Design and synthesis of benzenesulfonanilides active against methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus

Article abstract of DOI:10.1016/j.bmc.2008.04.040

Vancomycin is mainly used as an antibacterial agent of last resort, but recently vancomycin-resistant bacterial strains have been emerging. Although new antimicrobials have been developed in order to overcome drug-resistant bacteria, many are structurally

Full text of DOI:10.1016/j.bmc.2008.04.040

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 6131–6144
Design and synthesis of benzenesulfonanilides active
against methicillin-resistant Staphylococcus aureus
and vancomycin-resistant Enterococcus
Kensuke Namba, Xiaoxia Zheng, Kazunori Motoshima, Hidetomo Kobayashi,
Akihiro Tai, Eizo Takahashi, Kenji Sasaki, Keinosuke Okamoto and Hiroki Kakuta*
Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Division of Pharmaceutical Sciences,
1-1-1, Tsushima-Naka, Okayama 700-8530, Japan
Received 24 March 2008; accepted 16 April 2008
Available online 24 April 2008
Abstract—Vancomycin is mainly used as an antibacterial agent of last resort, but recently vancomycin-resistant bacterial strains
have been emerging. Although new antimicrobials have been developed in order to overcome drug-resistant bacteria, many are
structurally complex b-lactams or quinolones. In this study, we aimed to create new anti-drug-resistance antibacterials which can
be synthesized in a few steps from inexpensive starting materials. Since sulfa drugs function as p-aminobenzoic acid mimics and
inhibit dihydropteroate synthase (DHPS) in the folate pathway, we hypothesized that sulfa derivatives would act as folate metab-
olite-mimics and inhibit bacterial folate metabolism. Screening of our sulfonanilide libraries, including benzenesulfonanilide-type
cyclooxygenase-1-selective inhibitors, led us to discover benzenesulfonanilides with potent anti-methicillin-resistant Staphylococcus
aureus (MRSA)/vancomycin-resistant Enterococcus (VRE) activity, that is, N-3,5-bis(trifluoromethyl)phenyl-3,5-dic-
hlorobenzenesulfonanilide (16b) [MIC = 0.5 lg/mL (MRSA), 1.0 lg/mL (VRE)], and 3,5-bis(trifluoromethyl)-N-(3,5-dichloro-
phenyl)benzenesulfonanilide (16c) [MIC = 0.5 lg/mL (MRSA), 1.0 lg/mL (VRE)]. These compounds are more active than
vancomycin [MIC = 2.0 lg/mL (MRSA), 125 lg/mL (VRE)], but do not possess an amino group, which is essential for DHPS inhi-
bition by sulfa drugs. These results suggested that the mechanism of antibacterial action of compounds 16b and 16c is different from
that of sulfa drugs. We also confirmed the activity of these compounds against clinical isolates of Gram-positive bacteria.
ꢀ 2008 Elsevier Ltd. All rights reserved.
1. Introduction
organ abscesses. In the United States, the number of
MRSA-infected patients in 2005 was estimated to be
94,000, and the number of deaths was estimated to be
18,000, exceeding the number of deaths due to AIDS.6
In England and Wales, mortality from MRSA rose from
50 in 1993 to 1600 in 2005.7 In the 1940s, infection with
S. aureus was cured with penicillin. Thereafter, penicil-
lin-resistant S. aureus emerged and spread around the
world as an increase in the use of penicillin. Methicillin
(1) (Fig. 1) was developed in the 1960s to treat penicillin-
resistant S. aureus and came into wide use in Europe and
the United States, but resistance quickly developed.8
Infectious diseases are a major cause of death,1 espe-
cially in developing countries.2,3 These diseases are kill-
ing about 15 million people every year, which
correspond to about one third of the numbers of
deaths.1 Nosocomial infectious diseases, such as methi-
cillin-resistant Staphylococcus aureus (MRSA),4,5 pres-
ent a particular threat to postsurgical or elderly
patients. MRSA is thought to be one of the resident mic-
robiota, which is harmless to healthy people, but intrac-
table in immunosuppressed patients, and has been
implicated in postoperative wound infection, bone infec-
tion (osteomyelitis), infectious endocarditis (IE), and
b-Lactam antibiotics, including penicillin and methicillin
(1), act by inhibiting transpeptidases (also known as pen-
icillin binding protein; PBP), which play a role in pepti-
doglycan synthesis for construction of the bacterial
cell-wall. MRSA is characterized by production of
PBP20, an enzyme to which b-lactams do not bind.9 Van-
comycin (2, VCM) (Fig. 1), a glycopeptide-type antibi-
otic that is effective in the treatment of Gram-positive
Keywords: Methicillin-resistant Staphylococcus aureus; Anti-MRSA;
Vancomycin-resistant Enterococcus; Anti-VRE; Sulfa drugs;
Benzenesulfonanilides.
*
Corresponding author. Tel.: +81 86 251 7977; fax: +81 86 251
7926; e-mail: kakuta@pharm.okayama-u.ac.jp
0968-0896/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.04.040

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K. Namba et al. / Bioorg. Med. Chem. 16 (2008) 6131–6144
Figure 1. Chemical structures of antibacterial agents 1–5, and our COX-1 selective inhibitors 6 and 7.
bacterial infections, was developed for the treatment of
MRSA10–12; it is also useful in patients who are allergic
to b-lactam antibiotics.12 However, vancomycin-resis-
tant Enterococcus (VRE) has emerged.4,13 Consequently,
many new antibacterials have been developed to treat
drug-resistant bacteria. However, most are structurally
complex b-lactam or quinolone antimicrobials which
are not easy or cheap to synthesize.14 Thus, we aimed
to create new agents with simple structures that would
be easily synthesizable from inexpensive starting materi-
als, and that would exhibit antimicrobial activity
through a different mechanism from that of currently
used drugs.
selective toxicity. In addition, trimethoprim (5), which
is effective against sulfa-resistant strains, inhibits dihy-
drofolate reductase (DHFR) (Fig. 2),18 so that
DHFR inhibitors are also of interest as candidate
antimicrobials.
We hypothesized that sulfa derivatives would act as
folate metabolite-mimics and inhibit folate metabolism.
Previously, we developed benzenesulfonanilide-type
cyclooxygenase-1 (COX-1)-selective inhibitors 6 and 7
(Fig. 1), which resemble sulfa derivatives.19 Thus, we
examined the antibacterial activities of these compounds
and their derivatives against MRSA and vancomycin-
resistant Enterococcus (VRE), and found that benze-
nesulfonanilides 16b and 16c have potent anti-MRSA/
VRE activity. These compounds can be synthesized in
a single step from commercially available reagents. Here
we describe the design and synthesis of these com-
pounds, and the results of a structure–activity relation-
ship study. We also confirmed the activity of these
compounds against clinical isolates of Gram-positive
bacteria.
For this purpose, we focused on sulfonamides, often
called simply sulfa drugs [e.g., sulfapyridine (3), sulfa-
dimethoxine (4) (Fig. 1)], which possess a simple
chemical structure with a 4-aminobenzenesulfonamide
skeleton. They have been developed early by G. Do-
magk, but have been hardly used owing to the emer-
gence of sulfa-resistant strains.15 It is well known that
sulfa drugs act as dihydropteroate synthase (DHPS)
inhibitiors in the synthetic pathway of folate, thereby
causing DNA and RNA synthesis inhibition,16,17 be-
cause they act as analogues of p-aminobenzoic acid
(PABA), a substrate of DHPS (Fig. 2). Mammals,
including humans, cannot biosynthesize folate, which
is an essential nutrient, to lack an enzyme necessary
for folate biosynthesis. They take in folate as vitamin
from eatables and drinkables. The difference in folate
requirement between bacteria and mammals results in
2. Results and discussion
Compounds 8–11 were obtained previously during our
search for COX-1-selective inhibitors (Table 1).19 Deriv-
atives of them were prepared according to Schemes 1–3,
mostly in a single step. N-alkylation of sulfonamide was
carried out by reaction of compounds 16b and 16c with

K. Namba et al. / Bioorg. Med. Chem. 16 (2008) 6131–6144
6133
Figure 2. Schematic illustration of relationships between the folate biosynthetic pathway and antimicrobial agents.
methyl iodide after treatment with NaH in anhydrous
DMF. As shown in Scheme 2, compound 21a, in which
the anilino moiety is replaced with a pyridine ring, was
prepared by coupling reaction with 2-chloro-5-(trifluo-
within which the test strain did not grow, was measured
and categorized as follows: minus (no inhibitory), plus/
minus (0–1 mm), plus 1 (1–15 mm), plus 2 (15–20 mm),
and plus 3 (>20 mm).
romethyl)pyridine,
(20), and potassium carbonate using copper as a catalyst
in anhydrous DMF.
3,5-dichlorobenzenesulfonamide
Compound 9h, whose anilino ring possesses bis-trifluo-
romethyl groups at meta positions, showed moderate
anti-MRSA activity, but most compounds were inactive
(Table 1). Further structure development of compound
9h led to the series 12, within which most compounds
showed anti-MRSA activity (Table 2). Interestingly, it
was found that the anti-MRSA activity tended to in-
crease with increasing electron-withdrawing character
(Table 2).
At the first screening, anti-MRSA activities of com-
pounds 8–11 were assessed by the disc diffusion method.
Briefly, to a paper disc on agar medium containing bac-
terial test strain was added 10 lL of a solution of test
compound in DMSO at 20 mM, and the plate was incu-
bated at 37 ꢁC for 20 h. The size of the inhibitory zone,

