Synthesis and redox-enzyme modulation by amino-1,4-dihydro-benzo[d][1,2]dithiine derivatives

Article abstract of DOI:10.1016/j.tetlet.2009.03.209

A convenient method to prepare a series of benzodithiine derivatives was developed, via the synthesis of cyclic disulfide building blocks containing an amino-group linker. Some of the novel cyclic disulfide compounds are shown to modulate the activity of the redox-enzyme glutathione reductase.

Full text of DOI:10.1016/j.tetlet.2009.03.209

Tetrahedron Letters 50 (2009) 3023–3026
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Synthesis and redox-enzyme modulation by
amino-1,4-dihydro-benzo[d][1,2]dithiine derivatives
Sandraliz Espinosa a, Melissa Solivan a, Cornelis P. Vlaar a,
*
a School of Pharmacy, Medical Sciences Campus, University of Puerto Rico, PO Box 365067, San Juan PR00936, Puerto Rico
a r t i c l e i n f o
a b s t r a c t
Article history:
A convenient method to prepare a series of benzodithiine derivatives was developed, via the synthesis of
cyclic disulfide building blocks containing an amino-group linker. Some of the novel cyclic disulfide com-
pounds are shown to modulate the activity of the redox-enzyme glutathione reductase.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 6 November 2008
Revised 28 March 2009
Accepted 30 March 2009
Available online 5 April 2009
The redox activities of cyclic disulfides and their reactivity with
thiols and relative redox potentials have been the subject of de-
tailed physico-chemical studies.1 More recently, the utility of the
cyclic disulfide group has found interest in a variety of applica-
tions. For example, cyclic disulfides have been used to serve as a
bridging molecule between a gold layer and a single-walled carbon
nanotube through a thioalkylthiol linkage2, to attach an oligonu-
cleotide to a gold nanoparticle providing increased stability3, or
as a linker to connect folic acid to gold nanoparticles for potential
use as drug delivery vehicles.4 Although the role of the dithiolane
out via investigation of their potential for modulation of the re-
dox-enzyme glutathione reductase.
A general synthetic scheme is provided in Figure 1. Starting
with either 3- or 4-nitro substituted ortho-xylene, both 5-amino-
and 6-amino substituted benzodithiine derivatives 6 were synthe-
sized, respectively. In the first step, the nitroxylene 1 is brominated
with bromine in a biphasic methylene chloride/water mixture,
from which the
a,
a0-dibromide 2 can be isolated and purified via
a single crystallization. Nucleophilic substitution with potassium
thioacetate in methanol provides bis-thioacetate 3. Direct hydroly-
sis of 3 with sodium hydroxide in different solvents led to insoluble
products, most likely polymeric polysulfides. However, when the
hydrolysis was carried out with ammonium hydroxide in high
dilution (0.01 M) in methanol, concomitant air oxidation provided
the cyclic disulfide 4 in good yields.8,9 A last challenge was to re-
duce the nitro-group of compound 4 to an amino group, while
maintaining the reduction-prone cyclic disulfide group intact.
Reducing agents such as Fe/HCl, Zn/H2NNH2, Ni/HCOOH, or Pd/C/
H2, either did not react, or provided a mixture of unidentified or
polymeric products. However, reduction with sodium dithionite
successfully provided the desired amino-substituted dihydroben-
zodithiins 5.12 Subsequently, the amino group could be reacted
a-lipoic acid in biological processes is well established, the study
of synthetic cyclic disulfides remains largely unexplored. Recent
examples include the unsubstituted benzodithiine 4 (NO2 replaced
by H), which inhibits Respiratory Syncytial Virus replication5 and
oxidized dithiothreitol, which interferes with HIV-replication by
ejecting zinc from Zinc-finger protein via interaction with active-
site sulfhydryl groups.6
In view of the above, a systematic exploration of a broader
range of compounds containing cyclic disulfide building blocks
could prove interesting. Although various non-cyclic disulfide
compounds have been demonstrated to interact with active-site
cysteine residues of a number of proteins7, they are prone to intra-
cellular reductive inactivation. The 1,4-dihydro-benzo[d][1,2]dithi-
ines have a much more negative reduction potential than linear
disulfides1, which provides increased stability. Therefore, we se-
lected the benzodithiines for further exploration. The most conve-
nient way to potentially synthesize a wide variety of cyclic
disulfide derivatives would be via a benzodithiine core that has a
handle for further derivatization. We now present the synthesis
of substituted 1,4-dihydro-benzo[d][1,2]dithiines that contain an
amino group as a linking unit. These novel building blocks have
been reacted with various electrophilic compounds and prelimin-
ary studies of their potential biological activity have been carried
O
CH3
Br2
H2O/
KSCOCH3
Br
Br CH3OH
O2N
S
S
CH3
CH3
CH
3 CH2Cl2
O2N
O2N
2 (40%)
1
3 (88%)
O
S
S
Na2S2O4
S
S
NH4OH
CH3OH
"air"
R-X
S
S
HN
R
THF/
O2N
H2N
H2O/
4 (84%)
5 (40%)
6 (29-76%)
CH3OH
* Corresponding author. Tel.: +1 787 758 2525x5432; fax: +1 787 767 2796.
