Benzosultines as sulfur dioxide (SO2) donors

Article abstract of DOI:10.1021/ol400190f

In order to understand precise biological roles of sulfur dioxide (SO ₂), reliable SO₂ donors, compounds that produce SO ₂ under physiological conditions, are necessary. The design and development of 1-phenyl-benzosultine as an efficient SO₂ donor is reported. This compound undergoes cycloreversion to generate SO₂ upon dissolution in aqueous buffer at 37 C with a yield of 89% and a half-life of 39 min and shows SO₂-like biological activity in a DNA cleavage assay.

Full text of DOI:10.1021/ol400190f

ORGANIC
LETTERS
2013
Vol. 15, No. 5
1116–1119
Benzosultines as Sulfur Dioxide
(SO2) Donors
Satish R. Malwal, Mahesh Gudem, Anirban Hazra,* and Harinath Chakrapani*
Department of Chemistry, Indian Institute of Science Education and Research Pune,
Pune 411 008, Maharashtra, India
ahazra@iiserpune.ac.in; harinath@iiserpune.ac.in
Received January 22, 2013
ABSTRACT
In order to understand precise biological roles of sulfur dioxide (SO2), reliable SO2 donors, compounds that produce SO2 under physiological
conditions, are necessary. The design and development of 1-phenyl-benzosultine as an efficient SO2 donor is reported. This compound undergoes
cycloreversion to generate SO2 upon dissolution in aqueous buffer at 37 °C with a yield of 89% and a half-life of 39 min and shows SO2-like
biological activity in a DNA cleavage assay.
Sulfur dioxide (SO2), a byproduct of combustion of
fossil fuels, volcanic emissions, and industrial processes,
is a toxic environmental pollutant with a profound impact
on habitat suitability for plant as well as animal life.1
Human exposure to SO2 occurs through inhalation of
gaseous SO2 and through food and cosmetics. Its hydrated
forms bisulfite, HSO3ꢀ, and sulfite, SO32ꢀ, find routine use
as antibiotics, preservatives, and antioxidants.2 SO2 is also
produced in cells during oxidation of hydrogen sulfide
(H2S) and decomposition of β-sulfinylpyruvate to pyruvate.3
Some recent evidence supports potential regulatory roles for
this gas in the cardiovascular system including during
hypoxia and inflammatory responses.
Despite the importance of SO2 for living systems, the
molecular mechanisms of SO2 biology remain poorly under-
stood.3 Because SO2 is a reactive gas, obtaining biological
data with it is cumbersome. For mechanistic studies, typi-
cally either gaseous SO2 or a mixture of sulfite and bisulfite is
utilized as a surrogate.1 While useful, such exogenous sources
lack temporal control over SO2 generation and, thus, may not
be effective in mimicking endogenously produced SO2. Re-
cently, 2,4-dinitrophenylsulfonamides were reported as a
class of thiol-mediated SO2 donors.4 However, the use of thiol
as a trigger in 2,4-dinitrophenylsulfonamides may complicate
mechanistic studies because targets of SO2 include biologically
relevant disulfides and thiols.3 Thus, organic compounds that
can be used for controlled generation of SO2 under none-
nzymatic and physiological conditions are not yet available.
1,4-Dihydro-2,3-benzoxathiin-3-oxides (benzosultines)
offer the potential for efficient generation of SO2 through
a thermal retro-DielsꢀAlder reaction (Table 1). Perturba-
tion of sterics and electronics of the incipient 1,3-diene
(such as 1b) might provide opportunities for tuning SO2
release profiles from benzosultines (such as 1a). Herein, we
have presented the results of our work toward the develop-
ment of a new series of benzosultines as sulfur dioxide donors
with potential for use as tools in biochemical studies.
Previous reports have shown that benzosultines such as
1a are stable up to 80 °C.5 In order for benzosultines to be
useful as organic SO2 donors, their cycloreversion must
occur at physiological temperature of 37 °C. Clearly, lower
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Pelletier, M.; Lavastre, V.; Girard, D. Toxicol. Sci. 2002, 69, 210.
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Singleton, V. L.; Webb, A. D. Technology of Wine Making, 4th ed.; AVI
Publishing Co., Inc.: Westport, CT, 1980. (b) Ough, C. S.; Crowell, E. A.
