Synthesis of radioiodine labeled dibenzyl disulfide for evaluation of tumor cell uptake

Article abstract of DOI:10.1016/j.bmc.2004.01.002

Benzyl 4-halobenzyl and ally benzyl disulfide were synthesized as diallyl disulfide analogues and their tumor growth inhibitory effects on the cancer cells (SNU C5 and MCF-7) were comparable to that of diallyl disulfide, indicating that the disulfide func

Full text of DOI:10.1016/j.bmc.2004.01.002

Bioorganic & Medicinal Chemistry 12 (2004) 859–864
Synthesis of radioiodine labeled dibenzyl disulfide for evaluation
of tumor cell uptake
Eun Kyoung Ryu,a Yearn Seong Choe,a,* Sang Sung Byun,a Kyung-Han Lee,a
Dae Yoon Chi,b Yong Choia and Byung-Tae Kima
aDepartment of Nuclear Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, 50 Ilwon-dong,
Kangnam-ku, Seoul 135–710, South Korea
bDepartment of Chemistry, Inha University, 253 Yonghyundong, Namgu, Inchon, 402–751, South Korea
Received 17 September 2003; revised 26 December 2003; accepted 5 January 2004
Abstract—Benzyl 4-halobenzyl and ally benzyl disulfide were synthesized as diallyl disulfide analogues and their tumor growth
inhibitory effects on the cancer cells (SNU C5 and MCF-7) were comparable to that of diallyl disulfide, indicating that the disulfide
functional groupwas responsible for the tumor growth inhibitory effects. Cu(I)-assisted radioiodination of benzyl 4-bromobenzyl
disulfide gave benzyl 4-[123I/125I]iodobenzyl disulfide in 30–40% radiochemical yield. The radiolabeled disulfide was taken upby the
cancer cells in a time-dependent manner, and the uptake was inhibited by the pretreatment of S-methyl methanethiosulfonate
(MMTS), phorone and diallyl disulfide. This study suggested that the radiolabeled dibenzyl disulfide was taken up by the cancer
cells via thiol-disulfide exchange and retained inside the cells.
# 2004 Elsevier Ltd. All rights reserved.
1. Introduction
6 was shown to inhibit the growth of canine mammary
tumor cells (CMT-13) in vitro and N-methyl-N-nitro-
sourea-induced rat mammary tumors, whereas water-
soluble organosulfur compounds were not effective.14ꢀ16
Disulfide 6 also markedly inhibits the growth of human
colon tumor cells (HCT-15).17 Therefore, diallyl dis-
ulfide 6, a major organosulfur compound in garlic,
appears to be the most effective in growth inhibition of
human cancer cells such as skin, breast, and colon can-
cer cells.17
Garlic has been a focus of attention as the dietary
anticarcinogen, since the recent studies implicating the
correlation between consumption of garlic and low
incidence of cancer.1 In addition to this anticarcinogenic
property, garlic has been known to possess antibiotic,
fungicidic, antihelmentic, antithrombotic, and anti-
oxidant properties.1ꢀ3 The anticarcinogenicity of garlic
has been therefore evaluated in the studies using chemi-
cally induced tumors in animals.4ꢀ6 Garlic was shown to
inhibit the promotion phase of carcinogenesis in skin
tumors induced by 7,12-dimethylbenz[a]anthracene.7
Reduction of breast cancer risk was also shown related
to the consumption of garlic.8,9 It was suggested that the
anticarcinogenic property of the garlic derives from a
complex mixture of organosulfur compounds, such as
oil-soluble diallyl sulfide, diallyl disulfide (6), diallyl tri-
sulfide, and water-soluble S-allyl cysteine.10,11 Dietary
allyl sulfur compounds of garlic reduced tumors in rats
treated with the typical colon carcinogen, 1,2-dimethyl-
hydrazine.12,13 The oil-soluble organosulfur compound
The anticarcinogenicity of the organosulfur compounds
seems to derive from more than one mechanism,
because the organosulfur compounds can inhibit acti-
vation and carcinogenicity of chemicals such as diverse
carcinogens and also influence in various tissues such as
skin, breast, colon, etc.18 Suppression of cell division
rate and induction of apoptosis were reported as
another mechanisms for the anticarcinogenicity of 6.19
It is likely that the disulfide functional groupof 6 is
responsible for its anticarcinogenicity. In this study,
therefore, benzyl halobenzyl and allyl benzyl disulfide
(Fig. 1) were synthesized as diallyl disulfide analogues
and evaluated for their inhibitory effects on growth of
human breast cancer (MCF-7) cells and colon cancer
(SNU C5) cells. Their tumor cell uptake and retention
mechanisms were investigated using the radiolabeled
Keywords: Benzyl 4-[123I/125I]iodobenzyl disulfide; Diallyl disulfide;
Radioiodine; Tumor growth inhibition; Tumor cell uptake.