E-mail address: cornelis.vlaar@upr.edu (C.P. Vlaar).
Figure 1. Synthesis of cyclic disulfides depicted in Table 1. In brackets: yields for 6-
substituted derivatives.
0040-4039/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2009.03.209

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S. Espinosa et al. / Tetrahedron Letters 50 (2009) 3023–3026
Table 1
Preparation of benzodithiine derivatives and their effects on glutathione reductase activity
Entry
Electrophile
Producta
Yield (%)
76
% Enzyme activity at 50
144.5 13.3
l
Mc
% Enzyme activity at 25
90.5 6.5
l
Mc
1
O
O
S
S
Cl
N
H
7
2
67
45
55
93
18.5 1.9
59.4 14.7
66.0 2.5
90.5 6.9
46.9 5.2
72.7 9.8
91.0 4.4
91.3 15.1
O
O
S
S
Cl
Cl
N
H
8
3
O
O
N
H
S
S
9
4
O
S Cl
O
O
S
O
S
S
N
H
10
517
O
O
O
S
S
N
O
H
OH
11
O
6
35
29
100.0 11.9
101.8 6.2
O
O
S
Br
O
S
N
H
O
12
7b
90.4 7.7
76.2 4.2
Cl
S
S
HN
Cl
N
13
Cl
N
a
The products were formed by adding the electrophile to a solution of 5- or 6-amino-benzodithiine at 0 °C in THF in the presence of 1.2 equiv triethyl amine.
The product was obtained via CuI/N,N-dimethylglycine/Cs2CO3 catalyzed amination reaction in DMSO15 with microwave heating to 120 °C for 1 h.
Activity of yeast glutathione reductase compared with vehicle [activity = 100%] (for method used see Ref. 16).
b
c
with a number of electrophilic compounds to provide derivatives 6.
The products synthesized are summarized in Table 1.
dithiol-disulfide couple, formed by two active-site cysteine resi-
dues provides the key-functionality in the oxidation–reduction
process. Based on the reactivity of thiols with disulfides, our novel
benzodithiine derivatives seem especially suitable for enzyme
modulation, and are intended to be able to interact with the ac-
tive-site sulfhydryl groups of the enzyme (Fig. 3). Depending on
enzyme kinetics, the benzodithiines could prove to be reversible
covalent inhibitors, or alternatively function as competitive sub-
strates, leading to dithiol products. Although an extensive pharma-
cological study is beyond the scope of the present Letter, a proof-
of-principle for the structure-dependent activity of benzodithiine
derivatives to serve as modulators of cysteine-containing redox en-
zymes was evidenced by their effect on yeast glutathione reduc-
tase. The interference with enzymatic activity of the novel
benzodithiine products is summarized in Table 1.
In order to provide a preliminary investigation of their biologi-
cal potential, the interaction of the new cyclic disulfide compounds
7–13 with commercially available yeast glutathione reductase was
investigated. The redox-enzyme glutathione reductase is responsi-
ble for the reduction of oxidized glutathione (GSSG) to two mole-
cules of reduced glutathione (GSH), with NADPH as the co-
reductant (Fig. 2). The enzyme maintains GSH concentrations,
and possible inhibitors of this enzyme have been indicated as po-
tential anti-malarial13 or anti-cancer agents.14
A reversible
glutathione
reductase
GS-SG + NADPH + H+
2 GS-H + NADP+
As can be seen from Table 1, at a concentration of 50
lM, three
Figure 2. Reaction catalyzed by glutathione reductase.
compounds (8–10) reduce the activity of yeast glutathione reduc-

S. Espinosa et al. / Tetrahedron Letters 50 (2009) 3023–3026
3025
H
H
H
k2
k1
S
SH
S
HS
N R
N R
S
S
HS
HS
SH
SH
S
S
N R
ENZ
+
ENZ
ENZ
+
k-2
k-1
Figure 3. Possible interaction of benzodithiine derivatives with the active-site cysteine residues of glutathione reductase.
