Synthesis, characterization, mechanism of decomposition, and antiproliferative activity of a class of PEGylated benzopolysulfanes structurally similar to the natural product varacin

Article abstract of DOI:10.1021/jo100870q

Benzopolysulfanes, 4-CH₃(OCH₂CH₂) ₃NHC(O)-C₆H₄-1,2-S_x (x = 3-7 and 9) were synthesized with a PEG group attached through an amide bond and examined for water solubility, antitumo

Full text of DOI:10.1021/jo100870q

pubs.acs.org/joc
Synthesis, Characterization, Mechanism of Decomposition, and
Antiproliferative Activity of a Class of PEGylated Benzopolysulfanes
Structurally Similar to the Natural Product Varacin
Adaickapillai Mahendran,† Angela Vuong,† David Aebisher,† Yaqiong Gong,‡
Robert Bittman,‡ Gilbert Arthur,§ Akira Kawamura, and Alexander Greer*,†
†Department of Chemistry, Brooklyn College of The City University of New York, Brooklyn, New York 11210,
‡Department of Chemistry and Biochemistry, Queens College of The City University of New York, Flushing,
New York 11367, §Department of Biochemistry and Medical Genetics, University of Manitoba, Winnipeg,
Manitoba, Canada R3E 0W3, and Department of Chemistry, Hunter College of The City University of
New York, New York, New York 10065
agreer@brooklyn.cuny.edu
Received May 4, 2010
Benzopolysulfanes, 4-CH3(OCH2CH2)3NHC(O)-C6H4-1,2-Sx (x = 3-7 and 9) were synthesized with a
PEG group attached through an amide bond and examined for water solubility, antitumor activity, and
propensity to equilibrate and desulfurate. LCMS and HPLC data show the PEG pentasulfane ring
structure predominates, and the tri-, tetra-, hexa-, hepta-, and nonasulfanes were present at very low
concentrations. The presence of the PEG group improved water solubility by 50-fold compared to the
unsubstituted benzopolysulfanes, C6H4Sx (x=3, 5, and 7), based on intrinsic solubility measurements.
Polysulfur linkages in the PEG compounds decomposed in the presence of ethanethiol and hydroxide ion.
The PEG pentathiepin desulfurated rapidly, and an S3 transfer reaction was observed in the presence of
norbornene; no S2 transfer reaction was observed with 2,3-dimethylbutadiene. The antitumor activities of
the PEG-substituted benzopolysulfane mixtures were analyzed against four human tumor cell lines PC3
(prostate), DU145 (prostate), MDA-MB-231 (breast), and Jurkat (T-cell leukemia). The PEG-conjugated
polysulfanes had IC50 values 1.2-5.8 times lower than the parent “unsubstituted” benzopolysulfanes.
Complete cell killing was observed for the PEG polysulfanes at 4 μM for PC3 and DU145 cells and at
12 μM for MDA-MB-231 cells. The results suggest that solubilization of the polysulfur linkage is a key
parameter to the success of these compounds as drug leads.
Introduction
N-dimethyl-5-(methylthio)varacin (3) (Scheme 1).1-6 Unnatu-
ral benzopolysulfanes have also been synthesized, e.g., 6-(2-
aminoethyl)benzopentathiepin (4)7 and the parent benzopenta-
thiepin (5B)8,9 (alphabetical labels will be given to some polysul-
fanes, as will be elaborated on below). Benzopolysulfanes
represent an attractive target but are an understudied class of
Tunicates or their associated microorganisms produce benzo-
polysulfanes, such as varacin (1), lissoclinotoxin A (2), and N,
(1) Davidson, B. S.; Molinski, T. F.; Barrows, L. R.; Ireland, C. M. J. Am.
Chem. Soc. 1991, 113, 4709–4710.
(2) Liu, H.; Fujiwara, T.; Nishikawa, T.; Mishima, Y.; Nagai, H.; Shida,
T.; Tachibana, K.; Kobayashi, H.; Mangindaane, R. E. P.; Namikoshi, M.
Tetrahedron Lett. 2005, 61, 8611–8615.
(3) Litaudon, M.; Guyot, M. Tetrahedron Lett. 1991, 32, 911–914.
(4) Compagnone, R. S.; Faulkner, D. J.; Carte, B. K.; Chan, G.; Hemling,
M. A.; Hofmann, G. A.; Mattern, M. R. Tetrahedron 1994, 50, 12785–12792.
(5) Searle, P. A.; Molinski, T. F. J. Org. Chem. 1994, 59, 6600–6605.
(6) Makarieva, T. N.; Stonik, V. A.; Dmitrenok, A. S.; Grebnev, B. B.;
Iskov, V. V.; Rebachyk, N. M. J. Nat. Prod. 1995, 58, 254–258.
(7) Sato, R.; Ohyama, T.; Ogawa, S. Heterocycles 1995, 41, 893–896.
(8) Feher, F.; Langer, M. Tetrahedron Lett. 1971, 24, 2125–2126.
(9) Chenard, B. L.; Harlow, R. L.; Johnson, A. L.; Vladuchick, S. A.
J. Am. Chem. Soc. 1985, 107, 3871–3879.
DOI: 10.1021/jo100870q
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Published on Web 07/26/2010
J. Org. Chem. 2010, 75, 5549–5557 5549
2010 American Chemical Society

Mahendran et al.
JOCArticle
SCHEME 1. Natural and Unnatural Benzopolysulfanes
SCHEME 2. Equilibration Between Tri-, Penta-, and Hepta-
sulfanes
PEG group replaced the naturally occurring dopamine core
1-3. The aims of the present work were to determine (1) whether
benzopolysulfanes could be synthesized with a PEG side group,
(2) whether the pentasulfur species predominates, (3) whether
the polysulfur linkage(s) are unstable to medium effects, (4)
whether the PEGylated benzopolysulfanes decompose at dif-
ferent rates and transfer sulfur to norbornene and butadiene
traps, (5) the extent the PEG group enhances water solubility,
(6) whether the polysulfur ring is essential for bioactivity, and (7)
whether enhanced benzopolysulfane water solubility is corre-
lated with an enhanced pharmacological activity against human
tumor cells. We synthesized a mixture of PEG-benzopoly-
sulfane conjugates 4-CH3(OCH2CH2)3NHC(O)-C6H4-1,2-Sx
(x=3-7 and 9) 6A-F and explored their stability and activity
in a variety of tumor cell lines [PC3 (prostate), DU145 (prostate),
MDA-MB-231 (breast), and Jurkat (T-cell leukemia)]. In 6, the
labels A-E and F correspond to compounds with 3-7 and 9
sulfur atoms, respectively. The octasulfane species was not
detected.
