Synthesis and antimycobacterial activity of prodrugs of sulfur dioxide (SO2)

Article abstract of DOI:10.1016/j.bmcl.2012.04.048

Here, we synthesized and studied a library of 2,4-dinitrophenylsulfonamides that closely resembled N-benzyl-2,4-dinitrophenylsulfonamide (1), a thiol-activated prodrug of sulfur dioxide (SO₂) which has shown high potency as a Mycobacterium tuberculosis (Mtb) inhibitory agent. The ability of these compounds to generate SO₂ in the presence of a thiol was evaluated. A good correlation between pK_aH of the corresponding amine and reactivity with thiols to generate SO₂ was found suggesting that the rate determining step of SO₂ generation involved protonation of the amine. Amongst analogues with measurable MICs, we also found a correlation between ability to generate SO₂ and Mtb growth inhibitory activity. Together, we report several thiol-mediated prodrugs of SO₂ which strongly inhibited Mtb growth (MIC <1 μg mL^-1) with potential for further development as tuberculosis drug candidates.

Full text of DOI:10.1016/j.bmcl.2012.04.048

Bioorganic & Medicinal Chemistry Letters 22 (2012) 3603–3606
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and antimycobacterial activity of prodrugs of sulfur dioxide (SO2)
Satish R. Malwal a, Dharmarajan Sriram b, Perumal Yogeeswari b, Harinath Chakrapani a,
⇑
a Department of Chemistry, Indian Institute of Science Education and Research, Pune 411 008, India
b Department of Pharmacy, Birla Institute of Technology & Science-Pilani, Hyderabad Campus, Jawahar Nagar, Hyderabad 500 078, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Here, we synthesized and studied a library of 2,4-dinitrophenylsulfonamides that closely resembled
N-benzyl-2,4-dinitrophenylsulfonamide (1), a thiol-activated prodrug of sulfur dioxide (SO2) which has
shown high potency as a Mycobacterium tuberculosis (Mtb) inhibitory agent. The ability of these com-
pounds to generate SO2 in the presence of a thiol was evaluated. A good correlation between pKaH of
the corresponding amine and reactivity with thiols to generate SO2 was found suggesting that the rate
determining step of SO2 generation involved protonation of the amine. Amongst analogues with measur-
able MICs, we also found a correlation between ability to generate SO2 and Mtb growth inhibitory activity.
Together, we report several thiol-mediated prodrugs of SO2 which strongly inhibited Mtb growth (MIC
Received 18 February 2012
Revised 17 March 2012
Accepted 10 April 2012
Available online 17 April 2012
Keywords:
Tuberculosis
Mycobacterium tuberculosis
Sulfur dioxide
Thiol
<1 l
g mLÀ1) with potential for further development as tuberculosis drug candidates.
Ó 2012 Elsevier Ltd. All rights reserved.