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K. Namba et al. / Bioorg. Med. Chem. 16 (2008) 6131–6144
Table 1. Anti-MRSA activities of compounds 8–11 which were prepared during our search for cyclooxygenase-1 inhibitorsa
R
SO2
NH
3
R'
4
6
5
Compound
R0
Inhibitory zone at 200 lmol
8 (R = CO2H)
9 (R = NO2)
10 (R = NH2)
11 (R = NHMs)
a
b
c
d
e
f
H
n.t.b
—
—
—
—
—
—
—
—
—
+1
—
—
—
—
—
—
—
—
—
2-Me
3-Me
4-Me
3,5-diMe
4-OMe
4-CF3
3,5-bisCF3
4-Cl
n.t.
+1
—
n.t.
—
—
—
—
—
g
h
i
n.t.
n.t.
n.t.
—
—
+3
n.t.
n.t.
j
4-I
n.t.
a Anti-MRSA activities were determined by the disc diffusion method as described in Section 4.
b n.t.: not tested.
Next, the minimum inhibitory concentration (MIC) of
each compound bearing a 3,5-bis-trifluoromethylphenyl
group against MRSA was determined by means of the
microdilution susceptibility test. Again, anti-MRSA
Scheme 1. Reagents and yields: (a) pyridine, 11–99%; (b) H2, Pd/C, EtOH or Sn, HCl, THF, 39–99%; (c) MeI, NaH, DMF, 33–58%.

K. Namba et al. / Bioorg. Med. Chem. 16 (2008) 6131–6144
6135
Scheme 2. Reagents and yields: (a) NH3 aq, 88%; (b) 2-chloro-5-(trifluoromethyl)pyridine, K2CO3, Cu, DMF, 32%.
VRE activities, introduction of either halogens or tri-
fluoromethyl groups at the meta positions seems opti-
mal. Compounds 18a and 18b, N-methylated
derivatives of 16b and 16c, showed no activity (Table
3), indicating that these compounds require NH-sulfon-
amide structure for anti-MRSA/VRE activity. Further
structural development by the introduction of a hydro-
xyl group at the para position of the anilino moiety of
19b resulted in less potent anti-MRSA/VRE activity
than that of 16c (Table 3). Replacement of phenyl
rings with nitrogenous heterocycles was unfavorable
(Table 4). The most potent compounds in this study
were 16b and 16c. In summary, as shown in Figure 3,
Scheme 3. Reagent and yield: (a) pyridine, 22–65%.
benzenesulfonanilide-type
anti-MRSA/VRE
com-
activity tended to increase with increasing electron-with-
drawing character. Compounds 13 with the sulfonamide
group reversed in the structure also showed a similar
tendency. Among these compounds, 12d possessing a
chloro substituent at the para position of the benzene-
sulfonyl group exhibited potent anti-MRSA activity
(Table 3). We then examined compounds 14 and 15,
which possess chlorine substituents in place of the bis-
trifluoromethyl groups of 12 and 13 (Table 3). Their
anti-MRSA activities were similar to those of 12 and
13, and also tended to increase with increasing elec-
tron-withdrawing character.
pounds require an NH-sulfonamide moiety with halo-
gen or trifluoromethyl substituents at the meta
positions of both phenyl rings.
We next evaluated the antibacterial activities of com-
pounds 16b and 16c against strains isolated from patients
with urinary tract infection at Okayama University hos-
pital. These compounds were effective against staphylo-
cocci and enterococci, while sulfa drugs were ineffective
(Table 5). These compounds do not possess an amino
group on the benzenesulfonyl moiety, though an amino
group is essential for DHPS inhibition by sulfa drugs
(Fig. 2). These results suggested that the mechanism of
the antibacterial action of 16b and 16c is different from
that of sulfa drugs. In an acute toxicity study, compounds
16c and 17g were administered at the dose of 100 mg kgꢀ1
ip to mice, and no toxic symptoms were observed up to 14
days. Although these compounds were developed on the
basis of our hypothesis that they would be folate metab-
olism inhibitors, further studies will be needed to confirm
this and to establish the action mechanism in detail.
The anti-MRSA activities of 16a, a regioisomer of 12d,
and 16b, a 3,5-dichlorobenzenesulfonanilide derivative
of 12d, were evaluated (Table 3). Surprisingly, 16b exhib-
ited more potent anti-MRSA activity (MIC = 0.5 lg/mL)
than vancomycin (2) (MIC = 2.0 lg/mL). Then, the anti-
MRSA activity of 16c, in which the the sulfonamide group
is reversed compared with the structure of 16b, as well as
16d and 16e, which are regioisomers of 16c, was evaluated
(Table 3). Although compounds 16d and 16e, which pos-
sess chloro substituents at ortho and para, or bis-ortho
positions on anilino moieties, showed poor anti-MRSA
activity, 16c showed potent activity (MIC = 0.5 lg/mL).
3. Conclusion
We discovered the potent benzenesulfonanilide-type
anti-MRSA/VRE compounds 16b and 16c, which are
available by one-step synthesis from commercially avail-
able reagents, based on our hypothesis that they would
be folate metabolism inhibitors. Structure–activity rela-
tionship study revealed that a NH-sulfonamide moiety
with halogen atoms or trifluoromethyl groups at the
meta positions of both phenyl rings is required for po-
tent anti-MRSA/VRE activity. These compounds do
not possess an amino group on the benzenesulfonyl moi-
ety, indicating that their mechanism of antibacterial ac-
tion is different from that of sulfa drugs.
Since compounds 16b and 16c showed more potent anti-
MRSA activity than vancomycin (2), their anti-vanco-
mycin-resistant Enterococcus (anti-VRE) activity was
evaluated. Compounds 16b and 16c both showed potent
anti-VRE activity (MIC = 1.0 lg/mL). Next, compound
17 bearing di-fluorine substituents were evaluated (Table
3). As in the cases of 16d and 16e, di-fluoro substitution
at ortho and para, or bis-ortho positions on the anilino
moiety negated the anti-MRSA and anti-VRE activities.
Considering that the most potent compound in Table 3
is 17g, and that 16b and 16c show potent anti-MRSA/

6136
K. Namba et al. / Bioorg. Med. Chem. 16 (2008) 6131–6144
Table 2. Anti-MRSA activity of compounds 12 bearing trifluoro-
methyl groupsa
4.3.2. GP-B-2. To a solution of substituted nitro-
benzenesulfonanilide (1.0 mmol) in THF (4 mL) were
added tin powder (3.5 mmol) and concd HCl, then
the mixture was refluxed for 30 min. After the reac-
tion was finished, the mixture was basified with 2 N
NaOH solution. The mixture was filtered through
Celite and extracted with EtOAc. The organic layer
was washed with H2O and brine, and dried over
MgSO4. The solvent was removed under reduced
pressure. The residue was purified by flash column
chromatography or recrystallized using an appropri-
ate solvent to give corresponding substituted
aminobenzenesulfonanilide.
R
SO2
F3C
NH
CF3
Compound
R
Inhibitory zone
at 200 lmol
a
b
c
d
e
f
NH2
OMe
Me
+1
+1
—
4.4. General procedure for synthesis of N-methylated
benzenesulfonanilides (GP-C)
Cl
+2
+3
+3
CF3
NO2
To a solution of sodium hydride (2.0 mmol) (60% in
mineral oil, washed with n-hexane) in DMF (3 mL)
was added a solution of substituted benzenesulfonani-
lide (1.0 mmol) in DMF (3 mL). The mixture was stirred
at room temperature for 15 min, then methyl iodide
(1.2 mmol) was added, and stirring was continued at
room temperature overnight. To the reaction mixture
was added water, and the organics were extracted with
EtOAc. The extract was washed with water and brine,
dried over MgSO4, and concentrated under reduced
pressure. The residue was purified by flash column
chromatography or recrystallized using an appropriate
solvent to give the corresponding N-methylated
benzenesulfonanilide.
a Anti-MRSA activity was determined by the disc diffusion method as
described in Section 4.
4. Experimental
4.1. General methods
Melting points were determined with a Yanagimoto hot-
stage melting point apparatus and are uncorrected. IR
spectra were recorded on JASCO FT/IR350 (KBr). H
1
NMR spectra were recorded on a VarianVXR-300
(300 MHz) or VarianVXR-500 (500 MHz) spectrome-
ter. Proton chemical shifts were referenced to the TMS
internal standard. Elemental analysis was carried out
with a Yanagimoto MT-5 CHN recorder elemental ana-
lyzer and results were within 0.4% of the theoretical.
FAB-MS was carried out with a VG70-SE. TLC analy-
4.4.1. 4-Amino-N-3,5-bis(trifluoromethyl)phenylbenzene-
sulfonanilide (12a). According to the general procedure
(GP-B-1), 12a was obtained in 84% yield as colorless so-
lid after column chromatography (eluent: MeOH/
sis was carried out on Merck Kieselgel 60F254
.
CHCl3 = 1:100). Mp 171.5–172.5 ꢁC; IR (KBr) cmꢀ1
:
3508, 3410 (NH2), 3245 (NH), 1376, 1189 (SO2); 1H
NMR (300 MHz, DMSO-d6) d: 10.80 (s, 1H), 7.66 (s,
1H), 7.60 (s, 2H), 7.43 (d, 2H, J = 8.7 Hz), 6.55 (d,
2H, J = 8.7 Hz), 6.12 (s, 2H); FAB-MS m/z: 384 [M]+,
385 [M+H]+. Anal. Calcd for C14H11F6N2O2S: C,
43.76; H, 2.62; N, 7.29. Found: C, 43.83; H, 2.83; N,
7.22.
4.2. General procedure for synthesis of substituted
benzenesulfonanilides from anilines (GP-A)
To a solution of aniline (0.5 mmol) in pyridine (1 mL)
was added benzenesulfonyl chloride (0.5 mmol) at
0 ꢁC. The mixture was stirred at room temperature over-
night, then acidified with 2 N HCl solution, and ex-
tracted with EtOAc. The organic layer was washed
with H2O and brine, and dried over MgSO4. The solvent
was removed under reduced pressure. The residue was
purified by flash column chromatography or recrystal-
lized using an appropriate solvent to give a substituted
benzenesulfonanilide.
4.4.2. N-3,5-Bis(trifluoromethyl)phenyl-4-methoxybenzene-
sulfonanilide (12b). According to the general procedure
(GP-A), 12b was obtained in 75% yield as yellow needles
after recrystallization from CH2Cl2/c-hexane. Mp
100.5–101.5 ꢁC; IR (KBr) cmꢀ1: 3223 (NH), 1378,
1
1129 (SO2); H NMR (300 MHz, DMSO-d6) d: 11.09
(s, 1H), 7.75 (d, 2H, J = 8.2 Hz), 7.74 (s, 1H), 7.64 (s,
2H), 7.11 (d, 2H, J = 8.2 Hz), 3.80 (s, 3H); FAB-MS
m/z: 399 [M]+, 400 [M+H]+. Anal. Calcd for
C15H11F6NO3S: C, 45.12; H, 2.78; N, 3.51. Found: C,
45.05; H, 2.90; N, 3.65.
4.3. General procedure for reduction of nitro-substituted
benzenesulfonanilides
4.3.1. GP-B-1. Nitro-substituted benzenesulfonanilide
(2.5 mmol) was dissolved in EtOH (10 mL) and hydro-
genated (1 bar H2) over 10% palladium on charcoal.
The mixture was filtered through a pad of Celite, and
the filtrate was evaporated under reduced pressure.
The residue was purified by flash column chromatogra-
phy or recrystallized from an appropriate solvent to give
an amino-substituted benzenesulfonanilide.
4.4.3. N-3,5-Bis(trifluoromethyl)phenyl-4-methylbenzene-
sulfonanilide (12c). According to the general procedure
(GP-A), 12c was obtained in 99% yield as white powder
after recrystallization from EtOAc/n-hexane. Mp 148.0–
150.0 ꢁC; IR (KBr) cmꢀ1: 3249 (NH), 1379, 1136 (SO2);
1H NMR (300 MHz, CDCl3) d: 7.72 (d, 2H, J = 8.2 Hz),

