
were statistically significant (p < 0.05) and heavier than
those of the control and Ex-RAD groups between 10 d and 30
d post-irradiation. The results showed that 8n could prolong
the survival of mice after exposure to ionizing radiation.
performed on silica gel glass plates containing 60 GF-254. Vi-
sualization was achieved using UV light (λmax = 254 or 365
nm). The purification of compounds was performed using sil-
ica gel (200–300 mesh) column chromatography. 1H-NMR
and 13C-NMR spectra were recorded using Bruker (Palo Alto,
CA, USA) AV-400 spectrometers in DMSO-d6 with TMS as the
internal standard. Mass spectral data were obtained using
electron spray ionization on a Micromass ZabSpec high-
resolution mass spectrometer (Karlsruhe, Germany).
4.1.1. General procedure for the synthesis of compounds
(8d, 8g, 8j, 8l, 8n and 8p). To an ice cold solution of 7 (1.0
mmol) in 50 mL acetic acid was added 2.0 mmol 30% H2O2,
then the mixture was stirred at room temperature for about
3–5 h. After the completion of the reaction (monitored by
TLC), the mixture was poured into ice water, and the formed
white precipitate was filtered, washed with water and dried
under vacuum to get the target compounds (8).
3. Conclusions
The structure–activity relationship (SAR) study revealed that
most sulfoxides exhibited better radioprotective activity than
the corresponding sulfones. The introduction of halogen
groups at the 4-position of the benzene ring enhanced the
biological activity to a much greater extent.
In summary, we report on the design and synthesis of
some 2-naphthyl benzyl sulfoxide (sulfone) derivatives for use
as radioprotective drugs. Evaluations of their radioprotective
activities in vitro indicated that some derivatives such as 8d,
8g, 8j, 8l, 9l, 8n, 9o, 8p and 9t have a high radioprotective ac-
tivity and no cytotoxicity for HUVECs. Among them, 8n
exhibited a significant radioprotective effect on cell survival
and DNA damage. In addition, 8n also showed remarkable
biological activity by significantly increasing the survival rate
of mice after being exposed to irradiation compared to Ex-
RAD. The findings strongly suggest the great potential of this
class of compounds as radioprotective drugs for radiation
therapy and other purposes. Further detailed research will be
conducted to evaluate the molecular mechanism underlying
the radioprotective activity of these compounds.
2-((4-Fluorobenzyl)sulfinyl)naphthalene (8d). Obtained in
87.5% yield, white solid, m.p. 206–208 °C. 1H NMR (400
MHz, DMSO-d6) δ (ppm): 4.16(d, 1H, J = 12.9 Hz), 4.39(d, 1H,
J = 12.9 Hz), 7.05–7.13(m, 4H), 7.60–7.66(m, 3H), 7.99–
8.09(m, 4H); 13C-NMR (100 MHz, DMSO-d6) δ (ppm): 163.13,
160.71, 140.53, 138.86, 132.42, 132.34, 132.23, 128.85, 128.39,
128.00, 127.74, 127.25, 126.58, 126.56, 124.72, 120.64, 115.07,
114.86, 59.92; HRMS-ESI (m/z) calcd. for C17H14FOS [M + H]+:
285.0749, found: 285.0744.
2-((4-Chlorobenzyl)sulfinyl)naphthalene (8g). Obtained in
93.0% yield, white solid, m.p. 214–216 °C. 1H NMR (400
MHz, DMSO-d6) δ (ppm): 4.18(d, 1H, J = 12.6 Hz), 4.42(d, 1H,
J = 12.9 Hz), 7.08(d, 2H, J = 8.4 Hz), 7.30(d, 2H, J = 8.4 Hz),
7.60–7.67(m, 3H), 7.99–8.02(m, 4H); 13C-NMR (100 MHz,
DMSO-d6) δ (ppm): 140.45, 138.83, 132.63, 132.19, 132.12,
129.32, 128.82, 128.34, 128.02, 127.95, 127.71, 127.21, 124.69,
120.56, 59.95; HRMS-ESI (m/z) calcd. for C17H14ClOS [M +
H]+: 301.0454, found: 301.0448.
