Development of novel antibacterial agents against methicillin-resistant Staphylococcus aureus

Article abstract of DOI:10.1016/j.bmc.2012.06.018

Methicillin-resistant Staphylococcus aureus (MRSA) poses a serious threat to public health because of its resistance to multiple antibiotics most commonly used to treat infection. In this study, we report the unique ability of the cyclooxygenase-2 (COX-2) inhibitor celecoxib to kill Staphylococcus aureus and MRSA with modest potency. We hypothesize that the anti-Staphylococcus activity of celecoxib could be pharmacologically exploited to develop novel anti-MRSA agents with a distinct mechanism. Examination of an in-house, celecoxib-based focused compound library in conjunction with structural modifications led to the identification of compound 46 as the lead agent with high antibacterial potency against a panel of Staphylococcus pathogens and different strains of MRSA. Moreover, this killing effect is bacteria-specific, as human cancer cells are resistant to 46. In addition, a single intraperitoneal administration of compound 46 at 30 mg/kg improved the survival of MRSA-infected C57BL/6 mice. In light of its high potency in eradicating MRSA in vitro and its in vivo activity, compound 46 and its analogues warrant continued preclinical development as a potential therapeutic intervention against MRSA.

Full text of DOI:10.1016/j.bmc.2012.06.018

Bioorganic & Medicinal Chemistry 20 (2012) 4653–4660
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Development of novel antibacterial agents against methicillin-resistant
Staphylococcus aureus
Hao-Chieh Chiu a, , Su-Lin Lee b, , Naval Kapuriya b, Dasheng Wang b, Yi-Ru Chen a, Sung-Liang Yu a,
Samuel K. Kulp b, Lee-Jene Teng a, Ching-Shih Chen b,c,
⇑
a Department of Clinical Laboratory Sciences and Medical Biotechnology, National Taiwan University College of Medicine, Taipei 100, Taiwan
b Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State University, 500 West 12th Avenue, Columbus, OH 43210, United States
c Institute of Basic Medical Sciences, National Cheng-Kung University, Tainan 701, Taiwan
a r t i c l e i n f o
a b s t r a c t
Article history:
Methicillin-resistant Staphylococcus aureus (MRSA) poses a serious threat to public health because of its
resistance to multiple antibiotics most commonly used to treat infection. In this study, we report the
unique ability of the cyclooxygenase-2 (COX-2) inhibitor celecoxib to kill Staphylococcus aureus and MRSA
with modest potency. We hypothesize that the anti-Staphylococcus activity of celecoxib could be pharma-
cologically exploited to develop novel anti-MRSA agents with a distinct mechanism. Examination of an
in-house, celecoxib-based focused compound library in conjunction with structural modifications led
to the identification of compound 46 as the lead agent with high antibacterial potency against a panel
of Staphylococcus pathogens and different strains of MRSA. Moreover, this killing effect is bacteria-spe-
cific, as human cancer cells are resistant to 46. In addition, a single intraperitoneal administration of com-
pound 46 at 30 mg/kg improved the survival of MRSA-infected C57BL/6 mice. In light of its high potency
in eradicating MRSA in vitro and its in vivo activity, compound 46 and its analogues warrant continued
preclinical development as a potential therapeutic intervention against MRSA.
Received 3 April 2012
Revised 1 June 2012
Accepted 8 June 2012
Available online 15 June 2012
Keywords:
Staphylococcus aureus
Anti-MRSA agents
Anti-infection agents
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
which has become endemic in many hospitals worldwide. In addi-
tion to b-lactam antibiotics, S. aureus has also developed resistance
Staphylococcus aureus, a gram-positive bacterium, is one of the
leading causes of hospital- and community-acquired infections in
developed countries.1 It is estimated that S. aureus is commensally
found on nasal passages, skin and mucous membranes of 20–30%
of the human population.2,3 S. aureus can cause infection of the
bloodstream, lower respiratory track, skin and soft tissue, leading
to bacteremia, pneumonia, endocarditis and osteomyelitis.1,4
Initially, S. aureus infections could be successfully treated with
b-lactam antibiotics, like penicillin and methicillin. However, since
the mid-1900s, the emergence of resistant strains of S. aureus has
been reported,5,6 including methicillin-resistant S. aureus (MRSA),
to several other classes of antibiotics, including aminoglycosides,
macrolides, lincosamides, chloramphenicol, sulfonamides, strepto-
mycin and tetracycline.4,7 The capability of S. aureus to resist multi-
ple antibiotics has rendered its treatment difficult, leading to a
higher mortality in patients. Thus, development of new antibacte-
rial agents against S. aureus, especially strains resistant to multiple
antibiotics, has become an urgent public health issue.