TABLE 1. GC-MS Detection of Parent Benzopolysulfanes 5A-Ca
molecular weights
no. of S
atoms
MS tR
(min)b
compound
calcd
foundc
o-C6H4S3 5A
o-C6H4S5 5B
o-C6H4S7 5C
3
5
7
14.2
26.5
39.1
172
236
300
172
236
300
aReference 12. See also ref 13. bGC-MS retention time. cLow-
resolution GC-MS. Support for the GC-MS peak assignments came
from spectroscopic comparisons of 5A-C, which were independently
synthesized and examined shortly after their purification, i.e., before
equilibration was pronounced, which took 1-3 days.
compounds for antitumor drug discovery. High nanomolar and
low micromolar antiproliferative IC50 values have been re-
ported for benzopolysulfanes.1-7,10 It was suggested that the
bioactivity of varacin derives from DNA damage because of an
observed difference in toxicity toward the CHO cell line EM9
(chlorodeoxyuridine sensitive) compared to BR1.1
Results and Discussion
Synthesis and Characterization. Pentasulfane 6C was synthe-
sized as the major constituent of a mixture of polysulfanes
4-CH3(OCH2CH2)3NHC(O)-C6H4-1,2-Sx (x=3-7 and 9) in
8 steps and 1.5% overall yield. A procedure developed by
Lienard et al.14 was used for the conversion of 3,4-dihydroxy-
ꢀ
Benzopolysulfanes have not been studied widely in the con-
text of drug discovery because of the instability of the polysulfur
ring. Even determining the number of sulfur atoms in o-benzo-
polysulfanes has not been a trivial task.11-13 MS fragmentation
patterns can be difficult to interpret; for example, the structure
of the natural product lissoclinotoxin A was assigned first as a
trisulfane3 and later revised as a pentasulfane.11 In 2007, the syn-
thesis and purification of parent o-C6H4S5 (5B) revealed an
equilibration involving elemental sulfur, S8 (Scheme 2).12
A facile equilibration took place between the pentasulfane and
the tri- and heptasulfanes (o-C6H4S3 and o-C6H4S7): the ratio
5A:B:C was 49:45:6 in CH2Cl2 over 1-3 days. In 5, the labels A,
B, and C correspond to compounds with 3, 5, and 7 sulfur
atoms, respectively. Analysis of GC-MS retention times re-
vealed that 5A and 5B differed by 12.3 min, and the retention
times of 5B and 5C differed by 12.6 min (Table 1).
benzoic acid (7) to 3,4-disulfuranylbenzoic acid (11) (steps i-iv,
Scheme 3). Dithiastannole-5-carboxylate anion 12 was gener-
ated under basic conditions by the reaction of dimethyltin chlo-
ride with 11 using a modified procedure by Sato et al.15 Stannole
12 reacted with p-nitrophenol, DCC, and DMAP to yield
4-nitrophenyl ester stannole 13 in 72% yield. Stannole 13 reacted
with disulfur dichloride giving 4-nitrophenyl ester benzopenta-
sulfane (14) in 27% yield. Amino-terminated poly(ethylene
glycol) (15) was prepared in 2 steps by the method of Dombi
et al.16 and PEGylated to benzopentasulfane 14 at the 7-position
of the benzene ring. It is possible that other cyclic polysulfanes
related to benzopentasulfane 14 were formed in ∼1-10% yields,
but this was not determined. The resulting mixture was purified
by column chromatography to afford 6C in 93% purity. In the
1H NMR, 13C NMR, COSY, HMBC, and HSQC spectra, the
benzene and PEG portions of the structure were confirmed
(Supporting Information). For example, in COSY NMR, a
strong 3Jcorrelation was found between C9-HandC8-H, and
between N-H and C13-H, and a weak 4J-W correlation found
between C8-H andC6-H (Scheme 4). The HMBC and HSQC
NMR data further bolstered the structural assignment of the
Because natural benzopolysulfanes are in short supply and
their stability is difficult to assess, we synthesized benzopoly-
sulfanes with a short-chain PEG, in which the benzene ring and
(10) Liu, H.; Mishima, Y.; Fujiwara, T.; Nagai, H.; Kitazawa, A.; Mine,
Y.; Kobayashi, H.; Yao, X.; Yamada, J.; Oda, T.; Namikoshi, M. Mar.
Drugs 2004, 2, 154–163.
ꢀ
(11) Guyot, M. Pure Appl. Chem. 1994, 66, 2223–2226.
(12) Aebisher, D.; Brzostowska, E. M.; Sawwan, N.; Ovalle, R.; Greer, A.
J. Nat. Prod. 2007, 70, 1492–1494.
(14) Lienard, B. M. R.; Selevsek, N.; Oldham, N. J.; Schofield, C. J.
ChemMedChem 2007, 2, 175–179.
(15) Ogawa, S.; Yomoji, N.; Chida, S.; Sato, R. Chem. Lett. 1994, 507–510.
(16) Dombi, K. L.; Griesang, N.; Richert, C. Synthesis 2002, 6, 816–824.
(13) Brzostowska, E. M.; Greer, A. J. Org. Chem. 2004, 69, 5483–5485.
5550 J. Org. Chem. Vol. 75, No. 16, 2010

Mahendran et al.
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SCHEME 3. Synthesis of PEG Conjugated Benzopentasulfane 6Ca
aReagents and conditions: (i) HCl (cat.), MeOH, reflux 80 °C, 7 h; (ii) Me2NC(dS)Cl, DABCO, DMF, rt, 30 min; (iii) Ph2O, 230 °C, 40 min; (iv) (a)
aqueous NaOH, under N2, 70 °C, 4 h, (b) 1 N HCl; (v) (a) Me2SnCl2, KOH, EtOH, water, (b) 1 N HCl; (vi) p-nitrophenol, DCC, DMAP, CH2Cl2, 1 d;
(vii) S2Cl2, CH2Cl2, 0 °C, 30 min, then warmed to rt, 24 h; (viii) H2N-(CH2CH2O)3-CH3 15 THF, rt, 12 h.
SCHEME 4. COSY NMR Couplings of Benzopentasulfane 6C
TABLE 2. Mass Spectrometry Data for PEGylated Benzopolysulfanes
6A-F
experimental
calculated mass [(M þ Hþ) error
a
tR
(min)
compound
formula
mass
- Hþ]b
(ppm)
6A
6B
6C
6D
6E
6F
C14H19NO4S3 4.6
C14H19NO4S4 5.6
C14H19NO4S5 6.5
C14H19NO4S6 7.1
C14H19NO4S7 7.8
C14H19NO4S9 9.2
361.0476
393.0197
424.9918
456.9638
488.9359
552.8800
361.0478
393.0193
424.9926
456.9636
488.9354
552.8787
0.62
-1.00
1.99
-0.60
-1.08
-2.34
aLCMS retention time. Chromatography was performed on a SB-C18
3.5 μm column using water containing 0.1% formic acid and 5 mM
ammonium formate (solvent A) and methanol containing 0.1% formic acid
and 5 mM ammonium formate (solvent B) at a flow rate 0.5 mL/min. The
gradient program was as follows: 15-85% B (0-13 min), 85% B (13-
15 min), 85-15% B (1 min). bThe experimental exact mass is calculated by
the subtraction of a proton (Hþ; 1.00728 Da) from the measured m/z value
of the [M þ Hþ] ion for the molecular formula of interest.