K. Namba et al. / Bioorg. Med. Chem. 16 (2008) 6131–6144
6137
Table 3. Anti-MRSA/VRE activities of compounds 12–19 evaluated with the microdilution susceptibility testa
3
4
R
5
SO2
2
N
3
X
R'
4
6
5
Compound
R
R0
—
X
MIC (lg/mL)
MRSAb
VREc
Vancomycin (2)
—
—
H
H
H
H
H
H
2.0
125
62.5
62.5
62.5
7.8
12a
12b
12c
12d
12e
12f
4-NH2
4-OMe
4-Me
4-Cl
3,5-bisCF3
3,5-bisCF3
3,5-bisCF3
3,5-bisCF3
3,5-bisCF3
3,5-bisCF3
125
62.5
62.5
7.8
4-CF3
4-NO2
15.6
31.3
7.8
3.9
13a
13b
13c
13d
13e
13f
3,5-bisCF3
3,5-bisCF3
3,5-bisCF3
3,5-bisCF3
3,5-bisCF3
3,5-bisCF3
4-NH2
4-OMe
4-Me
H
H
H
H
H
H
>250
>250
>250
31.3
7.8
>250
125
>250
62.5
3.9
4-Cl
4-CF3
4-NO2
7.8
7.8
14a
14b
14c
14d
14e
14f
4-NH2
4-OMe
4-Me
3,5-diCl
3,5-diCl
3,5-diCl
3,5-diCl
3,5-diCl
3,5-diCl
H
H
H
H
H
H
250
250
31.3
125
62.5
62.5
7.8
125
4-Cl
4-CF3
4-NO2
31.3
15.6
>250
62.5
15a
15b
15c
15d
15e
15f
15g
3,5-diCl
3,5-diCl
3,5-diCl
3,5-diCl
3,5-diCl
3,5-diCl
3,5-diCl
4-NH2
4-OMe
4-Me
H
H
H
H
H
H
H
>250
125
>250
125
>250
>250
125
62.5
125
4-Cl
3-CF3
4-CF3
4-NO2
62.5
31.3
15.6
31.3
7.8
16a
16b
16c
16d
16e
3-Cl
3,5-diCl
3,5-bisCF3
3,5-bisCF3
3,5-diCl
H
H
H
H
H
31.3
0.5
15.6
1.0
3,5-bisCF3
3,5-bisCF3
3,5-bisCF3
0.5
>250
>250
1.0
>250
>250
2,4-diCl
2,6-diCl
17a
17b
17c
17d
17e
17f
17g
17h
4-F
3,5-bisCF3
2,3-diF
2,4-diF
2,5-diF
2,6-diF
3,4-diF
3,5-diF
3,5-diF
H
H
H
H
H
H
H
H
15.6
15.6
>250
15.6
>250
3.9
62.5
62.5
>250
62.5
>250
7.8
3,5-bisCF3
3,5-bisCF3
3,5-bisCF3
3,5-bisCF3
3,5-bisCF3
3,5-bisCF3
3,5-diCl
2.0
31.3
7.8
7.8
18a
18b
3,5-diCl
3,5-bisCF3
3,5-bisCF3
3,5-diCl
Me
Me
>250
>250
>250
>250
19a
19b
3,5-diCl
3,5-bisCF3
3,5-diCl, 4-OH
3,5-diCl, 4-OH
H
H
31.3
125
31.3
62.5
a The microdilution susceptibility test was performed as described in Section 4.
b Anti-MRSA activity was examined against methicillin-resistant Staphylococcus aureus OM584.
c Anti-VRE activity was examined against Enterococcus faecium FN-1.
7.57 (s, 1H), 7.52 (s, 2H), 7.29 (d, 2H, J = 8.2 Hz), 7.03
(s, 1H), 2.41 (s, 3H); FAB-MS m/z: 383 [M]+, 384
[M+H]+. Anal. Calcd for C15H11F6NO3S: C, 47.00; H,
2.89; N, 3.65. Found: C, 47.09; H, 3.01; N, 3.60.
4.4.4. N-3,5-Bis(trifluoromethyl)phenyl-4-chlorobenzene-
sulfonanilide (12d). According to the general procedure
(GP-A), 12d was obtained in 48% yield as white needles
after recrystallization from EtOAc/n-hexane. Mp

6138
K. Namba et al. / Bioorg. Med. Chem. 16 (2008) 6131–6144
Table 4. Anti-MRSA/VRE activities of compounds 21 evaluated with the microdilution susceptibility testa
3
4
R
5
SO2
NH
5
6
4
R'
1Y
X3
2
Compound
R
R0
X
Y
MIC (lg/mL)
MRSAb
VREc
Vancomycin (2)
—
—
CH
—
N
—
C–CF3
N
2.0
125
>250
7.8
a
b
c
3,5-diCl
3,5-diCl
3,5-bisCF3
>250
15.6
15.6
2,6-diCl
2,6-diCl
CH
CH
N
7.8
a The microdilution susceptibility test was performed as described in Section 4.
b Anti-MRSA activity was examined against methicillin-resistant Staphylococcus aureus OM584.
c Anti-VRE activity was examined against Enterococcus faecium FN-1.
403 [M]+, 404 [M+H]+, 405 [M+2]+, 406 [M+2+H]+.
Anal. Calcd for C14H8ClF6NO2S: C, 41.65; H, 2.00;
N, 3.47. Found: C, 42.01; H, 2.27; N, 3.31.
4.4.5. N-3,5-Bis(trifluoromethyl)phenyl-4-trifluoromethyl-
benzenesulfonanilide (12e). According to the general pro-
cedure (GP-A), 12e was obtained in 99% yield as white
solid after recrystallization from EtOAc/n-hexane. Mp
122.0–124.0 ꢁC; IR (KBr) cmꢀ1: 3269 (NH), 1325,
1161 (SO2); 1H NMR (300 MHz, CDCl3) d: 7.95 (d,
2H, J = 8.3 Hz), 7.78 (d, 2H, J = 8.3 Hz), 7.65 (s, 1H),
7.55 (s, 2H), 7.05 (s, 1H); FAB-MS m/z: 437 [M]+, 438
[M+H]+. Anal. Calcd for C15H8F9NO2S: C, 41.2; H,
1.84; N, 3.20. Found: C, 41.06; H, 1.94; N, 3.40.
Figure 3. Important structural factors for antibacterial activity of
benzenesulfonanilide derivatives.
115.0–117.0 ꢁC; IR (KBr) cmꢀ1: 3265 (NH), 1345, 1157
1
4.4.6. N-3,5-Bis(trifluoromethyl)phenyl-4-nitrobenzenesul-
fonanilide (12f). According to the general procedure
(GP-A), 12f was obtained in 76% yield as colorless solid
(SO2); H NMR (300 MHz, DMSO-d6) d: 11.29 (br s,
1H), 7.82 (dd, 2H, J = 6.6, 2.1 Hz), 7.79 (s, 1H), 7.68
(dd, 2H, J = 6.6, 2.1 Hz), 7.66 (s, 2H); FAB-MS m/z:
Table 5. Antibacterial activities of our compounds and sulfa drugs on clinical isolatesa
3
H2N
4
H2N
R
5
3
SO2
NH
SO2
NH
SO2
NH
O
N
N
R'
4
N
5
Me
14e, 15f-g, 16b-c
Sulfadiazine (22)
Sulfamethoxazole (23)
Compound
R
R0
Staphylococci
(13)b
Enterococci
(12)
Pseudomonas aeruginosa
E. coli
(14)
(11)
14e
15f
15g
16b
16c
22
4-CF3
3,5-diCl
3,5-diCl
3,5-diCl
3,5-bisCF3
—
3,5-diCl
4-CF3
4-NO2
3,5-bisCF3
3,5-diCl
—
13c
13
13
13
13
0
12
9
1
0
0
0
2
1
5
7
0
0
5
2
1
2
1
10
12
0
23
—
—
0
0
a The assay was performed by the disc diffusion method as described in Section 4.
b The number of strains.
c The number of strains against which the compounds showed confirmed antibacterial activity.

