Synthesis and biological activity of sulfur-containing aryl-aldehyde Schiff bases.

Article abstract of DOI:10.1248/cpb.51.1307

A series of chemically modified aryl-aldehyde Schiff bases has been synthesized and tested for their antioxidant activity and radiation protection. We observed that disulfide-containing aryl-aldehyde Schiff base 6c exhibited potent free radical scavenging

Full text of DOI:10.1248/cpb.51.1307

November 2003
Notes
Chem. Pharm. Bull. 51(11) 1307—1310 (2003)
1307
Synthesis and Biological Activity of Sulfur-Containing Aryl-aldehyde
Schiff Bases
a
b
,c
d
,a
*
*
Jyh-Jia JIANG, Tsu-Chung CHANG, Wen-lin HSU, Jing-Min HWANG, and Ling-Yih HSU
a School of Pharmacy; b Department of Biochemistry, National Defense Medical Center; d Department of Radiation
Oncology, Tri-Service General Hospital; Taipei, Taiwan 114, Republic of China: and c Cancer Medical Center and
Department of Radiation Oncology, Buddhist Tzu Chi Medical Center; Hualien, Taiwan 970, Republic of China.
Received May 19, 2003; accepted July 26, 2003
A series of chemically modified aryl-aldehyde Schiff bases has been synthesized and tested for their antioxi-
dant activity and radiation protection. We observed that disulfide-containing aryl-aldehyde Schiff base 6c exhib-
ited potent free radical scavenging, antioxidation, and radioprotection activities.
Key words aryl-aldehyde Schiff base; antioxidant; radioprotection
The interaction of ionizing radiation with the human body, and (2-mercapto-phenyl)-methanol, respectively. Thiophenol
arising either from external sources outside the body or from was reacted with 2-chlorobenzaldehyde in hexamethylphos-
internal contamination of the body by radioactive substances, phoramide (HMPA) in the presence of sodium carbonate at
leads to biological effects which may later show up as clini- 85 °C for 5 h producing the aldehyde 1 in a good yield. Re-
cal symptoms. In general, these symptoms result from dam- duction of 2-mercaptobenzoic acid with lithium aluminum
age to the blood forming organs, digestive system, or central hydride gave (2-mercapto-phenyl)-methanol which was used
nervous system, depending on the radiation dose.1—3) There- for the subsequent preparation of 3 and 5. Treatment of (2-
fore, protecting the body from the toxic effects of radiation mercapto-phenyl)-methanol with 2-chlorobenzaldehyde in
has been a major concern in public health. Patt et al.4) re- HMPA in the presence of sodium carbonate at 100 °C for 5 h
ported that the administration of the sulfurhydryl amino acid
cysteine, given to rats before an 8-Gy dose of whole-body X-
irradiation, significantly increased the animals’ resistance to
the cytotoxic effects of the exposure. Since this initial find-
ing, new agents have been developed for cytoprotection. As
reviewed5,6) by Foye, these agents include mercaptans, di-
and trisulfides, phosphorothioates, acid hydrazides, imida-
zoles, and amine oxides. Although these compounds are too
toxic to be used in the clinic, the need still exists for com-
pounds that are more effective and less toxic for both mili-
tary and emergency use. Recently, the fact that amifostine
(WR2721; NH2–(CH2)3–NH–(CH2)2–SPO3H2) has been used
to protect7) cancer patients against the cytotoxicity of ioniz-
ing radiation in the clinic has gained much more attention in
the search for new cytoprotective agents. Substituted
anilines8) were reported to possess significant radioprotective
activity, but some of them are toxic. Blickenstaff et al.9) re-
ported the Schiff base products of substituted anilines and
aryl-aldehyde could reduce the toxicity of substituted ani-
lines but preserve the radioprotective activity. Since the radi-
cal scavenging ability of mercaptans and disulfides is well-
known, we speculated that the introduction of these impor-
tant functional groups into the Schiff base products might
provide potent antioxidative and radioprotective activity. In
this work, the sulfur-containing Schiff bases of substituted
anilines and benzaldehyde have been synthesized and their
free radical scavenging, antioxidation, and radioprotection
effects were also investigated.