* Corresponding author. Tel.: +82-2-3410-2623; fax: +82-2-3410-
2639; e-mail: yschoe@samsung.co.kr
0968-0896/$ - see front matter # 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.01.002

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E. K. Ryu et al. / Bioorg. Med. Chem. 12 (2004) 859–864
disulfide, because little is known about the mechanisms
of the disulfide. The disulfides containing benzene rings
were desirable for radiolabeling, due to facile introduc-
tion of radiohalogen to a benzene ring and a long syn-
thetic route required for the synthesis of radioiodine
labeled diallyl disulfide. The slight modification in ben-
zyl halobenzyl and allyl benzyl disulfide does not seem
to induce a significant change of the physicochemical
properties of 6. Among these disulfides, dibenzyl dis-
ulfide was radiolabeled with 123I/125I and its tumor cell
uptake and retention mechanisms were evaluated.
hydroxyl groupfor displacement with a sulfur nucleo-
phile,20 thioacetylation of the alcohol followed by clea-
vage of the thioacetate to thiol under basic condition
provided thiol and disulfide in low yields which were
also limited in separation due to their similar polarities.
Therefore, the hydroxyl groupwas activated by conver-
sion to the methanesulfonate 7, followed by displace-
ment with a suitable sulfur nucleophile. Reduction of
the resulting thioester 8 with LiAlH4 gave the sole
product 9.21
Synthesis of the unsymmetric disulfide was carried out
in high yield using 2,20-dithiobis(benzothiazole) (10) as
the intermediate disulfide.22 This symmetric disulfide
was reacted with one equivalent of benzyl thiol under
the mild conditions to provide unsymmetric disulfides,
11 and 12. Further thiol-disulfide exchange reaction of
this disulfide with 4-halobenzyl thiol or allyl thiol yiel-
ded the desired unsymmetric disulfides, 1, 2, 4 and 5
(Figs 2 and 3).22,23 When 4-halobenzyl thiol was reacted
with disulfide 10, the resulting unsymmetric disulfide
was not readily separated from 10. Therefore, benzyl
thiol was converted to 2-benzothiazolyl benzyl disulfide
(11) that was then reacted with 4-halobenzyl thiol to
give the desired unsymmetric disulfide in high yield
(>89%) and with high purity (>99%).
Figure 1. Structures of disulfides: benzyl 4-iodobenzyl disulfide (1),
benzyl 4-bromobenzyl disulfide (2), dibenzyl disulfide (3), allyl 4-
iodobenzyl disulfide (4), allyl benzyl disulfide (5) and diallyl disulfide (6).
2.2. Radiochemical Synthesis of [123I/125I]1
Radioiodine labeled disulfide was prepared by halogen
exchange reaction of benzyl 4-bromobenzyl disulfide in
the presence of Na123I/125I and CuCl at 150 ꢁC for 1 h
(Fig. 4). It was suggested that Cu(I) ion forms a tricyclic
intermediate between Br and the aromatic carbon which
allows substitution of Br with I.24 Progress of the reac-
tion was monitored by radio-TLC (Fig. 5). At the end
of the reaction, the product was purified by reverse
phase HPLC (tR=10–11 min) (Fig. 6). When a normal
2. Results and discussion
2.1. Synthesis of disulfides
4-Halobenzyl thiol (9) was readily prepared from 4-
halobenzyl alcohol in three steps (Fig. 2). Although the
Mitsunobu reaction does not require activation of a
Figure 3. Synthetic pathways of allyl 4-iodobenzyl disulfide (4) and
allyl benzyl disulfide (5).