K. Namba et al. / Bioorg. Med. Chem. 16 (2008) 6131–6144
6139
after column chromatography (eluent: MeOH/CHCl3 =
1:100). Mp 143.5–144.0 ꢁC; IR (KBr) cmꢀ1
3242
2H, J = 8.7 Hz), 7.09 (br s, 1H); FAB-MS m/z: 437
[M]+. Anal. Calcd for C15H8F9NO2S: C, 41.20; H,
1.84; N, 3.20. Found: C, 40.92; H, 1.95; N, 3.34.
:
1
(NH), 1348, 1170 (SO2); H NMR (300 MHz, DMSO-
d6) d: 11.53 (s, 1H), 8.40 (d, 2H, J = 9.0 Hz), 8.06 (d,
2H, J = 9.0 Hz), 7.82 (br s, 1H), 7.68 (br s, 2H); FAB-
MS m/z: 414 [M]+, 415 [M+H]+. Anal. Calcd for
C14H8F6N2O4S: C, 40.59; H, 1.95; N, 6.76. Found: C,
40.56; H, 2.13; N, 6.65.
4.4.12. 3,5-Bis(trifluoromethyl)-N-4-nitrophenylbenzene-
sulfonanilide (13f). According to the general procedure
(GP-A), 13f was obtained in 53% yield as yellow needles
after recrystallization from EtOAc/n-hexane. Mp 183.0–
185.0 ꢁC; IR (KBr) cmꢀ1: 3332 (NH), 1366, 1183 (SO2);
1H NMR (300 MHz, DMSO-d6) d: 11.55 (br s, 1H), 8.51
(s, 1H), 8.41 (s, 2H), 8.16 (dd, 2H, J = 7.1, 2.3 Hz), 7.36
(dd, 2H, J = 7.1, 2.3 Hz); FAB-MS m/z: 414 [M]+, 415
[M+H]+. Anal. Calcd for C14H8F6N2O4S: C, 40.59; H,
1.95; N, 6.76. Found: C, 40.80; H, 2.25; N, 6.70.
4.4.7. N-4-Aminophenyl-3,5-bis(trifluoromethyl)benzenesul-
fonanilide (13a). According to the general procedure
(GP-B-2), 13a was obtained in 39% yield as pale yellow
solid after recrystallization from EtOAc/n-hexane. Mp
181.5–182.5 ꢁC; IR (KBr) cmꢀ1: 3471, 3357 (NH2),
3239 (NH), 1362, 1171 (SO2); 1H NMR (300 MHz,
DMSO-d6) d: 9.79 (s, 1H), 8.47 (s, 1H), 8.12 (br s,
2H), 6.63 (dd, 2H, J = 8.6, 2.0 Hz), 6.41 (dd, 2H,
J = 8.6, 2.0 Hz), 5.09 (s, 1H); FAB-MS m/z: 384 [M]+,
385 [M+H]+. Anal. Calcd for C14H10F6N2O2S: C,
43.76; H, 2.62; N, 7.29. Found: C, 43.68; H, 2.72; N,
7.25.
4.4.13. 4-Amino-N-3,5-dichlorophenylbenzenesulfonani-
lide (14a). According to the general procedure (GP-B-
1), 14a was obtained in 99% yield as white needles after
recrystallization from CH2Cl2/n-hexane. Mp 120.0–
122.0 ꢁC; IR (KBr) cmꢀ1: 3395 (NH), 1316, 1160
1
(SO2); H NMR (300 MHz, DMSO-d6) d: 10.46 (br s,
1H), 7.42 (dd, 2H, J = 6.9, 1.8 Hz), 7.17 (t, 1H,
J = 1.8 Hz), 7.03 (d, 2H, J = 1.8 Hz), 6.57 (dd, 2H,
J = 6.9, 1.8 Hz), 6.10 (s, 2H); FAB-MS m/z: 316 [M]+,
317 [M+H]+, 318 [M+2]+, 319 [M+2+H]+. Anal. Calcd
for C12H10Cl2N2O2S: C, 45.44; H, 3.18; N, 8.83. Found:
C, 45.72; H, 3.14; N, 9.16.
4.4.8. 3,5-Bis(trifluoromethyl)-N-4-methoxyphenylbenzene-
sulfonanilide (13b). According to the general procedure
(GP-A), 13b was was obtained in 89% yield as white nee-
dles after recrystallization from EtOAc/n-hexane. Mp
87.0–90.4 ꢁC; IR (KBr) cmꢀ1: 3286 (NH), 1331, 1139
(SO2); 1H NMR (300 MHz, CDCl3) d: 8.08 (s, 2H),
8.03 (s, 1H), 6.97 (d, 2H, J = 6.8, 2.3 Hz), 6.83 (d, 2H,
J = 6.8, 2.3 Hz), 6.68 (br s, 1H), 3.78 (s, 3H); FAB-MS
m/z: 399 [M]+, 400 [M+H]+. Anal. Calcd for
C15H11F6NO3S: C, 45.12; H, 2.78; N, 3.51. Found: C,
44.98; H, 2.91; N, 3.42.
4.4.14. N-3,5-Dichlorophenyl-4-methoxybenzenesulfonan-
ilide (14b). According to the general procedure (GP-A),
14b was obtained in 94% yield as white needles after
recrystallization from MeOH. Mp 128.0–130.0 ꢁC; IR
1
(KBr) cmꢀ1: 3315 (NH), 1332, 1162 (SO2); H NMR
(300 MHz, DMSO-d6) d: 10.71 (br s, 1H), 7.73 (d, 2H,
J = 8.9 Hz), 7.22 (s, 1H), 7.10 (d, 2H, J = 8.9 Hz), 7.07
(s, 2H), 3.81 (s, 3H); FAB-MS m/z: 331 [M]+, 332
[M+H]+, 333 [M+2]+, 334 [M+2+H]+, 335 [M+4]+,
336 [M+4+H]+. Anal. Calcd for C13H11Cl2NO3S: C,
47.00; H, 3.34; N, 4.22. Found: C, 47.31; H, 3.55; N,
4.21.
4.4.9. 3,5-Bis(trifluoromethyl)-N-4-methylphenylbenzene-
sulfonanilide (13c). According to the general procedure
(GP-A), 13c was obtained in 67% yield as white needles
after recrystallization from EtOAc/n-hexane. Mp 109.0–
112.0 ꢁC; IR (KBr) cmꢀ1: 3237 (NH), 1362, 1148 (SO2);
1H NMR (300 MHz, CDCl3) d: 8.09 (s, 2H), 8.02 (s,
1H), 7.11 (d, 2H, J = 8.3 Hz), 6.93 (d, 2H, J = 8.3 Hz),
6.44 (s, 1H), 2.38 (s, 3H); FAB-MS m/z: 383 [M]+, 384
[M+H]+. Anal. Calcd for C15H11F6NO2S: C, 47.00; H,
2.89; N, 3.65. Found: C, 47.00; H, 2.97; N, 3.57.
4.4.15. N-3,5-Dichlorophenyl-4-methylbenzenesulfonani-
lide (14c). According to the general procedure (GP-A),
14c was obtained in 90% yield as white columnar crys-
tals after recrystallization from EtOAc/n-hexane. Mp
146.0–147.0 ꢁC; IR (KBr) cmꢀ1: 3232 (NH), 1327,
4.4.10. 3,5-Bis(trifluoromethyl)-N-4-chlorophenylbenzene-
sulfonanilide (13d). According to the general procedure
(GP-A), 13d was was obtained in 78% yield as white
powder after recrystallization from EtOAc/n-hexane.
Mp 148.0–151.0 ꢁC; IR (KBr) cmꢀ1: 3226 (NH), 1282,
1140 (SO2); 1H NMR (300 MHz, CDCl3) d: 8.15 (s,
2H), 8.06 (s, 1H), 7.29 (d, 2H, J = 6.8, 2.0 Hz), 7.03
(d, 2H, J = 6.8, 2.0 Hz), 6.63 (s, 1H); FAB-MS m/z:
403 [M]+. Anal. Calcd for C14H8ClF6NO2S: C, 41.65;
H, 2.00; N, 3.47. Found: C, 41.86; H, 2.20; N, 3.29.
1
1166 (SO2); H NMR (300 MHz, DMSO-d6) d: 10.83
(br s, 1H), 7.70 (d, 2H, J = 8.5 Hz), 7.41 (d, 2H,
J = 8.5 Hz), 7.23 (t, 1H, J = 2.0 Hz), 7.08 (d, 2H,
J = 2.0 Hz), 2.36 (s, 3H); FAB-MS m/z: 315 [M]+, 316
[M+H]+, 317 [M+2]+, 318 [M+2+H]+, 319 [M+4]+,
320 [M+4+H]+. Anal. Calcd for C13H11Cl2NO2S: C,
49.38; H, 3.51; N, 4.43. Found: C, 49.14; H, 3.43; N,
4.13.
4.4.16. 4-Choro-N-3,5-dichlorophenylbenzenesulfonani-
lide (14d). According to the general procedure (GP-A),
14d was obtained in 92% yield as white needles after
recrystallization from MeOH/CH2Cl2. Mp 130.0–
132.0 ꢁC; IR (KBr) cmꢀ1: 3224 (NH), 1335, 1169
4.4.11. 3,5-Bis(trifluoromethyl)-N-4-trifluoromethylphenyl-
benzenesulfonanilide (13e). According to the general pro-
cedure (GP-A), 13e was obtained in 40% yield as white
needles after recrystallization from EtOAc/n-hexane.
Mp 142.0–143.0 ꢁC; IR (KBr) cmꢀ1: 3283 (NH), 1325,
1124 (SO2); 1H NMR (300 MHz, CDCl3) d: 8.21 (s,
2H), 8.07 (s, 1H), 7.58 (d, 2H, J = 8.7 Hz), 7.22 (d,
1
(SO2); H NMR (300 MHz, DMSO-d6) d: 10.95 (br s,
1H), 7.81 (dd, 2H, J = 6.7, 2.0 Hz), 7.68 (dd, 2H,
J = 6.7, 2.0 Hz), 7.27 (t, 1H, J = 1.8 Hz), 7.09 (d, 2H,