4. Experimental section
4.1. Chemistry
2-((4-Bromobenzyl)sulfinyl)naphthalene (8j). Obtained in
93.3% yield, white solid, m.p. 219–220 °C. 1H NMR (400
MHz, DMSO-d6) δ (ppm): 4.16(d, 1H, J = 12.8 Hz), 4.41(d, 1H,
J = 12.8 Hz), 7.03(d, 2H, J = 8.5 Hz), 7.44(d, 2H, J = 8.5 Hz),
7.60–7.67(m, 3H), 8.00–8.03(m, 3H), 8.09(d, 1H, J = 8.5 Hz);
All chemicals and reagents were purchased from commercial
suppliers, of reagent grade and used without further purifica-
tion. Melting points were recorded in an open capillary tube
and uncorrected. Reactions were monitored using TLC and
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13C-NMR (100 MHz, DMSO-d6) δ (ppm): 140.46, 132.45,
132.19, 131.23, 130.95, 129.74, 128.83, 128.34, 127.95, 127.71,
127.21, 124.69, 121.25, 120.56, 60.03; HRMS-ESI (m/z) calcd.
for C17H14BrOS [M + H]+: 346.9928, found: 346.9923.
127.23, 126.56, 124.27, 123.15, 58.16; HRMS-ESI (m/z) calcd. for
C17H12BrClKO2S [M + K]+: 434.9047, found: 434.9041.
2-Bromo-6-((4-bromobenzyl)sulfonyl)naphthalene
(9t).
Obtained in 100% yield, white solid, m.p. 203–205 °C. 1H
NMR (400 MHz, DMSO-d6) δ (ppm): 4.80(s, 2H), 7.10(d, 2H, J
= 8.5 Hz), 7.49(d, 2H, J = 8.3 Hz), 7.79–7.84(m, 2H), 8.12–
8.15(m, 2H), 8.42(m, 2H); 13C-NMR (100 MHz, DMSO-d6) δ
(ppm): 135.43, 135.40, 132.59, 131.12, 130.85, 130.37, 129.69,
129.49, 129.27, 127.99, 127.62, 123.74, 122.56, 121.55, 59.34;
HRMS-ESI (m/z) calcd. for C17H12Br2KO2S [M + K]+: 478.8541,
found: 478.8551.
2-Bromo-6-((2-fluorobenzyl)sulfinyl)naphthalene
(8l).
Obtained in 70.0% yield, white solid, m.p. 235–237 °C. 1H
NMR (400 MHz, DMSO-d6) δ (ppm): 4.27(d, 1H, J = 13.2 Hz),
4.41(d, 1H, J = 13.2 Hz), 7.03–7.33(m, 4H), 7.68–7.75(m, 2H),
7.95–8.07(m, 3H), 8.33(s, 1H); 13C-NMR (100 MHz, DMSO-d6)
δ (ppm): 161.98, 159.53, 141.28, 135.10, 132.85, 132.82,
130.74, 130.59, 130.32, 130.24, 129.95, 129.36, 128.99, 128.09,
124.79, 124.12, 124.09, 121.75, 121.14, 117.20, 117.05, 115.20,
114.99, 54.37; HRMS-ESI (m/z) calcd. for C17H13BrFOS [M +
H]+: 362.9855, found: 362.9849.
Note: Except for some target compounds with better bio-
logical activity, the synthesis of all other compounds men-
tioned in Scheme 1 is described in the ESI.†
2-Bromo-6-((4-fluorobenzyl)sulfinyl)naphthalene
(8n).