Chart 1
Chemistry
The synthetic routes to the target compounds (2a—c),
(4a, b) and (6a, c) are outlined in Charts 1—3. Initially, the
key intermediates, 2-(phenylthio)benzaldehyde (1), 2,2Ј-
dibenzaldehyde sulfide (3), and 2,2Ј-dithiodibenzaldehyde
(5) were prepared from thiophenol, 2-mercaptobenzoic acid,
Chart 2
To whom correspondence should be addressed. e-mail: hly@ndmctsgh.edu.tw
© 2003 Pharmaceutical Society of Japan

1308
Vol. 51, No. 11
followed by oxidation with pyridine chlorochromate (PCC)
The radiation protection activities of these test compounds
yielded 3. (2-Mercapto-phenyl)-methanol could also be oxi- were assessed using cultured human HeLa tumor cells. The
dized with PCC to produce 5. Seven Schiff bases 2a—c, cells were irradiated in the absence or presence of 10Ϫ4 M test
4a, b and 6a, c were obtained using mono- and bis-benzalde- compounds and the percent of cell survival after 7 d, summa-
hyde (1, 3, 5) with aniline, p-anisidin, and 2,4-dichloraniline, rized in Table 3, was in the following the order of activity:
respectively, in the presence of magnesium sulfate at reflux 6cϾ4aϾ2aϭ6a. These results indicated that all of the test
temperature.
compounds exhibited a good radioprotective effect compared
to the untreated control cells. The radioprotection was most
Results and Discussion
The model of scavenging DPPH free radical10) is a simple
method to evaluate the antioxidative activity of antioxidants.
As shown in Table 1, these compounds displayed various de-
grees of free radical scavenging activity, with decreasing ac-
tivity in the following order: 6aϾ6cϾ2bϾ4bϾ4aϾ2cϾ2a.
Among them, compounds 6a and 6c, containing disulfide
structures, were the most potent, having antiradical effects
comparable to that of trolox.
Table 1. Free Radical Scavenging Activity of Compounds for DPPH Radi-
cal
Compound name
IC50a) (mM)
Inhibition (%)b)
Trolox
WR2721
Glutathione
34.5
106.5
96.5
880
116
185
155
128
44.4
35.5
62.9
24.3
38.6
5.8
20.7
12.8
14.4
17.9
49.8
72.0
2a
2b
2c
4a
4b
6a
6c
The in vitro model using AAPH-induced lipid peroxida-
tion of Tween-emulsified linoleic acid is a common method11)
used to measure the antioxidative activity of synthetic antiox-
idants. In this assay, the oxidation is carried out under condi-
tions relatively similar to in vivo biological systems. The in-
hibitory effects on lipid peroxidation or the antioxidative ac-
tivity of these compounds are listed in Table 2. We defined
a) IC50: The concentration of test compounds needed to reduce DPPH absorption by
50% at 517 nm. The values were calculated from the slope equation of the dose–re-
sponse curves. Values are means of three independent determinations. b) Inhibition
the antioxidative activity of these compounds as the relative (%) indicates the percent inhibition at 50 mM of antioxidant.
rate of lipid peroxidation initiated by AAPH radical. The
stronger the antioxidative activity, the smaller the rate of lipid
peroxidation. The inhibitory activity of these compounds in
decreasing order was 6cϾ4aϾ2aϾ6a. Compound 6c exhib-
ited antioxidative activity that was comparable to that of
trolox.