Sulfonamide
Tuberculosis causes millions of fatalities each year and in combi-
nation with HIV is proving to be a significant threat to global
health.1,2 Although several new promising drug candidates are
under consideration for clinical use, the increasing incidences of
multi-drug resistant (MDR) and extensively drug resistant (XDR)
strains of Mycobacterium tuberculosis (Mtb) necessitates new strate-
gies for targeting this pathogen.3–5 Recently, we reported that small
amounts of sulfur dioxide (SO2) inhibited Mtb growth.6 SO2 is an
environmental pollutant that is toxic to humans at elevated levels;7
chronic exposure to SO2 induces oxidative stress and asthma-like
symptoms.8 SO2 has diverse documented biological effects includ-
ing damage to biomacromolecules such as proteins, lipids and
DNA.9–11 Oxidation of sulfur dioxide by metal ions produces sulfur
trioxide radical, which possibly mediates damage to biomacromol-
ecules.11 Furthermore, sulfite, the anionic form of sulfur dioxide can
break disulfide linkages to produce S-sulfonates.10 Thus, SO2 can
participate in oxidative as well as reductive12–14 processes under
physiological conditions and may perturb redox equilibrium in
cells. Recently, alteration of redox homeostasis in Mtb has been
proposed as an effective mechanism for targeting this bacterium.15
Thus, sulfur dioxide may have diverse mechanisms for cytotoxicity
induction and multiple biological targets. Despite such well-docu-
mented deleterious effects, SO2 (in the form of bisulfite, metabisul-
fite and sulfite) has also been routinely used as an antibiotic and
antioxidant in the food industry16–18 and is well tolerated in most
individuals.9,10 Thus, we proposed that SO2 could be used as an
anti-bacterial agent. In order to test this hypothesis, our strategy
was to mask SO2 as 2,4-dinitrobenzenesulfonamides,19 which are
candidates of thiol-activated SO2 generation (Scheme 1).6 We
reported that amongst the compounds tested, the most potent
Mtb inhibitor was found to be N-benzyl-2,4-dinitrophenylsulfona-
mide (1, Table 1) with a MIC of 0.05
better than the MIC of clinically used isoniazid (0.05
0.37 M). This compound did not show significant toxicity against
human embryonic renal cells at MIC (0.15 M) against Mtb. Taken
lg/mL (0.15
lM), which was
lg/mL,
l
l
together, our results showed that SO2 could be used as an anti-
mycobacterial agent when masked as a suitable prodrug form.
Our preliminary results indicated that the rate of SO2 genera-
tion affected inhibitory activity of this class of compounds. We
found that small modifications to the structure of the amine
resulted in a significant change in the half-lives of SO2 generation
(t1/2 = 2–63 min). Thus, varying the amine structure could poten-
tially help modulate anti-mycobacterial activity. Here, we pro-
posed to synthesize and study a library of structural analogues of
1 in order to further understand the relationship between thiol-
mediated sulfur dioxide generation and Mtb inhibitory activity. In
order to study the effects of varying sterics and/or electronics on
chemical and biological properties, analogues with comparable
clogPs to 1 were synthesized.
O
S
R'
R
R"SH
RNR'H
+
SO2
+
O2N
SR
NO2
O2N
N
O
NO2
⇑
Corresponding author. Tel.: +91 20 2590 8090; fax: +91 20 2589 9790.
E-mail address: harinath@iiserpune.ac.in (H. Chakrapani).
Scheme 1. 2,4-Dinitrophenylsulfonamides are thiol-activated sources of sulfur
dioxide (SO2).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.04.048

3604
S. R. Malwal et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3603–3606
We prepared the benzylamine analogues 2–5 (Table 1, entries
(10 equiv). SO2 was determined as sulfite in pH 7.4 using an ion
chromatograph equipped with a conductivity-based detector.20
Under these reaction conditions, the benzylamine derivative 1 pro-
duced 100% SO2 in 30 min (Table 1, entry 1). Presence of an electron
withdrawing or electron donating group did not significantly affect
the yield of SO2 in comparison with 1 (Table 1, entries 2–5). The sul-
2–5) with electron donating and electron withdrawing groups
using a previously reported method of reaction of the correspond-
ing amine with 2,4-dinitrophenylsulfonyl chloride (DNsCl).
Similarly, aniline derivatives 6–11 were also prepared using the
aforementioned procedure with substituents capable of perturbing
the electronics of the amine (Table 1, entries 6–11). In order to
study the effect of changing sterics around the nitrogen bearing
fanilide 6 produced 55 lM (55% yield) SO2 under comparable reac-
tion conditions (Table 1, entry 6). Amongst the aniline derivatives,
we found a strong electronic effect on the yield of sulfur dioxide
during decomposition of these derivatives. For example, the pres-
ence of an electron donating group enhanced the yield of SO2 in
comparison with aniline (Table 1, entries 7 and 8). Conversely,
when an electron withdrawing group is present on the aromatic
ring of aniline, the yield of SO2 decreased in comparison with ani-
line (Table 1, entries 9–11). The effect was particularly strong in
the presence of a 4-cyano group (11), whose sulfur dioxide yield
(30 min) was 5% (Table 1, entry 11). In the presence of an additional
the DNs group, we prepared 12 and 13 with an a-methyl substitu-
ent (Table 1, entries 12–13). The 1-phenyl-2-aminoethyl derivative
14 was prepared by treatment of the corresponding amine with
DNsCl (Table 1, entry 14). Finally, the alkylated derivatives 15–20
were prepared by treatment of the corresponding alkyl halide with
a suitable 2,4-dinitrosulfonamide (Table 1, entries 15–20).
