Electron-withdrawing substituted benzenesulfonamides against the predominant community-associated methicillin-resistant Staphylococcus aureus strain USA300

Article abstract of DOI:10.1007/s00706-013-0937-3

A small focused chemical library constituted of sulfonamides was synthesized. These compounds were designed to lack the p-aminobenzene moiety typically found in sulfonamide antibiotics. Antimicrobial activities of these synthetic compounds were investigated against global predominant methicillin-resistant Staphylococcus aureus (MRSA) strain USA300 (SF8300) and control strains of Staphylococcus aureus (S. aureus) ATCC 25923 and ATCC 29213 using disk diffusion and microdilution assays. Based on susceptibility results, potent S. aureus and MRSA USA300 growth inhibitors such as N-[3,5- bis(trifluoromethyl)phenyl]-4-bromobenzenesulfonamide with minimum inhibitory concentration (MIC) as low as 5.6 μg/cm³ along with other effective sulfonamides were discovered. Structure-activity correlations revealed that these desamino-benzenesulfonamides required electron-withdrawing substituents to be effective inhibitors of bacterial pathogen growth. In addition, their ability to inhibit growth of S. aureus strains was retained even when bacterial folate synthetic intermediate, p-aminobenzoic acid (PABA), was supplemented, whereas PABA supplementation completely diminished the antibacterial activity of the known sulfa drug tested, sulfamethoxazole. The sulfa-resistant MRSA strain COL also showed great susceptibility to these desamino-benzenesulfonamides. These results imply a unique mechanism of growth inhibition by these potent desamino-benzenesulfonamides, different from the well-known folate pathway target of sulfonamide antibiotics.

Full text of DOI:10.1007/s00706-013-0937-3

Monatsh Chem (2013) 144:461–471
DOI 10.1007/s00706-013-0937-3
ORIGINAL PAPER
Electron-withdrawing substituted benzenesulfonamides against
the predominant community-associated methicillin-resistant
Staphylococcus aureus strain USA300
•
Wanida Phetsang Soraya Chaturongakul
•
Chutima Jiarpinitnun
Received: 8 November 2012 / Accepted: 24 January 2013 / Published online: 19 March 2013
Ó Springer-Verlag Wien 2013
Abstract A small focused chemical library constituted of
sulfonamides was synthesized. These compounds were
designed to lack the p-aminobenzene moiety typically
found in sulfonamide antibiotics. Antimicrobial activities
of these synthetic compounds were investigated against
global predominant methicillin-resistant Staphylococcus
aureus (MRSA) strain USA300 (SF8300) and control
strains of Staphylococcus aureus (S. aureus) ATCC 25923
and ATCC 29213 using disk diffusion and microdilution
assays. Based on susceptibility results, potent S. aureus
and MRSA USA300 growth inhibitors such as N-[3,5-
bis(trifluoromethyl)phenyl]-4-bromobenzenesulfonamide
with minimum inhibitory concentration (MIC) as low as
5.6 lg/cm3 along with other effective sulfonamides were
discovered. Structure–activity correlations revealed that
these desamino-benzenesulfonamides required electron-
withdrawing substituents to be effective inhibitors of
bacterial pathogen growth. In addition, their ability to
inhibit growth of S. aureus strains was retained even when
bacterial folate synthetic intermediate, p-aminobenzoic acid
(PABA), was supplemented, whereas PABA supplementation
completely diminished the antibacterial activity of the
known sulfa drug tested, sulfamethoxazole. The sulfa-
resistant MRSA strain COL also showed great suscepti-
bility to these desamino-benzenesulfonamides. These
results imply a unique mechanism of growth inhibition by
these potent desamino-benzenesulfonamides, different
from the well-known folate pathway target of sulfonamide
antibiotics.
Keywords Antibiotics Á Sulfonamides Á
Drug-resistant Staphylococcus aureus
Introduction
Compounds in the sulfonamide class have been well rec-
ognized for their antibacterial activity [1–3]. Several
antibiotics clinically used for treatment of staphylococcal
infections possess sulfonamide core structures, for exam-
ple, sulfapyridine, sulfadimethoxine, and sulfamethoxazole
(SMZ). Recently, sulfonamide analogs also showed other
diverse pharmacological activities including carbonic
anhydrase inhibition [4, 5], anticancer [4–7], and anti-
inflammatory activities [7]. Nevertheless, much interest has
still been focused on structural modification of sulfon-
amides to achieve highly effective antibacterial agents or to
broaden their spectrum of activity [8–10]. Such structural
modifications are often based on one of the first sulfon-
Electronic supplementary material The online version of this
article (doi:10.1007/s00706-013-0937-3) contains supplementary
material, which is available to authorized users.
W. Phetsang Á C. Jiarpinitnun (&)
Department of Chemistry and Center for Innovation
in Chemistry (PERCH-CIC), Faculty of Science,
Mahidol University, Rama VI Road,
Bangkok 10400, Thailand
amide antibiotics, sulfanilamide, sharing
a common
structure with p-aminobenzenesulfonamide [1, 8, 9]. This
particular arylamine was anticipated to mimic p-amino-
benzoic acid (PABA), the natural substrate of bacterial
dihydropteroate synthase (DHPS) enzyme required for
synthesis of folate, an important intermediate for thymidine
production [11, 12]. Inhibition of DHPS enzyme leads to
e-mail: chutima.jia@mahidol.ac.th
S. Chaturongakul
Department of Microbiology and Center for Emerging Bacterial
Diseases, Faculty of Science, Mahidol University,
Rama VI Road, Bangkok 10400, Thailand
123

462
W. Phetsang et al.
inhibition of DNA and RNA synthesis, resulting in cell death.
Based on computational and mutagenesis studies, the aryl-
amine is involved with the DHPS-catalyzed condensation
reaction with 6-hydroxymethyl-7,8-dihydropterin pyrophos-
phate (DHPP) [12]. Emergence of sulfa-resistance, however,
influences the infrequent use of this particular class of anti-
biotics [13, 14]. Sulfa-resistance could occur due to mutations
in the dhps gene, alteration of DHPS enzymes, or hyperpro-
duction of the DHPS enzyme natural substrate PABA [15]. In
addition, the risk of hypersensitivity reactions has also led to a
decrease in clinical use of sulfonamide antibiotics. The sul-
fonamide antibiotic-induced hypersensitivity reaction is
mediated by a series of complicated metabolic and immuno-
logical events. Previous studies suggested that the aromatic
amine moiety is considered to be responsible for the allergic
reactions [16–18].
Herein, we investigated the potential of sulfonamides
lacking the p-aminobenzene substitution as antibacterial
agents. We synthesized a small focused library, constituted
of sulfonamides that lacked arylamine substitution. Each
compound was tested for its ability to inhibit growth of
typical S. aureus control strains and clinically predominant
MRSA PFGE type USA300 strain (SF8300). Susceptibility
of S. aureus and MRSA SF8300 to the synthetic com-
pounds was first determined via disk diffusion, and the
compounds exhibiting potent antimicrobial activity were
further quantitatively evaluated for their efficiency via
microdilution methods. Based on our structural analysis,
we discovered potent sulfonamides lacking the p-amino-
benzene moiety that could inhibit growth of typical
methicillin-susceptible S. aureus (MSSA) strains and that
of our MRSA strain USA300. More interestingly, the
results revealed the structural requirements for growth
inhibition. In addition, we further tested the susceptibility
of a sulfa-resistant S. aureus strain to the potent sulfon-
amides in our library. These potent sulfonamides showed
strong ability to inhibit the growth of the sulfa-resistant
S. aureus strain, suggesting a mode of action different from
that of typical sulfonamide antibiotics. These results sug-
gest the structural features of sulfonamides crucial for the
development of therapeutics against multidrug-resistant,
specifically methicillin and sulfa drugs, S. aureus.