Table 2. Antioxidant Activity of the Compounds on 2,2Ј-Azobis(2-amino-
propane)dihydrochloride (AAPH)-Induced Lipid Peroxidation of a Tween-
Emulsified Linoleic Acid System
Rate of peroxidation
Inhibition of
Compound name
(DA500/min)
peroxidation (%)a)
Control
Trolox
WR2721
Glutathione
2a
4.30ϫ10Ϫ3
7.54ϫ10Ϫ4
2.48ϫ10Ϫ3
1.75ϫ10Ϫ2
1.92ϫ10Ϫ3
1.25ϫ10Ϫ3
2.99ϫ10Ϫ3
7.67ϫ10Ϫ4
0
82.5
13.9
59.3
55.3
70.9
30.5
82.2
4a
6a
6c
a) Inhibition of peroxidation (%)ϭ(1Ϫrate of test compound/rate of control)ϫ
100%. Peroxidation was initiated by the addition of AAPH (0.1 M) solution to the
Tween-emulsified linoleic acid mixture. Degree of peroxidation was estimated by mea-
suring absorption at 500 nm for the formation of complex [Fe(SCN)]2ϩ. Trolox and glu-
tathione (GSH) served as reference compounds. All tests were performed in triplicate.
Data shown here represent the slope of the time course-absorption curves of each com-
pound analyzed. The control for the assay was carried out identically but in the absence
of the test compound and the slope set as 100%.
Chart 3
Table 3. Radiation Protection of Compounds in Human Cells
Cell survival (%)
Treatment
Fold of protection
0 Gy
2 Gy
5 Gy
10 Gy
at 10 Gy
Untreated
Trolox
WR2721
2a
0 M
10Ϫ4
10Ϫ4
10Ϫ4
10Ϫ4
10Ϫ4
10Ϫ4
100.0
100.0
100.0
100.0
100.0
100.0
100.0
51.4
92.0
48.6
71.3
76.8
73.5
91.8
29.0
57.5
27.0
52.1
57.1
40.5
71.2
16.6
56.2
16.8
28.0
41.0
27.9
69.2
1.0
M
M
M
M
M
M
3.38
1.01
1.69
2.47
1.68
4.17
4a
6a
6c
HeLa cells were treated in the absence or presence of indicated synthetic or standard compounds for 30 min before being irradiated with various doses of 137Cs. The treated cells
were then split and seeded in identical cell numbers into appropriate cultured vessels. After seven days, the number of viable cells was assessed. Results shown here represent the
average of two independent experiments.

November 2003
1309
7.15 (d, 2H, Jϭ6.9 Hz, ArH), 7.42—7.51 (m, 4H, ArH), 7.94 (dd, 2H,
Jϭ7.35, 1.6 Hz, ArH), 10.33 (s, 2H, CHO). 13C-NMR (CDCl3) d: 128.47,
132.32, 133.15, 135.21, 135.62, 139.16, 191.99. IR (KBr) cmϪ1: 1674
(CϭO). MS m/z: 243 (MϩH)ϩ. Anal. Calcd for C14H10O2S2: C, 69.40; H,
4.16. Found: C, 69.20; H, 4.25.
2,2؅-Dithiodibenzaldehyde A mixture of pyridine chlorochromate (13
g, 60 mmol) in dry dichloromethane (60 ml) under nitrogen atmosphere was
added dropwise to the mixture of (2-mercapto-phenyl)-methanol (3.37 g, 24
mmol) in dry dichloromethane (50 ml). After stirring at room temperature
for 4 h, the reaction mixture was filtered. The residue was washed with
dichloromethane (3ϫ30 ml) and ether (30 ml). The filtrate and washings
were combined, and then concentrated to give the solid product. Pure com-
pound was obtained by crystallization with ethanol (3.1 g, 47%), mp: 145—
148 °C (lit14) mp 145 °C).
Schiff Base Formation, General Procedure The substituted anilines
and anhydrous magnesium sulfate were added under a nitrogen atmosphere
to a stirred solution of sulfur-containing aryl-aldehyde in absolute ethanol.
The reaction mixture was heated under reflux for 4 or 14 h and then filtered.
The residue was washed with absolute ethanol. The combined solution of fil-
trate and washings were combined, concentrated and then purified by chro-
matography (silica gel, n-hexane/ethyl acetateϭ15/1 or 5/1) to give the cor-
responding products.
prominent with compound 6c, which is greater than that of
the reference compound trolox. The lack of a radioprotective
effect for WR2721 may be due to the lack of alkaline phos-
phatase in the medium that is required to convert WR2721 to
its thiol active form. It should be noted that the efficacy of
the test compounds in terms of radioprotective activity corre-
lates well with the antioxidative activity as shown in Table 2.