Previously, we reported that the cyclooxygenase-2 (COX-2)
inhibitor celecoxib and its derivatives exhibited unique antimicro-
bial activities against various pathogenic bacteria in vitro, including
Salmonella and Francisella.8–10 This antibacterial effect, however,
was not noted with rofecoxib, a more potent COX-2 inhibitor, sug-
gesting the dissociation of these two pharmacological activities. In
addition, celecoxib was recently reported to inhibit multidrug resis-
tance in pathogenic bacteria,11 and has been used to develop a new
class of efflux pump inhibitors.12 In this study, we further demon-
strated the unique ability of celecoxib to directly suppress, though
with modest potency, the growth of S. aureus, S. epidermidis and
MRSA. As celecoxib has been shown to suppress cancer cell prolifer-
ation, in part, by competing ATP binding of certain signaling kinases,
such as phosphoinositide-dependent kinase-1 (PDK-1)13 and
cyclin-dependent kinases (CDKs),14 and endoplasmic reticulum
Abbreviations: MRSA, methicillin-resistant Staphylococcus aureus; COX-2,
cyclooxygenase-2; LB, Luria Bertani; CAMHB, cation-adjusted Muller Hinton broth;
MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium; MIC, minimum inhibitory
concentration; IC50, 50% inhibitory concentration; CFU, colony-forming unit; PDK-1,
phosphoinositide-dependent kinase-1; CDK, cyclin-dependent kinases; SCCmec,
staphylococcal chromosome cassette mec.
⇑
Corresponding author. Tel.: +1 614 688 4008; fax: +1 614 688 8556.
E-mail addresses: chen.844@osu.edu, chen@pharmacy.ohio-state.edu (C.-S.
Chen).
These authors contributed equally to this work.
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.06.018

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H.-C. Chiu et al. / Bioorg. Med. Chem. 20 (2012) 4653–4660
Ca2+-ATPases,15 we hypothesize that celecoxib mediates the bacte-
rial killing by blocking ATP-dependent enzymes or transporters that
are crucial to cell survival. Pursuant to this premise, we conducted a
screening of an in-house celecoxib-based focused compound li-
brary, followed by structural optimization, to identify novel agents
that exhibit high anti-MRSA potencies without acute cytotoxicity
against human cells.
optimization of celecoxib to develop novel PDK-1 inhibitors,13,16
we generated a series of derivatives with varying degrees of
antiproliferative potency against cancer cells. Of these derivatives,
we chose 40 representative celecoxib derivatives for screening
against S. aureus and S. epidermidis (1–40; Fig. 2), which are note-
worthy because they represent the most common causes of medi-
cal device-associated infections.17 This screening netted compound
36 as the lead anti-Staphylococcus agent, followed by compound 9.
Further structural modifications of 36 by substituting the phenan-
threne ring with various aromatic structures yielded 41–47, of
which compound 46 was identified as the optimal agent. General
procedures for the synthesis of compounds 1–47 are depicted in
Figure 1B.
2. Results and discussion
In this study, we screened a celecoxib-based focused compound
library to identify candidate anti-Staphylococcus agents for lead
optimization (Fig. 1A). Previously, during the course of our lead
Figure 1. Chemical structures of celecoxib and compounds 1–40 in the celecoxib-based focused compound library.

H.-C. Chiu et al. / Bioorg. Med. Chem. 20 (2012) 4653–4660
4655
Figure 2. (A) Chemical structures of compounds 41–47. (B) General synthetic procedures for compounds 1–47.
2.1. Suppressive effect of celecoxib on the growth of
Staphylococcus bacteria
35984). Of these derivatives, compounds 1–8 and 10 of the carbox-
amide series, 12–15 of the sulfonamide series, 16–20 of the amine
series, 21, 22, 32, and 37–40 did not exhibit appreciable activity at
Pursuant to our previous finding that celecoxib inhibited the
proliferation of Francisella directly in culture medium,10 we exam-
ined its suppressive effect on the growth of S. aureus (ATCC 29213),
S. epidermidis (ATCC 35984), and two different strains of MRSA
(ATCC 33592 and SCCmec VT). Celecoxib exhibited a clear, though
modest, activity against these staphylococcal bacteria with the
64 lg/mL or improved activity relative to celecoxib (data not
shown). However, other derivatives exhibited multi-fold increases
in anti-Staphylococcus potency (Table 1), providing a proof-of-con-
cept of our premise that celecoxib could be structurally modified to
enhance its anti-Staphylococcus activity. Among these more active
derivatives, compound 36, followed by compound 9, represented
minimum inhibitory concentrations (MIC) of 32
and both strains of MRSA, and 16 g/mL for S. epidermidis. Expo-
sure of S. aureus to celecoxib at the MIC of 32 g/mL after 24 h re-
sulted in a 6-log decrease in CFU relative to that of control (data
not shown). In light of the absence of COX-2-like gene in bacteria,11
this finding suggests that celecoxib’s anti-Staphylococcus activity
was dissociated from it effects on COX-2.
l
g/mL for S. aureus
the lead agents with MIC values of 62
and S. epidermidis.
lg/mL against both S. aureus
l
l
As celecoxib is cytotoxic to cancer cells,18–20 the growth inhib-
itory activities of celecoxib and selected derivatives were assessed
in HT-29 human colon adenocarcinoma cells after 24-h exposure.
As shown in Table 1, most of the active anti-Staphylococcus com-
pounds examined suppressed the viability of HT-29 cells with high
potency (low IC50 values), resulting in low selectivity ratios,
defined as IC50/MIC against S. aureus. It is noteworthy that com-
pounds 9 and 36, the lead anti-Staphylococcus derivatives, showed
lower cytotoxic activity against HT-29 cells relative to the other
compounds resulting in the highest selectivity ratios (6 and 12,
respectively), indicating a better selectivity in suppressing the
growth of Staphylococcus versus HT-29 cells.