Recently, the sulfonamides were suggested to interfere
with bacterial pathways other than the typical folate syn-
thesis [19]. Therefore, we envisioned that derivatives
that lacked arylamine could perhaps, nevertheless, still
inhibit bacterial growth. Fluorophenyl-styrene-sulfonamide
(FPSS), for example, was discovered as an inhibitor of
cellular stress and virulence modulator, alternative tran-
scription sigma factor B (SigB), provoking the alternate
functions of sulfonamides [19]. FPSS showed ability in
reducing gene expression from Listeria monocytogenes SigB-
dependent promoters, resulting in a transcription profile sim-
ilar to that of the sigB null mutation strain. This evidence
suggested that the sulfonamide FPSS could inhibit SigB
activity and that a selected group of sulfonamides tar-
get alternative mechanisms leading to bacterial growth
inhibition. Although that study was performed in Listeria
monocytogenes, SigB is conserved across low G ? C content
Gram-positive bacteria, including Staphylococci.
Results and discussion
Chemistry
Synthesis of sulfonamides 1–10, 15–42 was achieved in a
single step via reaction of the corresponding commercially
available sulfonylchloride derivatives and primary amine
counterparts in the presence of base. Sulfonamides 11–14
were obtained by FeCl3-catalyzed bromination reaction of
their parent compounds 7–10 using N-bromosuccinimide
(NBS) as bromine source. Each synthetic compound was
isolated and characterized.
New antimicrobial agents that could strongly inhibit
growth of MRSA strain USA300, as designated on the
basis of pulsed-field gel electrophoresis (PFGE) typing, are
of particular interest as USA300 has emerged as the pre-
dominant community-associated MRSA (CA-MRSA)
strain, causing staphylococcal infectious diseases in com-
munity settings, especially in the USA [20]. Though
typically responsible for skin and soft tissue infections,
USA300 isolates were also reportedly recovered from
invasive cases [21]. USA300 isolates have dramatically
spread and are currently recognized as causes of infections
in many continents globally [22]. More importantly, sev-
eral USA300 isolates showed reduced susceptibility to
vancomycin in addition to occasional resistance to com-
monly used combination therapies for MRSA such as
trimethoprim–sulfamethoxazole [23–25]. The alarming
spread of CA-MRSA strain USA300 and the emergence of
resistance to the currently used antibiotics have greatly
heightened the need for discovery of novel therapeutics.
Biological assays
The synthesized sulfonamides were first tested for their
ability to inhibit growth of S. aureus control strain ATCC
25923 and MRSA PFGE type USA300 strain SF8300 via disk
diffusion method [16]. Compounds that exhibited inhibition
zones were quantitatively evaluated using microdilution sus-
ceptibility testing to determine their minimum inhibitory
concentration (MIC) and minimum bactericidalconcentration
(MBC) values for further analysis [26].
Ampicillin at 10 lg and oxacillin at 1 lg were
employed to determine the susceptibility of the strains
123

Electron-withdrawing substituted benzenesulfonamides
463
towards beta-lactam antibiotics [26]. The results of disk
diffusion tests showed that neither ampicillin nor oxacillin
could inhibit growth of SF8300 (average zone diameter
6 mm) while they completely inhibited growth of the
control strain ATCC 25923 (average zone diameter
26.6 0.41 mm for ampicillin and 19.33 0.03 mm for
oxacillin), confirming that SF8300 is completely resistant
to beta-lactam antibiotics. Quantitatively, the microdilution
results also validated the resistance of SF8300 to ampicillin
and oxacillin, as the MIC values of the two compounds
were above the breakpoint concentrations at [35 and
C8 lg/cm3, respectively (see ESM) [26].
Based on the results of disk diffusion tests, of all 42
compounds synthesized, 10 compounds resulted in com-
plete inhibition zones in both S. aureus control strain
ATCC 25923 and MRSA epidemic strain SF8300
(Table 1). Specifically, these 10 compounds were com-
pounds 3, 4, 6, 10, 13, 14, and 18–21. All of our
compounds that resulted in growth inhibition of
MSSA control strains and MRSA strain SF8300 were
nonheterocyclic sulfonamides. The effectiveness of these
sulfonamides, at 30 lg, appeared to be diverse, as sug-
gested by the resulting clear inhibition zone measurements.
To obtain quantitative results for further analysis, we per-
formed microdilution susceptibility testing to determine
their MIC and MBC values.
A similar trend was also observed with the series of
compounds 11–14, which share the structure of a bromine-
substituted naphthalene ring. Compound 14 with two nitro
moieties substituted at the 2- and 4-positions of the anilino
ring exhibited the strongest growth inhibition, as indicated
by the complete inhibition zone of approximately 16 mm
in diameter for both ATCC 25293 and SF8300 strains and
MIC and MBC values in the range of 11.3–22.6 lg/cm3.
With one fewer nitro substituent, compound 13 exhibited a
slight decrease in potency. Comparable results were obtained
with the electron-withdrawing bromine substituent. The
electron-donating group, however, dramatically decreased
the potency as evidently observed in case of compound 12,
which has –CH3 substituted on the benzene ring. Compound
12 could not inhibit growth of either ATCC 25923 or MRSA
SF8300 strain.
Regarding the structural analysis of the substituents on
the sulfonyl side (R–SO2–), we compared the series of
compounds 11–14, which contain a bromine-substituted
naphthalene ring, with the series of compounds 7–10,
which lack bromine substitution. Among the series of
compounds 7–10, only compound 10 resulted in a complete
inhibition zone. However, compound 10 was clearly less
potent when compared with its similar derivative 14. The
importance of electron-withdrawing substitution was further
emphasized when comparing the activities of compounds 9
and 13. Compounds 9 and 13 are similar in their structures
with the only exception that compound 9 lacks bromine on
the naphthalene ring. This particular difference between these
two structures resulted in remarkable distinction in their
activities. Compound 13 could inhibit growth of both
S. aureus strains to a certain extent while compound 9 could
not inhibit growth of either strain.
The compounds with abilities to inhibit growth of
S. aureus and MRSA in this study interestingly shared
common structure of phenyl-benzenesulfonamide. The
correlation between quantitative and qualitative results
with chemical structures suggested that, though either
benzene ring could be substituted, the choice of substituent
seemed to be a key factor. The compounds that resulted in
growth inhibition of MSSA strain ATCC 25923 and MRSA
strain SF8300 required electron-withdrawing substituents.