6140
K. Namba et al. / Bioorg. Med. Chem. 16 (2008) 6131–6144
J = 2.0 Hz); FAB-MS m/z: 335 [M]+, 336 [M+H]+, 337
[M+2]+, 338 [M+2+H]+, 339 [M+4]+, 340 [M+4+H]+.
Anal. Calcd for C12H8Cl3NO2S: C, 42.82; H, 2.40; N,
4.16. Found: C, 43.07; H, 2.64; N, 4.12.
1H, J = 2.0 Hz), 7.66 (d, 2H, J = 2.0 Hz), 7.09 (d, 2H,
J = 8.5 Hz), 6.97 (d, 2H, J = 8.5 Hz), 2.21 (s, 3H);
FAB-MS m/z: 315 [M]+, 316 [M+H]+, 317 [M+2]+,
318 [M+2+H]+, 319 [M+4]+, 320 [M+4+H]+. Anal.
Calcd for C13H11Cl2NO2S: C, 49.38; H, 3.51; N, 4.43.
Found: C, 49.52; H, 3.73; N, 4.24.
4.4.17. N-3,5-Dichlorophenyl-4-trifluoromethylbenzene-
sulfonanilide (14e). According to the general procedure
(GP-A), 14e was obtained in 60% yield as white needles
after recrystallization from CHCl3/c-hexane. Mp 118.0–
119.0 ꢁC; IR (KBr) cmꢀ1: 3255 (NH), 1326, 1132 (SO2);
1H NMR (300 MHz, DMSO-d6) d: 11.11 (s, 1H), 8.02 (s,
4H), 7.29 (t, 1H, J = 1.9 Hz), 7.11 (d, 2H, J = 1.9 Hz);
FAB-MS m/z: 369 [M]+, 370 [M+H]+, 371 [M+2]+,
372 [M+2+H]+. Anal. Calcd for C13H8Cl2F3NO2S: C,
42.18; H, 2.18; N, 3.78. Found: C, 42.43; H, 1.88; N,
3.44.
4.4.22. N-4-Chlorophenyl-3,5-dichlorobenzenesulfonani-
lide (15d). According to the general procedure (GP-A),
15d was obtained in 65% yield as pale red solid after
recrystallization from CHCl3/c-hexane. Mp 145.5–
146.0 ꢁC; IR (KBr) cmꢀ1: 3275 (NH), 1339, 1170
(SO2); 1H NMR (300 MHz, DMSO-d6) d: 10.63 (s,
1H), 7.97 (t, 1H, J = 1.9 Hz), 7.71 (d, 2H, J = 1.9 Hz),
7.35 (dd, 2H, J = 8.9, 2.1 Hz), 7.12 (dd, 2H, J = 8.9,
2.1 Hz); FAB-MS m/z: 335 [M]+, 336 [M+H]+, 337
[M+2]+, 338 [M+2+H]+, 339 [M+4]+, 340 [M+4+H]+,
341 [M+6]+. Anal. Calcd for C12H8Cl3NO2S: C, 42.82;
H, 2.40; N, 4.16. Found: C, 42.79; H, 2.22; N, 4.06.
4.4.18. N-3,5-Dichlorophenyl-4-nitrobenzenesulfonanilide
(14f). According to the general procedure (GP-A), 14f
was obtained in 99% yield as white needles after recrys-
tallization from EtOAc/n-hexane. Mp 186.0–188.0 ꢁC;
IR (KBr) cmꢀ1: 3265 (NH), 1350, 1176 (SO2); 1H
NMR (300 MHz, DMSO-d6) d: 11.19 (br s, 1H), 8.41
(dd, 2H, J = 7.0, 2.0 Hz), 8.06 (dd, 2H, J = 7.0,
2.0 Hz), 7.31 (t, 1H, J = 2.0 Hz), 7.11 (d, 2H,
J = 2.0 Hz); FAB-MS m/z: 346 [M]+, 347 [M+H]+, 348
[M+2]+, 349 [M+2+H]+, 350 [M+4]+, 351 [M+4+H]+.
Anal. Calcd for C12H8Cl2N2O4S: C, 41.51; H, 2.32; N,
8.07. Found: C, 41.33; H, 2.22; N, 8.16.
4.4.23. 3,5-Dichloro-N-3-trifluoromethylphenylbenzene-
sulfonanilide (15e). According to the general procedure
(GP-A), 15e was obtained in 64% yield as white solid
after recrystallization from CH2Cl2/n-hexane. Mp
139.5–141.5 ꢁC; IR (KBr) cmꢀ1: 3283 (NH), 1325,
1
1170 (SO2); H NMR (300 MHz, DMSO-d6) d: 10.89
(br s, 1H), 7.97 (t, 1H, J = 2.0 Hz), 7.72 (d, 2H,
J = 2.0 Hz), 7.47 (m, 4H); FAB-MS m/z: 369 [M]+, 370
[M+H]+, 371 [M+2]+, 372 [M+2+H]+. Anal. Calcd for
C13H8Cl2F3NO2S: C, 42.18; H, 2.18; N, 3.78. Found:
C, 42.35; H, 2.27; N, 3.72.
4.4.19. N-4-Aminophenyl-3,5-dichlorobenzenesulfonani-
lide (15a). According to the general procedure (GP-B-
2), 15a was obtained in 76% yield as white needles after
recrystallization from CHCl3/n-hexane. Mp 175.0–
176.0 ꢁC; IR (KBr) cmꢀ1: 3396, 3333 (NH2), 3063
4.4.24. 3,5-Dichloro-N-4-trifluoromethylphenylbenzene-
sulfonanilide (15f). According to the general procedure
(GP-A), 15f was obtained in 65% yield as white solid
after recrystallization from CHCl3/c-hexane. Mp
145.5–146.0 ꢁC; IR (KBr) cmꢀ1: 3282 (NH), 1324,
1
(NH), 1335, 1166 (SO2); H NMR (300 MHz, DMSO-
d6) d: 9.69 (s, 1H), 7.93 (t, 1H, J = 1.9 Hz), 7.59 (d,
2H, J = 1.9 Hz), 6.66 (dd, 2H, J = 8.8, 2.2 Hz), 6.42
(dd, 2H, J = 8.8, 2.2 Hz), 5.06 (s, 2H); FAB-MS m/z:
316 [M]+, 317 [M+H]+, 318 [M+2]+, 319 [M+2+H]+,
320 [M+4]+, 321 [M+4+H]+. Anal. Calcd for
C12H10Cl2N2O2S: C, 45.44; H, 3.18; N, 8.83. Found:
C, 45.32; H, 2.88; N, 8.45.
1
1144 (SO2); H NMR (300 MHz, DMSO-d6) d: 11.06
(s, 1H), 7.98 (t, 1H, J = 1.8 Hz), 7.79 (d, 2H,
J = 1.8 Hz), 7.65 (d, 2H, J = 8.3 Hz), 7.31 (d, 2H,
J = 8.3 Hz); FAB-MS m/z: 369 [M]+, 370 [M+H]+, 371
[M+2]+, 372 [M+2+H]+, 373 [M+4]+, 374 [M+4+H]+.
Anal. Calcd for C13H8Cl2F3NO2S: C, 42.18; H, 2.18;
N, 3.78. Found: C, 42.35; H, 2.05; N, 3.59.
4.4.20. 3,5-Dichloro-N-4-methoxyphenylbenzenesulfonan-
ilide (15b). According to the general procedure (GP-A),
15b was obtained in 90% yield as pale red plates after
recrystallization from MeOH. Mp 88.0–90.0 ꢁC; IR
4.4.25. 3,5-Dichloro-N-4-nitrophenylbenzenesulfonanilide
(15g). According to the general procedure (GP-A), 15g
was obtained in 69% yield as yellow plates after recrys-
tallization from EtOAc/n-hexane. Mp 190.0–192.0 ꢁC;
IR (KBr) cmꢀ1: 3298 (NH), 1335, 1174 (SO2); 1H
NMR (300 MHz, DMSO-d6) d: 11.45 (s, 1H), 8.17
(dd, 2H, J = 9.3, 2.2 Hz), 8.00 (t, 1H, J = 2.0 Hz), 7.85
(d, 2H, J = 2.0 Hz), 7.35 (dd, 2H, J = 9.3, 2.2 Hz);
FAB-MS m/z: 346 [M]+, 347 [M+H]+, 348 [M+2]+,
349 [M+2+H]+, 350 [M+4]+, 351 [M+4+H]+. Anal.
Calcd for C12H8Cl2N2O4S: C, 41.51; H, 2.32; N, 8.07.
Found: C, 41.65; H, 2.23; N, 7.85.
1
(KBr) cmꢀ1: 3294 (NH), 1340, 1173 (SO2); H NMR
(300 MHz, DMSO-d6) d: 10.12 (br s, 1H), 7.94 (t, 1H,
J = 2.0 Hz), 7.62 (d, 2H, J = 2.0 Hz), 6.97 (d, 2H,
J = 9.2 Hz), 6.85 (d, 2H, J = 9.2 Hz), 3.69 (s, 3H);
FAB-MS m/z: 331 [M]+, 332 [M+H]+, 333 [M+2]+,
334 [M+2+H]+, 335 [M+4]+, 336 [M+4+H]+. Anal.
Calcd for C13H11Cl2NO3S: C, 47.00; H, 3.34; N, 4.22.
Found: C, 46.91; H, 3.16; N, 4.02.
4.4.21. 3,5-Dichloro-N-4-methylphenylbenzenesulfonani-
lide (15c). According to the general procedure (GP-A),
15c was obtained in 99% yield as white needles after
recrystallization from MeOH. Mp 123.0–125.0 ꢁC; IR
4.4.26. N-3,5-Bis(trifluoromethyl)phenyl-3-chlorobenzene-
sulfonanilide (16a). According to the general procedure
(GP-A), 16a was obtained in 63% yield as white granules
after recrystallization from EtOAc/n-hexane. Mp 135.0–
137.0 ꢁC; IR (KBr) cmꢀ1: 3271 (NH), 1341, 1132 (SO2);
1
(KBr) cmꢀ1: 3210 (NH), 1340, 1163 (SO2); H NMR
(300 MHz, DMSO-d6) d: 10.29 (br s, 1H), 7.93 (td,