2.2. Identification of novel anti-Staphylococcus agents
The dissociation of these two pharmacological activities
(antibacterial vs anti-COX-2) provided
a molecular basis for
the pharmacological exploitation of celecoxib to develop novel
anti-Staphylococcus agents. As the target for celecoxib’s anti-
Staphylococcus activity remained unknown, we used an in-house,
celecoxib-based focused compound library consisting of 40 deriva-
tives with modifications to the terminal aromatic group (R) and
polar side chain (Fig. 2), which were screened for growth inhibitory
activities against S. aureus (ATCC 29213) and S. epidermidis (ATCC
The above findings underscore the translational potential of
compounds 9 and 36 to develop potent anti-Staphylococcus agents.
Based on these results, we hypothesized that there existed inter-
play between the terminal aromatic system and the hydrophilic
side chain to mediate the anti-Staphylococcus activity through the

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H.-C. Chiu et al. / Bioorg. Med. Chem. 20 (2012) 4653–4660
Table 1
aromatic ring. For example, the phenanthrene ring could be re-
placed by biphenyl or substituted biphenyls (42–44) without com-
promising the anti-Staphylococcus activity. Of particular interest is
the substitution with an anthracen-9-yl moiety (compound 46),
which gave rise to a twofold increase in the anti-Staphylococcus po-
Anti-Staphylococcus (MIC) versus antiproliferative (IC50) activities of test agents
Compound
MIC (
l
g/mL)
IC50
HT-29 cells
(
l
g/mL)
Selectivity
ratioa
S. aureus
(ATCC
S. epidermidis
(ATCC 35984)
29213)
tency with MIC of 0.5
exhibited lower antiproliferative potency against HT-29 cancer
cells (IC50, 20 g/mL), providing a selectivity ratio of 40 relative
to that of 12 for compound 36. It is noteworthy that compound
46 represents a hybrid of compounds 9 and 36, underlying a subtle
structure-activity relationship in interacting with the bacterial
target.
lg/mL (Table 2). Moreover, compound 46
Celecoxib
9
11
23
24
25
26
27
28
29
30
31
33
34
35
36
32
2
4
4
4
4
4
4
4
4
4
4
4
4
2
1
16
2
8
4
4
4
4
4
4
4
4
4
4
4
4
2
18
12
0.6
6
l
12.5
3.6
6.2
6.5
4.2
6
7.5
7.2
3.2
17.5
3.3
19.5
5.2
12
3.1
0.9
1.6
1.6
1.1
1.5
1.9
1.8
0.8
4.4
0.8
4.9
2.6
12
2.3. Antibacterial spectra of compounds 9, 36, and 41–46 against
different Staphylococcus species
As different strains/species of Staphylococcus might respond dif-
ferently to the antibacterial effects of these novel agents, we
further expanded our investigation to include a panel of represen-
tative Staphylococcus pathogens, consisting of different strains of S.
aureus, S. epidermidis and MRSA, as well as S. haemolyticus, S. hom-
inis, S. intermedius, S. saprophyticus, and S. lugdunesis.
a
Selectivity ratio = IC50/MIC against S. aureus.
As shown in Table 3, the inhibitory potencies of these test
agents against the three strains of MRSA, including the multi-
drug-resistant community-associated MRSA that carries the novel
staphylococcal chromosome cassette mec (SCCmec) subtype VT,21
were consistent with those of S. aureus and S. epidermidis. Among
these derivatives, compound 46 represented the optimal anti-
Table 2
Antibacterial activities (MIC) of test agents vis-à-vis ampicillin and chloramphenicol
against S. aureus and S. epidermidis and selectivity over the antiproliferative activities
against HT-29 human colorectal cancer cells
Compound
MIC (
S. aureus
l
g/mL)
IC50
HT-29 cells
(
l
g/mL)
Selectivity
ratioa
MRSA agent with MIC of 0.5
lg/mL, followed by compounds 36
S. epidermidis
(ATCC
and 42–44, all of which exhibited an MIC value of 1
lg/mL. More-
(ATCC
29213)
35984)
over, as these MRSA strains have been reported to resist different
classes of antibiotics,7,21,22 this finding suggests that a novel anti-
bacterial target is involved in the mechanism of action of these
agents.
Other non-Staphylococcus aureus species examined, with the
exception of S. intermedius, showed a lesser degree of susceptibility
to compounds 36 and 46, with MIC around 2 lg/mL. In contrast,
the potency of compound 9 remained relatively unchanged across
different Staphylococcus species.
9
36
41
42
43
44
45
46
2
1
4
1
1
1
1
0.5
4
2
2
4
2
2
2
2
1
4
12
12
28
16
9
10
19
20
11
—
6
12
7
16
9
10
19
40
2.8
—
47
Ampicillin
Chloramphenicol
8
8
64
8
—
—
2.4. Compounds 36 and 46 are bactericidal against S. aureus
a
Selectivity ratio = IC50/MIC against S. aureus.