Figure 4. Synthetic pathway of [123I/125I]1.
Figure 2. Synthetic pathway of benzyl 4-halobenzyl disulfide (1 and 2).

E. K. Ryu et al. / Bioorg. Med. Chem. 12 (2004) 859–864
861
Table 1. Percentage of growth inhibition on MCF-7 and SNU C5
cells by disulfide analoguesa
Disulfidea
1 (%)
2 (%)
3 (%)
4 (%)
5 (%)
6 (%)
MCF-7
SNU C5
96
100
90
103
94
101
97
—
91
101
100
95
a Disulfide: benzyl 4-iodobenzyl disulfide (1), benzyl 4-bromobenzyl
disulfide (2), dibenzyl disulfide (3), allyl 4-iodobenzyl disulfide (4),
allyl benzyl disulfide (5) and diallyl disulfide (6). Data are expressed
as mean (n=3).
Figure 5. Radio-TLC of the radioiodination mixture. Developing sol-
vents: 49:1 hexane–ethyl acetate.
Uptake of the radiolabeled disulfide by the cancer cells
showed a time-dependent increase as shown in Figure 7,
demonstrating a two-fold increase at 1 h compared to
15 min by both cells and a four-fold increase at 2 h by
SNU C5 cells. This result demonstrated that the radio-
tracer was more efficiently taken upby SNU C5 cells
than by MCF-7 cells.
2.5. Inhibition of the uptake by various compounds
Morphologic change of the cells was observed at the
concentrations higher than 500 mM of the disulfide,
which was consistent with the results reported in the
literature.16 Thus the concentrations lower than 500 mM
were used in this study. Pretreatment of MMTS, phor-
one, and 6 reduced the uptake of [125I]1 by the cancer
cells in a dose-dependent manner (Fig. 8), showing a
similar pattern of the inhibition in both cancer cells.
Percentage of the inhibition was well correlated with the
concentration of the compounds added (5, 50, and 500
mM). This result can be explained by dose-dependent
reduction of the intracellular enzyme-SH and glu-
tathione (GSH) level by MMTS, a temporary blocking
agent for enzyme-SH groups, and phorone, a GSH
depleting agent, respectively,25 which leads to lowering
the uptake of the radiolabeled disulfide by the cancer
cells. It was reported that tumor growth inhibitory effect
of 6 was modified by intracellular GSH level.16 Attempts
were therefore made to find the direct effect of intracel-
lular GSH on the uptake of [125I]1. The cancer cells were
pretreated with GSH for 30 min prior to the incubation
with [125I]1, however, there was no change in the uptake
(data not shown), probably due to impermeability of
GSH through the cell membranes. Although the uptake
mechanism of the disulfide is not well known, dose-
dependent inhibition of the uptake of [125I]1 by MMTS
and phorone suggested that the uptake was mediated by
Figure 6. Semipreparative HPLC traces of [123I/125I]1. HPLC solvents:
85:15 CH3OH–H2O; HPLC column: C18 silica gel; tR=10.4 min; flow
rate: 4 mL/min; UV detector (UV) and a NaI(Tl) radioactivity detector.
phase HPLC system was used for the purification, the
product was coeluted with the precursor 2. Therefore, a
reverse phase HPLC system was applied to the pur-
ification of the final product using a 85:15 mixture of
methanol and water. Removal of the solvents (70 ꢁC,
N2) was troublesome, because this procedure not only
required a long period of time for removal of the sol-
vents but also accompanied simultaneous evaporation
of the product. The product from the desired HPLC
fraction was therefore extracted with CH2Cl2 and the
solvents were carefully removed at low temperature
(40 ꢁC) under a gentle stream of N2. [123I/125I]1 was
obtained in 30–40% radiochemical yield (decay-cor-
rected) and with high specific activity (420–660 GBq/
mmol). The identity of [123I/125I]1 was confirmed by
coelution with the nonradioactive authentic standard (1)
as determined by HPLC.