(4) (a) Malwal, S. R.; Sriram, D.; Yogeeswari, P.; Konkimalla, V. B.;
Chakrapani, H. J. Med. Chem. 2012, 55, 553. (b) Malwal, S. R.; Sriram,
D.; Yogeeswari, P.; Chakrapani, H. Bioorg. Med. Chem. Lett. 2012, 22,
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Soc. 1973, 95, 3420. (c) Sharma, N. K.; Jung, F.; Durst, T. Tetrahedron
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J. Food Sci. 1987, 52, 386. (c) Carrete, R.; Vidal, M. T.; Bordons, A.;
Constantı, M. FEMS Microbiol. Lett. 2002, 211, 155.
(3) (a) Liu, D.; Jin, H.; Tang, C.; Du, J. Mini-Rev. Med. Chem. 2010,
10, 1039. (b) Li, J.; Meng, Z. Nitric Oxide Biol. Chem. 2009, 20, 166. (d)
Lu, W.; Sun, Y.; Tang, C.; Ochs, T.; Qi, J.; Du, J.; Jin, H. Exp. Biol. Med.
2012, 237, 867. (c) Balazy, M.; Abu-Yousef, I. A.; Harpp, D. N.; Park, J.
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r
10.1021/ol400190f
Published on Web 02/20/2013
2013 American Chemical Society

respect to the plane of the ring. The methyl and phenyl
derivaties 2a and 3a were found to have seven stable
diastereomers each (see the Supporting Information (SI),
Figures S1 and S2). The greater number of isomers in these
cases as compared to 1a is due to the two different orienta-
tions of the ꢀR group attached to the carbon atom next to
the oxygen. The prefix cis or trans is used to differentiate the
relative orientation of ꢀR with respect to the SdO bond.
For a particular ꢀR, the individual cis isomers can inter-
convert among themselves and so can the trans isomers, but
the cis isomers cannot convert to the trans isomers and vice
versa without cycloreversion to the 1,3-diene and SO2. In all
cases, 1a, 2a, and 3a, IRC calculations and comparison of
Gibbs free energy values of the transition state (TS) struc-
tures suggest that two of the stereoisomers, namely the cis-
boat-ax (boat-ax in case of 1a) and trans-boat-eq (boat-eq
in case of 1a) stereoisomers have significantly lower bar-
riers for cycloreversion than the other isomers and are the
only ones likely to be mechanistically relevant. These in-
volve the endo or exo stereoisomers of the TS structures
rather than planar TS structures (see the SI), which corre-
spond to higher energy pathways. Moreover, the diene
formed on cycloreversion of the sultine is in the (E) form,
consistent with prior studies of hetero-DielsꢀAlder reac-
tions.6 The structures of the cis-boat-ax and trans-boat-eq
sultines, their cycloreversion products, and corresponding
TSs for R = Ph are shown in Figure 2. The reaction
energies (ΔErxn), Gibbs free energies (ΔGrxn), and Gibbs
free-energy barriers (ΔG‡) of the mechanistically relevant
species calculated at 37 °C are presented in Table 1.
Table 1. Calculated Thermodynamic and Kinetic Data at 37 °C in
kcal/mol for Conversion of Benzosultines 1aꢀ3a or Sulfones 1cꢀ3c
To Produce 1,3-Dienes 1bꢀ3b Respectively and Sulfur Dioxidea
b
b
reactant species
ΔErxn
ΔGrxn
ΔG‡c
ΔΔG‡d
boat-ax-1a
boat-eq-1a
1c
33.9
35.4
37.6
36.3
38.2
39.8
34.5
34.6
37.6
15.3
16.4
18.3
16.6
18.4
19.6
15.9
16.3
18.6
29.5
31.0
33.5
28.1
30.4
32.9
26.3
27.9
29.5
0
1.5
4.0
cis-boat-ax-2a
trans-boat-eq-2a
cis-2c
ꢀ1.4
ꢀ0.9
3.4
cis-boat-ax- 3a
trans-boat-eq-3a
cis-3c
ꢀ3.2
ꢀ1.5
0
a The data are for two lowest barrier pathways for benzosultines and
the lowest barrier pathway for sulfones. Energy values in bold correspond to
the most favorable pathway (lowest activation barrier) for the cyclorever-
sion of 1aꢀ3a. b The significant difference between ΔErxn and ΔGrxn is
because of the increase in the number of molecular species during the cyclo-
reversion leading to a large translational and rotational entropy contribu-
tion to the driving force of the reaction. c Vibrational entropy and zero-point
energy contributions lower the reaction barriers as compared to the pure
electronic barrier (see the Supporting Information, Tables S1ꢀS3). This is
because of the relatively floppy nature of the transition state with respect to
the reactant, which leads to a lower zero-point energy and higher vibrational
entropy. d Calculated by using boat-ax-1a as the reference.