An antibacterial agent is defined as bactericidal when it exhibits
the distinctive endpoint of causing a 99.9% reduction in bacterial
inoculum within a 24-h period of exposure.23 Otherwise, it is con-
sidered bacteriostatic. To category these novel agents, represented
by compounds 9, 36, and 46, in this regard, their time-killing kinet-
ics were assessed in S. aureus ATCC 29213 over a 24-h treatment
period as compared to the known bactericidal and bacteriostatic
agents ampicillin and chloramphenicol, respectively. Overnight-
grown bacteria were inoculated in cation-adjusted Muller Hinton
interactions with target protein(s). In light of the modestly im-
proved antibacterial activity of compound 36 relative to compound
9, we used the former compound as a scaffold for structural mod-
ifications by replacing the 2-phenanthrenyl ring with various aro-
matic systems, generating compounds 41–47 (Fig. 1A). These
derivatives were assessed for their antibacterial activities against
S. aureus (ATCC 29213) and S. epidermidis (ATCC 35984), which
revealed some degree of flexibility in altering the size of the
Table 3
Antibacterial spectra of compounds 9, 36, and 41–47 vis-à-vis ampicillin and chloramphenicol against a panel of Staphylococcus pathogens
Staphylococcus species
MIC (
l
g/mL)
45
9
36
41
42
43
44
46
47
Amp
0.25
16
>64
>64
64
Cm
S. aureus (ATCC 12598)
S. epidermidis (ATCC 12228)
MRSA (ATCC 33592)
MRSA (SCCmec VT)
MRSA (ATCC 49476)
S. haemolyticus (ATCC 29970)
S. hominis (ATCC 27844)
S. intermedius (ATCC 29663)
S. saprophyticus (ATCC 15305)
S. lugdunesis (NTUH isolate)
4
4
4
4
4
4
2
4
4
4
1
1
1
1
1
2
2
1
2
2
4
4
4
4
4
16
8
8
8
16
1
2
1
1
1
2
2
2
2
2
1
1
1
1
1
2
2
2
2
2
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
4
4
2
4
4
0.5
0.5
0.5
0.5
0.5
2
2
0.5
2
4
2
2
2
2
16
2
4
4
2
64
32
4
4
2
4
4
0.25
<0.0625
0.125
0.5
8
2
4
32
2

H.-C. Chiu et al. / Bioorg. Med. Chem. 20 (2012) 4653–4660
4657
Figure 3. The viability of S. aureus ATCC 29213 after exposure to various concentrations (2ꢀ, 4ꢀ, and 8ꢀ MIC) of compound 9 (MIC, 2
l
g/mL), 36 (MIC, 1
l
g/mL), 46 (MIC,
0.5 lg/mL), ampicillin (MIC, 8 lg/mL), and chloramphenicol (MIC, 8 lg/mL) for 2, 4, 8 and 24 h in CAMHB. Numbers of viable bacteria in the broth after each exposure period
were enumerated by CFU assay, and the results expressed as CFU/mL. Points indicate means, and bars indicate SD (n = 3). The dashed line represents 99.9% cell killing. Ctl,
control.
broth (CAMHB) at a concentration of 5 ꢀ 105 CFU/mL followed by
exposure to individual compounds at 2- to 8-fold their respective
MIC values. As shown in Figure 3, compounds 9, 36, and 46 caused
time-dependent killing of S. aureus, achieving reductions in CFU of
99.14%, 99.91%, and 99.99% respectively, after 24-h exposure to 8
times the MIC. In comparison to ampicillin and chloramphenicol,
the bacterial killing effect of compounds 36 and 46 were charact-
ristic of bactericidal, while the antibacterial effect of compound 9
was not sufficient to be defined as bactericidal.
2.5. Intraperitoneal administration of compound 46 improves
the survival of MRSA-infected C57BL/6 mice
Figure 4. Intraperitoneal administration of compound 46 improves the survival of
To further evaluate the therapeutic potential of compounds 36
and 46 against MRSA, inbred C57BL/6 mice were intraperitoneally
injected with a lethal dose (7 ꢀ 107 CFU) of MRSA (ATCC 33592),
followed by a single, intraperitoneal administration of vehicle con-
trol or compound 36 or 46 at 30 mg/kg at 1 h post-infection (N = 5
for each group). Mice treated with vehicle or compound 36 rapidly
developed signs of severe infection that included weight loss of
over 20%, significant decrease in body temperature, and lethargy.
Survival time for these mice was no more than 2 days (Fig. 4A).
Among the five mice that received a single dose of compound 46
at 30 mg/kg, three mice died during the first day post-infection
(Fig. 4A). However, two of the compound 46-treated mice survived
for at least 7 days post-infection, exhibiting decreases of body
weight at 1 day post-infection that returned to pre-infection levels
after 3–6 days post-infection (Fig. 4B). It is noteworthy that, other
than body weight loss, no other signs of illness were observed in
these two mice. Together, these findings provided a proof-of-con-
cept that compound 46 exhibited anti-Staphylococcus activity in
vivo.
MRSA-infected mice. (A) Effect of intraperitoneal administration of compound 36
and compound 46 on survival of MRSA-infected mice. Female C57BL/6 mice were
inoculated intraperitoneally with 7 ꢀ 107 CFU of MRSA (ATCC 33592). At 1 h post-
infection, mice were treated intraperitoneally once with vehicle (filled circle),
compound 36 (open circle) or compound 46 (filled triangle) at 30 mg/kg. (B) Body
weight changes in individual compound 46-treated MRSA-infected mice. The body
weights of two out of five MRSA-infected mice (#2 and #4) that survived to 7 days
post-infection were monitored during the experiment. Mouse #2 (filled circle),
mouse #4 (open circle).