These results demonstrate the Tween-emulsified lipid peroxi-
dation system used in our study could reflect the in vivo cell
system.
In summary, we have synthesized sulfur-containing aryl-
aldehyde Schiff bases in the present study. The results have
demonstrated that these sulfur-containing aryl-aldehyde
Schiff bases exhibit evident antioxidative activity that en-
ables them to be potential antioxidative, anti-inflammatory,
or radioprotective agents. Further experiments evaluating the
radioprotective effects of 6c in normal cell or mice are cur-
rently in progress.
[N-2-(Phenylthio)benzylidene]aniline (2a): Yield: 40%. Rf 0.5 (n-
hexane/ethyl acetateϭ7/1). 1H-NMR (CDCl3) d: 7.24—7.53 (m, 13H, ArH),
8.39 (d, 1H, Jϭ7.4 Hz, ArH), 9.16 (s, 1H, CNH). 13C-NMR (CDCl3) d:
121.80, 126.85, 127.62, 128.78, 128.92, 129.26, 129.42, 130.10, 130.42,
130.53, 130.74, 131.01, 132.30, 134.43, 137.01, 137.35, 152,68, 159.27. IR
(KBr) cmϪ1: 1618 (CϭN). UV lmax (EtOH) nm (log e): 289 (3.90). FAB-MS
m/z: 290 (MH)ϩ. Anal. Calcd for C19H15NS: C, 78.86; H, 5.22; N, 4.84.
Found: C, 78.60; H, 5.37; N, 4.82.
Experimental
General Melting points (mp) were taken on a BUCHI 530 apparatus
and are uncorrected. Merck Art No. 105554 plates precoated with Silica gel
60 containing fluorescent indicator were used for thin-layer chromatography,
and Silica gel 60 (Merck Art No 109385, 230—400 mesh) was employed for
column chromatography. Evaporations were carried out at Ͻ50 °C using a
rotary evaporator at reduced pressure (water aspirator). 1H- and 13C-NMR
spectra were obtained with a Varian 300 NMR spectrometer at 300 and
75 MHz, respectively. Where necessary, deuterium exchange experiments
were used to obtained proton shift assignments. Mass spectra were recorded
on a JEOL J.M.S-300 spectrophotometer. Analytical samples were dried
under reduced pressure at 78 °C in the presence of P2O5 for at least 12 h un-
less otherwise specified. Elemental analyses were obtained using a Perkin-
Elmer 2400 Elemental Analyzer.
2-(Phenylthio)benzaldehyde (1) The reaction mixture of 2-chlorobenz-
aldehyde (15 g, 107 mmol), thiophenol (15 g, 136 mmol), sodium carbonate
(20 g), and hexamethylphosphoramide (31 g, 173 mmol) was heated at 85 °C
for 5 h. After cooling, the mixture was poured into water (100 ml). After ex-
traction with ether and drying with magnesium sulfate, the mixture was con-
centrated under reduced pressure to a residue. The residue was crystallized
with n-hexane to give pure 1 (18.5 g, 80%), mp: 47—48 °C (lit.12) 50—
51 °C).
(2-Mercapto-phenyl)-methanol The suspension of LiAlH4 (2 g, 52
mmol) in dry tetrahydrofuran (THF) (80 ml) was added dropwise to a solu-
tion of 2-mercaptobenzoic acid (4.36 g, 28.4 mmol) in dry THF (50 ml).
After stirring at room temperature for 24 h, 10% H2SO4 (40 ml) and ethyl ac-
etate (40 ml) were added carefully and the reaction mixture was filtered. The
residue was washed with ethyl acetate (3ϫ10 ml). The filtrate and washings
were combined, dried with magnesium sulfate, and concentrated under re-
duced pressure to give an oily product (2 g, 50%), which became solid after
cooling. Pure compound was obtained by crystallization with a mixture of n-
hexane and ethyl acetate, mp: 31—32 °C (lit.13) 32—32 °C).
