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K. A. Pardeshi et al. / Bioorg. Med. Chem. Lett. 25 (2015) 2694–2697
Among the bis(2,4-dinitrophenylsulfonamides) 9a–9i, all com-
When tested against MRSA, we found that 5b and 5e were
excellent inhibitors with an MIC of 4 g/mL (Table 3, entries 13
pounds gave nearly 2 mol of SO2 per mol of compound (Table 2,
entries 27–35). Consistent with previous observations that both
nitro groups were necessary for decomposition, we found that
9j–9l gave 1 mol of SO2 per mol of compound (Table 2, entries
36–38).22,23
Using a standard microbroth dilution protocol, the DNs deriva-
tives synthesized in this study were tested for their inhibitory
activity against methicillin sensitive S. aureus (MSSA) and MICs
were recorded (Table 2).24 The SO2 donor 1a which had high
l
and 16). In order to determine if these compounds had a broad-
spectrum antibacterial activity, this library was tested against
Enterococcus faecalis (E. faecalis) and Escherichia coli (E. coli). A
majority of the compounds in the 1–8 series tested were found
to have moderate MICs against E. faecalis but none of the com-
pounds were capable of inhibiting E. coli. Compounds 4g, 5b and
5e were found to be good inhibitors of E. faecalis with MICs of
8 lg/mL (Table 3, entries 10, 13 and 16). Based on these results,
potency against Mtb was inactive against MSSA at 16
lg/mL
5e was identified as the lead compound and further studies were
(Table 2, entry 1). Other derivatives containing a primary amine
DNs functional group were similarly inactive or were found to be
moderate or good inhibitors of MSSA with MICs of 68 lg/mL
(Table 2, entries 2, 3, 6, 7, 11, 13, 16, 17 and 20–24). The best inhi-
bitors of MSSA were found to be 1c, 4g and 5e all containing a
propargyl substituent (Table 2, entries 3, 17 and 23). The MICs of
directed toward this SO2 donor.
Our hypothesis was that the antibacterial activity of SO2 pro-
drugs is mediated by compound’s entry into cells followed by acti-
vation by thiols to generate SO2, which can possibly induce stress
through enhanced oxidative species. If the SO2 donor permeated
bacterial cells, depletion of intracellular thiols might result by reac-
tion with the 2,4-dinitroaryl group. In addition, thiols are primary
responders to oxidative stress and are converted to disulfides.
Depletion of free thiols might thus occur as a response to enhanced
oxidative species as well. A monobromobimane (mBBr) protocol
was used to assess levels of free thiols within cells.25 Here, the
weakly fluorescent mBBr reacts with thiols and is converted to a
highly fluorescent adduct. Hence, increased fluorescence response
in this assay is an indicator of greater free thiol levels inside cells
and vice versa. When MRSA cells were incubated with varying con-
centrations of 5e, we found a nearly dose-dependent decrease in
fluorescence, compared to untreated control indicating that 5e is
capable of depleting thiols in bacteria (Fig. 1).
the remaining compounds in this series were all P8 lg/mL
(Table 2). None of the bis(2,4-dinitrophenylsulfonamides) synthe-
sized in this study were found to have significant inhibitory
activity against MSSA (Table 2, entries 27–35). Similarly com-
pounds 9j–9l were incapable of inhibiting MSSA growth (Table 2,
entries 36–38). The presence of a propargyl group appears to
enhance efficacy. For example, in the cases of the benzylamine
derivative 1a, a significant increase in potency was seen when a
propargyl group (1c) was introduced (Table 2, entries 1 and 3). A
similar result was recorded with 2a and 2b; 5a and 5e (Table 2,
entries 5, 6, 19 and 20). The presence of the allyl group on the other
hand no significant effect on the efficacy (Table 2, entries 11, 13, 19
and 22). The partition coefficients were calculated (clogP) as an
estimate of permeability of these compounds. However, no signif-
icant trend between clogP and MIC was observed in this series.
Together, these data indicate no observable correlation between
the ability of the compound to generate SO2 in a test tube (see
Table S1, Supporting information), or its calculated partition coef-
ficient (clogP) and its inhibitory potency but show some sensitivity
to introduction of key substituents such as the propargyl group.
In a similar experiment, N-ethylmaleimide (NEM) a known thiol
depleting agent was also used and we find a diminished fluores-
cence that is consistent with decreased thiol levels. Under these
conditions, 8b and 9d, both with poor MRSA inhibitory activity
(Table 3, entries 18 and 21), we find diminished capacity to deplete
thiols (see Supporting information Fig. S1). These data indicate that
the capacity of the compound to permeate cells to deplete free thi-
ols played a significant role in the observed inhibitory potency.
SO2 is known to generate oxidative species resulting in
biomacromolecular damage.11,15,26,27 We tested the ability of 5e
to produce oxidative species intracellularly using a reported 2,7-
dichlorodihydrofluorescein-diacetate (DCFH2-DA) assay.28,29 We
found increased fluorescence levels intracellularly in S. aureus upon
incubation with 5e (Fig. 2). This result is indicative of production of
oxidative species and perhaps, induction of oxidative stress18,19,30–
Table 3
MICs against MRSA, E. faecalis and E. coli
Entry
Compd
MICa, MRSA
MICa, E. faecalis
MIC, E. coli
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
1c
1d
2b
4a
4b
4c
4d
4e
4f
4g
4h
5a
5b
5c
5d
5e
4
8
8
8
16
8
>32
>32
4
16
32
16
16
16
>32
>32
>32
>32
8
>32
>32
8
8
16
8
>32
>32
>32
>32
>32
2
—
—
—
—
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
—
is a mechanism of action.33,34 The results of this assay are also
32
consistent with the thiol depletion assay described previously.
Next, we exploited the presence of a propargyl group in 5e to
independently determine if the compound permeated cells. A
fluorescent azide was synthesized and using a copper-catalyzed
8
>32
32
4
8
8
4
8
8a
8b
9b
9c
16
>32
>32
>32
1
0.25
2
16
>32
9d
Linezolid
Ciprofloxacin
Vancomycin
Meropenem
Tobramycin
60.00075
—
—
Figure 1. Intracellular thiol depletion in S. aureus upon treatment with the 5e (0,
—
50, 100 and 150
lM) measured after 60 min of incubation at 37 °C using
a
MIC was determined using a broth dilution method and are reported in lg/mL.
monobromobimane (mBBr) based fluorescence assay.