cycloreversion barriers as compared to 1a are desired, and
the literature suggests that a way to achieve this is by
suitable substitution on the carbon atom adjacent to
oxygen in the sultine ring.6 Keeping this design principle
in mind, we calculated the Gibbs free energy barriers for
the cycloreversion of the unsubstituted and substituted
benzosultines 1a, 2a, and 3a to the corresponding dienes
1b, 2b, and 3b, and SO2 using ab initio theoretical methods.
The optimized structures, energies, vibrational frequencies,
and intrinsic reaction coordinates (IRCs) were all calculated
at the MøllerꢀPlesset second-order perturbation (MP2)
level of theory with the 6-31G(d,p) basis set. All calculations
were performed using the GAMESS program package.7
Compound 1a was found to have four stable conforma-
tions (Figure 1). In two of the conformers, the six-membered
sultine ring is in the pseudochair conformation, while, in the
two others, the sultine ring is in the pseudoboat conforma-
tion. The notation “ax” (axial) or “eq” (equatorial) in the
figure labels refers to the orientation of the SdO bond with
Figure 1. Stable conformations of 1a.
The most favorable path for cycloreversion of the benzo-
sultines is through the endo (E) TS starting from the cis-boat-
ax stereoisomers. For this stereoisomer, comparing across 1a,
2a, and 3a, we can see that methyl substitution (2a) reduces
the cycloreversion barrier by 1.4 kcal/mol possibly due to the
electron-donating effect of a methyl group, while phenyl sub-
stitution (3a) reduces the barrier by 3.2 kcal/mol possibly due
to extended conjugation (Table 1). At 37 °C, this barrier
reduction corresponds to an ∼10-fold rate enhancement in
the rate of SO2 generation in the case of methyl substitution
(2a) and an ∼200-fold rate enhancement in the case of
phenyl substitution (3a) as compared to 1a.
(6) (a) Vogel, P.; Sordo, J. A. Curr. Org. Chem. 2006, 10, 2007. (b)
Roversi, E.; Monnat, F.; Vogel, P.; Schenk, K.; Roversi, P. Helv. Chim.
Acta 2002, 85, 733.
(7) (a) Gordon, M. S.; Schmidt, M. W. In Theory and Applications of
Computational Chemistry: The First Forty Years; Dykstra, C. E., Frenking,
G., Kim, K. S., E.Scuseria, G., Eds.; Elsevier: Amsterdam, 2005; p 1167. (b)
Schmidt, M. W.; Baldridge, K. K.; Boatz, J. A.; Elbert, S. T.; Gordon, M. S.;
Jensen, J. H.; Koseki, S.; Matsunaga, N.; Nguyen, K. A.; Su, S.; Windus,
T. L.; Dupuis, M.; Montgomery, J. A. J. Comput. Chem. 1993, 14, 1347.
1,3-Dienes such as 1b undergo cheletropic addition of
SO2 to produce a sulfone (1c). As in the case of sultines,
calculationsof1cꢀ3cwereperfomed(Table1andFigure2;
see SI Figures S3ꢀS5). From IRC calculations, we found
Org. Lett., Vol. 15, No. 5, 2013
1117

Figure 2. Gibbs free energy profiles for decomposition of 3a to produce 3b and SO2 and its conversion to 3c at 37 °C.
only one mechanistically relevant pathway for sulfone for-
mation in all cases. The corresponding product has the SO2
Table 2. Synthesis of 1aꢀ10a
moiety and the ꢀR group both on the same side of the five-
membered ring and is therefore referred to as cis. The
sulfones 1cꢀ3c are found to be both thermodynamically and
kinetically more stable than the corresponding sultines
(Table 1). The 1,3-diene and SO2, once formed, can react
to form either the sultine or the sulfone: the ΔG‡ for forma-
tion of 3a or 3c from 3b is sufficiently low (<11 kcal/mol)
that both reactions occur easily at 37 °C (Figure 2). How-
ever, once formed, the ΔG‡ for extrusion of SO2 from 3c is
relatively large (29.5 kcal/mol) and the reaction would be
∼200 times slower than the cycloreversion of 3a. We thus
predict that 3a eventually completely converts to 3c.5
Benzosultines 1aꢀ3a were synthesized in several steps
from commercially available phthalide (see the SI) with a
sulfuryl chloride mediated cyclization reaction as the final
step (Table 2, entries 1ꢀ3).