4-Methoxyl-[N-(2-phenylthio)benylidene]aniline (2b): Yield: 38%. Rf
1
0.47 (n-hexane/ethyl acetateϭ7/1). H-NMR (CDCl3) d: 3.82 (s, 3H, CH3),
6.93—6.97 (q, 2H, ArH), 7.20—7.47 (m, 10H, ArH), 8.33 (d, 1H, Jϭ7.4 Hz,
ArH), 9.10 (s, 1H, CHN), 13C-NMR (CDCl3) d: 56.07, 115.02, 123.09,
127.45, 128.88, 129.07, 123.0, 130.52, 131.89, 134.45, 136.81, 157.09. IR
(KBr) cmϪ1: 1618 (CϭN). UV lmax (EtOH) nm (log e): 289 (3.91). FAB-MS
m/z: 320 (MH)ϩ. Anal. Calcd for C20H17NOS: C, 75.20; H, 5.36; N, 4.39.
Found: C, 75.06; H, 5.46; N, 4.28.
2,4-Dichloro-[N-(2-phenylthio)benylidene]aniline (2c): Yield: 42%. Rf
0.37 (n-hexane/ethyl acetateϭ13/1). 1H-NMR (CDCl3) d: 6.69 (d, 2H,
Jϭ8.5 Hz, ArH), 7.12 (dd, 2H, Jϭ8.4, 2.2 Hz, ArH), 7.28—7.38 (m, 4H,
ArH), 7.44—7.46 (m, 3H, ArH), 8.35 (dd, 1H, Jϭ6.2, 2.4 Hz, ArH), 8.98 (s,
1H, CHN). 13C-NMR (CDCl3) d: 121.33, 127.66, 128.36, 128.95, 129.33,
129.86, 130.10, 130.27, 130.68, 131.79, 132.82, 134.35, 136.70, 136.86,
137.68, 148.60, 161.23. IR (KBr) cmϪ1: 1618 (CϭN). UV lmax (EtOH) nm
(log e): 289 (3.82). FAB-MS m/z: 358 (Mϩ). Anal. Calcd for C19H13NCl2S:
C, 63.69; H, 3.66; N, 3.91. Found: C, 63.39; H, 3.51; N, 3.73.
2,2Ј-Di(phenylaminobenzylidene)sulfide (4a): Yield: 21%. Rf 0.4 (n-
hexane/ethyl acetateϭ15/1). 1H-NMR (CDCl3) d: 7.24—7.35 (m, 10H,
ArH), 7.40—7.46 (m, 6H, ArH), 8.29 (dd, 2H, Jϭ9.5, 2.5 Hz, ArH), 9.08 (s,
2H, CNH). 13C-NMR (CDCl3) d: 121.71, 126.89, 128.51, 129.76, 129.81,
132.39, 136.68, 137.87, 152.48, 158.71. IR (KBr) cmϪ1: 1618 (CϭN). UV
lmax (EtOH) nm (log e): 321 (3.91), 289 (3.94). MS: FAB-MS m/z: 393
(MH)ϩ. Anal. Calcd for C19H13NCl2S: C, 79.56; H, 5.14; N, 7.14. Found: C,
79.43; H, 5.22; N, 7.01.
2-(2-Hydroxymethylphenylthio)benzaldehyde The reaction mixture of
2-chlorobenzaldehyde (6.6 g, 47 mmol), (2-mercapto-phenyl)-methanol (6.8
g, 49 mmol), 63% NaOH (3 ml), and hexamethylphosphoramide (12 g, 69
mmol) was heated at 100 °C for 5 h. After cooling, the mixture was poured
into water (100 ml). After extraction with benzene and drying with magne-
sium sulfate, the mixture was concentrated under reduced pressure to a
residue (5.4 g, 80%). Pure compound was obtained by chromatography (sil-
ica gel, n-hexane/ethyl acetateϭ3/1) as an oily product. Rf 0.27 (n-hexane/
ethyl acetateϭ3/1). 1H-NMR (CDCl3) d: 3.31 (s, 1H, OH), 4.69 (d, 2H,
Jϭ18 Hz, CH2), 6.77—7.76 (m, 8H, ArH), 10.19 (s, 1H, CHO).