Obtained in 89.1% yield, white solid, m.p. 195–197 °C. 1H
NMR (400 MHz, DMSO-d6) δ (ppm): 4.17(d, 1H, J = 12.9 Hz),
4.41(d, 1H, J = 12.9 Hz), 7.06–7.08(m, 4H), 7.68–7.75(m, 2H),
7.97–8.08(m, 3H), 8.33(s, 1H); 13C-NMR (100 MHz, DMSO-d6)
δ (ppm): 163.10, 160.67, 141.26, 134.93, 132.35, 132.27,
130.74, 130.50, 130.18, 129.89, 127.98, 126.33, 126.30, 124.76,
121.84, 121.00, 115.00, 114.78, 59.74; HRMS-ESI (m/z) calcd.
for C17H13BrFOS [M + H]+: 362.9855, found: 362.9849.
4.2. Biological evaluation
4.2.1. Cell culture and irradiation. Human umbilical vein
endothelial cells (HUVECs) were used for in vitro studies.
HUVECs were cultured aseptically in Roswell Park Memorial
Institute-1640 (RPMI-1640) supplemented with 10% (v/v) fetal
bovine serum (FBS) and penicillin (100 units per mL)/strepto-
mycin (100 μg mL−1) at pH 7.2 and a 5% CO2 humidified at-
mosphere at 37 °C. After attaining 80% confluence, the cells
were trypsinized with 0.25% trypsin–EDTA and diluted with
media to a fixed number of cells. The cell proliferation capac-
ity was determined by the MTS tetrazolium assay in the pres-
ence of target compounds, according to the protocol of the
manufacturer (Promega, USA). Monolayer cells were incu-
bated with the target compounds for 24 h in 96-well plates
before irradiation. The plates were placed under separate
Plexiglas covers and irradiated with the needed dose (8.0 Gy
for cell survival and 6.0 Gy for the comet assay) at a dose rate
of 0.93 Gy min−1. Cell irradiation was done at the 60Co
γ-radiation facility of the Beijing Institute of Radiation Medi-
cine, Beijing, China.
4.2.2. Cell survival. The cell survival after irradiation was
measured by the standard MTS tetrazolium assay. The cells
were seeded into 96-well plates containing the medium at a
density of 4000 cells per mL (100 μL per well). The com-
pounds were dissolved in DMSO to a concentration of 100
mM, and diluted in culture medium to the concentrations
needed. After 24 h, the cultured cells were treated with the
synthesized compounds (40 μM or 20 μM) for 24 h. After 24
h of incubation, the cells were exposed to 8.0 Gy of 60Co
γ-irradiation. The cells were continued to be incubated for 4
days after radiation and the supernatant was replaced by
fresh medium (100 μL per well) every two days. After 4 days
of incubation, the supernatant was replaced by fresh medium
(100 μL per well) once more and 10 μL MTS reagent ([3-(4,5-
dimethyl thiazol-2-yl)-5-(3-carboxy methoxy phenyl)-2-(4-
sulfophenyl)-2H-tetrazolium, inner salt]) was added to each
well. The plate was further incubated for 3 h at 37 °C in 5%
CO2. The optical absorbance in individual wells was deter-
mined at 492 nm using a Microplate Reader. The inhibition
rates were calculated using the following formula. The final
2-Bromo-6-((3-chlorobenzyl)sulfinyl)naphthalene
(8p).
Obtained in 86.4% yield, white solid, m.p. 149–150 °C. 1H
NMR (400 MHz, DMSO-d6) δ (ppm): 4.18(d, 1H, J = 12.9 Hz),
4.43(d, 1H, J = 12.6 Hz), 6.99(d, 1H, J = 7.6 Hz), 7.14(s, 1H),
7.25(t, 1H, J = 7.6 Hz), 7.33(d, 1H, J = 7.3 Hz), 7.70–7.76(m,
2H), 7.97–8.09(m, 3H), 8.34(s, 1H); 13C-NMR (100 MHz,
DMSO-d6) δ (ppm): 141.15, 134.97, 132.59, 132.55, 130.74,
130.52, 130.21, 130.10, 129.88, 129.79, 128.98, 128.03, 127.70,
124.78, 121.79, 121.04, 59.96; HRMS-ESI (m/z) calcd. for
C17H13BrFOS [M + H]+: 380.9539, found: 380.9527.