entry
R
compd
product
yield (%)
1
2
H
1d
2d
3d
4d
5d
6d
7d
8d
9d
10d
1a
2a
3a
4a
5a
6a
7a
8a
9a
10a
60
88
43a
42
49
51
38
32
38
35
Me
Ph
3
4
4-FPh
5
4-ClPh
6
4-CNPh
4-MePh
3-OMePh
3-CNPh
2-NO2Ph
7
8
9
10
HPLC analysis of acetonitrile solutions of 1aꢀ3a at
70 °C for 3 h showed no significant decomposition of 1a
(<5%), whereas, during this time, 2a was 41% decom-
posed while 3a was completely decomposed in this time
period (see the SI, Table S5). These results are in agreement
with the predicted barriers for cycloreversion being 3a >
2a > 1a (Table 1) and similar to a previous report of rates
of reactions of 1aꢀ3a at 80 °C being 3a > 2a > 1a.5
When a similar decomposition experiment was conducted
at a physiological temperature of 37 °C in MeCN, 3a de-
composed (k = 2.9 ꢁ 10ꢀ3 minꢀ1) to produce the sulfone
3c (4.5 ꢁ 10ꢀ3 minꢀ1) in 92% yield (see the SI, Figure S6).
No significant differences in the rates of decomposition of
the major or minor diastereomers of 3a were found sug-
gesting that both cis and trans isomers decompose at com-
parable rates (see the SI, Figure S6). Under similar conditions,
sultines 1a and 2a were found to be unreactive. No significant
decomposition of the sulfone 3c at 37 °C was observed
supporting the predicted mechanism that the formation
of 3c from 3a was an irreversible process.
a 1H NMR (12% minor) and HPLC (19% minor) analysis showed 3a
to be a mixture of cis and trans isomers. Computational studies identify
the most stable cis isomer as cis-boat-ax-3a and the two lowest energy
interconvertible trans isomers as boat trans-boat-eq-3a and trans-chair-ax-
3a (see the SI, Tables S1ꢀS3).
(Figure 3).8 The major inorganic product of this decom-
position was SO2, measured as sulfite (89% yield) with a
half-life of SO2 generation of 39 min (Table 3).
The suitability of 3a for use as a SO2 donor under biologi-
cal assay conditions was demonstrated using a pBR322 super-
coiled plasmid cleavage assay (Figure 4).9 A nearly identical
DNA damage profile in the presence of equimolar amounts of
Cu(II) and 3a or sulfites (1:3 Na2SO3 and NaHSO3) was
recorded (Figure 4). The DNA damage experiment was
(8) Stabilization of the activated complex by H-bonding was reported
to be responsible for acceleration of the retro-DielsꢀAlder of anthrace-
nedione in water in comparison with an aprotic organic solvent. A
similar H-bonding effect might explain the observed increased rate of
cycloreversion of 3a in aqueous media. Wijnen, J. W.; Engberts, J. B. F.
N. J. Org. Chem. 1997, 62, 2039.
At physiological pH 7.4 and 37 °C, 3a was nearly com-
(9) (a) Burrows, C. J.; Muller, J. G. Chem. Rev. 1998, 98, 1109. (b)
Shi, X.; Mao, Y. Biochem. Biophys. Res. Commun. 1994, 205, 141.
pletely decomposed with a rate constant k = 0.0140 minꢀ1
1118
Org. Lett., Vol. 15, No. 5, 2013

Figure 4. Nuclease activity of (100 μM of analyte) was deter-
mined by a pBR322 plasmid DNA (100 ng of form I DNA)
cleavage assay in pH 8.0 buffer, incubated at 37 °C for 5 h. An
aqueous solution of Na2SO3 and NaHSO3 in a 1:3 ratio is
labeled as Sulfites. Nicked DNA is represented by II.
Figure 3. Decomposition of 3a in pH 7.4 buffer at 37 °C
produced SO2.
conducted in two independent batches of 3a, each in duplicate,
and we found similar results in all cases demonstrating batch-
to-batch reproducibility in the use of 3a as a SO2 donor.