2.6. Discussion
Although hospitals have fought MRSA infections since the late
1960s, the past two decades have witnessed severe community-
associated MRSA cases affecting healthy, young individuals with
no link to the healthcare system.24 To date, S. aureus has developed
various mechanisms to evade the inhibitory effect of almost all
classes of antibiotics, as well as the third-line agent vancomy-
cin.25,26 Consequently, MRSA represents an impending public

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H.-C. Chiu et al. / Bioorg. Med. Chem. 20 (2012) 4653–4660
health crisis, and there is an urgent need to develop new anti-
MRSA agents with distinct modes of antibacterial action to
overcome the multi-drug resistance. Here, we report the pharma-
cological exploitation of the suppressive effect of the COX-2 inhib-
itor celecoxib on the growth of Staphylococcus bacteria to develop a
novel class of anti-MRSA agents with high potency.
In this study, we tested three different strains of MRSA, all of
which exhibit resistance to multiple antibiotics, including oxacil-
lin, clindamycin, sulfonamides, erythromycin, tetracycline, cotrim-
oxazole, gentamicin, chloramphenicol, and streptomycin.7,21,22
Despite this multi-drug resistant phenotype, all of the MRSA
strains were as sensitive to the inhibitory effects of compounds
46, 36, and 9 as the methicillin-sensitive S. aureus strains, with
MIC values in the range of 0.5 to 2 lg/mL. This lack of cross-resis-
tance suggests that the mode of action of these compounds is dif-
ferent from those of existing antibiotics.
Among the 47 derivatives examined, there exists a subtle
structure–activity relationship in inhibiting the growth of Staphy-
lococcus bacteria. Although the majority of the compounds of the
carboxamide (1–8, 10), sulfonamide (12–15), and amine (16–20)
4. Material and methods
4.1. Chemistry general methods
Celecoxib was prepared from CelebrexÒ capsules (Amerisource
Health, Malvern, PA) by solvent extraction followed by recrystalli-
zaion from a mixture of ethyl acetate and hexane.
Unless otherwise indicated, all anhydrous solvents were com-
mercially obtained and stored in Sure-seal bottles under nitrogen.
All other reagents and solvents were purchased as the highest
grade available and used without further purification. Flash
column chromatography was performed with silica gel (Sorbent
Technologies, 230–400 mesh). Nuclear magnetic resonance spectra
(1H NMR) were acquired on a Bruker DPX 300 model spectrometer.
Chemical shifts (d) were reported in parts per million (ppm) rela-
tive to the TMS peak. Coupling constants (J) were reported in Hertz
throughout. Electrospray ionization mass spectrometry analyses
were performed with a Micromass Q-Tof II high-resolution electro-
spray mass spectrometer. The purity of all tested compounds was
determined to be greater than 95% by elemental analyses, which
were performed by Atlantic Microlab, Inc. (Norcross, GA) and were
reported within 0.4% of calculated values. All synthesized com-
pounds were soluble in organic solvents, including DMSO.
series did not show improved activity over celecoxib (MIC, 32 lg/
mL), conversion of any of these three functional groups into an
aminosulfonamide moiety increased the anti-Staphylococcus po-
tency by multifold, that is, 1 and 16 versus 42, 4 and 15 versus
43, 20 versus 44, and 9 versus 46.
4.2. General procedure of carboxamide series (9) and the
aminosulfonamide series (36 and 41–47)
The evaluation of compounds 36 and 46 in MRSA-infected mice
demonstrated that compound 46 possesses promising in vivo anti-
MRSA activity. Despite infection with a lethal dose of MRSA and
treatment with only a single dose of drug, two of five compound
46-treated mice recovered and survived to the study endpoint,
while none of the vehicle-treated control mice survived for more
than two days post-infection. Studies to develop an optimal formu-
lation for in vivo administration of compound 46, as well as syn-
thetic strategies aimed at increasing aqueous solubility are
currently underway.
To shed light onto the potential antibacterial target of these
compounds, we are currently identifying bacterial proteins with
homology to mammalian targets of celecoxib using the pub-
lished proteome of S. aureus and S. epidermidis at the National
Center of Biotechnology Information (http://www.ncbi.nlm.nih.-
gov/).27 Previously, we demonstrated that celecoxib mediated
the antiproliferative effect in cancer cells by targeting a number
of non-COX enzymes, including certain signaling kinases, such as
PDK-113 and CDKs,14 and endoplasmic reticulum Ca2+-ATPases.15
Through BLASTP analysis, we have identified a number of bacte-
rial proteins of S. aureus and S. epidermidis with some degree of
homology to PDK-1 and endoplasmic reticulum calcium ATPases,
The sulfonamide series compounds 11–15, the amine series
compounds 16–20, and the amino acid series compounds
22–35 and 38–40 were synthesized as previously described.16,28
Syntheses of the active compounds of the carboxamide series
(i.e., 9) and the aminosulfonamide series (i.e., 36 and 41–47) are
illustrated by the syntheses of compounds 9 and 36, respectively,
as examples.
Step a. To a suspension of ethyl trifluoroacetate (850 mg,
6 mmol, 1.2 equiv) and NaH (150 mg, 6.25 mmol, 1.25 equiv) in
anhydrous THF, individual ketone substrates (5 mmol, 1 equiv) in
anhydrous THF were slowly added at 25 °C. The resulting mixture
was stirred for 5 h, concentrated, diluted with ethyl acetate,
washed, in tandem, with water, 1 N HCl and brine. The organic
phase was dried over sodium sulfate, filtered and concentrated.