2.3. Tumor growth inhibition by disulfides
Disulfides were evaluated for their abilities to inhibit the
growth of MCF-7 and SNU C5 cells in vitro. Treatment
of the cancer cells with the disulfide (500 mM) resulted in
significant inhibition on the growth of cancer cells,
showing growth inhibition (>90%) comparable to that
by 6 (Table 1). The result suggested that the presence of
disulfide functional groupmight play a major role in
inhibiting the growth of the cancer cells.
2.4. Uptake of [125I]1 by the cancer cells
Figure 7. Uptake of [125I]1 by MCF-7 cells (closed bar) and SNU C5
cells (open bar). Data are expressed as meanꢂSD (n=3, p<0.05).
[
125I]1 was incubated at 37 ꢁC with MCF-7 or SNU C5
cells which were grown at the same temperature for 48 h.

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4. Experimental
4.1. General methods
1H NMR spectra were performed on a JNM-LA 300
(JEOL Ltd) and Varian 500NB spectrometer. Chemical
shifts (d) were reported in ppm downfield from tetra-
methylsilane as an internal reference. Electron impact
(EI) and fast atom bombardment (FAB) mass spectra
were obtained on a JMS-700 Mstation (JEOL Ltd)
instrument in the positive ion mode. For the FAB mass
spectrometry, EtOAc or MeOH was used as the solvent
and 3-nitrobenzyl alcohol saturated with NaI as matrix.
HPLC was carried out on a Thermo Separation Pro-
ducts System using a semipreparative column (Alltech
Econosil, C18, 10 m, 10ꢃ250 mm) and an analytical
column (YMC, C18, 5 m, 4.6ꢃ250 mm). The eluant was
simultaneously monitored by a UV (254 nm) detector
and a NaI(Tl) radioactivity detector. Radio-TLC was
performed on a radio-TLC scanner (Bioscan). GSH was
purchased from Sigma Chemical Company and all
other chemicals from Aldrich Chemical Company. Sol-
vents were distilled prior to use.
4.2. Synthesis of disulfides
Figure 8. Effect of various compounds (5, 50, and 500 mM) on the
uptake of [125I]1 by MCF-7 cells (top) and SNU C5 cells (bottom).%
Inhibition of the uptake by the compounds was expressed relative to
the control (100%) at 2 h after administration of the radiotracer
(n=3); Control (closed bar), 6 (right-handed striped bar), MMTS
(open bar), and phorone (left-handed striped bar). Data are expressed
as meanꢂSD.
4.2.1. 4-Halobenzyl methanesulfonate (7). 4-Halobenzyl
alcohol (6.42 mmol) was dissolved in dry CH2Cl2 (30
mL), and to this was added N,N0-diisopropylethylamine
(1.5 mL, 8.61 mmol). The reaction mixture was allowed
to stir at rt for 30 min. At the end of the reaction, the
mixture was cooled to ꢀ20 ꢁC and then methanesulfonyl
chloride (0.6 mL, 7.75 mmol) was slowly added while
maintaining the temperature below ꢀ10 ꢁC. The result-
ing solution was stirred for 3 h and then quenched with
1 N HCl. The solution was then poured into water (15
mL) and extracted with CH2Cl2 (30 mLꢃ3). The product
isolation afforded a pale brown solid 7 in 99% yield.
thiol-disulfide exchange which has been proposed as a
mechanism of enzyme activity control.25,26 Once the
disulfide is taken upby the tumor cells via thiol-disulfide
exchange, it is likely that the radioactivity is trapped
inside the cells after binding to either enzyme-SH or
GSH. Inhibition of the uptake of [125I]1 by 6 might
result from competitive uptake between the two
disulfides.