Table 3. Decomposition Profiles of Benzosultines 3aꢀ10a
Prepared in This Study in pH 7.4 Buffer at 37 °C and Substituent
Constants used for Construction of Hammett Plot
k
t1/2
(min)b
max SO2
yield (%)c
compd
(minꢀ1 a
)
σd
σþe
Figure 5. Hammett plot of rate constants for decomposition of
3aꢀ9a in pH 7.4 buffer at 37 °C. Linear regression analysis
yielded F as ꢀ0.6 (R2 = 0.75).
3a
4a
5a
6a
7a
8a
9a
10a
0.0140
0.0244
0.0140
0.0093
0.0336
0.0104
0.0069
0.0500
39
14
34
38
13
56
68
10
89
83
79
76
80
73
59f
81
0
0
0.15
0.24
0.71
ꢀ0.14
0.12
0.62
ꢀ
ꢀ0.073
0.114
0.659
ꢀ0.311
0.047
0.562
ꢀ
F, was found to be ꢀ0.6 implying a weak electronic effect
on the reaction center wherein an electron-donating group
accelerated the cycloreversion.12
Here, we have presented a strategy for controlled and
efficient generation of SO2 under physiological pH and
temperature under nonenzymatic conditions. There is a
real need to understand precisely the biomolecular mech-
anisms of interaction of SO2, as increased industrialization
has resulted in larger populations being exposed to this gas1
and SO2’s unique vasodilatory3 and antimicrobial4 effects
might be exploited in developing novel therapeutic agents.
It is anticipated that 3a and its analogues, several of which
are stable powders that release SO2 in pH 7.4 buffer at
predictable rates via a well-understood mechanism, would
facilitate discerning these biological mechanisms.
a Determined by kinetic analysis of decomposition of sultine using
HPLC. All rate data obtained are for diastereomers where applicable.
b Determined kinetic analysis of formation of SO2 as sulfite by an ion
chromatograph attached with a conductivity detector. c The highest yield of
SO2 produced after complete decomposition of sultine. d Reference 10a.
e Reference 10b. f As no evidence for the formation of the corresponding
sulfone was found, the diminished yields of SO2 must be due to collateral
consumption of the sultine through other yet uncharacterized processes.
1-Aryl-benzosultines also offer scope for tuning rates of
SO2 generation through systematic structural modifica-
tions on the 1-aryl ring. Using a methodology similar to
that for the synthesis of 3a, we prepared 1-aryl-benzosultines
4aꢀ10a (Table 2) with electron-donating and -withdrawing
groups on the 1-aryl ring (see the SI, Table S4). All the
sultines tested were found to be labile in pH 7.4 buffer at
37 °C, and half-lives of SO2 generation varied from 10 to
68 min (Table 3). The maximum SO2 yields were in excess of
75% in the majority of the sultines tested (Table 3).
Acknowledgment. This work was supported in part by
the Department of Science and Technology (DST) India
(Grant No. SR/FT/CS-89/2010).
Supporting Information Available. Compound charac-
terization data, computational data, procedures for de-
composition and sulfur dioxide analysis, and NMR
spectra. This material is available free of charge via the
Internet at http://pubs.acs.org.
A nearly linear Hammett plot constructed using rate
constants for decomposition of 3aꢀ9a (Table 3) and sub-
stituent constant σþ suggested a predictable mechanism of
decomposition for this scaffold (Figure 5).10,11 The slope,
(12) Although a Hammett plot for a similar retro-DielsꢀAlder was not
available for comparison, a previous report of a DielsꢀAlder reaction of
1-aryl-1,3-butadienes with maleic anhydride at 35 °C found F as ꢀ0.621.
DeWitt, E. J.; Lester, C. J.; Ropp, G. A. J. Am. Chem. Soc. 1956, 78, 2101.
(10) (a) Isaacs, N. S. Physical Organic Chemistry, 2nd ed.; Pearson
Education Limited: 1995. (b) Brown, H. C.; Okamato, Y. J. Am. Chem. Soc.
1958, 80, 4979.
(11) A nearly identical F-value of ꢀ0.6 (R2 = 0.60) was obtained
when σ was used to construct the Hammett plot.
The authors declare no competing financial interest.
Org. Lett., Vol. 15, No. 5, 2013
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