Compound 19, structurally containing two trifluoromethyl
moieties substituted at the 3- and 5-positions of the anilino
ring and bromine substituted at the p-position of another
benzene ring, was the most potent compound in growth
inhibition of ATCC 25293 and SF8300 with complete
inhibition zone [20 mm in diameter. The MIC and MBC
of compound 19 were within the range of 5.6–11.2 lg/cm3
(in comparison with MIC breakpoints of 8 and 2 lg/cm3
for methicillin and vancomycin, respectively) [26]. On
replacement of the electron-withdrawing bromine at the p-
position of the benzene ring with another electron-with-
drawing nitro moiety, the activity of compound 20 was still
comparable to 19. On the contrary, compound 18, having
chemical structure similar to 19 and 20 with the only
exception that an electron-donating methylene moiety was
in place of the electron-withdrawing bromine in 19 or nitro
in 20, showed significant decrease in its ability to inhibit
S. aureus and MRSA with MIC elevated to 38.3 lg/cm3.
Such tendency for electron-withdrawing requirement was
observed across the board. In addition, the results implied
that the distance from the electron-withdrawing substituents
to the sulfonamide cores might influence the activity. Even
though compound 22 contained electron-withdrawing bro-
mine substituted on the benzene ring, having one methylene
unit (–CH2) inserted between the benzene ring and sulfonyl
amide nitrogen could influence the inhibition activity, as a
complete inhibition zone was no longer observed. However,
we could not overlook the electron-donating methyl sub-
stituent that is perhaps responsible for its inactivity. In
addition, within our small library, only compounds 23–28
contained one or two methylene units inserted between the
benzene ring and sulfonyl amide core, yet these compounds
were also substituted with strongly electron-donating moi-
eties such as methoxy group, which may also account for the
observed inability for growth inhibition. Further evaluation
on the effects of flexibility and distance of the electron-
withdrawing substituents from the sulfonamide core is cur-
rently ongoing.
123

464
W. Phetsang et al.
Table 1 Antibacterial activities of nonheterocyclic sulfonamides 1–28 determined by disk diffusion and microdilution susceptibility tests
O
O
n
S
N
R'
R
H
Compound
n
R
R’
Average SE inhibition zone (mm)
MIC (lg/cm3)/MBC (lg/cm3)
ATCC 25923
SF8300
ATCC 29213
SF8300
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
1
1
1
2
2
2
H
4-CH3
4-CH3
4-CH3
4-Br
6
6
–
–
2
3,4-diOCH3
4-Br
6
6
–
–
3
7.30 0.16
8.00 0.35
65.2/65.2
65.2/65.2
4
4-Br
9.94 0.33
9.94 0.48
39.1/39.1
39.1/39.1
5
4-Br
4-NO2
6
6
–
–
6
4-Br
2,4-diNO2
4-Br
12.55 0.22a
15.79 0.12
20.1/B80.4
20.1/B80.4
7
Fused-Ph (naphthalene)
Fused-Ph (naphthalene)
Fused-Ph (naphthalene)
Fused-Ph (naphthalene)
4-Br-(naphthalene)
4-Br-(naphthalene)
4-Br-(naphthalene)
4-Br-(naphthalene)
2-OH-4-NO2
2-OH-4-NO2
2-OH-4-NO2
3,5-diCF3
6
6
–
–
8
4-CH3
4-NO2
2,4-diNO2
4-Br
6
6
–
–
9
6
6
–
–
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
13.56 0.08a
6a
15.45 0.14
6a
18.7/B33.4
B18.7/18.7
5.5/88.2
B5.5/B44.1
4-CH3
4-NO2
2,4-diNO2
4-CH3
4-Br
6
6a
6
–
–
7.55 0.27a
10.2/B20.4
20.4/[81.5
11.3/B22.6
15.82 0.09
16.30 0.38
11.3/11.3
6
6
–
–
6
6
–
–
4-NO2
4-CH3
4-Br
6
6
–
–
11.81 0.24a
23.16 0.34a
12.84 0.07a
21.29 0.13a
38.3/38.3
38.3/38.3
3,5-diCF3
B11.2/B11.2
5.6/B11.2
3,5-diCF3
4-NO2
2,4-diNO2
4-CH3
4-CH3
4-Br
19.58 0.34
18.01 0.18
B10.4/10.4
B10.4/B10.4
3,5-diCF3
12.58 0.25
12.43 0.09
23.0/23.0
23.0/23.0
2-Br
6
6
6
6
6
6
6
6
6
6
6
6
6
6
–
–
–
–
–
–
–
–
–
–
3,4-diOCH3
3,4-diOCH3
3,4-diOCH3
3,4-diOCH3
3,4-diOCH3
3,4-diOCH3
4-NO2
4-CH3
4-Br
–
–
–
4-NO2
30 lg of test compound in dimethyl sulfoxide (DMSO) was used in disk diffusion tests. At least three independent experiments were performed,
and the average diameter of complete inhibition zones is reported with the standard error (SE). The Whatman disk has diameter of 6 mm;
therefore, an average inhibition zone of 6 mm indicates inability to inhibit growth
Each compound was two-fold diluted based on its stock concentration; the reported value is converted to lg/cm3
a
The value indicates the diameter of the visually complete inhibition zone. However, the compound also exhibited an incomplete zone of
inhibition
Among the heterocyclic compounds in our library
(Table 2), only the series of p-nitro-substituted benzene
rings (compounds 35–37) showed fair abilities to inhibit
growth of S. aureus. Interestingly, the clinically used
sulfonamide antibiotics such as sulfamethoxazole (SMZ),
sulfadimethoxine, and other recently reported antibacte-
rial sulfonamides also share similar structure of
heterocyclic sulfonamide [10, 27, 28]. Therefore, we
evaluated whether SMZ alone could inhibit S. aureus
control strains and MRSA strain SF8300. The results via
disk diffusion assay showed that SMZ (30 lg) could not
inhibit growth of all tested strains (see ESM). SMZ,
however, is generally used in combination with trimeth-
oprim (TMP); the SMZ–TMP combination is considered
as effective treatment for CA-MRSA infections, although
several reports indicated increasing resistance among
USA300 isolates to the combination [21]. Our results
validated the effectiveness of this combination (SMZ–
123

Electron-withdrawing substituted benzenesulfonamides
465
Table 2 Antibacterial activities of heterocyclic sulfonamides 29–42 determined by disk diffusion and microdilution susceptibility tests
O
O
n
X
S
N
Y
Z
R'
H
Compound
n
R
X
R’
Average SE inhibition zone (mm)
MIC (lg/cm3)/MBC (lg/cm3)
Y
Z
ATCC 25923
SF8300
ATCC 29213
SF8300
29
30
31
32
33
34
35
36
37
38
39
40
1
1
1
1
1
1
1
1
1
1
1
1
N
CH
N
CH
CH
N
4-CH3
4-CH3
4-CH3
4-Br
6
6
–
–
–
–
–
–
–
CH
CH
N
6
6
–
CH
CH
N
6
6
–
CH
CH
N
6
6
–
CH
CH
N
4-Br
6
6
–
CH
CH
N
4-Br
6
6
–
CH
CH
N
4-NO2
4-NO2
4-NO2
6a
6a
6a
6
6a
6a
6a
6
29.3/B58.7
B117.3/C117.3
CH
CH
N
N/A
N/A
CH
CH
N
B29.3/B29.3
B58.7/B58.7
CH
CH
N
2,4-diNO2
2,4-diNO2
2,4-diNO2
–
–
–
–
–
–
CH
CH
6
6
CH
6
6
O
O
S
R
R'
Compound
R
R’
Average SE inhibition zone (mm)
MIC (lg/cm3)/MBC (lg/cm3)
ATCC 25923
6
SF8300
6
ATCC 29213
–
SF8300
41
4-Br
–
N
N
N
N
42
4-NO2
6
6
–
–
30 lg of test compound in DMSO was used in disk diffusion tests. At least three independent experiments were performed, and the average
diameter of complete inhibition zones is reported with the standard error. The Whatman disk has a diameter of 6 mm; therefore, an average
inhibition zone of 6 mm indicates inability to inhibit growth
Each compound was two-fold diluted based on its stock concentration; the reported value is converted to lg/cm3
a
The value indicates the diameter of the visually complete inhibition zone. However, the compound also exhibited an incomplete zone of
inhibition. N/A not applicable as the compound was not soluble in MH broth
TMP) against S. aureus control strains, while also sug-
gesting resistance in MRSA strain SF8300.