4.1.2. General procedure for the synthesis of compounds
(9l, 9o and 9t). To an ice cold solution of 7 (1.0 mmol) in 50
mL acetic acid was added 6.0 mmol 30% H2O2, then the mix-
ture was heated to 50 °C and stirred for about 2–5 h. After
the completion of the reaction (monitored by TLC), the mix-
ture was poured into ice water, and the formed white precipi-
tate was filtered, washed with water and dried under vacuum
to get the target compounds (9).
2-Bromo-6-((2-fluorobenzyl)sulfonyl)naphthalene
(9l).
Obtained in 58.8% yield, white solid, m.p. 130–132 °C. 1H
NMR (400 MHz, DMSO-d6) δ (ppm): 4.79(s, 2H), 7.08–7.39(m,
4H), 7.81(t, 2H, J = 8.7 Hz), 8.11–8.14(m, 2H), 8.42(d, 2H, J =
6.7 Hz); 13C-NMR (100 MHz, DMSO-d6) δ (ppm): 162.07,
159.60, 135.91, 133.30, 131.63, 131.12, 131.04, 130.85, 130.18,
129.97, 129.87, 128.50, 124.41, 124.20, 123.11, 115.92, 115.77,
115.51, 115.30, 54.59; HRMS-ESI (m/z) calcd. for
C17H12BrFKO2S [M + K]+: 418.9342, found: 418.9339.
2-Bromo-6-((2-chlorobenzyl)sulfonyl)naphthalene
(9o).
Obtained in 70.4% yield, white solid, m.p. 136–138 °C. 1H NMR
(400 MHz, DMSO-d6) δ (ppm): 4.88(s, 2H), 7.31–7.37(m, 4H),
7.74–7.83(m, 2H), 8.12(t, 2H, J = 8.7 Hz), 8.41(s, 2H); 13C-NMR
(100 MHz, DMSO-d6) δ (ppm): 136.00, 135.96, 134.52, 133.48,
131.65, 130.85, 130.59, 130.24, 130.08, 129.97, 129.48, 128.54,
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Scheme 1 Reagents and conditions: (a) Br2, acetic acid, reflux, 3 h; (b) (i) NaH, DMF, 0 °C, 15 min, (ii) dimethyl carbamoyl chloride, 80 °C (2 h), r.t.
(15 h); (c) 220 °C, 6 h; (d) KOH, MeOH, 80 °C, 2.5 h; (e) NaOH, EtOH, reflux, 3 h; (f) H2O2, acetic acid, r.t. or 40 °C, 4–5 h.
survival rates reported here represent the average of three in-
dependent experiments. The survival rate calculation was cal-
culated using GraphPad Prism Software (GraphPad Prism 5,
Version 5.01).
horizontal electrophoresis apparatus at 1.0 V cm−1 at a cur-
rent of 300 mA for 20 min. After electrophoresis, the slides
were dried in 70% ethanol for 5 min, air-dried (1–2 h), and
stained for 5 min with vista green DNA dye (1/10 000 dilution
of stock supplied by Cell Biolabs). The slides were observed
at 400× magnification under an epifluorescence microscope
equipped with an excitation filter of 494–521 nm. A total of
50 randomly captured comets from each slide were exam-
ined. To quantify the DNA damage, the tail lengths were eval-
uated. The tail length (length of DNA migration) is related di-
rectly to the DNA fragment size and is measured in
micrometers. Lengths were calculated and analyzed with
Comet Assay Software Project Lab (CASP1.2.3 beta 2). The ex-
periment was repeated three times.
4.2.4. Mice and irradiation. 20–24 g male C57/BL mice
were purchased from SPF (Beijing) Biotechnology (China)
and were housed five per cage in an air-conditioned facility
at the Laboratory Animal Center of Academy of Military Medi-
cal Sciences (Beijing, China). The holding rooms for the mice
were maintained at a temperature of 25°C with 10–15 hourly
cycles of fresh air and a relative humidity of 50% 10%. All
mice were kept in rooms with a 12 h light/dark cycle with
lights on from 08:00 to 20:00. Before starting the experi-
ments, all mice were allowed one week of acclimatization in
the facility. Mice were irradiated in well-ventilated Lucite
Survival rate (%) = (ODsample − ODblank)(ODnegative control*
ODblank) × 100%.