K. Namba et al. / Bioorg. Med. Chem. 16 (2008) 6131–6144
6141
1H NMR (300 MHz, DMSO-d6) d: 11.61 (br s, 1H),
8.18–8.15 (m, 1H), 7.72 (s, 1H), 7.70–7.66 (m, 2H),
7.62 (s, 2H), 7.61–7.57 (m, 1H); FAB-MS m/z: 403
[M]+, 404 [M+H]+, 405 [M+2]+, 406 [M+2+H]+. Anal.
Calcd for C14H8ClF6NO2S: C, 41.65; H, 2.00; N, 3.47.
Found: C, 41.95; H, 2.31; N, 3.47.
(dd, 2H, J = 8.9, 2.1 Hz), 7.75 (s, 1H), 7.64 (s, 2H), 7.44
(dd, 2H, J = 8.9, 2.1 Hz); FAB-MS m/z: 387 [M]+, 388
[M+H]+. Anal. Calcd for C14H8F7NO2S: C, 43.42; H,
2.08; N, 3.62. Found: C, 43.12; H, 2.17; N, 3.59.
4.4.32. 3,5-Bis(trifluoromethyl)-N-2,3-difluorophenylben-
zenesulfonanilide (17b). According to the general procedure
(GP-A), 17b was obtained in 49% yield as white solid
after recrystallization from EtOAc/n-hexane. Mp 113.0–
4.4.27. N-3,5-Bis(trifluoromethyl)phenyl-3,5-dichlorobenz-
enesulfonanilide (16b). According to the general proce-
dure (GP-A), 16b was obtained in 64% yield as white
powders by flash column chromatography (eluent:
EtOAc/n-hexane = 1:8). Mp 110.0–111.0 ꢁC; IR
115.0 ꢁC; IR (KBr) cmꢀ1
: 3299 (NH), 1358, 1164
1
(SO2); H NMR (300 MHz, DMSO-d6) d: 10.87 (br s,
1H), 8.56 (s, 1H), 8.26 (s, 2H), 7.38–7.04 (m, 3H);
FAB-MS m/z: 405 [M]+. Anal. Calcd for C14H7F8NO2S:
C, 41.49; H, 1.74; N, 3.46. Found: C, 41.25; H, 1.90; N,
3.46.
1
(KBr) cmꢀ1: 3258 (NH), 1345, 1163 (SO2); H NMR
(300 MHz, DMSO-d6) d: 10.15 (s, 1H), 8.02 (t, 1H,
J = 1.8 Hz), 7.86 (s, 1H), 7.78 (d, 2H, J = 1.8 Hz), 7.67
(s, 2H); FAB-MS m/z: 437 [M]+, 438 [M+H]+ 439
[M+2]+, 440 [M+2+H]+, 441 [M+4]+, 442 [M+4+H]+.
Anal. Calcd for C14H7Cl2F6NO2S: C, 38.38; H, 1.61;
N, 3.20. Found: C, 38.36; H, 1.77; N, 3.08.
4.4.33. 3,5-Bis(trifluoromethyl)-N-2,4-difluorophenylben-
zenesulfonanilide (17c). According to the general proce-
dure (GP-A), 17c was obtained in 84% yield as white
needles after recrystallization from EtOAc/n-hexane.
Mp 121.0–123.0 ꢁC; IR (KBr) cmꢀ1: 3249 (NH), 1353,
4.4.28. 3,5-Bis(trifluoromethyl)-N-3,5-dichlorophenylbenz-
enesulfonanilide (16c). According to the general procedure
(GP-A), 16c was obtained in 79% yield as white solid
after recrystallization from CHCl3/n-hexane. Mp 128.0–
128.5 ꢁC; IR (KBr) cmꢀ1: 3261 (NH), 1361, 1173 (SO2);
1H NMR (300 MHz, DMSO-d6) d: 11.09 (s, 1H), 8.13
(s, 1H), 8.32 (br s, 2H), 7.36 (t, 1H, J = 1.8 Hz), 7.11
(d, 2H, J = 1.8 Hz); FAB-MS m/z: 437 [M]+, 438
[M+H]+, 439 [M+2]+, 440 [M+2+H]+, 441 [M+4]+, 442
[M+4+H]+. Anal. Calcd for C14H7Cl2F6NO2S: C,
38.38; H, 1.61; N, 3.20. Found: C, 38.47; H, 1.84; N, 3.05.
1
1169 (SO2); H NMR (300 MHz, DMSO-d6) d: 10.61
(br s, 1H), 8.54 (s, 1H), 8.21 (s, 2H), 7.32–7.05 (m,
3H); FAB-MS m/z: 405 [M]+, 406 [M+H]+. Anal. Calcd
for C14H7F8NO2S: C, 41.49; H, 1.74; N, 3.46. Found: C,
41.45; H, 1.89; N, 3.45.
4.4.34. 3,5-Bis(trifluoromethyl)-N-2,5-difluorophenylben-
zenesulfonanilide (17d). According to the general proce-
dure (GP-A), 17d was obtained in 41% yield as white
needles after recrystallization from EtOAc/n-hexane.
Mp 121.0–123.0 ꢁC; IR (KBr) cmꢀ1: 3254 (NH), 1349,
1171 (SO2); 1H NMR (300 MHz, DMSO-d6) d: 10.87 (s,
1H), 8.54 (s, 1H), 8.29 (s, 2H), 7.30–7.10 (m, 3H); FAB-
MS m/z: 405 [M]+. Anal. Calcd for C14H7F8NO2S: C,
41.49; H, 1.74; N, 3.46. Found: C, 41.30; H, 2.04; N,
3.37.
4.4.29. 3,5-Bis(trifluoromethyl)-N-2,4-dichlorophenylbenz-
enesulfonanilide (16d). According to the general proce-
dure (GP-A), 16d was obtained in 47% yield as white
granules after recrystallization from EtOAc/n-hexane.
Mp 110.0–112.0 ꢁC; IR (KBr) cmꢀ1: 3259 (NH), 1281,
1
1177 (SO2); H NMR (300 MHz, DMSO-d6) d: 10.70
(br s, 1H), 8.56 (s, 1H), 8.23 (s, 2H), 7.63 (d, 1H,
J = 2.5 Hz), 7.44 (dd, 1H, J = 8.7, 2.5 Hz), 7.31 (d, 1H,
J = 8.7 Hz); FAB-MS m/z: 437 [M]+, 438 [M+H]+, 439
4.4.35. 3,5-Bis(trifluoromethyl)-N-2,6-difluorophenylben-
zenesulfonanilide (17e). According to the general proce-
dure (GP-A), 17e was obtained in 55% yield as white
solid after recrystallization from EtOAc/n-hexane. Mp
150.0–152.0 ꢁC; IR (KBr) cmꢀ1: 3257 (NH), 1352,
[M+2]+,
440
[M+2+H]+.
Anal.
Calcd
for
C14H7Cl2F6NO2S: C, 38.38; H, 1.61; N, 3.20. Found:
C, 38.47; H, 1.75; N, 2.95.
1
1167 (SO2); H NMR (300 MHz, DMSO-d6) d: 10.63
(br s, 1H), 8.55 (s, 1H), 8.28 (s, 1H), 7.40 (t, 1H,
J = 8.6 Hz), 7.14 (t, 2H, J = 8.6 Hz); FAB-MS m/z:
405 [M]+, 406 [M+H]+. Anal. Calcd for C14H7F8NO2S:
C, 41.49; H, 1.74; N, 3.46. Found: C, 41.30; H, 1.88; N,
3.54.
4.4.30. 3,5-Bis(trifluoromethyl)-N-2,6-dichlorophenylbenz-
enesulfonanilide (16e). According to the general proce-
dure (GP-A), 16e was obtained in 11% yield as white
granules after recrystallization from EtOAc/n-hexane.
Mp 146.0–148.0 ꢁC; IR (KBr) cmꢀ1: 3264 (NH), 1281,
1
1137 (SO2); H NMR (300 MHz, DMSO-d6) d: 10.75
4.4.36. 3,5-Bis(trifluoromethyl)-N-3,4-difluorophenylben-
zenesulfonanilide (17f). According to the general proce-
dure (GP-A), 17f was obtained in 93% yield as white
needles after recrystallization from EtOAc/n-hexane.
Mp 97.0–100.0 ꢁC; IR (KBr) cmꢀ1: 3216 (NH), 1281,
(br s, 1H), 8.55 (s, 1H), 8.30 (s, 2H), 7.54 (d, 2H,
J = 8.5 Hz), 7.38 (t, 1H, J = 8.5 Hz); FAB-MS m/z:
437 [M]+, 438 [M+H]+, 439 [M+2]+, 440 [M+2+H]+.
Anal. Calcd for C14H7Cl2F6NO2S: C, 38.38; H, 1.61;
N, 3.20. Found: C, 38.25; H, 1.77; N, 3.03.
1
1165 (SO2); H NMR (300 MHz, DMSO-d6) d: 10.74
(br s, 1H), 8.51 (s, 1H), 8.26 (s, 2H), 7.41–6.88 (m,
3H); FAB-MS m/z: 405 [M]+, 406 [M+H]+. Anal. Calcd
for C14H7F8NO2S: C, 41.49; H, 1.74; N, 3.46. Found: C,
41.52; H, 1.95; N, 3.54.
4.4.31. N-3,5-Bis(trifluoromethyl)phenyl-4-fluorobenzene-
sulfonanilide (17a). According to the general procedure
(GP-A), 17a was obtained in 86% yield as white needles
after recrystallization from EtOAc/n-hexane. Mp 117.0–
119.0 ꢁC; IR (KBr) cmꢀ1: 3267 (NH), 1382, 1130 (SO2);
1H NMR (300 MHz, DMSO-d6) d: 11.24 (br s, 1H), 7.88
4.4.37. 3,5-Bis(trifluoromethyl)-N-3,5-difluorophenylben-
zenesulfonanilide (17g). According to the general