pathogens via another mechanism rather than that known
for SMZ and other typical sulfa-drugs. The amine moiety
substituted on the benzenesulfonamide plays a crucial role
in mimicking the DHPS-mediated catalysis of PABA,
leading to inhibition of folate production. Hence, our
potent benzenesulfonamides, which lack the crucial amine
The resistance of MRSA strain SF8300 to SMZ and
the SMZ–TMP combination implies that our synthetic
desamino-benzenesulfonamides that resulted in growth
inhibition of SF8300 could possibly inhibit growth of the
123

466
W. Phetsang et al.
Table 3 Susceptibility of MSSA ATCC 25923 to potent desamino-benzenesulfonamides and sulfamethoxazole determined by disk diffusion
Concentration of
PABA supplemented (lg/cm3)
Inhibition zone (mm)
SMZa
4
6
10
14
18
19
20
21
0
21.34
11.26
10.45
10.51
11.42
9.48
12.57
13.79
13.48
11.93
13.26
14.19
12.84
13.05
12.51
12.53
12.96
13.27
10.68
12.76
12.56
13.38
16.18
18.02
18.02
17.28
17.78
18.16
16.94
16.92
14.41
13.95
15.38
14.16
12.93
13.79
13.90
13.71
21.11
21.39
21.42
20.57
20.99
20.9
23.07
23.31
23.06
23.44
22.64
24.15
24.38
22.67
13.07
12.94
13.16
13.27
12.75
13.07
12.27
12.50
1
6
6
6
6
6
6
6
5
10
20
50
100
200
10.81
11.24
11.42
21.76
21.14
30 lg of test compound (unless otherwise noted) diluted in DMSO was used in disk diffusion tests. At least three independent experiments were
performed, and the average diameter of complete inhibition zones is reported. The Whatman disk has diameter of 6 mm; therefore, an average
inhibition zone of 6 mm indicates inability to inhibit growth
a
250 lg sulfamethoxazole was used
moiety, understandably should not interfere with DHPS
catalysis of PABA. Susceptibility experiments in which the
growth medium was supplemented with PABA [29] were
performed. For the sulfonamide antibiotics that inhibit
growth by mimicking PABA, the elevated concentration of
PABA supplemented should clearly affect its ability to
inhibit bacterial growth. The susceptibility of the control
strain ATCC 25923 to SMZ and our potent desamino-
benzenesulfonamides was first determined via disk diffu-
sion followed by microdilution assays. The results shown
in Tables 3 and 4 indicate that the antibacterial activities of
these potent benzenesulfonamides from our library were
not affected even with high concentration of supplemented
PABA (up to 200 lg/cm3). The MIC and MBC values
when the growth medium was supplemented with PABA
were clearly in the same range as without PABA supple-
mented. However, SMZ only exhibited ability to inhibit
growth without PABA supplementation. Addition of only
5 lg/cm3 PABA to the growth medium could completely
diminish the antibacterial activity of SMZ. These results
evidently suggest that these desamino-benzenesulfonamides
inhibit growth of MSSA and MRSA via a mechanism dif-
ferent from the classical interference with the folate pathway.
We further investigated the susceptibility of another
known sulfa-resistant S. aureus strain, COL, to our iden-
tified potent sulfonamides. The clinical isolate S. aureus
strain COL was considered closely related to the USA300
strain [30, 31]. The core genome of USA300 and strain
COL only differ by 678 single-nucleotide polymorphisms
(SNPs), while the USA300 genome is different from
USA200 strains by approximately 44,964 SNPs [30].
Nonetheless, the mobile genetic elements that these two
strains carry are different. COL carries SCCmecI, whereas
USA300 carries SCCmecIV. The resistance of strain COL
to SMZ and SMZ–TMP was first verified. Similar to SF8300
strain, when strain COL was treated with 250 lg SMZ, no
inhibition zone was observed (Table 5). However, SMZ–
TMP treatment resulted in an inhibition zone, albeit visually
incomplete. Therefore, based on the standard breakpoint, it is
considered a sulfa-resistant strain. A total of nine compounds
from our focused library were selected to test against the
methicillin- and sulfa-resistant strain COL. These compounds
exhibited growth inhibition of MRSA strain COL as well as
the control strain ATCC 25923 as shown by complete inhi-
bition zones on disk diffusion assay. These results also
support the hypothesis that these sulfonamides may inhibit
growth of S. aureus normal strain and MRSA strains via
mechanisms different from the usual inhibition of folate
pathway proposed in other sulfonamide antibiotics. The
bacterial growth inhibition mechanisms of these sulfon-
amides are currently under investigation.
Table 4 Antibacterial activities of potent desamino-benzenesulfon-
amides and sulfamethoxazole determined by microdilution
susceptibility tests
Compound MIC/MBC (lg/cm3)
ATCC 29213 without ATCC 29213 with 5 (lg/cm3)
PABA supplemented
PABA supplemented
SMZ
4
100
[1.6 mg/cm3 a
78.2/78.2
39.1/39.1
20.1/B80.4
18.7/B37.4
10.2/B20.4
11.3/11.3
38.3/38.3
B11.2/B11.2
B10.4/10.4
23.0/23.0
6
20.1/40.2
10
13
14
18
19
20
21
18.7/B37.4
20.4/40.7
11.3/B22.6
38.3/38.3
11.2/11.2
10.4/10.4
23.0/23.0
Each compound was twofold diluted based on its stock concentra-
tion. The reported value is converted to lg/cm3
a
MBC of SMZ could not be determined
123

Electron-withdrawing substituted benzenesulfonamides
467
addition, investigation of the susceptibility of MSSA with
PABA supplementation was performed, and these potent
desamino-benzenesulfonamides could inhibit growth of
S. aureus strains even with high concentration of PABA
supplemented. Clearly, addition of PABA did not perturb
the antibacterial activity of these sulfonamides. The
mechanisms underlying the growth inhibition via these
sulfonamides are currently being investigated.
Table 5 Susceptibility of sulfa-resistant MRSA to potent benzene-
sulfonamides and known antibiotics determined by disk diffusion
Compound
Average SE inhibition zone (mm)
SF8300
COL
Known antibioticsa
OXA (1 lg)
SMZ
6 (R)
6 (R)
6c (R)
6c (R)
12.54 0.59 (I)
6 (R)
SMZ (250 lg)
SMZ–TMPb
6 (R)
6d (R)
Potent benzenesulfonamides from library
Experimental
4
9.94 0.48
10.23 0.13
13.30 0.30
11.77 0.21
10.43 0.62
14.58 0.34
15.13 1.03
23.21 0.08
22.08 0.59
13.41 0.41
6
15.79 0.12
15.45 0.14
7.55 0.27c
16.30 0.38
12.84 0.07c
21.29 0.13c
18.01 0.18
12.43 0.09
All materials, unless otherwise noted, were obtained from
commercial suppliers and used without further purification.
Nonaqueous reactions were conducted under inert atmo-
sphere of nitrogen. Reactions were monitored by thin-layer
chromatography (TLC). Analytical TLC was performed
on Merck TLC alumina sheet precoated with 0.25 mm
silica gel 60 F254, and compounds were visualized using
an ultraviolet (UV) lamp and appropriate TLC stains. Col-
umn chromatography was performed using silica gel 60
(0.063–0.200 mm), 70–230 mesh ASTM. Merck silica gel 60
PF254 was used for preparative thin-layer chromatography.