SCHEME 5. Equilibration of Pentasulfane 6C with Minor
Amounts of Tri-, Tetra-, Hexa-, Hepta-, and Nonasulfanes in
Methanol/Water (1:1 v/v)
polysulfane masses and retention times to compare the number
of sulfur atoms in the compounds. The LCMS spectra of poly-
sulfanes 6A-F contained peaks spaced by ∼0.7 min per addi-
tional sulfur atom (Figure 1). For example, a minor amount of a
sulfur-rich compound was assigned as nonasulfane 6F (HRMS
calcd for C14H19NO4S9 = 552.8800, found 552.8788). The ratio
6A:B:C:D:E:F was 1.5:1.5:93:0.3:0.1:0.1 by HPLC (methanol/
water), and there was ∼2% uncharacterized material and
∼1.5% of elemental sulfur. The moderate solubility of the ele-
mental sulfur in methanol/water suggested it to be a residue or
colloid particles of the orthorhombic cyclo-S8 form, but neither
the monoclinic S8 ring form (usually found at ∼95 °C) nor the
polymeric, oligomeric, or amorphous forms (insoluble mate-
rials).17-19 The overlaid-ion extracted LCMS of 6 in Figure 1
displayed a series of polysulfanes, analogous to that seen for
parent 5by SIM-GC-MS.12 Preference for odd-membered ring
nonsulfur portion of 6C. LC/MS data indicated that 6C con-
tained five sulfur atoms (HRMS calcd for C14H19NO4S5 =
424.9918, found 424.9926).
Lability of the Pentasulfur Ring. Polysulfanes are chal-
lenging structures to study because of their instability. Few
reports on benzopolysulfanes describe the distribution of the
polysulfanes or their equilibration and often incorrectly assume
the pentathiepin to be the sole compound present.12 We found
that over a 12-24 h period 6C equilibrates S8 and forms low
concentrations of structurally related polysulfanes in aqueous
methanol at room temperature (Scheme 5). Table 2 lists the
(17) Donaldson, G. W.; Johnston, F. J. J. Phys. Chem. 1968, 72, 3552–
3558.
(18) Tuller, W. N. Elemental Sulfur. In The Analytical Chemistry of Sulfur
and its Compounds. Part 1; Karchmer, J. H, Ed.; Wiley & Sons: New York,
1970; pp 1-86.
(19) Steudel, R.; Eckert, B. Top. Curr. Chem. 2003, 230, 1–79.
J. Org. Chem. Vol. 75, No. 16, 2010 5551

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FIGURE 1. Ion extracted [M þ Hþ] LCMS spectra of compounds 6A-F. Chromatograms were normalized to the largest peak 6C.
The “counts” on the y-axis are equal to the number of ions detected.
TABLE 3. Polysulfane Distribution as a Function of HPLC Solvent
Elution Conditiona
polysulfanes and elemental sulfur
MeOH/H2O
methodb ratio (v/v)
6A
6B
6C
6D
6E
6F
S8
1
2
3
4
5
50/50
60/40
75/25
80/20
85/15
2.2
2.5
4.3
4.7
5.3
1.8
2.1
3.2
4.8
5.7
93.5
94.7
84.6
83.6
79.0
0.4
0.3
0.6
0.9
0.3
1.2
0.2
1.6
1.5
1.0
3.4
0.5
0.4
0.1
3.4
5.1
5.3
aHPLC analysis at 254 nm at room temperature with a flow rate of 1 mL/
min in water/methanol mixtures. bHPLC methods: Method 1 consisted of a
gradient of methanol from 10% to 90% over 53 min and maintained for
2 min before reverting to 10% methanol over 1 min. Method 2 consisted of
60% methanol run isocratically over 91 min. Method 3 consisted of a gradi-
ent of methanol from 60% to 95% over 30 min, which was maintained for
10 min before reverting to 15% methanol over 5 min. Method 4 consisted of
agradientofmethanolfrom15%to95%over 30 min, which was maintained
for 15 min before reverting to 15% methanol over 3 min. Method 5 consisted
of a gradient of methanol from 15% to 95% over 15 min, which was main-
tained for 15 min before reverting to 15% methanol over 2 min. Method
6 consisted of water (0.1% formic acid and 5 mM ammonium formate in
water) and methanol (0.1% formic acid and 5 mM ammonium formate in
MeOH) with a gradient of methanol from 15% to 85% over 13 min, which
was maintained for 2 min before reverting to 15% methanol over 1 min.
FIGURE 2. HPLC chromatogram of polysulfanes; peaks include 1,
6A; 2, 6B; 3, 6C; 4, 6D; 5, 6E; 6, 6F. Elemental sulfur S8 elutes in the
11-13 min range. Peaks marked with an asterisk belong to un-
identified substances.
Increasing the methanol concentration in water produces
more pronounced polysulfane equilibration. When eluting
at 85:15 methanol/water, the ratio 6A:B:C:D:E:F:S8 was
5.3:5.7:79.0:3.4:1.2:0.1:5.3 (Method 5, Table 3). The HPLC
measurements do not ensure that thermodynamic conditions
have been reached. Equilibrium at a new condition is established
in 1-3daysinCH2Cl212 and probably much longer in methanol/
water. Our results are similar to observations of 7-methyl-
benzopolysulfane equilibrations enhanced in polar solvents
with higher solvation compared to nonpolar solvents.22 Ele-
mental S8 also involves equilibration with S6 and S7, which was
more pronounced in polar than nonpolar organic solvents,23
and sulfur-reactive solvents such as pyridine or other amines
that dissolve elemental sulfur readily.18 The enhanced equilibra-
tion in methanol compared to that in methanol/water is likely a
result of the increase in the solubility of S8, which is pivotal to the
reversible addition reactions leading to the ring compounds
6A-F (Figure 2).
compounds was observed experimentally for the parent system
o-C6H4Sx (x=3, 5, 7) in CH2Cl2.12 Gas-phase DFT calculations
showed the odd-membered o-C6H4Sx rings to be conforma-
tionally stable with gauche adjacent lone-pair electron inter-
actions, whereas eclipsing lone-pair electron interactions occurred
in the even-numbered cases.13 It may be noted that Nakayama
et al. also observed odd-membered ring products (namely, the
trithiolane and penathiepan) in the reaction of elemental sulfur
with a benzobarrelene compound and an acenaphthylene
compound.20,21 However, the preference for odd-membered
polysulfur rings does not apply to the PEG benzopolysulfanes,
which is likely because the equilibrium is at an early stage and
has not been reached in aqueous methanol, even after 24 h. The
effect of solvent or HPLC conditions on the equilibrium of
6A-F and S8 was investigated next.
Influence of Solvent on Equilibration of Polysulfanes
6A-F. To better understand the equilibration between
6A-F and S8, the HPLC elution methods were modified.
When eluting at 1:1 methanol/water, the ratio 6A:B:C:D:E:
F:S8 was 2.2:1.8:93.5:0.4:0.3:0.2:1.6 (Method 1, Table 3).
Desulfuration of 6A-F in the Presence of Nucleophiles and
Trapping Agents. The PEGylated benzopolysulfanes 6A-F
(20) Sugihara, Y.; Takeda, H.; Nakayama, J. Tetrahedron Lett. 1998, 39,
2605–2608.
(21) Nakayama, J. J. Sulfur Chem. 2009, 30, 393–468.
(22) Brzostowska, E. M.; Greer, A. J. Am. Chem. Soc. 2003, 125, 396–404.
(23) Tebbe, F. N.; Wasserman, E.; Peet, W. G.; Vatvars, A.; Hayman,
A. C. J. Am. Chem. Soc. 1982, 104, 4971–4972.
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TABLE 4. Decomposition of PEGylated Benzopolysulfanes 6A-F as a
quickly compared to other polysulfanes, which led us to trap-
ping studies to analyze the sulfur-transfer reaction with buta-
diene and norbornene traps.