Next, we evaluated cysteine-mediated SO2 yields from this
library of compounds (Table 1) by recording the yield of SO2
30 min after commencement of the reaction with cysteine
Table 1
Synthesis, calculated partition coefficients (clogP), thiol-mediated sulfur dioxide generation and anti-mycobacterial activities of 2,4-dinitrosulfonamides prepared in this study
and related compounds
H
N
DNs
N
H
DNs
N
H
DNs
N
H
CF3
DNs
N
H
DNs
N
H
DNs
Cl
F3C
MeO
3
4
5
6
1
2
Me
H
H
N
OMe
H
H
N
F
N
DNs
H
DNs
N
DNs
N
DNs
DNs
N
DNs
H
NC
MeO
F
7
8
12
9
10
11
R
N
Me
N
H
N
Me
N
R
DNs
DNs
DNs
DNs
N
H
DNs
15
; R = Me
19
; R = Me
16; R = n-Pr
17; R = Ph
18
14
20; R = Bn
13
Yielda (%)
clogPb
SO2 yield, 30 min (
l
M)c
SO2 yield, 5 min (
l
M)c
pKaH
MIC (l
g mLÀ1
)
MIC (lM)
d
e
Entry
Compd
Mol. Wt.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
1
2
3
4
5
6
7
8
337.05
367.33
371.75
405.31
405.31
323.28
353.30
353.30
341.27
341.27
348.29
351.33
351.33
351.33
351.33
379.38
413.40
337.31
365.56
441.46
137.13
204.31
123.11
41
33
54
43
54
30
66
70
70
48
47
47
38
79
86
73
47
81
95
80
—
2.87
2.78
3.58
3.75
3.75
2.76
2.69
2.69
2.93
2.92
2.24
3.17
3.17
3.19
2.41
3.47
3.87
2.10
2.74
4.50
À0.66
2.08
À0.67
100
84
96
75
84
55
80
86
24
55
5
97
94
100
100
96
94
94
84
75
—
83
74
77
79
81
37
39
57
5
9.51
9.50
9.44
9.45
9.23
4.64
5.11
4.42
3.22
3.80
1.63
9.73
9.73
9.79
9.70
9.90
3.92
5.64
10.13
9.88
—
0.05f
0.25
0.4
0.15
0.68
1.07
0.98
3.84
9.69
4.4
0.4
1.56
3.13f
1.56f
3.13
25f
8.85
73
9
10
11
12
13
14
15
16
17
18
19
20
Isoniazid
18
6
6.25
>50
18.31
>100
4.44
4.44
1.13
1.10
8.25
4.44
2.31
2.13
7.09
0.37
7.63
50.8
80
78
76
79
68
44
89
79
37
—
1.56
1.56
0.4
0.4f
3.13f
1.56
0.78
0.78
3.13
0.05
1.56
6.25
Ethambutol
Pyrizinamide
—
—
—
—
—
—
—
—
a
b
c
Yield is for reaction of the amine with 2,4-dinitrophenylsulfonyl chloride or alkylation of 2,4-dinitrophenylsulfonamide.
Calculated using Chembiodraw Ultra.
Sulfur dioxide as sulfite was quantified using an ion chromatograph equipped with a conductivity detector: yields are 5 min and 30 min after treatment of compound
M) with 10 equiv of cysteine in pH 7.4 phosphate buffer.
(100
l
d
Values are for the corresponding amine and were calculated using Marvinsketch 5.7.1.
Minimum inhibitory concentration (MIC) is the minimum concentration of the compound required to inhibit 99% of bacterial growth and was found against Myco-
e
bacterium tuberculosis H37Rv strain.
f
Previously reported in Ref. 6.