−
*The negative control group was composed of normal cul-
tured cells without irradiation.
4.2.3. Single-cell electrophoresis (comet assay). The alka-
line comet assay was performed to determine the radiopro-
tective effect of Ex-RAD on DNA damage. The assay was
performed according to instructions provided by the manu-
facturer (OxiSelect™ Comet Assay Kit, Cell Biolabs). Briefly,
24 h after irradiation (6.0 Gy), the cells (control or treated)
were seeded at a concentration of 1 × 105 mL−1, combined
with comet agarose at a ratio of 1 : 10 (v/v), titrated to mix,
and immediately 75 μL per well was onto the 3-well comet
slides. The slides were maintained at 4 °C for 15 min in the
dark for gel solidification. The slides were then submerged
horizontally in the precooled provided lysis buffer in the dark
at 4 °C for 30 min. Finally, the slides were submerged in an
alkaline buffer (300 mM NaOH and 1 mM EDTA, pH > 13) at
4 °C for 30 min. Electrophoresis was conducted in a fresh
chilled alkaline electrophoresis buffer (300 mM NaOH and 1
mM EDTA, pH > 13). The slides were placed side by side in a
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boxes at a dose rate of 0.62 Gy min−1. After irradiation, the mice
were returned to their cages and monitored for 30 day survival.
Mice irradiation was done at the 60Co γ-radiation facility of the
Beijing Institute of Radiation Medicine, Beijing, China.
21272273) for its financial support for this project. Tremen-
dous thanks are owed to the Beijing Institute of Pharmacol-
ogy and Toxicology for helping with 1H NMR, 13C NMR and
HR-MS spectra analyses.
Ethical statement: All animal procedures were performed
according to a protocol approved by the Committee on the
Ethics of Animal Experiments of the Animal Center at the Bei- References
jing Institute of Radiation Medicine (IACUC of AMMS 2013022).
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4.2.5. Survival study in mice. Mice were divided into four
groups: irradiated vehicle-treated (Vehicle), irradiated Ex-
RAD-treated (Ex-RAD), irradiated nylestriol-treated (Nylestriol)
and irradiated 8n-treated (8n). Each group had 10 mice. Each
mouse in group 8n and Vehicle received 0.25 mL of either 8n
(300 mg kg−1) or a vehicle i.p. 24 h and 15 min (two doses)
before irradiation. Each mouse in group Ex-RAD received
0.25 mL of Ex-RAD (300 mg kg−1) s.c. 24 h and 15 min (two
doses) before irradiation. Each mouse in group Nylestriol re-
ceived 0.25 mL of nylestriol (5 mg kg−1) i.g. 24 h (one dose)
before irradiation. Compound 8n and Ex-RAD were
suspended in a vehicle consisting of 20% HPCD in normal
saline. Nylestriol was suspended in 0.5% CMCNa. All mice
were whole-body irradiated with 8.0 Gy. The survival was
monitored for 30 days post-radiation.
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4.3. Statistical analysis
For data of cell survival, comet assay and mice weight, statis-
tical analysis to find the significance between two groups was
performed using the two tailed paired Student's t-test, and p
< 0.05 was taken as statistically significant. Error bars repre-
sent standard error of mean (SEM). A Kaplan–Meier sur-
vival plot was drawn using GraphPad Prism Software. For
mice survival data, the Fisher's exact test was used to com-
pare the survival at 30 days and a log-rank test was used to
compare survival curves, and p < 0.05 was taken as statisti-
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Conflicts of interest
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Chem., 2010, 833–845.
The authors declare no conflict of interest.
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2011, 4693–4698.
Acknowledgements
The authors are grateful to the National Natural Science
Foundation of China (Grant No. 81273431, 21102176 and
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