4. Dixit, V.; Van den Bossche, J.; Sherman, D. M.; Thompson, D. H.; Andres, R. P.
Bioconjugate Chem. 2006, 17, 603–609.
5. Sudo, K.; Watanabe, W.; Konno, K.; Sato, R.; Kajiyashiki, T.; Shigeta, S.; Yokota,
T. Antimicrob. Agents Chemother. 1999, 43, 752–757.
tase to 19%, 59%, and 66%, respectively, compared with the baseline
enzymatic activity. Thus, at this concentration, compound 8 inhib-
its more than 80% of enzyme activity, as measured by NADPH con-
6. Rice, W. G.; Baker, D. C.; Schaeffer, C. A.; Graham, L.; Bu, M.; Terpening, S.;
Clanton, D.; Schultz, R.; Bader, J. P.; Buckheit, R. W., Jr; Field, L.; Singh, P. K.;
Turpin, J. A. Antimicrob. Agents Chemother. 1997, 41, 419–426.
7. (a) Baum, E.; Siegel, M. M.; Bebernitz, G. A.; Hulems, J. D.; Sridharan, L.; Sun, L.;
Tabei, K.; Johnston, S. H.; Wildey, M. J.; Nygaard, J.; Jones, T. R.; Gluzman, Y.
Biochemistry 1996, 35, 5838–5846; (b) Boerzel, H.; Koeckert, M.; Bu, W.;
Spingler, B.; Lippard, S. J. Inorg. Chem. 2003, 42, 1604–1615.
sumption. At a concentration of 25 lM, as can be expected, the
effect of all three compounds on enzyme activity is much reduced.
Nevertheless, even at this reduced concentration, compound 8
shows an enzyme inhibitory activity of about 50%. For comparison,
a bis-dithiocarbamate18 was recently revealed as a covalent irre-
versible inhibitor of yeast glutathione reductase with Ki = 56
and kinact = 0.1 minꢀ1
In contrast to the above compounds, at both 25 and 50
lM
8. Synthesis
of
6-nitro-1,4-dihydro-benzo[d][1,2]dithiine
4
(m-
.
nitrobenzodithiine): In
a
2 L erlenmeyer flask, 2.99 g (10.0 mmol)
3
was
dissolved in 1000 mL methanol. To this solution was added 5.8 mL NH4OH and,
while maintaining the vessel at open air, the solution was stirred vigorously for
16 h at room temperature, to obtain a dark-brown solution. The methanol was
removed in vacuo on the rotavap (can be recycled for a subsequent batch of the
reaction) and the remaining solid was extracted with water and ethyl acetate
(2ꢁ) (THF may be added to aid in dissolution of the solids). Extraction of the
combined organic phases with brine, drying on Na2SO4 and removal of the
l
M con-
centrations, the modulatory effect of compounds 11 and 12 on en-
zyme activity is only moderate at most. These data indicate that
the observed effects are not only the result of interaction with
the cyclic disulfide group, but are also indeed dependent on the
complete molecular architecture of the modulating compounds.
solvents gave
a dark-brown solid that was purified by silica gel column
chromatography (hexanes/ethyl acetate, 6:1) to provide 1.79 g (8.4 mmol) of a
yellowish solid which by TLC (hexanes/ethyl acetate = 6:1: Rf = 0.78) contained
a minor impurity (Rf = 0.72) (probably a dimeric tetrasulfide product, as more
of it is observed when the reaction is carried out at higher concentrations).
Although further purification by column chromatography is possible, the
product can be utilized as obtained for the next step. 1H NMR (DMSO-d6) d 4.28
(2H, s), 4.30 (2H, s), 7.47 (1H, d, J = 8.4 Hz), 8.06 (1H, dd, J = 8.6 Hz, J = 2.4 Hz),
8.11 (1H, d, J = 2.4 Hz); 13C NMR d 34.1, 34.7, 114.0, 116.1, 122.8, 131.0, 133.5,
144.9; HRMS m/z calcd for [C8H7O2N32S2+H]+ 213.9991, found 213.9992.