1
4.2.2. 4-Iodobenzyl methanesulfonate. H NMR (CDCl3,
500 MHz) d 7.74 (d, J=8.5 Hz, 2H), 7.15 (d, J=8.5 Hz,
2H), 5.17 (s, 2H), 2.93 (s, 3H); MS (FAB) m/z 335
(M++Na); HRMS calcd for C8H9IO3SNa 334.9215,
found 334.9216.
3. Conclusion
Diallyl disulfide found in garlic has been known to
inhibit the growth of various human cancer cells. In this
study, therefore, benzyl 4-halobenzyl and allyl benzyl
disulfide were synthesized as analogues of 6 and they
were all effective on the growth inhibition of MCF-7
and SNU C5 cells, indicating that the disulfide func-
tional groupmight be responsible for the tumor growth
inhibitory effects. Unsymmetric radioiodine labeled dis-
ulfide, [123I/125I]1 was synthesized in high radiochemical
yield and taken upby the cancer cells in a time-depen-
dent manner. The uptake of [125I]1 was inhibited by
pretreatment of MMTS and phorone, suggesting that
the uptake was mediated by thiol-disulfide exchange. It
is highly probable that this exchange allows the reten-
tion of the radioactivity inside the cells, which would
facilitate the tumor imaging by this radiotracer. Further
studies are warranted to investigate the biodistribution
of [123I]1 in tumor cell implanted animal models using
single photon emission computed tomography (SPECT).
4.2.3. 4-Bromobenzyl methanesulfonate. 1H NMR
(CDCl3, 500 MHz) d 7.54 (d, J=8.5 Hz, 2H), 7.29 (d,
J=7.5 Hz, 2H), 5.18 (s, 2H), 2.94 (s, 3H); MS (FAB) m/
z 289 (M++Na, 81Br), 287 (M++Na, 79Br); HRMS
calcd for C8H979BrO3SNa 286.9353, found 286.9374.
4.2.4. 4-Halobenzyl thioacetate (8). 4-Halobenzyl
methanesulfonate (6.79 mmol) was dissolved in DMF
(32 mL) and Cs2CO3 (6.64 g, 20.37 mmol) was added.
After the reaction mixture was stirred at rt for 30 min,
thiolacetic acid (1.46 mL, 20.37 mmol) was added. The
mixture was stirred for another 2 h and then extracted
with ethyl acetate (30 mLꢃ3), and the combined organic
extracts were then dried over anhydrous MgSO4. Flash
column chromatography (3:1 hexane–ethyl acetate)
afforded a yellow solid 8 in 95% yield.
4.2.5. 4-Iodobenzyl thioacetate. 1H NMR (CDCl3,
500 MHz) d 7.64 (s, 2H), 7.06 (s, 2H), 4.06 (d, J=26 Hz,

E. K. Ryu et al. / Bioorg. Med. Chem. 12 (2004) 859–864
863
2H), 2.36 (s, 3H); MS (FAB) m/z 293 (M++H); HRMS
calcd for C9H10IOS 292.9497, found 292.9503.
stirred at rt for 1 h. The product isolation and purifica-
tion (25:1 hexane–ethyl acetate) gave 12 (350 mg, 89%)
as a colorless oil. H NMR (CDCl3, 500 MHz) d 7.86
(dd, J=4.8, 3 Hz, 1H), 7.79 (dd, J=7.8, 0.5, 1H), 7.42
(td, J=8.3, 1.5 Hz, 1H), 7.32 (td, J=8, 1 Hz, 1H), 5.88
(m, 1H), 5.21 (ddd, J=34.5, 17, 10 Hz, 2H), 3.58 (d,
J=7.5, 2H); MS (FAB) m/z 240 (M++H); HRMS
calcd for C10H10NS3 239.9975, found 239.9976.
1
4.2.6. 4-Bromobenzyl thioacetate. 1H NMR (CDCl3,
300 MHz) d 7.39 (s, 2H), 7.14 (s, 2H), 4.01 (d, J=15.0
Hz, 2H), 2.32 (s, 3H); MS (EI) m/z 244 (M+) (Sigma
Chemical Company).