1H nuclear magnetic resonance (NMR) spectra were
obtained using either a Bruker DPX-300 (300 MHz), or a
Bruker Avance-300, or a Bruker Avance-500 spectrometer.
13C NMR spectra were obtained using a Bruker Avance-
300 (75 MHz) or a Bruker Avance-500 spectrometer
(125 MHz) spectrometer. Chemical shifts (d) are reported
relative to residual solvent signals in parts per million
(CDCl3: 1H: 7.27 ppm, 13C: 77.23 ppm; CD3OD: 1H:
10
13
14
18
19
20
21
30 lg of test compound (unless otherwise noted) diluted in DMSO
was used in disk diffusion tests. At least three independent experi-
ments were performed, and the average diameter of complete
inhibition zones is reported with the standard error. R indicates
resistance while I indicates intermediate resistance to the antibiotics
a
The amount of known antibiotic used was based on their break-
points and is indicated in parenthesis
b
25 lg of sulfamethoxazole (SMZ) and trimethoprim (TMP) (1:20)
mixture was used
c
The value indicates the diameter of the visually complete inhibi-
tion zone. However, the compound also exhibited an incomplete zone
of inhibition
3.31 ppm, 13C: 49.15 ppm; DMSO-d6: H: 2.50 ppm, 13C:
1
39.51 ppm). H NMR data were assumed to be first order
1
Conclusions
with apparent doublets and triplets reported as d and t,
respectively. Multiplets are reported as m, and resonances
that appeared broad are designated as br. High-resolution
mass spectra (HRMS) were obtained on a Bruker micro
TOF spectrometer in electrospray ionization (ESI) and
atmospheric-pressure chemical ionization (APCI) mode.
Infrared spectra were recorded with a PerkinElmer 683 GX
FTIR system spectrometer.
We synthesized a small focused chemical library contain-
ing sulfonamides bearing various substituents but not the
4-aminobenzene typically found in known sulfonamide
antibiotics. The results from susceptibility tests with con-
trol S. aureus and the global epidemic methicillin-resistant
MRSA PFGE type USA300 strains revealed that the syn-
thesized benzenesulfonamides substituted with electron-
withdrawing moieties showed potent abilities in inhibiting
pathogen growth. In addition, we compared the efficacies
of our potent sulfonamides with the clinically used anti-
biotic SMZ as well as its combination therapy with TMP
(SMZ–TMP). We found that neither SMZ nor SMZ–TMP
could completely inhibit growth of MRSA strains, SF8300
and COL, implying that these two MRSA strains are sulfa-
resistant. However, the potent sulfonamides from our
library were able to inhibit growth of these sulfa-resistant
strains effectively, suggesting that these sulfonamides may
affect pathways other than interfering with folate synthesis,
typically known as a target for sulfonamide antibiotics. In
The detailed syntheses and characterization (via nuclear
magnetic spectroscopy and mass spectroscopy) of all
compounds are described in the ESM. Following is the
detailed syntheses and characterization of the sulfonamides
not yet described in literature.
N-(Naphthalene-1-yl)-2,4-dinitrobenzenesulfonamide
(10, C16H11N3O6S)
Naphthalene-1-amine (2 mmol) and 1 g anhydrous NaHCO3
were stirred neat at room temperature. The reaction mixture
was kept dried under vacuum. To the resulting fine powder
was added 2,4-dinitrobenzenesulfonyl chloride (2 mmol) at
123

468
W. Phetsang et al.
room temperature without use of any solvent. The reaction
mixture was stirred and monitored periodically by TLC. Upon
completion, the reaction mixture was quenched by addition of
water, followed by extraction three times with CH2Cl2. The
combined organic layers were washed with saturated NaCl
solution and dried over Na2SO4. The yellow solid product was
obtained in 50 % yield. 1H NMR (300 MHz, CDCl3,
CD3OD): d = 8.64 (d, J = 2.02 Hz, 1H), 8.25 (dd,
J = 8.66, 2.14 Hz, 1H), 8.06 (d, J = 7.92 Hz, 1H), 7.87–
7.81 (m, 3H), 7.50–7.41 (m, 4H) ppm; 13C NMR (75 MHz,
CDCl3, CD3OD): d = 149.56, 147.89, 138.19, 134.16,
132.65, 130.06, 129.91, 128.39, 128.12, 126.70, 126.29,
126.14, 125.14, 125.05, 121.67, 119.89 ppm; IR (KBr):
1H), 8.03 (d, J = 8.35 Hz, 1H), 7.94 (d, J = 8.85 Hz, 2H),
7.82 (d, J = 8.02 Hz, 1H), 7.68 (t, J = 7.94 Hz, 1H), 7.53
(t, J = 8.19 Hz, 1H), 7.08 (d, J = 8.03 Hz, 1H) ppm; 13C
NMR (75 MHz, DMSO-d6): d = 149.76, 145.13, 131.91,
131.73, 130.79, 129.74, 128.39, 128.29, 127.34, 126.72,
ꢀ
124.63, 124.58, 123.79, 120.69 ppm; IR (KBr): m = 3,331,
3,102, 1,938, 1,891, 1,605, 1,552, 1,349, 1,178, 828, 764,
615 cm-1
;
APCI–MS: calc. for C16H12BrN2O4S
[M ? H]? 406.9701, found 406.9622.
N-(4-Bromonaphthalen-1-yl)-2,4-dinitrobenzenesulfon-
amide (14, C16H10BrN3O6S)
Based on general procedure I, compound 14 was synthe-
sized from compound 10. The yellow solid product was
1
obtained in 47 % yield. H NMR (300 MHz, DMSO-d6):
ꢀ
m = 3,328, 3,096, 3,075, 1,945, 1,607, 1,552, 1,538, 1,346,
1,170, 901, 834, 801, 749 cm-1; ESI–MS: calc. for
d = 8.87 (d, J = 2.26 Hz, 1H), 8.48 (dd, J = 8.71,
2.32 Hz, 1H), 8.14–8.02 (m, 3H), 7.85 (d, J = 8.01 Hz,
1H), 7.68 (dt, J = 7.39, 1.06 Hz, 1H), 7.57 (dt, J = 7.65,
1.05 Hz, 1H), 7.21 (d, J = 8.03 Hz, 1H) ppm; 13C NMR
(75 MHz, DMSO-d6): d = 149.92, 147.32, 136.75, 131.92,
131.73, 131.15, 129.76, 128.30, 127.53, 127.06, 126.74,
C16H11N3NaO6S [M ? Na]? 396.0266, found 396.0313.
General procedure I for synthesis of sulfonamides
11, 13, and 14
To a solution of NBS (1 mmol) in CH2Cl2 a catalytic
amount of FeCl3 (0.05 mmol) was added, followed by
addition of sulfonamide (1 mmol) in CH2Cl2 under nitro-
gen atmosphere. The reaction was stirred overnight at room
temperature. Upon completion the reaction was quenched
by addition of water. The quenched reaction was extracted
with CH2Cl2 (three times). The organic phase was washed
with saturated NaCl solution and dried over Na2SO4. The
solvent was removed under reduced pressure, affording the
crude product. Recrystallization with appropriate solvents
(typically methanol and dichloromethane) yielded pure
product.