The residue was purified by flash column chromatography
(EtOAc–hexane, 1:4) to afford pure 1,3-diketone in fair to good
yields.
Step b. A mixture of the 1,3-diketone from step a (3 mmol,
1 equiv), individual hydrazaine substrates (3.75 mmol, 1.25 equiv),
and concentrated HCl (0.4 mL, 1.5 equiv) in ethyl alcohol was re-
fluxed until the reaction completed (monitored with TLC; EtOAc–
hexane, 3:7). The resulting mixture was concentrated, diluted with
ethyl acetate, and washed with water and brine. The organic phase
was dried over sodium sulfate, filtered and concentrated. The
residue was purified by flash column chromatography (EtOAc–
hexane, 3:7) to yield pure pyrazole ring derivatives.
including
a serine/threonine kinase (NP_764450), the copper
transporter ATPase copA, potassium transporter ATPase subunit
B, and cadmium transporting ATPase. Evaluation of the involve-
ment of these putative targets in the anti-Staphylococcus effects
of compounds 9, 36, and 46, in conjunction with genomic anal-
ysis of drug-resistant mutants, is currently underway. From a
translational perspective, understanding the mode of action of
these novel agents will foster new strategies for the treatment
of staphylococcal infections.
Step c. To a solution of 4-(5-anthracen-9-yl-3-trifluoromethyl-
pyrazol-1-yl)-benzonitrile generated from step
b
(206 mg,
0.5 mmol, 1 equiv) in DMSO (1 mL) were added Na2CO3 (106 mg,
1 mmol, 2 equiv) and H2O2 (30%, 0.2 mL, 3 equiv) at 0 °C. The reac-
tion mixture was stirred at 20 °C for 3 h, and water (3 mL) was
added. The white precipitate was filtered, washed with water,
and dried over sodium sulfate, and filtered to afford compound 9
(177 mg) as off-white crystal in 82% yield.
Step d. A reaction mixture of various 5-aryl-1-(4-nitrophenyl)-
3-(trifluoromethyl)-1H-pyrazole derivatives (3 mmol, 1 equiv),
generated from step b, platinum oxide (PtO2, 23 mg, 0.1 mmol,
0.03 equiv) in EtOH was stirred overnight under H2 atmosphere,
3. Conclusion
The high potency of compounds 36 and 46 in eradicating MRSA
in vitro justifies continued preclinical development of these com-
pounds in animal models as potential therapeutic agents against
infection with MRSA. In addition, identification of the molecular
target by which these agents kill Staphylococcus bacteria will help
design more potent anti-MRSA agents for clinical use.

H.-C. Chiu et al. / Bioorg. Med. Chem. 20 (2012) 4653–4660
4659
filtered, and washed with ethyl acetate. The combined filtrate was
4.2.6. N-{4-[5-(40-Bromobiphenyl-4-yl)-3-trifluoromethyl-
concentrated and the residue was purified with flash column chro-
matography (CH2Cl2–NH4OH, 99.9:0.1) to give the corresponding
amines with quantitative yields.
pyrazol-1-yl]-phenyl}-aminosulfonamide (44)
Melting point: 157–158 °C. 1H NMR (DMSO-d6) d 7.18–7.49 (m,
11H), 7.68–7.22 (m, 4H), 9.93 (s, 1H). 13C NMR (CDCl3) d 151.5,
150.8, 143.7, 140.8, 140.3, 137.7, 137.0, 132.0, 129.3 (2ꢀ), 127.2
(2ꢀ), 126.5 (2ꢀ), 126.5, 126.0 (2ꢀ), 122.2, 117.0, 116.1 (2ꢀ),
102.3. HRMS exact mass of C22H16BrF3N4O2S, (M+Na)+, 559.0027
amu; found: 559.0038 amu. Anal. calcd C 49.17, H 3.00, N 10.43;
found: C 49.31, H 3.14, N 10.18.
4.2.1. 4-(5-Anthracen-9-yl-3-trifluoromethyl-pyrazol-1-yl)-
benzamide (9)
1H NMR (DMSO-d6) d 7.16 (s, 1H), 7.19 (s, 1H), 7.34 (br s, 1H),
7.41 (s, 1H), 7.57–7.53 (m, 8H), 7.79 (br s, 1H), 8.17 (m 2H), 8.83
(s, 1H). HRMS exact mass of C25H16F3N3O, (M+Na)+, 454.1143
amu; found 454.1136 amu. Anal. calcd C 69.60, H 3.74, N 9.74;
found C 69.42, H 3.85, N 9.84.