Function of Hydroxide Ion Equivalents Addeda
polysulfanes and
elemental sulfur
The reaction of benzopolysulfanes 6A-F with ethanethiol
and norbornene (16) led to the formation of norbornenetrithio-
lane (17) and desulfurated or polymerized PEGylated benzo-
polysulfanes (Scheme 6). Unlike 6C, the concentrations of 6A,
6B, and 6D-F did not change significantly over the course of
the trapping experiment. Thus, we propose that 6C is the most
reactive polysulfane and responsible for the S3-transfer to nor-
bornene. Interestingly, the decomposition of benzopolysulfanes
6A-F did not show an S2 transfer reaction. The sulfuration of
2,3-dimethylbutadiene (18) by 6A-F with ethanethiol did not
yield the disulfide product 19. A possible mechanism for the
desulfuration of pentathiepin 6C is shown in Scheme 7, which
begins with an apical attack of ethanethiolate ion to the sulfur
atom (S1) adjoining the aryl ring. A previous DFT study
showed that attack of the HS- nucleophile at the S1 position
of a pentathiepin was preferred to S2.24 The resulting open-
chain polysulfide anion (20) has the potential for thiozone (S3)
elimination driven by the delocalization of the negative charge in
the remaining carbon-sulfur fragment (21)24,25 followed by
thiozonation of norbornene. Ab initio calculations predict the
open C2v zwitterionic form26 of thiozone S3 to be energetically
preferred to the cyclic D3h form.27,28
distributionb
hydroxide internal
ion equiv standard 6A 6B þ Xc 6C
6D
6E 6F
S8
0.25
0.5
100
100
100
100
100
10.9
13.2
14.2
14.2
14.9
11.2
19.6
38.1
64.9
85.5
362.4 2.3
289.2 2.1
222.9 1.8
163.9 1.5
113.6 0.7
3.0 2.1 12.3
2.8 3.7 23.1
4.7 3.8 26.7
1.6 3.1 32.4
0.75
1.0
1.0d
2.2
3.1
aReaction of benzopolysulfanes 6A-F (2.5 mM) and hydroxide ion in
the presence of internal standard acetanilide (0.25 mM) in methanol/water
(99.9:0.1 v/v). bPolysulfane ratios were determined by HPLC monitoring at
254 nm at room temperature with the flow rate 1 mL/min using method 4
(Table 3). HPLC analysis was carried out after 1 h of equilibration between
c
benzopolysulfanes 6A-F and hydroxide ion. X is an unidentified com-
pound that was eluted with 6B. dAnalysis was done after 24 h.
SCHEME 6. PEGylated Benzopolysulfanes 6A-F as
Sulfur-Transfer Reagents to Norbornene but Not to
2,3-Dimethylbutadiene
Intrinsic Solubilities. Elemental S8 suffers from poor solu-
bility.19,29,30 We investigated the intrinsic solubilities of ele-
mental S8, 5A-C, and 6A-F (Table 5). Aliquots of methanol
or water were added to 1-3 mg sample quantities, and the
solutions were vortexed (2 min) and stirred (10-15 min) at
room temperature. As expected, the presence of the PEG group
at the 7-position of 6A-F significantly increased their solubi-
lities. By comparison to 6A-F, elemental sulfur S8 and 5A-C
had ∼50-fold lower solubility. Our aqueous solubility measure-
ments are consistent with previous values for elemental sulfur
S8 reported in the literature, namely, the solubility of S8 in water
was reported to be 0.4 μg/mL.29 The solubility of S8 in ethanol
was reported to be 0.51 mg/mL.30
Antiproliferative Activities of Polysulfanes. We examined
the effects of 1,2-benzenedithiol, o-benzopolysulfanes 5A-C,
and PEGylated benzopolysulfanes 6A-F on the proliferation of
several cancer cell lines. Their bioactivities are shown in Table 6
and Figure 3. 1,2-Benzenedithiol was nontoxic; it stimulated the
growth of PC3 cells and had a minimal effect on the proliferation
of MDA-MB-231 cells. However, at concentrations of 10 μM
and above, it inhibited the proliferation of DU145 cells by
∼40%. In a MTS assay (3-(4,5-dimethylthiazol-2-yl)-5-(3-car-
decomposed in the presence of NaOH or ethanethiol (Table 4),
which led to an increase in elemental S8 and uncharacterized
products, such as oligomers and/or polymers of desulfurated
PEGylated benzopolysulfanes. The reaction of hydroxide ion
with benzopolysulfanes 6A-F was comparatively slower than
that with ethanethiol and could be monitored by HPLC. As can
be seen in Table 4, the decomposition rate of pentasulfane 6C
was rapid, whereas the concentrations of the other benzopoly-
sulfanes were barely reduced. Determining whether the concen-
tration of tetrasulfane 6B increased or decreased was not
possible because it eluted along with an unidentified compound
(X in Table 4), and the two peaks could not be deconvoluted.
The data suggested that pentasulfane 6C desulfurated more
SCHEME 7. Potential Mechanism for the Triatomic Sulfur Transfer of Benzopentasulfane 6C
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JOCArticle
FIGURE 3. Effect of 1,2-benzenedithiol, o-benzopolysulfanes 5A-C, and PEGylated benzopolysulfanes 6A-F on the growth of human
cancer cell lines. Prostate cancer cell lines DU145 and PC3 cells, breast cancer cell line MDA-MB-231, and Jurkat cells, a leukemia cell line, were
grown in 96-well plates. When the cells were in exponential growth, they were incubated with different concentrations of 1,2-benzenedithiol,
5A-C, or 6A-F for 48 h (DU145, PC3, MDA-MB-231) or 72 h (Jurkat). The increase in cell numbers relative to controls was determined by the
CyQuant assay (A-C) and by the MTS assay (D) as described in the Experimental Section. The values were expressed as the increase in cell
numbers relative to controls without any compound. Data are the mean ( SE (n = 6-8).
TABLE 5. Experimental Solubility Valuesa
TABLE 6. Inhibition of Cancer Cell Gowth by 1,2-Benzenedithiol,
o-Benzopolysulfanes 5A-C, and PEGylated Benzopolysulfanes 6A-F
solvent
IC50 (μM)a
methanol
(mg/mL)
water
(μg/mL)
reagent
MDA-
DU145 MB-231 Jurkat
compound
PC3
elemental sulfur, S8
o-benzopolysulfanes
5A-C (97%)
PEGylated benzopolysulfanes
6A-F (93%)
aEquilibrium time of 24 h was used in the solubility study. Measurements
were conducted three times, and the solubility value was averaged.
0.33 ( 0.03
0.37 ( 0.02
0.14 ( 0.02
0.18 ( 0.02
no effect >30
>30
60
19.00 ( 0.05
8.70 ( 0.25
1,2-benzenedithiol
o-benzopolysulfanes
5A-C (97% purity)
10.5 (20)b 4.9 (6)b 27
0.5
0.4
boxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium in the
presence of phenazine methosulfate),31 1,2-benzenedithiol was
PEGylated benzopolysulfanes 1.8 (4)b
3 (4)b
5.5 (12)b
6A-F (∼98% total purity)
(24) Greer, A. J. Am. Chem. Soc. 2001, 123, 10379–10386.