2,2Ј-Di(4-methoxylphenylaminobenzylidene)sulfide (4b): Yield: 35%. Rf
0.4 (n-hexane/ethyl acetateϭ15/1). mp: 91—92 °C. 1H-NMR (CDCl3) d:
3.82 (s, 6H, CH3), 6.89 (d, 4H, Jϭ8.9 Hz, ArH), 7.12—7.27 (m, 6H, ArH),
7.30—7.41 (m, 4H, ArH), 8.19 (dd, 2H, Jϭ7.2, 1.9 Hz, ArH), 9.02 (s, 2H,
CHN). 13C-NMR (CDCl3) d: 56.05, 114.0, 122.88, 122.99, 128.20, 128.35,
129.28, 131.87, 131.92, 133.90, 133.25, 136.84, 137.49, 156.54. IR (KBr)
cmϪ1: 1618 (CϭN). UV lmax (EtOH) nm (log e): 337 (4.28). MS: FAB-MS
m/z: 453 (MH)ϩ. Anal. Calcd for C28H24N2S: C, 74.31; H, 5.34; N, 6.19.
Found: C, 74.07; H, 5.36; N, 6.12.
2,2Ј-Di-phenylaminobenzylidene Disulfide (6a): Yield: 50%. Rf 0.35 (n-
1
2,2؅-Dibenzaldehyde Sulfide (3) A mixture of pyridine chlorochromate
(10 g, 46 mmol) in dry dichloromethane (60 ml) under nitrogen atmosphere
was added dropwise to a mixture of 2-(2-hydroxymethylphenylthio)benzal-
dehyde (3.37 g, 24 mmol) in dry dichloromethane (50 ml). After stirring at
room temperayure for 4 h, the reaction mixture was filtered. The residue was
washed with dichloromethane (3ϫ30 ml) and ether (30 ml). The filtrate and
washings were combined, and then concentrated to give the solid product
(3.5 g, 88%). Pure compound was obtained by crystallization with ethanol,
hexane/ethyl acetateϭ5/1). H-NMR (CDCl3) d: 7.24—7.44 (m, 7H, ArH),
7.83 (d, 2H, Jϭ7.26 Hz, ArH), 7.96 (d, 2H, Jϭ7.35 Hz, ArH), 8.86 (s, 2H,
CHN). 13C-NMR (CDCl3) d: 121.65, 121.74, 126.88, 127.91, 129.72,
130.29, 130.36, 130.78, 130.92, 131.76, 158.74. IR (KBr) cmϪ1: 1625
(CϭN). UV lmax (EtOH) nm (log e): 297 (3.78), 289 (3.78). MS: FAB-MS
m/z: 423 (Mϩ). Anal. Calcd for C28H24N2S: C, 73.55; H, 4.75; N, 6.60.
Found: C, 73.44; H, 4.89; N, 6.43.
2,2Ј-Di(2,4-dichlorophenylaminobenzylidene)disulfide (6c): Yield: 52%.
1
1
mp: 96—97 °C. Rf 0.42 (n-hexane/ethyl acetateϭ3/1). H-NMR (CDCl3) d:
Rf 0.35 (n-hexane/ethyl acetateϭ3/1). mp: 132—134 °C. H-NMR (CDCl3)

1310
Vol. 51, No. 11
d: 6.95 (d, 3H, Jϭ8.46 Hz, ArH), 7.21—7.47 (d, 3H, Jϭ8.46 Hz, ArH), 7.78
(d, 2H, Jϭ7.68 Hz, ArH), 7.95 (dd, 2H, Jϭ7.41, 1.29 Hz, ArH), 8.73 (s, 1H,
CHN), 13C-NMR (CDCl3) d: 121.16, 128.21, 128.28, 130.33, 131.33,
132.37, 160.83. IR (KBr) cmϪ1: 1624 (CϭN). UV lmax (EtOH) nm (log e):
296 (4.00). MS: FAB-MS m/z: 560.9 (Mϩ). Anal. Calcd for C28H24N2S: C,
55.53; H, 2.87; N, 4.98. Found: C, 55.59; H, 3.03; N, 4.87.