Interestingly, at a concentration of 50 lM, compound 7 seems to
exhibit an increase in activity of glutathione reductase, as mea-
sured by an increased rate of NADPH consumption.19 A similar in-
crease in NADPH consumption was observed in the interaction of
human glutathione reductase with either ajoene (50% inhibition
within 15 min. at 200 l l
M)21 or with fluoro-M5 (IC50 = 4.1 M).22
These compounds, respectively, thioalkylate or alkylate an active-
site cysteine residue of the enzyme, thereby inhibit the reduction
of GSSG to GSH. Nevertheless, the covalently inhibited enzyme
shows an increased NADPH-oxidase activity, with a faster turnover
of NADPH than in the non-inhibited enzyme. In this case the sub-
strate is not GSSG, but rather, either molecular oxygen or a naph-
9. A possible alternative method could be to convert the a,
a0-dibromide 2 directly
into cyclic disulfide 4 via reaction with Na2S210, if needed in the presence of a
phase transfer catalyst.10b However, as aqueous polysulfide Sn
consists of
2ꢀ
various homologous anions in equilibrium (n = 1,2,3,. . .), the resulting product
mixture might contain products with different amounts of Sn which are
difficult to separate at a preparative scale.11 As our further investigations
required cyclic disulfides without the possibility of contamination with mono-,
tri-, or polysulfides, we preferred the above-described multi-step procedure
(Fig. 1).
thoquinone derivative, that presumably binds to
a second,
unidentified binding site. We suggest that the observed increased
activity of compound 7, when compared to enzyme activity in
the absence of 7, is due to a similar increase in oxidase activity,
leading to the observed NADPH consumption.23
10. (a) Harpp, D. N.; Gleason, J. G. J. Org. Chem. 1970, 35, 3258–3263; (b) Sonavane,
S. U.; Chidambaram, M.; Khalil, S.; Almog, J.; Sasson, Y. Tetrahedron Lett. 2008,
49, 520–522. and references cited.
11. (a) Rys, A. Z.; Abu-Yousef, I. A.; Harpp, D. N. Tetrahedron Lett. 2008, 49, 6670–
6673; (b) Steudel, R. Chem. Rev. 2002, 102, 3905–3946; (c) Kamyshny, A.;
Ekeltchlk, I.; Gun, J.; Lev, O. Anal. Chem. 2006, 78, 2631–2639.
12. Synthesis of 6-amino-1,4-dihydro-benzo[d][1,2]dithiine 5. A solution of 1.65 g
(7.7 mmol) 4 in methanol/THF/water (60 mL/20 mL/20 mL) was heated in an
oil bath of 60 °C. To this solution was added in one portion 5.53 g of
(27.0 mmol) 85% Na2S2O4 (should be fresh). After 30 min, the reaction was
shown to be complete by TLC, and after cooling to room temperature, CH2Cl2
and 10% K2CO3 were added. The precipitated solids were removed by vacuum
filtration, and the aqueous phase was extracted 2ꢁ with CH2Cl2. The combined
organic phases were extracted with water, dried on Na2SO4 and after removing
the solvent, the obtained solid was purified by silica gel column
chromatography (hexanes/ ethyl acetate 3:1) to obtain 0.60 g (3.3 mmol) of
an off-white solid. 1H NMR (CDCl3) d 3.59 (2H, br s), 3.95 (2H, s), 3.96 (2H, s),
6.40 (d, J = 2.4 Hz), 6.52 (1H, dd, J = 8.4 Hz, J = 2.4 Hz), 6.86 (1H, d, J = 8.4 Hz);
13C NMR d 34.1, 34.7, 114.0, 116.1, 122.8, 131.0, 133.5, 144.9; HRMS m/z calcd
for [C8H9N32S2+H]+ 184.0249, found 184.0250.
In summary, we have developed a procedure for the easy gener-
ation of a library of cyclic disulfides, via the preparation of amino-
benzodithiine building blocks. These can easily be connected to a
variety of electrophiles, some examples of which have been pre-
sented. Potential applications of the presented, and of other benzo-
dithiine derivatives, could be in the fields of nanotechnology, as
well as in the modulation of redox enzymes. Some of the examples
prepared have been shown to interact with glutathione reductase.
Possible interactions of other compounds with this novel pharma-
cophoric group, targeted to other enzymes with active-site cys-
teine residues will be investigated.
13. (a) Sarma, G. N.; Savvides, S. N.; Becker, K.; Schirmer, M.; Karplus, P. A. J. Mol.
Biol. 2003, 328, 893–907; (b) Friebolin, W.; Jannack, B.; Wenzel, N.; Furrer, J.;
Oeser, T.; Sanchez, C. P.; Lanzer, M.; Yardley, V.; Becker, K.; Davioud-Charvet, E.