4.2.7. 4-Halobenzyl thiol (9). 4-Halobenzyl thioacetate
(2.15 mmol) in dry THF (3 mL) was added dropwise to
a slurry of LiAlH4 in dry THF (12 mL). The reaction
mixture was stirred at rt for 1 h. The mixture was
cooled, treated with water and then filtered. The filtrate
was washed with water and dried over MgSO4. Flash
column chromatography (5:1 hexane–ethyl acetate)
afforded a white solid 9 in 83% yield. 4-Iodobenzyl thiol
was used without analysis due to its instability.
4.2.14. Allyl 4-iodobenzyl disulfide (4). To 12 (182 mg,
0.76 mmol) in CHCl3 (15 mL) was added dropwise 4-
iodobenzyl thiol (190 mg, 0.76 mmol) in CHCl3 (3 mL)
with vigorous stirring. After stirred at rt for 4 h, the
product isolation and purification (hexane) gave allyl 4-
iodobenzyl disulfide (4) as a white solid in 95% yield:
1H NMR (CDCl3, 500 MHz) d 7.65 (d, J=10.5 Hz, 2H),
7.06 (d, J=8.5 Hz, 2H), 5.73–5.78 (m, 1H), 5.12 (s, 1H),
5.08 (dd, J=18.5, 1.5 Hz, 1H), 3.82 (s, 2H), 3.08 (dd,
J=28.5, 7.0 Hz, 2H); MS (FAB) m/z 322 (M+); HRMS
calcd for C10H11IS2 321.9347, found 321.9331.
4.2.8. 4-Bromobenzyl thiol. 1H NMR (CDCl3, 500 MHz)
d 7.45 (s, 2H), 7.19 (s, 2H), 3.67 (d, J=7.5 Hz, 2H), 1.79
(t, J=7.0 Hz, 1H); MS (EI) m/z 201 (M+) (Sigma
Chemical Company).
4.2.15. Allyl benzyl disulfide (5). Allyl thiol (0.13 mL,
1.66 mmol) in CHCl3 (3 mL) was added dropwise to 11
(1.66 mmol) in CHCl3 (21 mL) with vigorous stirring.
After stirred at rt for 5 h, the product isolation and
purification (hexane) gave allyl benzyl disulfide (5) as a
4.2.9. 2-Benzothiazolyl benzyl disulfide (11). Benzyl thiol
(0.12 mL, 1.02 mmol) in CHCl3 (2.5 mL) was added
dropwise to a suspension of 2,20-dithiobis(benzothia-
zole) (10, 339.1 mg, 1.02 mmol) in CHCl3 (14 mL) with
stirring at rt.4,22 After stirred for 1 h, the reaction mix-
ture was washed successively with 5% NaOH (aq, 15
mLꢃ2) and water (15 mLꢃ2) and then dried over
MgSO4. The solvent was removed in vacuo and the
residue was purified by flash column chromatography
(25:1 hexane–ethyl acetate) to give 11 (242 mg, 92.7%)
1
yellow oil in 95% yield: H NMR (CDCl3, 300 MHz) d
7.25–7.33 (m, 5H), 5.73–5.75 (m, 1H), 5.11 (s, 1H), 5.05
(dd, J=17.4, 1.2 Hz, 1H), 3.90 (s, 2H), 3.01 (d, J=7.5
Hz, 2H); MS (EI) m/z 196 (M+); HRMS calcd for
C10H12S2 196.0380, found 196.0385.
4.2.16. Benzyl 4-[123I/125I]iodobenzyl disulfide ([123I/125I]1).
Benzyl 4-bromobenzyl disulfide (2, 1 mg, 3.1 mmol) was
dissolved in dioxane (100 mL), and to this solution were
added CuCl (76 mg, 0.08 mmol) in DMSO (20 mL) and
an appropriate amount of Na123I/125I. The reaction
mixture was heated at 150 ꢁC for 1 h. The mixture was
extracted with CH2Cl2 (1.5ꢃ3 mL) and the solvents
were removed under a gentle stream of N2. The residue
was redissolved in 1 mL of HPLC solvents and purified
by HPLC using a 85:15 mixture of methanol and water
at a flow rate of 4 mL/min. The desired product was
eluted at the retention time of 10–11 min. [123I/125I]1
collected from HPLC was extracted with CH2Cl2 and
then concentrated at 40 ꢁC under a gentle stream of N2.