Determination of the Scavenging Effect on 1,1-Diphenyl-2-pycryl-hy-
drazyl radical (DPPH·) The reactivity of title compounds with DPPH
was determined from the change in absorbance at 517 nm, according to the
method of Ohnishi et al.10) For radical scavenging measurements, 2 ml of the
compound solution under study was added to an ethanolic solution of
DPPH· and then the change in absorbance was measured after 30 min. All
tests were performed in triplicate.
of stock cultures and cell counts were determined using a hemocytometer.
Cells were washed with PBS and adjusted to achieve a concentration of
1ϫ106 cells/ml of medium. Cells exposed to 0.1 mM of test compounds were
treated at 37 °C for 30 min prior to irradiation. Immediately following irradi-
ation, the cells were washed free of drug with PBS, and along with the un-
treated control groups were counted diluted, and seeded in triplicate into 60-
mm-diameter dishes containing 3 ml of standard growth medium. The cells
were allowed to grow for 7 d and cell survival and proliferation were ana-
lyzed using a Cell Counting Kit-8 (CCK-8, Dojin East, Tokyo, Japan). Cell
survival was expressed as the ratio of absorbance of the drug-treated cells to
that of the untreated cells. Trolox and amifostine (WR2721) were used as
standard compounds for assessing the efficacy of the synthetic test com-
pounds. All assays were performed in triplicate.
Determination of Antioxidative Activity The antioxidative activity
was evaluated by using 2,2Ј-azobis(2-amidinopropane) dihydrochloride
(AAPH)-induced lipid peroxidation of a Tween-emulsified linoleic acid sys-
tem and measured by the ferric thiocyanate assay as described.10) Briefly, 0.2
ml of distilled water, 0.5 ml of 0.2 M phosphate buffer (pH 7.0), and 0.5 ml of
0.25% Tween-20 (in buffer solution) were mixed with 0.5 ml of 2.5% (w/v)
linoleic acid in ethanol. The mixture was then stirred for 1 min. The peroxi-
dation was initiated by the addition of 50 ml of AAPH solution (0.1 M). The
stock solution of antioxidant or test compounds in DMSO (final concentra-
tions for the test compounds and DMSO are 10Ϫ4 M and 0.1%, respectively)
was then added, and the reaction was carried out at 37 °C for 375 min in the
dark. The degree of inhibition of oxidation was measured by the ferric thio-
cyanate method for each interval of 75, 150, 225, 300, and 375 min. To 0.1
ml of peroxidation reaction mixture at each interval, 0.1 ml of 30% ammo-
nium thiocyanate and 0.1 ml of 2ϫ10Ϫ2 M freshly prepared FeCl2 (in 3.5%
aqueous HCl) were added. Precisely 3 min after addition, the absorbance of
the red complex [Fe(SCN)]2ϩ was measured at 500 nm. The control for the
assay was prepared in the same manner by mixing all of the chemicals and
reagents except the test compound. All tests were performed in triplicate.
Cell Culture Human cervical carcinoma (HeLa) cells were cultured at
37 °C in DMEM medium supplemented with 15 mM Hepes, 26 mM sodium
bicarbonate, 2 mM L-glutamine, 100 mg/ml streptomycin, 100 unit/ml peni-
cillin, and 10% fetal bovine serum. Cell number was determined by counting
the viable cells by trypan blue dye exclusion using a hemocytometer. Cells
in the logarithmic growth phases were used in this study.
Acknowledgments We thank the National Science Council (NSC) of
the Republic of China for its general financial support (NSC 91-2623-7-010-
001-NU to T.-C. Chang and NSC 91-2623-7-016-002-NU to L. Y. Hsu) and
Ms. Shu-Yun Sun and Ching-Wei Lu, Instrumentation Center, NSC for their
help in obtaining the mass spectra and elemental analysis.
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