S. R. Malwal et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3603–3606
3605
Figure 2. Kinetics of decomposition of
Disappearance of
Production of sulfite was monitored by ion chromatography analysis. Curve fitting
afforded first order rate constant for: appearance of 21 as 0.011 minÀ1
and
appearance of sulfite as 0.014 minÀ1
7
to produce sulfur dioxide and 21.
7
and appearance of 21 was followed by HPLC analysis.
;
.
Figure 1. Relationship between sulfur dioxide yield generated during cysteine-
mediated decomposition of 2,4-dinitrophenylsulfonamides prepared in this study
and their Mtb inhibitory activity. (a) SO2 yield was after 30 min; Spearman rank
correlation analysis of SO2 yield and MIC gave a Spearman correlation coefficient of
À0.50 (P-value = 0.01). (b) SO2 yield was after 5 min; Spearman rank correlation
analysis of SO2 yield and MIC gave
q = À0.69 (P-value = 0.001).
alkyl, aryl, or methylene group, either comparable or slightly dimin-
ished yield of SO2 in comparison with 1 was observed (Table 1,
entries 12–20).
The ability of compounds in this library to inhibit Mycobacte-
rium tuberculosis (H37Rv) growth was evaluated using a reported
protocol.6 We found minimum inhibitory concentrations (MICs)
ranged from 0.05 to >100
best Mtb inhibitor in this series, several derivatives 2–4, 14, 15,
18 and 19 had potent Mtb inhibitory activities with MICs <1 g/
mL (Table 1, entries 2–4, 14–15, 18 and 19); these MICs were bet-
ter than those of ethambutol and pyrizinamide, both clinically
used tuberculosis drugs, evaluated under similar assay conditions
(Table 1, entries 22 and 23).
Data analysis revealed that the most potent Mtb inhibitors gen-
erated elevated levels of SO2 in 30 min (P75% yield). Spearman
rank correlation analysis of MICs with sulfur dioxide yields
(Fig. 1a) revealed a moderate negative correlation value of Spear-
lg/mL (Table 1). While 1 was still the
Figure 3. Relationship between sulfur dioxide generated during cysteine-mediated
decomposition of 2,4-dinitrophenylsulfonamides prepared in this study and pKaH of
the amine from which the corresponding sulfonamide was prepared. (a) SO2 yield
l
was after 5 min; Pearson correlation analysis of SO2 yield and pKaH gave
a
correlation coefficient r = À0.79 (P-value <0.001) (b) SO2 yield was after 30 min;
Pearson correlation analysis of SO2 yield and pKaH gave a correlation coefficient
r = À0.71 (P-value <0.0001).
Next, we performed a detailed kinetic analysis of thiol-mediated
decomposition and sulfite generation from 7. This compound was
chosen as the rate of decomposition was amenable to simultaneous
analysis of product decomposition and sulfur dioxide generation.
Several possible mechanisms for the decomposition of this com-
pound were considered (Scheme 2): path A, wherein thiol attack
produces a Jackson–Meisenheimer intermediate I, which then
rearranges via a transfer of proton to produce intermediate III,
which then decomposes to simultaneously produces sulfur dioxide,
man rank correlation coefficient
between MICs and SO2 yields. Similar data collected for SO2 yields
after 5 min (Table 1), however, correlated well with MICs (Fig. 1b)
q
= À0.50 (P-value = 0.02)
q
= À0.69 (P-value = 0.001). These results indicate a role for effi-
ciency of SO2 generation, which in turn is related to reactivity of
the compound with thiols, in the observed Mtb inhibitory activity
of this series of compounds.