ꢀ
125.42, 123.56, 121.18, 120.12 ppm; IR (KBr): m = 3,331,
3,103, 1,938, 1,605, 1,552, 1,542, 1,349, 1,179, 828,
616 cm-1
;
ESI–MS: calc. for C16H10BrN3NaO6S
[M ? Na]? 473.9371, found 473.9372.
General procedure II for synthesis of sulfonamides
16–21, 38, and 39
To a solution of amine derivative (0.6 mmol) and
triethylamine (1.2 mmol) in CH2Cl2 was added benzene-
sulfonyl chloride derivative (0.5 mmol) at 4 °C. The
mixture was stirred at room temperature overnight. Sat-
urated NH4Cl solution was added to quench the reaction,
and the reaction mixture was subsequently extracted three
times with CH2Cl2. The combined organic layers were
dried over Na2SO4. The solvent was removed under
reduced pressure, affording the crude product, which was
purified by column chromatography to yield sulfonamide
derivatives.
4-Bromo-N-(4-bromonaphthalen-1-yl)benzenesulfonamide
(11, C16H11Br2NO2S)
Compound 11 was synthesized from compound 7 based on
general procedure I. The clear solid product was obtained
1
in 87 % yield. H NMR (300 MHz, CDCl3): d = 8.25 (d,
J = 8.41 Hz, 1H), 7.84 (d, J = 8.40 Hz, 1H), 7.72 (d,
J = 8.01 Hz, 1H), 7.64–7.60 (m, 3H), 7.56–7.49 (m, 3H),
7.26 (t, J = 8.02 Hz, 1H), 7.03 (s, 1H) ppm; 13C NMR
(75 MHz, CDCl3): d = 138.05, 132.57, 132.35, 130.77,
130.26, 129.44, 128.79, 128.23, 127.95, 127.89, 127.66,
4-Bromo-N-(2-hydroxy-4-nitrophenyl)benzenesulfonamide
(16, C12H9BrN2O5S)
Compound 16 was synthesized from 4-bromobenzenesulfo-
nyl chloride and corresponding 2-amino-5-nitrophenol
based on general procedure II. The yellow solid product
ꢀ
123.82, 122.27, 121.94 ppm; IR (KBr): m = 3,257, 3,096,
1,918, 1,861, 1,595, 1,328, 1,164, 825, 760, 602 cm-1
;
ESI–MS: calc. for C16H11Br2NNaO2S [M ? Na]?
1
was obtained in 88 % yield. H NMR (300 MHz, CDCl3):
463.8754, found 463.8794.
d = 8.02 (dd, J = 9.00, 2.43 Hz, 1H), 7.84–7.75 (m, 5H),
6.76 (d, J = 8.95 Hz, 1H) ppm; 13C NMR (75 MHz,
CDCl3): d = 145.90, 134.44, 133.91, 133.02, 130.68,
129.86, 124.53, 124.49, 119.55, 115.06 ppm; IR (KBr):
N-(4-Bromonaphthalen-1-yl)-4-nitrobenzenesulfonamide
(13, C16H11BrN2O4S)
Compound 13 was synthesized from compound 9 based on
general procedure I. The clear solid product was obtained
in 76 % yield. 1H NMR (300 MHz, DMSO-d6): d = 10.75
(s, 1H), 8.35 (d, J = 8.84 Hz, 2H), 8.12 (d, J = 8.41 Hz,
ꢀ
m = 3,479, 3,378, 3,087, 1,956, 1,595, 1,510, 1,344, 1,326,
1,195, 1,165, 826, 810, 557 cm-1; ESI–MS: calc. for
C12H9BrN2NaO5S [M ? Na]? 394.9313, found 394.9300.
123

Electron-withdrawing substituted benzenesulfonamides
469
N-(2-Hydroxy-4-nitrophenyl)-4-nitrobenzenesulfonamide
(17, C12H9N3O7S)
CD3OD): d = 150.52, 144.33, 137.99, 133.04 (q,
J = 0.45 Hz, 2C), 128.42, 124.62, 122.63 (q, J = 3.62
Hz, 2C), 119.95 (m, 2C), 118.66 (m, 1C) ppm; 19F NMR
(470 MHz, CDCl3): d = -63.18 ppm; IR (KBr): ꢀm =
3,270, 3,109, 1,948, 1,608, 1,526, 1,380, 1,349, 1,281,
1,171, 898, 837 cm-1; ESI–MS: calc. for C14H7F6N2O4S
[M - H]- 413.0031, found 413.0029.
Compound 17 was synthesized from 4-nitrobenzenesulfo-
nyl chloride and corresponding 2-amino-5-nitrophenol
based on general procedure II. The light-brown solid
1
product was obtained in 98 % yield. H NMR (500 MHz,
CDCl3, CD3OD): d = 8.39 (d, J = 8.87 Hz, 2H), 8.12 (d,
J = 8.84 Hz, 2H), 7.93 (dd, J = 9.00, 2.55 Hz, 1H), 7.80
(d, J = 2.54 Hz, 1H), 6.70 (d, J = 9.03 Hz, 1H) ppm; 13C
NMR (125 MHz, CDCl3, CD3OD): d = 151.24, 146.44,
140.33, 137.22, 133.61, 129.90, 124.71, 124.50, 119.22,
N-[3,5-Bis(trifluoromethyl)phenyl]-2,4-dinitrobenzenesul-
fonamide (21, C14H7F6N3O6S)
Compound21 was synthesized from 2,4-dinitrobenzenesulfo-
nyl chloride and corresponding 3,5-bis(trifluoromethyl)
aniline based on general procedure II. The yellow solid
ꢀ
114.88 ppm; IR (KBr): m = 3,469, 3,370, 3,114, 1,952,
1,596, 1,510, 1,351, 1,328, 1,198, 1,166, 863, 825,
810 cm-1; ESI–MS: calc. for C12H9N3NaO7S [M ? Na]?
362.0059, found 362.0050.
1
product was obtained in 21 % yield. H NMR (500 MHz,
CDCl3, CD3OD): d = 8.61 (d, J = 2.21 Hz, 1H), 8.47 (dd,
J = 8.63, 2.23 Hz, 1H), 8.25 (d, J = 8.64 Hz, 1H), 7.69 (s,
2H), 7.61 (s, 1H) ppm; 13C NMR (125 MHz, CDCl3,
CD3OD): d = 150.07, 148.30, 138.13, 137.80, 132.99,
132.86 (q, J = 0.27 Hz, 2C), 126.70, 122.65 (q,
J = 2.17 Hz, 2C), 120.87, 120.50, 118.64 ppm; 19F NMR
N-[3,5-Bis(trifluoromethyl)phenyl]-4-methylbenzenesulfon-
amide (18, C15H11F6NO2S)
Compound 18 was synthesized from 4-methylbenzenesulfonyl
chloride and corresponding 3,5-bis(trifluoromethyl)aniline
based on general procedure II. The clear solid product was
obtained in 40 % yield. 1H NMR (300 MHz, CDCl3):
d = 7.89 (s, 1H), 7.78 (d, J = 8.34 Hz, 2H), 7.57 (s, 3H),
7.32 (d, J = 8.07 Hz, 2H), 2.42 (s, 3H) ppm; 13C NMR
(75 MHz, CDCl3): d = 145.09, 138.46, 135.07, 132.79 (q,
J = 0.45 Hz, 2C), 130.11, 127.28, 122.74 (q, J = 3.62 Hz,
2C), 119.77 (m, 2C), 118.11 (m, 1C), 21.55 ppm; 19F NMR
(470 MHz, CDCl3): d = -63.12 ppm; IR (KBr): ꢀm = 3,248,
3,100, 2,929, 2,849, 1,919, 1,598, 1,380, 1,284, 1,173, 885,
811 cm-1; ESI–MS: calc. for C15H11F6NNaO2S [M ? Na]?