4.2.7. N-[4-(5-Naphthalen-2-yl-3-trifluoromethyl-pyrazol-1-yl)-
phenyl]-aminosulfonamide (45)
Melting point: 190–191 °C. 1H NMR (CDCl3) d 5.02 (s, 2H), 6.83
(s, 1H), 7.09–7.18 (m, 4H), 7.26 (d, J = 6.6 Hz, 2H), 7.49–7.51 (m,
2H), 7.72–7.78 (m, 4H). 13C NMR (DMSO-d6) d.153.2, 141.4,
140.9, 139.6, 132.4, 130.7 (2ꢀ), 130.5, 129.6, 126.4 (2ꢀ), 127.5,
125.7, 124.8, 124.6, 122.2, 117.4, 116.7 (2ꢀ), 108.7. HRMS exact
4.2.2. N-[4-(5-Phenanthren-2-yl-3-trifluoromethyl-pyrazol-1-
yl)-phenyl]-aminosulfonamide (36)
Chlorosulfonyl isocyanate (142 mg, 1 mmol, 1 equiv) was added
dropwise to an ice-cold solution of t-BuOH (74 mg, 1 mmol,
1 equiv) in CH2Cl2, which was then added to a mixture of 4-(5-
phenanthren-2-yl-3-trifluoromethyl-pyrazol-1-yl)-phenylamine
(403 mg, 1 mmol, 1 equiv), generated from step d, and triethyl-
amine (152 mg, 1.5 mmol, 1.5 equiv) in CH2Cl2. The reaction mix-
ture was stirred at 25 °C for 1 h, and concentrated. The residue
was treated with 20% trifluoroacetic acid in CH2Cl2 for 3 h, washed
with 10% NaHCO3, dried over sodium sulfate, and concentrated.
The residue was purified by flash column chromatograph
(MeOH–CH2Cl2–NH4OH, 2:97.9:0.1) to give 36 (388 mg) as off-
white solid in 78% yield. 1H NMR (DMSO-d6) d 7.35–7.15 (m, 7H),
7.46 (dd, J = 1.8, 8.7 Hz, 1H), 7.73–7.65 (m, 2H), 7.88 (dd, J = 9,
33.6 Hz, 2H), 8.00–8.05 (m, 1H), 8.09 (d, J = 1.8 Hz, 2H), 8.82 (m
mass of
C
20H15F3N4O2S, (M+Na)+, 455.0766 amu; found:
455.0753 amu. Anal. calcd C 55.55, H 3.50, N 12.96; found: C
55.34, H 3.52, N 12.69.
4.2.8. N-[4-(5-Anthracen-9-yl-3-trifluoromethyl-pyrazol-1-yl)-
phenyl]- aminosulfonamide (46)
Melting point: 195–196 °C. 1H NMR (CD3OD)
d 6.83 (d,
J = 8.1 Hz, 2H), 6.99 (s, 1H), 7.06 (d, J = 8.4 Hz, 2H), 7.48 (s, 4H),
7.56 (s, 2H), 8.06 (d, J = 6.0 Hz, 2H), 8.61 (s, 1H). 13C NMR
(DMSO-d6) d 153.0, 141.4, 139.6, 132.4, 130.7 (2ꢀ), 130.5 (2ꢀ),
129.6, 128.8 (2ꢀ), 127.5 (2ꢀ), 125.7 (2ꢀ), 124.8 (2ꢀ), 124.6 (2ꢀ),
122.2, 117.6, 116.3 (2ꢀ), 108.7. HRMS exact mass of
2H), 9.89 (br s, 1H). HRMS exact mass of
C
24H17F3N4O2S,
C
24H17F3N4O2S, (M+Na)+, 505.0922 amu; found: 505.0905 amu.
(M+Na)+. 505.0922 amu; found: 505.0902 amu. Anal. calcd C
59.75, H 3.55, N 11.61; found C 59,98, H 3.71, N 11.51.
Compounds 41–47 were prepared by using the same procedure
as 36 with the following yields: 41 (277 mg, 70%); 42 (330 mg,
72%); 43 (354 mg, 75%); 44 (391.5 mg, 73%); 45 (303 mg, 70%);
46 (328 mg, 68%); 47 (343 mg, 71%).
Anal. calcd C 59.75, H 3.55, N 11.61; found: C 59.89, H 3.66, N
11.52.
4.2.9. N-[4-(5-Anthracen-2-yl-3-trifluoromethyl-pyrazol-1-yl)-
phenyl]-aminosulfonamide (47)
Melting point: 193–194 °C. 1H NMR (CD3OD) d 7.03 (s, 1H),
7.39–7.16 (m, 8H), 7.48 (s, 2H), 7.98 (s, 2H), 8.38 (d, J = 9.6 Hz,
1H). 13C NMR (DMSO-d6) d 153.2, 141.4, 139.2, 132.0, 130.7 (2ꢀ),
130.3 (2ꢀ), 129.6, 128.4 (2ꢀ), 127.5 (2ꢀ), 125.6 (2ꢀ), 124.8 (2ꢀ),
124.6, 123.3, 122.2, 117.6, 116.3 (2ꢀ), 106.7. HRMS exact mass of
4.2.3. N-[4-(5-p-Tolyl-3-trifluoromethyl-pyrazol-1-yl)-phenyl]-
aminosulfonamide (41)
Melting point: 199–200 °C. 1H NMR (CDCl3) d 2.30 (s, 3H), 7.27–
7.11 (m, 11 H), 9.88 (br s, 1H). 13C NMR (CDCl3) d 151.5, 143.7,
137.9, 131.4, 129.5 (2ꢀ), 127.0 (2ꢀ), 126.7 (2ꢀ), 126.5 (2ꢀ),
126.0 (2ꢀ), 116.3, 102.3, 21.1. HRMS exact mass of C17H15F3N4O2S,
(M+Na)+, 419.0766 amu; found: 419.0755 amu. Anal. calcd C 51.51,
H 3.81, N 14.13; found: C 51.30, H 3.79, N 14.08.