6142
K. Namba et al. / Bioorg. Med. Chem. 16 (2008) 6131–6144
procedure (GP-A), 17g was obtained in 86% yield as
white plates after recrystallization from CHCl3/n-hex-
ane. Mp 98.0–101.0 ꢁC; IR (KBr) cmꢀ1: 3234 (NH),
white solid after recrystallization from EtOAc/n-hexane.
Mp 155.0–156.0 ꢁC; IR (KBr) cmꢀ1: 3339, 3268 (OH),
3087 (NH), 1279, 1155 (SO2); 1H NMR (300 MHz,
CDCl3) d: 8.15 (s, 2H), 8.10 (s, 1H), 7.04 (s, 2H), 6.41
(br s, 1H), 5.86 (br s, 1H); FAB-MS m/z: 453 [M]+,
454 [M+H]+, 455 [M+2]+. Anal. Calcd for
C14H7Cl2F6NO3S: C, 37.02; H, 1.55; N, 3.08. Found:
C, 37.13; H, 1.83; N, 2.97.
1
1363, 1167 (SO2); H NMR (300 MHz, DMSO-d6) d:
11.10 (br s, 1H), 8.50 (s, 1H), 8.34 (s, 2H), 6.96 (tt,
1H, J = 9.2, 2.2 Hz), 6.80 (dd, 2H, J = 9.2, 2.2 Hz);
FABꢀMS m/z: 405 [M]+, 406 [M+H]+. Anal. Calcd for
C14H7F8NO2S: C, 41.48; H, 1.74; N, 3.46. Found: C,
41.25; H, 1.89; N, 3.40.
4.4.43. 3,5-Dichlorobenzenesulfonamide (20). 3,5-Dichlo-
robenzenesulfonylchloride (3.0 mmol) was added to
NH3 aq (8.0 mL). The mixture was stirred at room tem-
perature for 3 h, then extracted with EtOAc. The organ-
ic layer was washed with H2O and brine, and dried over
MgSO4. The solvent was removed under reduced pres-
sure. Compound 20 was obtained in 88% yield as white
needles after recrystallization from EtOAc/n-hexane.
Mp 158.0–160.0 ꢁC; 1H NMR (300 MHz, DMSO-d6)
d: 7.94 (td, 1H, J = 1.8, 0.3 Hz), 7.80 (dd, 2H, J = 1.8,
0.3 Hz), 7.66 (s, 2H).
4.4.38. 3,5-Dichloro-N-3,5-difluorophenylbenzenesulfo-
nanilide (17h). According to the general procedure
(GP-A), 17h was obtained in 80% yield as white plates
after recrystallization from CH2Cl2/n-hexane. Mp
115.0–118.0 ꢁC; IR (KBr) cmꢀ1: 3238 (NH), 1344,
1
1177 (SO2); H NMR (300 MHz, DMSO-d6) d: 11.07
(br s, 1H), 7.99 (td, 1H, J = 2.0, 0.8 Hz), 7.81 (dd, 2H,
J = 2.0, 0.8 Hz), 6.96 (tt, 1H, J = 9.2, 2.0 Hz), 6.80
(dd, 2H, J = 9.2, 2.0 Hz); FAB-MS m/z: 337 [M]+, 338
[M+H]+, 339 [M+2]+, 340 [M+2+H]+. Anal. Calcd for
C12H7Cl2F2NO2S: C, 42.62; H, 2.09; N, 4.14. Found:
C, 42.75; H, 1.74; N, 3.99.
4.4.44. 3,5-Dichloro-N-(5-trifluoromethyl-2-pyridinyl)ben-
zenesulfonanilide (21a). To mixture of 20 (1.4 mmol) and
2-chloro-5-(trifluoromethyl)pyridine (1.4 mmol) in dry
DMF (2.0 mL) were added K2CO3 (1.4 mmol) and Cu
(catalytic amount). The mixture was refluxed at 175 ꢁC
for 5 h, then poured into 10% AcOH. The whole was fil-
tered through a pad of Celite, and the filtrate was ex-
tracted with EtOAc (3· 30 mL). The organic layer was
collected, washed with H2O (2· 50 mL) and brine
(100 mL), and dried over MgSO4. The solvent was evap-
orated under reduced pressure. The residue was purified
by flash column chromatography (EtOAc/n-hex-
ane = 1:4) and obtained in 32% yield as yellow needles
after recrystallization from EtOAc/n-hexane. Mp
179.0–181.0 ꢁC; IR (KBr) cmꢀ1: 3083 (NH), 1332,
1128 (SO2); 1H NMR (300 MHz, CDCl3) d: 8.53 (s,
1H), 7.91 (d, 1H, J = 9.0 Hz), 7.81 (d, 2H, J = 1.8 Hz),
7.56 (s, 1H), 7.40 (d, 1H, J = 9.0 Hz); FAB-MS m/z:
371 [M+H]+, 372 [M+2]+, 373 [M+2+H]+. Anal. Calcd
for C12H7Cl2F3N2O2S: C, 38.83; H, 1.90; N, 7.55.
Found: C, 38.78; H, 1.85; N, 7.47.
4.4.39. N-3,5-Bis(trifluoromethyl)phenyl-3,5-dichloro-N-
methylbenzenesulfonanilide (18a)-methylbenzenesulfonan-
ilide (18a). According to the general procedure (GP-C),
18a was obtained in 33% yield as white granules after
recrystallization from EtOAc/n-hexane. Mp 105.0–
1
107.0 ꢁC; IR (KBr) cmꢀ1: 1279, 1175 (SO2); H NMR
(300 MHz, CDCl3) d: 7.85 (s, 1H), 7.63 (t, 1H,
J = 2.0 Hz), 7.58 (s, 2H), 8.10 (s, 1H, J = 0.8 Hz), 7.45
(s, 2H), 3.28 (s, 3H); FAB-MS m/z: 451 [M]+, 452
[M+H]+, 453 [M+2]+, 454 [M+2+H]+. Anal. Calcd for
C15H9Cl2F6NO2S: C, 39.84; H, 2.01; N, 3.10. Found:
C, 39.71; H, 2.18; N, 2.95.
4.4.40. 3,5-Bis(trifluoromethyl)-N-3,5-dichlorophenyl-N-
methylbenzenesulfonanilide (18b)-methylbenzenesulfonan-
ilide (18b). According to the general procedure (GP-C),
18b was obtained in 58% yield as white needles after
recrystallization from EtOAc/n-hexane. Mp 85.0–
1
86.0 ꢁC; IR (KBr) cmꢀ1: 1282, 1142 (SO2); H NMR
(500 MHz, CDCl3) d: 8.13 (s, 1H), 7.98 (s, 2H), 7.36 (t,
1H, J = 1.8 Hz), 7.00 (d, 2H, J = 1.8 Hz), 3.18 (s, 3H);
FAB-MS m/z: 451 [M]+, 452 [M+H]+, 453 [M+2]+, 454
[M+2+H]+. Anal. Calcd for C15H9Cl2F6NO2S: C,
39.84; H, 2.01; N, 3.10. Found: C, 39.60; H, 2.28; N, 2.99.
4.4.45. N-(2,6-Dichloro-4-pyridinyl)-3,5-dichlorobenzene-
sulfonanilide (21b). According to the general procedure
(GP-A), 21b was obtained in 22% yield as white solid
after recrystallization from EtOAc/n-hexane. Mp
155.0–156.0 ꢁC; IR (KBr) cmꢀ1: 3077 (NH), 1351,
1174 (SO2); 1H NMR (300 MHz, CDCl3) d: 7.78 (d,
2H, J = 1.8 Hz), 7.64 (t, 1H, J = 1.8 Hz), 7.01 (s, 2H);
FAB-MS m/z: 371 [M+H]+, 372 [M+2]+, 373
[M+2+H]+, 374 [M+4]+, 375 [M+4+H]+. Anal. Calcd
for C11H6Cl4N2O2S: C, 35.51; H, 1.63; N, 7.53. Found:
C, 35.24; H, 1.81; N, 7.15.
4.4.41. 3,5-Dichloro-N-3,5-dichloro-N -4-hydroxyphenyl-
benzenesulfonanilide (19a). According to the general pro-
cedure (GP-A), 19a was obtained in 89% yield as brown
needles after recrystallization from EtOAc/n-hexane.
Mp 207.0–210.0 ꢁC; IR (KBr) cmꢀ1: 3419, 3282 (OH),
3093 (NH), 1345, 1159 (SO2); 1H NMR (300 MHz,
DMSO-d6) d: 10.42 (s, 1H), 10.20 (s, 1H), 7.99 (s, 1H),
7.67 (s, 2H), 7.03 (s, 2H); FAB-MS m/z: 385 [M]+, 386
[M+H]+, 387 [M+2]+, 388 [M+2+H]+, 389 [M+4]+.
Anal. Calcd for C12H7Cl4NO3S: C, 37.24; H, 1.82; N,
3.62. Found: C, 32.28; H, 1.78; N, 3.57.
4.4.46. 3,5-Bis(trifluoromethyl)-N -(2,6-dichloro-4-pyridi-
nyl)benzenesulfonanilide (21c). According to the general
procedure (GP-A), 21c was obtained in 65% yield as
white granules after recrystallization from EtOAc/n-hex-
ane. Mp 171.0–172.0 ꢁC; IR (KBr) cmꢀ1: 3095 (NH),
1
4.4.42. 3,5-Bis(trifluoromethyl)-N-3,5-dichloro-N-4-hydrox-
yphenylbenzenesulfonanilide (19b). According to the gen-
eral procedure (GP-A), 19b was obtained in 69% yield as
1354, 1140 (SO2); H NMR (300 MHz, DMSO-d6) d:
8.54 (s, 1H), 8.48 (s, 2H), 7.12 (s, 2H); FAB-MS m/z:
439 [M+H]+, 440 [M+2]+, 441 [M+2+H]+, 442 [M+4]+,