(25) Brzostowska, E. M.; Paulynice, M.; Bentley, R.; Greer, A. Chem.
Res. Toxicol. 2007, 20, 1046–1052.
(26) Koch, W.; Natterer, J.; Heinemann, C. J. Chem. Phys. 1995, 102,
6159–6167.
aIC50 values were obtained for 72 h incubations with Jurkat cells and
48 h for PC3, DU145 and MDA-MB-231 cells. bConcentrations that
resulted in 100% cell kill are shown in parentheses. Experiments were
conducted in 96-well plates with 8 replicates for each concentration
(0-30 μM) of the indicated compounds.
(27) Rice, J. E.; Amos, R. D.; Handy, N. C.; Lee, T. J.; Schaefer, H. F., III
J. Chem. Phys. 1986, 85, 963–968.
(28) Peterson, K. A.; Lyons, J. R.; Francisco, J. S J. Chem. Phys. 2006,
125, 084314.
(29) Steudel, R.; Holdt, G. Angew. Chem., Int. Ed. 1998, 27, 1358–1359.
(30) Meyer, B. Chem. Rev. 1976, 76, 367–388.
(31) Wang, F.; Kawamura, A.; Mootoo, D. R. Bioorg. Med. Chem. Lett.
2008, 16, 8413–8418.
also not effective in inhibiting Jurkat cell growth (IC50 of 60 μM).
The requirement of the polysulfur ring was also observed by
Molinski et al.5 in the natural lissoclinotoxin A system when
judged against the corresponding benzenedithiol compound.
5554 J. Org. Chem. Vol. 75, No. 16, 2010

Mahendran et al.
JOCArticle
Parent polysulfanes 5A-C had a moderate antiprolifera-
tive effect on MDA-MB-231 cells, inhibiting the prolifera-
tion with an IC50 value of 28 μM. PC3 cell proliferation was
inhibited with an IC50 of 11 μM with complete cell killing at
20 μM. DU145 and Jurkat cells were sensitive to parent
polysulfanes 5A-C; 6 μM killed 100% of the DU145 cells,
and the IC50 was 0.5 μM for Jurkat cells. PEGylated
benzopolysulfanes 6A-F were the most potent of the three
compound classes for the four cell lines examined and were
toxic for all four cell lines. Complete cell killing was observed
for 6A-F at 4 μM for PC3 and DU145 cells and at 12 μM for
MDA-MB-231 cells. The PEG compounds 6A-F were
cytotoxic in Jurkat cells with an IC50 of 0.4 μM.
6A-F led to an S3-transfer to norbornene, but no S2-transfer
was observed to 2,3-dimethylbutadiene. (5) While the cause of
the poor solubility of benzopolysulfanes is the polysulfur linkage
itself, a key issue in the present study was the enhanced water
solubility the PEG group provided, which did not reduce the
prevalence of the pentasulfur ring species. (6) The results con-
firm the requirement of the polysulfur ring for low micromolar
antiproliferative IC50 values; 1,2-benzenedithiol showed little or
no antiproliferative activity. (7) The PEG polysulfanes 6A-F
were more water soluble and more active against four cancer
cell lines than the parent polysulfanes 5A-C, suggesting that
enhanced solubilization of benzopolysulfanes holds promise for
advancing these compounds as drug candidates.
The data in Table 6 indicate that the polysulfur ring is
significant in the enhancement of antiproliferative activity.
The data also demonstrate that the PEG group leads to an
increase in the antiproliferative effect. The PEGylated benzo-
polysulfanes 6A-F have higher water solubility compared to
that of the parent polysulfanes 5A-C and produced the
highest growth inhibition. It was previously reported that
lissoclinotoxin A 2 was cytotoxic against L1210 leukemia
cells (IC50 of 3.1 μM) and N,N-dimethylvaracin 3 was
cytotoxic against MDA-MB-231 cells (IC50 of 3.6 μM).10
Interestingly, varacin 1 showed greater cytotoxicity in HCT-
116 colon cancer cells (IC90 of 0.15 μM).1
Potential of Benzopolysulfanes as Drug Candidates. Even
though high nanomolar and low micromolar antiprolifera-
tive IC50 values for benzopolysulfanes were mentioned in the
Introduction1-7,10 and the above results for 6A-F appear
promising, in vivo stability and deliverability studies are
needed to evaluate benzopolysulfane lability to glutathione,
protein thiols, etc. Like some other drugs, benzopolysulfanes
are reductively activated. Our in vitro studies showed that
ethanethiol-induced desulfuration of 6C in aqueous meth-
anol took ∼2 min but in benzene took 4 h, suggesting that
greater thiol ionization/nucleophilicity increases the poly-
sulfane lability. Thus, hydrophobic or mild acidic conditions
that maintain the less reactive thiol form will preserve 6C,
but in the presence of thiolate ion 6C decomposes rapidly. In
the absence of thiol and thiolate ion, the solvent effect is in
the opposite direction; benzopolysulfane equilibria in organ-
ic solvents such as CH2Cl2 were more facile than in methanol
or methanol/water, driven by solvation of elemental sulfur
and the benzopolysulfanes (vide supra). Lastly, although a
DNA cleaving study of 7-methylbenzopentathiepin sug-
gested a metal- and oxygen-dependent Fenton pathway,32,33
more work is needed to evaluate a potential anaerobic S3-
transfer pathway that may underlie the antitumor activity.
Experimental Section
3,4-Dihydroxybenzoic acid, N,N-dimethylthiocarbamoyl chlor-
ide, DABCO, sodium hydroxide, potassium hydroxide, ethane-
thiol, acetanilide, formic acid, ammonium formate, dimethyltin
chloride, p-nitrophenol, DCC, S2Cl2, triethylene glycol mono-
methyl ether, phthalimide, triphenylphosphine, DIAD, hydrazine
monohydrate, elemental sulfur (S8), norbornene, 2,3-dimethyl-
butadiene, DMAP, sodium sulfate (anhydrous), magnesium sulfate
(anhydrous), NaCl, DMF, Na2CO3, NaHCO3, THF, CHCl3,
CH2Cl2, methanol, ethanol, ethyl acetate, hydrochloric acid (12
M), diphenyl ether, benzene, acetone-d6, CDCl3, CD3CN, CD3-
OD, and hexanes were used as received without further purifica-
tion. Purification of product mixtures was carried out by column
˚
chromatography using silica gel with 40-60 A particle size. TLC
was carried out using silica gel 60F 254 TLC plates. Proton NMR
data were acquired at 400 MHz, and 13C NMR data were acquired
at 100.6 MHz. HRMS, GC-MS, HPLC, and melting point data
were collected.
HPLC Instrumentation and Analysis. The HPLC instrument
was equipped with an autosampler and diode array detector.
The C18 column was 150 mm ꢀ 3.9 mm in size. The flow rate was
1 mL/min, and the injection volume was 50 μL. The mobile
phase consisted of methanol and water. Compounds were
detected by UV at 254 nm at rt.