406.0312, found 406.0323.
ꢀ
(470 MHz, CDCl3): d = -63.17 ppm; IR (KBr): m = 3,325,
3,113, 1,954, 1,608, 1,549, 1,380, 1,352, 1,280, 1,179, 906,
835 cm-1; ESI–MS: calc. for C14H6F6N3O6S [M - H]-
457.9881, found 457.9898.
2,4-Dinitro-N-(pyridin-2-ylmethyl)benzenesulfonamide
(38, C12H10N4O6S)
Compound38 was synthesized from 2,4-dinitrobenzenesulfo-
nyl chloride and corresponding pyridine-2-ylmethanamine
based on general procedure II. The yellow solid product
1
was obtained in 64 % yield. H NMR (300 MHz, CDCl3,
CD3OD): d = 9.17 (d, J = 2.64 Hz, 1H), 8.63 (d,
J = 4.80 Hz, 1H), 8.28 (dd, J = 9.50, 2.67 Hz, 1H), 7.79
(dt, J = 7.71, 1.71 Hz, 1H), 7.38 (d, J = 7.79 Hz, 1H), 7.34
(dd, J = 7.36, 5.24 Hz, 1H), 6.99 (d, J = 9.50 Hz, 1H), 4.78
(s, 2H) ppm; 13C NMR (75 MHz, CDCl3, CD3OD):
d = 155.01, 149.69, 148.20, 137.79, 136.57, 131.12,
130.58, 124.41, 123.44, 121.94, 114.85, 48.29 ppm; IR
N-[3,5-Bis(trifluoromethyl)phenyl]-4-bromobenzenesulfon-
amide (19, C14H8BrF6NO2S)
Compound 19 was synthesized from 4-bromobenzenesulfo-
nyl chloride and corresponding 3,5-bis(trifluoromethyl)
aniline based ongeneral procedure II. The white solid product
1
was obtained in 24 % yield. H NMR (300 MHz, CDCl3):
ꢀ
d = 7.74 (d, J = 8.65 Hz, 2H), 7.67 (d, J = 8.73 Hz, 2H),
7.63 (s, 1H), 7.57 (s, 2H) ppm; 13C NMR (75 MHz, CDCl3):
d = 137.94, 137.17, 133.05 (q, J = 0.45 Hz, 2C), 132.87,
129.24, 128.65, 122.65 (q, J = 3.62 Hz, 2C), 120.00 (m, 2C),
118.67 (m, 1C) ppm; 19F NMR (470 MHz, CDCl3):
(KBr): m = 3,298, 3,113, 2,920, 2,851, 1,819, 1,619, 1,583,
1,535, 1,377, 1,323, 1,145, 817, 776 cm-1; ESI–MS: calc. for
C12H11N4O6S [M ? H]? 339.0399, found 339.0466.
2,4-Dinitro-N-(pyridin-3-ylmethyl)benzenesulfonamide
(39, C12H10N4O6S)
ꢀ
d = -63.10 ppm; IR (KBr): m = 3,264, 3,087, 1,911,
Compound 39 was synthesized from 2,4-dinitro-
benzenesulfonyl chloride and corresponding pyridine-3-
ylmethanamine based on general procedure II. The yellow
solid product was obtained in 25 % yield. 1H NMR
(300 MHz, CDCl3, CD3OD): d = 9.16 (d, J = 2.66 Hz,
1H), 8.63 (d, J = 1.53 Hz, 1H), 8.58 (dd, J = 4.81,
1.29 Hz, 1H), 8.26 (dd, J = 9.45, 2.67 Hz, 1H), 7.75 (d,
J = 7.90 Hz, 1H), 7.40 (dd, J = 7.87, 4.90 Hz, 1H), 6.93
(d, J = 9.49 Hz, 1H), 4.73 (s, 2H) ppm; 13C NMR
(75 MHz, CDCl3, CD3OD): d = 149.24, 148.26, 147.69,
1,577, 1,384, 1,281, 1,155, 883, 818, 550 cm-1
.
N-[3,5-Bis(trifluoromethyl)phenyl]-4-nitrobenzenesulfon-
amide (20, C14H8F6N2O4S)
Compound 20 was synthesized from 4-nitrobenzenesulfo-
nyl chloride and 3,5-bis(trifluoromethyl)aniline based on
general procedure II. The clear solid product was obtained
in 25 % yield. 1H NMR (300 MHz, CDCl3, CD3OD):
d = 8.35 (d, J = 8.92 Hz, 2H), 8.05 (d, J = 8.88 Hz, 2H),
7.62 (s, 1H), 7.60 (s, 2H) ppm; 13C NMR (75 MHz, CDCl3,
123

470
W. Phetsang et al.
136.68, 135.07, 131.81, 130.95, 130.38, 124.11, 124.04,
ꢀ
agar was prepared and poured into Petri dishes to
approximately 4 mm thickness. After solidification, test
strains were spread on the agar surface by using sterile
swap. Inoculum was approximately 108 CFU/cm3. The
inoculated plate was allowed to stand at room temperature
for 5–10 min before disk application. Subsequently, small
filter paper disks (6 mm Whatman) were placed on the
agar, and a solution of test compound (30 lg, unless
otherwise noted) diluted in dimethyl sulfoxide (DMSO)
was transferred onto each paper disk. After 18 h of incu-
bation at 37 °C, the diameters of inhibition zones were
measured in millimeters using sliding callipers. The inhi-
bition zones were classified as complete and incomplete.
The complete zone margin was identified as the area where
no obvious visible growth was observed by the naked eye
[26]. Incomplete zone of inhibition was noted. The
experiments were performed at least three times. The
standard error is reported.
114.03, 44.80 ppm; IR (KBr): m = 3,363, 3,091, 2,951,
1,924, 1,618, 1,588, 1,523, 1,335, 1,319, 1,150, 823,
789 cm-1
.
Microbiology
Methicillin-resistant Staphylococcus aureus (MRSA)
strains SF8300 and COL were kindly provided by Dr. Binh
Diep, University of California at San Francisco. S. aureus
ATCC 29213 and ATCC 25923 (as control strains) were
received as gifts from Cornell University Food Safety Lab.
Overnight bacterial cultures were prepared in Tryptic soy
broth (TSB), and susceptibility tests (both disk diffusion and
microdilution assays) were performed using Mu¨ller–Hinton
broth (MHB) supplemented with Bacto agar. All media were
purchased from BD (Sparks, MD). Antibiotics (i.e., ampicil-
lin, oxacillin, sulfamethoxazole, and trimethoprim) werefrom
Sigma-Aldrich. Trimethoprim–sulfamethoxazole (SXT) disk
was purchased from Oxoid (Hampshire, UK).
Microdilution susceptibility test
Each member of the small chemical library was first
tested for antimicrobial activity, based on a protocol rec-
ommended by the Clinical and Laboratory Standards
Institute (CLSI) [26] using the disk diffusion method. All
compounds that exhibited ability to inhibit bacterial growth
were then further characterized for their antibacterial
potency by determining their minimum inhibitory con-
centration (MIC) and minimum bactericidal concentration
(MBC).