K. Namba et al. / Bioorg. Med. Chem. 16 (2008) 6131–6144
6143
443 [M+4+H]+. Anal. Calcd for C13H6Cl2F6N2O2S: C,
35.55; H, 1.38; N, 6.38. Found: C, 35.75; H, 1.73; N, 6.26.
test compound at which bacterial growth was clearly
inhibited. Controls with media, DMSO, and water
were run in parallel with the test compounds under
the same conditions. The experiment was performed
at least in duplicate.
4.5. Preparation of bacterial culture
Test strains were methicillin-resistant S. aureus OM584
as MRSA and Enterococcus faecium FN-1 as VRE. A
single colony of each strain was transferred into 3 mL
of nutrient broth (Eiken Chemical Co., Ltd, Tokyo, Ja-
pan) in a 15-mL test tube, and the tube was capped and
placed in incubator overnight at 35 ꢁC. After 12–18 h of
incubation, the broth was directly used for the disc dif-
fusion method. On the other hand, for the microdilution
susceptibility test, a 20 lL aliquot of the overnight broth
culture was transferred to 2 mL of sensitivity test broth
(Nissui Pharmaceutical, Tokyo, Japan) in a shaking
incubator (140 shakes/min) at 37 ꢁC for about 1.5 h for
MRSA and for about 4 h for VRE until the optical den-
sity at 620 nm reached 0.1. An optical density of 0.1 was
regarded as approximately equivalent to 108 CFU/mL
(colony-forming unit/mL).
4.8. Disc diffusion test against isolated clinical strains
Antimicrobial activities of the test compounds against
clinical isolates from patients with urinary tract infec-
tion at Okayama University hospital were assessed by
the disc diffusion method as described above.
4.9. Acute toxicity test in mice
Male mice, weighing 15–30 g, were acquired from Charles
River Co. Ltd. Only water was provided ad libitum during
the 12 h before experimentation. The study was con-
ducted according to internationally accepted principles
of laboratory animal use. Solutions of each compound
dissolved in 0.5% carboxymethyl cellulose (CMC) were
administered at doses up to 100 mg kgꢀ1 ip to mice
(n = 5/group). After drug administration, food, and water
was provided ad libitum. The weight gain of animals was
observed up to 14 days as an indicator of toxicity.
4.6. Disc diffusion method (first screening)
Muller-Hinton agar (Nissui Pharmaceutical, Tokyo, Ja-
pan) was prepared according to the supplier’s instruc-
tions. After autoclave treatment, the Muller-Hinton
agar (MH agar) was poured into 90-mm Petri dishes
(about 20 mL/dish). After solidification, on the surface
of the agar was spread 100 lL of overnight broth culture
as described above. Seven 8-mm paper discs were placed
on the agar, evenly spaced apart, and a solution of test
compound in DMSO (20 mM) was transferred onto
each paper disc. After overnight incubation of the plate
at 37 ꢁC, the diameters of the inhibitory zones were mea-
sured. The size was categorized into five classes: minus
(no inhibition zone), plus/minus (0–1 mm), plus 1 (1–
15 mm), plus 2 (15–20 mm), and plus 3 (>20 mm).
Acknowledgments
The authors are grateful to the SC-NMR Laboratory of
Okayama University for the NMR experiments. Ms
Ryoko Hirata (The University of Tokyo) obtained the
combustion analytic data. This research was partially
supported by Okayama Prefecture Industrial Promotion
Foundation and by a Center of Excellence (COE) pro-
ject at Okayama University and by the Program of
Founding Research Centers for Emerging and Reemerg-
ing Infectious Diseases, MEXT Japan.
4.7. The microdilution susceptibility test
References and notes
The microdilution susceptibility test in sensitivity test
broth was carried out for determination of the minimum
inhibitory concentration (MIC) according to Standard
method of Japan Society of Chemotherapy. Vancomy-
cin was used as a standard antibacterial agent. Solutions
of compounds in DMSO were prepared at a concentra-
tion of 10 mg/mL. Vancomycin was dissolved in water
at a concentration of 10 mg/mL. A 95 lL aliquot of
autoclaved sensitivity test broth was poured into the first
line wells of 96-well plates (Sumitomo Bakelite Co., Ltd,
Tokyo, Japan) and 50 lL of autoclaved sensitivity test
broth was poured into the remaining wells, then 5 lL
of test compound solution was poured into the first line
wells, and 50 lL of the mixture was transferred to the
adjacent wells successively to generate concentrations
of 500, 250,. . .,0.2 lg/mL. Finally, 50 lL of test strain
suspension, adjusted to ꢁ105 CFU/mL using the bacte-
rial cultures mentioned above and sensitivity test
broth, was inoculated into the corresponding wells.
Plates were incubated under sufficient humidity at
37 ꢁC for 20 h. After incubation, MIC was determined
by visual inspection as the lowest concentration of the
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