LCMS Instrumentation and Analysis. The LCMS system
consisted of a high-resolution TOF mass spectrometer attached
to an HPLC equipped with an autosampler, diode array detec-
tor, and binary pump. The chromatography was conducted with
a 2.1 mm ꢀ 30 mm SB-C18 3.5 μm column using water contain-
ing 0.1% formic acid and 5 mM ammonium formate (solvent A)
and methanol containing 0.1% formic acid and 5 mM ammo-
nium formate (solvent B) at a flow rate 0.5 mL/min. The
gradient program was as follows: 15-85% B (0-13 min),
85% B (2 min), 85-15% B (1 min). The mass spectra were
collected over a range of 100-1600 m/z. The reference masses
used were purine with (M þ Hþ) ion at 121.05087 m/z and HP-
922 with ion at 922.00980 m/z. They were infused into the spray
chamber using a calibrant delivery system.
Conclusion
GC-MS Instrumentation and Analysis. GC-MS samples
were ionized using the EI auto mode. The capillary column was
a VF-5 ms 30 m ꢀ 0.25 mm ꢀ 0.25 μm (where ID = 0.25 mm,
DF = 0.25 μm). The solvent delay was set to 3 min. Temperature
program was as follows: 80 °C (0-5 min), 80-250 °C (5-22 min,
at a rate 10 °C/min), 250 °C (22-28 min). Total run was 28 min.
The instrument parameters were set as follows: injector tempera-
ture 200 °C, column flow rate 1 mL/min. Data were collected with
the instrument set to mass range 40-650 m/z.
Equilibration and Solubility Determinations. To determine the
extent of polysulfane equilibration, a 6.4 mg sample of dry
6A-F was dissolved in 3 mL of methanol or methanol/water
mixtures by stirring for 2 min at rt. Then 0.2 mL was placed in a
vial and diluted to 1 mL with methanol. The solution was stirred
The following conclusions can be made: (1) The synthesis of
benzopolysulfanes 6A-F was accomplished with attached PEG
groups of 160 Da molecular weight via an amide linkage in 1.5%
overall yield. (2) Pentathiepin 6C was the main product, but even
after its purification, benzopolysulfanes 6A, 6B, 6D-F formed
in very low concentrations. (3) The pentasulfur linkage of 6C
was sensitive to the solvent composition in the HPLC experi-
ments. Methanol-rich elution conditions reduced the ratio of 6C
relative to the other polysulfanes. (4) Thiol-initiated reactions of
(32) Chatterji, T.; Gates, K. S. Bioorg. Med. Chem. Lett. 1998, 8, 535–538.
(33) Chatterji, T.; Gates, K. S. Bioorg. Med. Chem. Lett. 2003, 13, 1349–1352.
J. Org. Chem. Vol. 75, No. 16, 2010 5555

Mahendran et al.
JOCArticle
for 1 min, in which the concentration of each sample was
0.42 mg/mL. Finally, 50 μL of the solution was injected into the
HPLC autosampler and quantitated by the absorption signal at
254 nm. The solubilities of elemental S8, 5A-C, or 6A-F were
determined by adding 10-50 μL aliquots of methanol or water to
1-3 mg of the compounds. The solutions were vortexed for 2 min
and then stirred 10-15 min at rt. Polysulfane decompositions
were also conducted in the presence of sodium hydroxide. To a
solution of polysulfanes 6A-F (2.5 mM) and internal standard,
acetanilide (0.25 mM), was added NaOH (0.625, 1.25, 1.875, or
2.5 mM) in 0.1 mL of methanol. After certain periods of time, the
reaction was analyzed by HPLC by injecting 15 μL of the sample.
Sulfur Transfer and Trapping Studies. Trapping studies were
carried out in 0.2 mL of benzene solution. To a solution of
PEGylated benzopenthasulfanes 6A-F (2.3 mM) and norbor-
nene or 2,3-dimethylbutadiene (2.3 mM) was added ethanethiol
(2.3 mM). The reaction mixture was vortexed for 2 min and
analyzed by GC-MS by injecting 1 μL of the sample. Both scan
and single ion mode (SIM) analyses were conducted on the sample.
Cell Proliferation Assays. The cell lines were grown from
frozen stocks originally obtained from the American Type
Culture Collection. The prostate cancer cell lines PC3 and
DU145 cells were grown in F12K and DMEM, respectively.
MDA-MB-231 breast cancer cells were grown in DMEM. All
the media were supplemented with 10% FBS and penicillin/
streptomycin. Cell proliferation was assessed by a cell prolifera-
tion assay as previously described.34 Briefly, equal numbers of
cells were distributed in 96-well plates and the cells were
incubated at 37 °C in a 5% CO2 incubator until they were in log
phase growth. The medium was removed and replaced with a
growth medium containing the sulfur-containing compounds
(0-30 μM). After 48 h incubation, the media was removed, and
the plates were frozen at -80 °C. The assay was performed by
warming the plates to rt and followed by the addition of the
reagent to the wells. The fluorescence was measured with a plate
reader (excitation/emission, 485/530 nm).
The cytotoxicity assay was conducted as follows: Cells were
cultured in RPMI-1640 medium with 10% fetal bovine serum and
penicillin-streptomycin (100 U/mL and 100 μg/mL, respectively)
and maintained in a 37 °C humidified 5% CO2 incubator. On the
day before the drug treatment, cells were plated onto each well of
the 96-well plate at 2,000 cells/well (200 μL of the medium per well).
After 24 h, cells were treated with different concentrations of the
sulfur-containing compounds and incubated for 72 h. After the
incubation, cell growth was evaluated using a 96-titer solution cell
proliferation assay. UV absorption (490 nm) of each well was
quantified with a microplate reader.
N-(2-(2-(2-Methoxyethoxy)ethoxy)ethyl)benzo[d][1,2,3]trithiole-
5-carboxamide (6A). HPLC (150 mm ꢀ 3.9 mm C18 column,
90% acetonitrile in water, flow rate 1 mL/min): tR=20.3 min.
LCMS: tR = 4.6 min; HRMS (þESI) calcd for C14H19NO4S3 =
361.0476, found 361.0478.
with saturated aqueous NaHCO3 solution (3 ꢀ 30 mL), 1 M HCl
(3 ꢀ 30 mL), and water (3 ꢀ 30 mL). The organic solvent was evap-
orated and the crude product was chromatographed (CHCl3/
MeOH, 10:1) to yield 9.6 mg of 6C (93% purity): Rf = 0.57, 1H
NMR (CDCl3, 400 MHz) δ 8.30 (d, J=1.9 Hz, 1H), 7.89 (d, J=8.0
Hz, 1H), 7.78 (dd, J=7.90, 1.9 Hz, 1H), 7.18 (br s, 1H), 3.66 (m,
10H), 3.54 (m, 2H), 3.32 (s, 3H); 13C NMR (CDCl3, 100 MHz) δ
162.4, 155.2, 150.3, 145.8, 1145.0, 137.3, 136.3, 131.5, 130.2, 125.4,
122.5. HPLC (150 mm ꢀ 3.9 mm C18 column, 90% acetonitrile in
water, flow rate 1 mL/min): tR=28.7 min. LCMS: tR = 6.5 min;
HRMS (þESI) calcd for C14H19NO4S5 =424.9918, found
424.9926.
N-(2-(2-(2-Methoxyethoxy)ethoxy)ethyl)benzo[g][1,2,3,4,5,6]-
hexathiocine-8-carboxamide (6D). HPLC (150 mm ꢀ 3.9 mm
C18 column, acetonitrile/water 1:9 to 9:1 over 53 min, flow rate
at 1 mL/min): tR=30.0 min. LCMS: tR=7.1 min; HRMS (þESI)
calcd for C14H19NO4S6=456.9638, found 456.9636.