The residue was redissolved in ethanol and diluted with
saline to give a final solution of 18% ethanol in saline.
Specific activity of [123I/125I]1 was determined by using a
standard curve obtained from 1 with different con-
centrations injected on an analytical HPLC column
(flow rate: 1 mL/min) versus UV absorbance at 254 nm.
Another aliquot of [123I/125I]1 was coinjected with
unlabeled compound 1 on HPLC to confirm its identity.
1
as a white solid. H NMR (CDCl3, 500 MHz) d 7.87 (d,
J=7.5 Hz, 1H), 7.80 (d, J=7.0 Hz, 1H), 7.26–7.44 (m,
7H), 4.18 (s, 2H); MS (FAB) m/z 312 (M++Na);
HRMS calcd for C14H11NS3Na 311.9951, found
311.9957, identical with the literature values.22
4.2.10. Benzyl 4-halobenzyl disulfide (1 and 2). 4-Halo-
benzyl thiol (0.84 mmol) in CHCl3 (2.5 mL) was added
dropwise to 2-benzothiazolyl benzyl disulfide (11) (0.84
mmol) in CHCl3 (12 mL) with vigorous stirring at rt.
The reaction mixture was stirred for 4 h. The product
isolation and purification (hexane) afforded the unsym-
metric disulfides, 1 and 2 (95–97%).
4.2.11. Benzyl 4-iodobenzyl disulfide (1). 1H NMR
(CDCl3, 500 MHz) d 7.63 (d, J=6.5 Hz, 2H), 7.25–7.35
(m, 5H), 6.94 (d, J=6.5 Hz, 2H), 3.70 (s 2H); 3.50 (s,
2H); MS (FAB) m/z 373 (M++H); HRMS calcd for
C14H14IS2 372.9581, found 372.9573.
4.2.12. Benzyl 4-bromobenzyl disulfide (2). 1H NMR
(CDCl3, 300 MHz) d 7.42 (d, J=8.3 Hz, 2H), 7.24–7.36
(m, 5H), 7.06 (d, J=8.3 Hz, 2H), 3.66 (s, 2H), 3.48 (s,
2H); MS (FAB) m/z 326 (M+, 81Br), 324 (M+, 79Br);
HRMS calcd for C14H1379BrS2 323.9642, found 323.9652.
4.3. Tumor growth inhibition by disulfides
Cells plated from a single pellet into 25 cm2 tissue cul-
ture flasks were grown at 37 ꢁC under a humidified 5%
CO2 atmosphere in RPMI-1640 medium for 24 h.16
MCF-7 and SNU C5 cells were treated with disulfide
(500 mM) in DMSO and incubated at 37 ꢁC under a
humidified 5% CO2 atmosphere in RPMI-1640 medium
4.2.13. 2-Benzothiazolyl allyl disulfide (12). Allyl thiol
(1.3 mL, 1.66 mmol) in CHCl3 (4 mL) was added drop-
wise to a suspension of 10 (1.66 mmol) in CHCl3 (23
mL) with stirring at rt.4,22 The reaction mixture was

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E. K. Ryu et al. / Bioorg. Med. Chem. 12 (2004) 859–864
for 48 h. Control experiment was simultaneously carried
out in the absence of disulfide under the same condi-
tions. Cells were harvested by trypsinization (0.25%
trypsin-EDTA), diluted with RPMI-1640, removed by
gentle scraping and centrifuged. The cell pellet was sus-
pended in phosphate-buffered saline (pH 7.0) and trea-
ted with an equal volume of 0.4% trypan blue. The
mixture was allowed to incubate at rt for 3–5 min.
Viable cells were counted under hemocytometer.
Growth inhibition (%) of the cancer cells by disulfide
was determined according to Sundaram and Milner.16
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This work was supported by a grant from the Korea
Research Foundation (KRF-2001-015-DP0325).
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