The microdilution susceptibility test was performed based
on the previously reported protocol [26]. Stock solutions of
synthetic compounds that showed growth inhibition in the
disk diffusion tests were prepared in DMSO. The stock
solutions were then twofold diluted in sterilized Mu¨ller–
Hinton broth to obtain the tested concentrations. A 180-lL
aliquot of each compound at different dilutions was
transferred to sterilized 96-well plates. To each aliquot of
test compounds, 20 lL of test bacterial suspension was
added to final inoculum density of approximately 106 CFU/
cm3. The assay plates were incubated at 37 °C for 20 h.
After incubation, the optical density of each well was
measured using Wallac Victor2 V1420 Multilable HTS
Counter (PerkinElmer, USA).
Bacterial strains and inoculum preparation
The synthetic sulfonamides were tested against epidemic
MRSA PFGE type USA300 strain SF8300. Reference
strains of S. aureus ATCC 29213 and ATCC 25923 were
used as control strains in order to monitor the antimicrobial
microdilution test and disk diffusion susceptibility test,
respectively.
Based on the results from microdilution test, the solution
of microtiter well that appeared clear, or qualitatively
indicated by basal optical density, was spread on TSA agar
plate and subsequently incubated at 37 °C for 18–20 h. The
lowest concentration that showed no colony was recorded
as the minimal bactericidal concentration (MBC). The MIC
of each compound against tested strains and corresponding
MBC were determined and reported as ranges.
A single colony of each strain was picked and trans-
ferred into TSB (5 cm3) in a sterilized 15-cm3 test tube,
which was then capped and incubated at 37 °C for
15–18 h. The overnight culture was subsequently diluted in
phosphate buffered saline (PBS) to yield the appropriate
inoculum density of approximately 108 colony-forming
units CFU/cm3 for disk diffusion susceptibility test and
approximately 106 CFU/cm3 for microdilution assay. The
inoculum was used within 15 min after adjusting to its
appropriate density.
Susceptibility determination with p-aminobenzoic acid
(PABA) supplemented in the growth medium
(i) Disk diffusion method
To evaluate whether our synthetic sulfonamides inhibit
DHPS catalysis of PABA, disk diffusion assays were per-
formed with PABA supplemented in the medium. PABA
Disk diffusion method
The disk diffusion was performed based on previously
reported procedure [26]. Briefly, sterilized Mu¨ller–Hinton
¨
was added to sterilized Muller–Hinton agar to achieve a
final concentration ranging from 0 to 200 lg/cm3 and
123

Electron-withdrawing substituted benzenesulfonamides
471
9. Wang XL, Wan K, Zhou CH (2010) Eur J Med Chem 45:4631
10. Argyropoulou I, Geronikaki A, Vicini P, Zani F (2009) Arkivoc
6:89
11. Zander J, Besier S, Ackermann H, Wichelhaus TA (2010) Anti-
microb Agents Chemother 54:1226
subsequently poured into Petri dishes to approximately
4 mm thickness. The prepared agar surface was allowed to
solidify and subsequently used to determine the suscepti-
bility of the test strains via disk diffusion as described
previously [26].
12. Yun M-K, Wu Y, Li Z, Zhao Y, Waddell MB, Ferreira AM, Lee
RE, Bashford D, White SW (2012) Science 335:1110
¨
13. Skold O (2000) Drug Resist Updates 3:155
14. Huovinen P (2001) Clin Infect Dis 32:1608
(ii) Microdilution susceptibility test
Sterilized Mu¨ller–Hinton broth was prepared and supple-
mented with 5 lg/cm3 PABA. The PABA-supplemented
broth was used to dilute the stock solution of the tested
compounds to test their ability to inhibit growth of tested
strain via microdilution susceptibility test as described in
detail previously [26].
15. Al-Masaudi SB, Day MJ, Russell AD (1991) J Appl Bacteriol
70:279
16. Verdel BM, Souverein PC, Egberts ACG, Leufkens HGM (2006)
Ann Pharmacother 40:1040
17. Brackett CC, Singh H, Block JH (2004) Pharmacotherapy 24:856
18. Johnson KK, Green DL, Rife JP, Limon L (2005) Ann Phar-
macother 39:290
19. Palmer ME, Chaturongakul S, Wiedmann M, Boor KJ (2011)
mBio 2:e00241
Acknowledgments The authors are grateful for the financial sup-
port provided in part by the Thailand Research Fund and the Office of
the Higher Education Commission (MRG5280040 to C.J.), Mahidol
University under the National Research Universities Initiative, the
Center of Excellence for Innovation in Chemistry (PERCH-CIC), and
Center for Emerging Bacterial Diseases. W.P. was supported by a
Scientific Achievement Scholarship of Thailand (SAST). We thank
Prof. Vichai Reutrakul and Prof. Skorn Mongkolsuk for helpful sci-
entific discussions.
20. Tenover FC, Goering RV (2009) J Antimicrob Chemother 64:441
21. McDougal LK, Fosheim GE, Nicholson A, Bulens SN, Limbago
BM, Shearer JES, Summers AO (2010) Antimicrob Agents
Chemother 54:3804
22. Nimmo GR (2012) Clin Microbiol Infect 18:725
23. Hageman JC, Patel J, Franklin P, Miscavish K, McDougal L,
Lonsway D, Khan FN (2008) Diagn Microbiol Infect Dis 62:440
24. Graber CJ, Wong MK, Carleton HA, Perdreau-Remington F,
Haller BL, Chambers HF (2007) Emerg Infect Dis 13:491
25. McDougal LK, Fosheim GE, Nicholson A, Bulens SN, Limbago
BM, Shearer JE, Summers AO, Patel JB (2010) Antimicrob
Agents Chemother 54:3804
References
26. Clinical and Laboratory Standards Institute (2012) Performance
Standards for Antimicrobial Susceptibility Testing: Fifteenth
Informational Supplement M100–S22. CLSI, Wayne
´ ˇ
27. Zoumpoulakis P, Camoutsis C, Pairas G, Sokovic M, Glamoclija
J, Potamitis C, Pitsas A (2012) Bioorg Med Chem 20:1569
1. Linfield WM, Micich TJ, Montville TJ, Simon JR, Murray EB,
Bistline RG Jr (1983) J Med Chem 26:1741
2. Seydel JK (1968) J Pharm Sci 57:1455
3. Drews J (2000) Science 287:1960
4. Scozzafava A, Owa T, Mastrolorenzo A, Supuran CT (2003) Curr
Med Chem 10:925
´
28. Gobis K, Foks H, Wisniewska K, Da˛browska-Szponar M, Au-
gustynowicz-Kopec E, Napiorkowska A (2012) Arch Pharm
´
´
(Weinheim) 345:911
29. Iliades P, Meshnick SR, Macreadie IG (2004) Antimicrob Agents
Chemother 48:2617
5. Supuran CT, Briganti F, Tilli S, Chegwidden WR, Scozzafava A
(2001) Bioorg Med Chem 9:703
ˆ ´
6. Fortin S, Wei L, Moreau E, Lacroix J, Cote MF, Petitclerc E,
30. Diep BA, Gill SR, Chang RF, Phan TH, Chen JH, Davidson MG,
Lin F, Lin J, Carleton HA, Mongodin EF, Sensabaugh GF,
Perdreau-Remington F (2006) Lancet 367:731
Kotra LP, Gaudreault RC (2011) Eur J Med Chem 46:5327
7. Supuran CT, Casini A, Scozzafava A (2003) Med Res Rev
23:535
31. DeLeo FR, Chambers HF (2009) J Clin Invest 119:2464
8. Joshi S, Khosla N (2003) Bioorg Med Chem Lett 13:3747
123