J. Med. Chem. 2008, 51, 1260–1277.
14. (a) Seefeldt, T.; Dwivedi, C.; Peitz, G.; Herman, J.; Carlson, L.; Zhang, Z.; Guan, X.
J. Med. Chem. 2005, 48, 5224–5231; (b) Rice, K. P.; Penketh, P. G.; Shyam, K.;
Sartorelli, A. C. Biochem. Pharmacol. 2005, 69, 1463–1472.
Acknowledgments
This research was funded by NIH Grant 3-S06-GM08224-21 via
the MBRS-SCORE program. The authors thank Mr. Melvin de Jesús
at the University of Puerto Rico, Humacao Campus for his assis-
tance with the NMR measurements.
15. Zhang, H.; Cai, Q.; Ma, D. J. Org. Chem. 2005, 70, 5164–5173.
16. The reduction of GSSG by yeast glutathione reductase was determined similar
to that described in: Boese, M.; Keese, M. A.; Becker, K.; Busse, R.; Mülsch, A. J.
Biol. Chem., 1997, 272, 21767–21773. In short: The enzyme (Sigma–Aldrich)
was diluted (1 in 100) in assay buffer (200 mM KCl, 1 mM EDTA, 50 mM
potassium phosphate, pH 6.9). Separately, 1.85 mg NADPH was dissolved in
1 mL assay buffer and 14.2 mg GSSG in 2 mL assay buffer. Compounds 7–13
were dissolved in DMSO to obtain 5 mM and 2.5 mM solutions. At room
References and notes
1. Lees, W. J.; Whitesides, G. M. J. Org. Chem. 1993, 58, 642–647.
2. Nakamura, T.; Is ihara, T. O.; Hasegawa, Y. K. Diamond Relat. Mater. 2007, 16,
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Chem. 2000, 11, 289–291; (b) Li, Z.; Jin, R.; Mirkin, C. A.; Letsinger, R. L. Nucleic
Acids Res. 2002, 30, 1558–1562.
temperature, 790
by 50 enzyme solution, 50
solution (or 10 L DMSO for baseline enzyme activity). After 2 min., 100 lL
l
L assay buffer was transferred to a quartz cuvette, followed
l
L
l
L
NADPH solution, and 10 compound
lL
l

3026
S. Espinosa et al. / Tetrahedron Letters 50 (2009) 3023–3026
GSSG was added and the rate of consumption of NADPH was measured for
demonstrating that the observed increase in absorption is not due to substrate
interference in the assay procedure, but confirms an increase in NADPH
consumption.
3 min (scan every 5 s) via its absorption at 340 nm. Each compound
concentration was measured in triplicate, and the rate of NADPH
consumption was compared with baseline enzyme activity.
20. Ayers, J. T.; Anderson, S. R. Synth. Commun. 1999, 29, 351–358.
21. Ajoene: (E,Z)-4,5,9-trithiadodeca-1,6,11-triene 9-oxide): Gallwitz, H.; Bonse,
S.; Martinez-Cruz, A.; Schlichting, I.; Schumacher, K.; Krauth-Siegel, R. L. J. Med.
Chem. 1999, 42, 364–372.
17. For the synthesis of related N-phenylphtalanilic acids: Perry, C. J.; Parveen, Z. J.
Chem. Soc., Perkin Trans. 2 2001, 512–521.
18. Bisdithiocarbarmate inhibitor: 2-acetylamino-3-[4-(2-acetylamino-2-carbo-
xyethylsulfanylthiocarbonylamino)-phenylthiocarbamoylsulfanyl]propionic
acid: Seefeldt, T.; Zhao, Y.; Chen, W.; Raza, A. S.; Carlson, L.; Herman, J.;
Stoebner, A.; Hanson, S.; Foll, R.; Guan, X. J. Biol. Chem. 2009, 284, 2729–
2737.
22. Fluoro-M5:
6-[20-(30-fluoromethyl)-10,40-naphtoquinolyl]-hexanoic
acid:
Bauer, H.; Fritz-Wolf, K.; Winzer, A.; Khner, S.; Little, S.; Yardley, V.; Vezin,
H.; Palfey, B.; Schirmer, R. H.; Davioud-Charvet, E. J. Am. Chem. Soc. 2006, 128,
10784–10794.
19. The dithiol derivative of compound
7
was prepared independently via
23. The observed effect of increased NADPH consumption observed at the lower
concentration of compound 13 could be suggested to occur via a similar
mechanism.
reduction with tributylphosphine/H2O.20 Separate measurements indicated
very low absorption of both 7 and its dithiol reduction product at 340 nm,