N-(2-(2-(2-Methoxyethoxy)ethoxy)ethyl)benzo[h][1,2,3,4,5,6,7]-
heptathionine-9-carboxamide (6E). HPLC (150 mm ꢀ 3.9 mm
C18 column, acetonitrile/water 1:9 to 9:1 over 53 min, flow rate
at 1 mL/min): tR = 36.0 min. LCMS: tR = 7.8 min; HRMS
(þESI) calcd for C14H19NO4S7 = 488.9359, found 488.9354.
N-(2-(2-(2-Methoxyethoxy)ethoxy)ethyl)benzo[j][1,2,3,4,5,6,7,
8,9]nonathiacyclo-undecine-11-carboxamide (6F). HPLC (150 mm ꢀ
3.9 mm C18 column, acetonitrile/water 1:9 to 9:1 over 53 min,
flow rate 1 mL/min): tR = 40.8 min. LCMS: tR = 9.2 min; HRMS
(þESI) calcd for C14H19NO4S9 = 552.8800, found 552.8788. The
identification of peak 6F at 9.2 min in the spectrum is complicated
because the former is a shoulder in the chromatogram of 3A. Peak
6F has been assigned on the basis of the extracted ion chromato-
gram feature of the software.
Methyl 3,4-Dihydroxybenzoate (8). Yield 1.018 g (76%). 3,4-
Dihydroxybenzoic acid 7 (1.0 g, 6.49 mmol) was dissolved in
40 mL of MeOH. A catalytic amount of concentrated HCl
(5 drops) was added to the methanol solution, which was then
refluxed overnight at 80 °C. Water (300 mL) was added, and the
mixture extracted with EtOAc/hexane (60:40) and dried over
anhydrous MgSO4. The solvent was removed, affording a light
1
brown solid (mp 137-139 °C). H NMR (acetone-d6) δ 3.81 (s,
3H), 6.90 (d, J = 8.3 Hz, 1H), 7.45 (dd, J = 8.3, 2.0 Hz, 1H), 7.50
(d, J = 2.0 Hz, 1H), 8.47 (br s, 2H); 13C NMR (acetone-d6) δ 51.9,
115.8, 117.2, 123.0, 123.4, 145.6, 150.8, 167.1; HRMS (þESI) calcd
for C8H8O4 = 168.0423, found 168.0423.
Methyl 3,4-Bis[(dimethylamino carbothioyl)oxy]benzoate (9).
Yield 0.546 g (74%). Methyl-3,4-dihydroxybenzoate 8 (738 mg,
4.39 mmol), DABCO (17.6 mmol), and N,N-dimethylthio-
carbamoyl chloride (2.17 g, 17.6 mmol) were stirred in DMF
(10 mL) for 30 min. The white solid that formed was dissolved in
50 mL of water. The product was extracted with EtOAc (3 ꢀ
30 mL) and dried over anhydrous MgSO4. The solvent was
removed, affording a yellow-green oil. Benzoate 9 was obtained
by flash chromatography (EtOAc/hexanes, 2:3); Rf = 0.4 or via
recrystallization in 95% EtOH as white crystals (mp 110-
N-(2-(2-(2-Methoxyethoxy)ethoxy)ethyl)benzo[e][1,2,3,4]tetra-
thiine-6-carboxamide (6B). HPLC (150 mm ꢀ 3.9 mm C18
1
112 °C). H NMR (CDCl3) δ 8.01 (dd, J = 8.4, 2.0 Hz, 1H),
column, 90% acetonitrile in water, flow rate 1 mL/min): tR =
22.6 min. LCMS: tR=5.6 min; HRMS (þESI) calcd for C14H19-
7.85 (d, J = 2.0, 1H), 7.25 (d, J = 8.4, 1H), 3.90 (s, 3H), 3.43 (s,
3H), 3.30 (s, 6H); 13C NMR (CDCl3) δ 186.6, 186.1, 165.6,
149.4, 145.6, 128.7, 128.1, 125.9, 124.4, 52.3, 43.4, 43.3, 38.9,
38.8; HRMS (þESI) calcd for C14H18O4S2N2 calcd 342.0708,
found 342.0708. (lit. data for 9, ref 14); 1H NMR (CDCl3) δ 8.00
(dd, J = 8.5, J = 2.0 Hz, 1H), 7.86 (d, J = 2.0 Hz, 1H), 7.25 (d,
J = 8.5 Hz, 1H), 3.90 (s, 3H), 3.43, 3.42, 3.30, 3.29, 3.30 (4 ꢀ br s,
4 ꢀ 3H); 13C NMR (CDCl3) δ 186.4, 186.0, 165.6, 149.3, 145.5,
128.7, 128.2, 125.9, 124.4, 52.3, 43.4, 43.3, 38.9.
NO4S4 = 393.0197, found 393.0193.
N-(2-(2-(2-Methoxyethoxy)ethoxy)ethyl)benzo[f][1,2,3,4,5]-
pentathiepine-7-carboxamide (6C). Yield 9.6 mg (53%). A solu-
tion of 3,6,9-trioxadecylamine 15 (13.8 mg, 0.0847 mmol) in 1.5 mL
of THF was added to a solution of 4-nitrophenyl benzo[f]-
[1,2,3,4,5]pentathiepine-7-carboxylate 14 (17 mg, 0.0423 mmol) in
2.5 mL of THF. The reaction mixture was stirred under argon
atmosphere overnight. The solvent was evaporated, and the residue
was dissolved in 30 mL of CH2Cl2. The organic layer was washed
Methyl 2-Oxo-1,3-benzodithiole-5-carboxylate (10). Yield
0.079 g (40%). Methyl 3,4-bis([dimethylamino-carbothioyl]-
oxy)benzoate 9 (300 mg, 0.876 mmol) was heated at 240 °C in
10 mL of diphenyl ether for 30 min. The reaction mixture was
purified by flash chromatography using a gradient of EtOAc/
(34) Bittman, R.; Li, Z.; Samadder, P.; Arthur, G. Cancer Lett. 2007, 251,
53–58.
5556 J. Org. Chem. Vol. 75, No. 16, 2010

Mahendran et al.
JOCArticle
hexane (2:98 to 40:60) to afford 79 mg (40%) of 10 as a light brown
solid. Rf = 0.75 (EtOAc/hexane 1:2), mp 140-142 °C. 1H NMR
(CDCl3) δ 3.95 (s, 3H), 7.57 (d, J=8.5 Hz, 1H), 7.98 (dd, J=8.5,
J = 2.0 Hz, 1H), 8.18 (d, J = 2.0 Hz, 1H); 13C NMR (CDCl3) δ
52.5, 122.9, 124.1, 128.2, 129.2, 133.0, 138.0, 165.7, 188.8; MS (EI):
m/z=226 (M), 198 (M - CO), 167 (M - CO2Me) and 139 (M -
CO2Me - CO); molecular peak (C9H6O3S2, M=226) isotopic ratio
(calcd%, found%): [M þ 1] 227 (11.5, 11.4), [M þ 2] 228 (9.0, 9.8),
and [M þ 3] 229 (1.2, 1.5); HRMS (þESI) calcd for C9H6O3S2 =
225.9758, found 225.9757 (lit. ref 14); 1H NMR (CDCl3) δ 3.95 (s,
3H), 7.57 (d, J = 8.5 Hz, 1H), 7.98 (dd, J = 8.5, J = 2.0 Hz, 1H),
8.18 (d, J = 2.0 Hz, 1H); 13C NMR (CDCl3) δ 52.6, 122.9, 124.1,
127.8, 129.1, 133.0, 137.9, 165.7, 189.0.