Path A
~H+
RDS
H
R
S
O
OH
HS
pH 7.4
37
O2N
S
O
N
Me
O
NO
2 H
°
C
Me
O
HO
I
R
O
S
S
O
O2N
S
SO2
+
O2N
Me
O
7
N
NO2
H
NO
2 H
III
RDS
NH2
21
+H+
Path B
OH
+H+
SR
S
Me
O
O
Me
S
+ SO2
N
H
O2S
H2N
O
O2N
O
N
Me
O
RDS
pH 7.4
37
Path C
NO
II
°
C
2 H
21
IV
Scheme 2. Proposed mechanisms of decomposition of 7 to produce SO2.

3606
S. R. Malwal et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3603–3606
4-methoxyaniline and 2-hydroxyethylthio-2,4-dinitrobenzene
(21).
References and notes
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21. Under similar conditions, 7 produced 80% SO2 in the presence of cysteine
(Table 1, entry 7) whereas in the presence of mercaptoethanol, the yield of SO2
was 22%. This result indicates that the rate of SO2 generation was also
dependent on the nature of the thiol. Perhaps, decomposition of intermediate I
or II is accelerated in the presence of sterically encumbered cysteine in
comparison with 2-mercaptoethanol. It is also possible that attack by the
hydroxyl group of mercaptoethanol produces an O-sulfonate; this
intermediate may not form sulfur dioxide and this pathway could also
explain the diminished yield.
Path B is an alternative wherein the attack on the aryl ring is by
a thiolate to produce intermediate II, which then produces inter-
mediate III by a protonation (Scheme 2). pKas of thiols are typically
in the range of 9–10 and hence would be expected to be in the form
of thiolate ions in pH 7.4. However, protonation of II is still neces-
sary as decomposition of II would produce an amide (ArNÀ) anion
which may not have physiological relevance. Finally, path C was
considered wherein loss of 21 from III produces intermediate IV,
which then produces SO2 and 4-methoxyaniline by a slow decom-
position step that involves protonation. In order to understand
which pathway was dominant, 7 was treated with 2-mercap-
toethanol (10 equiv) in pH 7.4 at 37 °C. HPLC analysis of the reac-
tion mixture showed complete disappearance of 7 within 30 min of
commencement of reaction (Fig. 2). We monitored the generation
of 21 during the reaction course and we found a first order appear-
ance of this compound during 6 h (Fig. 2).
Sulfite generation during this time period closely followed the
generation of 21 suggesting that SO2 and 21 were produced in
the same decomposition step (Fig. 2). This observation suggests
that path C was disfavored. As 7 decomposed much faster than
appearance of SO2, path A or B would be preferred with intermedi-
ate III generation (either through proton transfer from I or addition
of proton to II) as the rate determining process (RDS). We found a
good positive correlation r = 0.79 (P-value <0.0001) between pKaH
of the amine and SO2 yields (5 min) and r = 0.71 (P-value <0.001)
for SO2 yields (30 min) during cysteine-mediated decomposition
of 2,4-dinitrosulfonamides supporting that the proposed mecha-
nism of formation of intermediate III as the rate determining step
could be a general mechanism of thiol-mediated SO2 generation
from 2,4-dinitrophenylsulfonamides (Fig. 3).21
Nitro group reduction plays a major role in the action of PA-824,
a nitroimidazole,22–26 dinitrobenzamides (DNBs), and benzothiazi-
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Mtb differ suggesting that reduction of an aromatic nitro group
may have wide-ranging consequences to Mtb growth. At this time,
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action of 2,4-dinitrophenylsulfonamides reported in this work is
unclear. However, as a majority of the potent Mtb inhibitors in
our study were nearly completely decomposed in 30 min, it is per-
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in Mtb.35 Thus, we report that anti-mycobacterial activity of closely
related structural analogues of 1 with comparable clogPs corre-
lated well with the analogue’s ability to generate sulfur dioxide
upon treatment with cysteine. Future work will focus on identifica-
tion of molecular targets and mechanisms of action of 2,4-dinitro-
phenylsulfonamides including 1.
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Acknowledgments
The authors thank IISER Pune and the Department of Science
and Technology (DST) India for funding (Grant number SR/FT/CS-
89/2010). SRM acknowledges a research fellowship from Council
for Scientific and Industrial Research (CSIR).
Supplementary data
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