0.465 mmol) was dissolved in dry CH2Cl2 (5 mL). To this
solution was added a catalytic amount of DMAP, with stirring
for 5 min. DCC (113 mg, 0.51 mmol) was added. After the
solution was stirred for 10 min, p-nitrophenol (138 mg, 0.93
mmol) was added, the solution was stirred overnight, and 30 mL
of CH2Cl2 was added. The white precipitate of urea was
removed by filtration. The organic layer was washed with con-
centrated citric acid solution, saturated aqueous NaHCO3 solu-
tion, and water, and the organic layer was dried over anhydrous
MgSO4. The solvent was removed, affording crude compound
13. The crude mixture was purified by column chromatography
using EtOAc/hexanes (2:3) to afford 10. Rf = 0.54, mp 198-199 °C;
1H NMR (CDCl3) δ 8.33 (d, J = 8.3 Hz, 2H), 8.30 (d, J =
1.6 Hz, 1H), 7.67 (dd, J = 8.1, 1.6 Hz, 1H), 7.59 (d, J = 8.1 Hz,
1H), 7.39 (d, J = 8.3 Hz, 2H), 1.09 (s, 6H); 13C NMR (CDCl3) δ
163.6, 155.9, 147.4, 145.4, 139.7, 131.2, 130.0, 125.6, 125.2,
124.4, 122.6, 3.2; HRMS (-ESI) calcd for C15H13NO4S2Sn =
446.9334, found 446.9324.
4-Nitrophenyl Benzo[f][1,2,3,4,5]pentathiepine-7-carboxylate
(14). Yield 9.5 mg (27%). Compound 14 was synthesized using
a modification of the procedure of Sato et al.15 To a solution of
10 (40 mg, 0.088 mmol), in 7 mL of dry CH2Cl2 at 0 °C, was
added dropwise a solution of S2Cl2 (23.8 mg, 0.176 mmol) in
3 mL of dry CH2Cl2 at 0 °C. The reaction mixture was allowed to
reach rt and stirred for 24 h. After 15 mL of CH2Cl2 was added,
the reaction mixture was washed with water (20 mL ꢀ 3). The
organic layer was dried with anhydrous MgSO4 and evaporated.
The product was purified by column chromatography using
EtOAc/hexanes (1:4) to afford 14. Rf = 0.44 (CHCl3/hexanes,
1:2); 1H NMR (CDCl3) δ 8.66 (d, J = 1.5 Hz, 1H), 8.36 (d, J =
8.1 Hz, 2H), 8.13 (dd, J = 8.1, 1.5 Hz, 1H), 8.02 (d, J = 8.1 Hz,
1H), 7.42 (d, J = 8.3 Hz, 2H); 13C NMR (CDCl3) δ 162.4, 155.2,
150.3, 145.8, 1145.0, 137.3, 136.3, 131.5, 130.2, 125.4, 122.5.
3,4-Disulfanylbenzoic Acid (11). Yield 0.52 mg (82%). An
aqueous solution of NaOH (1 N, 3 mL) was added to 10 (79 mg,
0.35 mmol). The resulting mixture was heated at 70 °C under
nitrogen atmosphere for 4 h. The reaction mixture was cooled to
rt and acidified with HCl (1 N). The white precipitate was
collected by filtration, washed several times with H2O, and dried
overnight to afford 11, mp 218-220 °C. 1H NMR (CD3CN) δ
9.50 (br s, 1H), 7.98 (d, J = 1.8 Hz, 1H), 7.66 (dd, J = 8.1, 1.8
1
Hz, 1H), 7.45 (d, J = 8.1 Hz, 1H), 4.34 (br s, 2H). H NMR
(CD3OD) δ 8.07 (d, J = 1.8 Hz, 1H), 7.73 (dd, J = 8.1, 1.8 Hz,
1H), 7.52 (d, J = 8.1 Hz, 1H), 13C NMR (CD3OD) δ 168.9,
140.4, 132.9, 131.5, 130.5, 129.5, 128.4. HRMS (-ESI): calcd for
C7H6O2S2 = 185.9809, found 185.9811. (lit. data for 11, ref 14);
1H NMR (CD3OD) δ 8.03 (d, J = 2 Hz, 1H), 7.70 (dd, J = 8, 2
Hz, 1H), 7.47 (d, J = 8 Hz, 1H), 4.05 (s, 1H), 3.67 (s, 1H); 13
C
NMR (CD3OD) δ 167.9, 139.4, 131.9, 130.4, 129.5, 128.5, 127.4.
2,2-Dimethylbenzo[d][1,3,2]dithiastannole-5-carboxylic Acid
(12). Compound 12 was synthesized using a modified literature
procedure by Lee et al.35 To an ethanolic solution (10 mL) of 11
(116 mg, 0.623 mmol) was added an ethanolic solution of NaOH
(5 mL, 0.2 M). After the mixture was stirred at rt for 15 min, an
aqueous solution (5 mL) of dimethyltin chloride (273 mg, 1.24
mmol) was added. The solution was stirredfor1.5h, acidifiedwith
1 N HCl, and extracted with CH2Cl2 (30 mL ꢀ 3). The resultant
organic layers were combined and washed with water and brine
solution. The organic layer was dried over anhydrous MgSO4, and
the solvent was removed, affording 155 mg (75%) of 12. 1H NMR
(CD3CN) 7.95 (d, J=1.6 Hz, 1H), 7.45 (d, J=8.1 Hz, 1H), 7.40
(dd, J = 8.1, 1.6 Hz, 1H), 0.96 (s, 6H); 13C NMR (CDCl3) δ 166.5,
146.5, 139.9, 129.7, 129.0, 125.1, 124.1 4.76; HRMS (þESI) calcd
for C9H10O2S2Sn = 325.9170, found 325.9171.
Acknowledgment. Grant support to Y.G. and R.B. was
provided by the National Institutes of Health (HL083187).
G.A. acknowledges support from the Manitoba Health
Research Council. A.M., A.V., D.A., and A.G. acknowledge
support from the National Institute of General Medical
Sciences (NIH SC1GM093830). The content is solely the
responsibility of the authors and does not necessarily repre-
sent the official views of the National Institute of General
Medical Sciences or the National Institutes of Health.
4-Nitrophenyl 2,2-Dimethylbenzo[d][1,3,2]dithiastannole-5-car-
boxylate (13). Yield 149 mg (72%). Compound 12 (155 mg,
Supporting Information Available: Spectroscopic data for
compounds 6A-F and 8-17. This material is available free of
charge via the Internet at http://pubs.acs.org.
(35) Lee, A. H. F.; Chen, J.; Liu, D.; Leung, T. Y. C.; Chan, A. S. C.; Li, T.
J. Am. Chem. Soc. 2002, 124, 13972–13973.
J. Org. Chem. Vol. 75, No. 16, 2010 5557