C
24H17F3N4O2S, (M+Na)+, 505.0922 amu; found: 505.0930 amu.
Anal. calcd C 59.75, H 3.55, N 11.61; found: C 59.93, H 3.59, N
11.68.
4.3. Bacteria stains
4.2.4. N-[4-(5-Biphenyl-4-yl-3-trifluoromethyl-pyrazol-1-yl)-
phenyl]-aminosulfonamide (42)
S. aureus strains ATCC 29213, ATCC 12598, MRSA strains ATCC
33592, ATCC 49476, S. epidermidis strains ATCC 35984 and ATCC
12228, S. haemolyticus strain ATCC 29970, S. hominis strain ATCC
27844, S. intermedius strain ATCC 29663, and S. saprophyticus strain
ATCC 15305 were obtained from American Type Culture Collection
(Manassas, VA). The clinically isolated S. lugdunesis and a MRSA
strain carrying SCCmec VT were obtained from the National Taiwan
University Hospital (Taipei, Taiwan).
Melting point: 170–171 °C. 1H NMR (CD3OD) d 6.96 (s, 1H),
7.25–7.41 (m, 5H), 7.44–7.51 (m, 4H), 7.60–7.63 (m, 4H). 13C
NMR (CDCl3) d 151.5, 150.8, 143.7, 140.8, 140.3, 132.3, 131.5,
128.7 (3ꢀ), 127.2 (2ꢀ), 126.9 (2ꢀ), 126.5 (2ꢀ), 126.0 (2ꢀ), 117.1,
116.1 (2ꢀ), 102.3. HRMS exact mass of C22H17F3N4O2S, (M+Na)+,
481.0922 amu; found: 481.0913 amu. Anal. calcd C 57.64, H 3.74,
N 12.22; found: C 57.69, H 3.78, N 12.16.
4.4. Antibacterial assays
4.2.5. N-{4-[5-(40-Methylbiphenyl-4-yl)-3-tirfluoromethyl-
pyrazol-1-yl]-phenyl}- aminosulfonamide (43)
The MIC of each agent was determined following the guidelines
for the broth microdilution method recommended by the Clinical
and Laboratory Standards Institute.29 Briefly, bacteria grown over-
night on Luria Bertani (LB) agar plates were suspended in phos-
phate-buffered saline (PBS) to an O.D. of 1.0 at 600 nm, which
was equivalent to 2 ꢀ 109 CFU/mL, and then diluted in CAMHB to
a final concentration of 5 ꢀ 105 CFU/mL. The bacterial suspensions
were exposed to the test agents and chloramphenicol at escalating
Melting point: 194–195 °C. 1H NMR (CD3OD) d 2.37 (s, 3H), 6.95
(s, 1H), 7.35–7.24 (m, 8H), 7.52 (d, J = 7.8 Hz, 2H), 7.61 (d,
J = 8.1 Hz, 2H). 13C NMR (CDCl3) d 151.5, 150.8, 143.7, 140.8,
140.3, 137.9, 137.0, 132.0, 131.5, 129.5 (2ꢀ), 127.0 (2ꢀ), 126.8
(2ꢀ), 126.5, 126.0 (2ꢀ), 117.2, 116.3 (2ꢀ), 102.3, 21.1. HRMS exact
mass of C23H19F3N4O2S, (M+Na)+, 495.1079 amu; found: 495.1061
amu. Anal. calcd C 58.47, H 4.05, N 11.86; found: C 58.28, H 4.06, N
11.84.
doses, ranging from 0.25 to 64 lg/mL, in triplicate in 96-well

4660
H.-C. Chiu et al. / Bioorg. Med. Chem. 20 (2012) 4653–4660
plates, and the plates were incubated at 37 °C for 24 h. The MIC of
each agent was defined as the lowest concentration at which no
growth of bacteria was observed. Stock solutions of the test agents
were made in DMSO at 100 mg/mL, and diluted in culture medium
to a final DMSO concentration of 0.1%.
and measurements of body weight were recorded daily. The
survival time of each mouse was recorded and was defined as
the time in days from the start of the study to when mice were sac-
rificed upon exhibiting signs of significant morbidity, which in-
cluded, though were not limited to, weight loss of 20% of initial
body weight.
4.5. Antiproliferative assay
Acknowledgments
The cytotoxicity of individual test agents in HT-29 human colon
adenocarcinoma cells was evaluated by using the 3-(4,5-dimethyl-
thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.
Briefly, HT-29 cells were seeded into 96-well plates at 1 ꢀ 104
cells/well (with a minimum of 6 wells per test condition) in RPMI
This work was supported by Award Number UL1RR025755
from the National Center for Research Resources, funded by the
Office of the Director, National Institutes of Health (OD) and sup-
ported by the NIH Roadmap for Medical Research to C.S.C. and by
the grant numbers NSC 99-2320-B-002-081 and NSC 100-2320-
B-002-104 from National Science Council, Taiwan to H.C.C. The
content is solely the responsibility of the authors and does not nec-
essarily represent the official views of the National Center for Re-
search Resources, the National Institutes of Health, or the
National Science Council, Taiwan.
1640 medium supplemented with 10% FBS and 10 lg/mL of genta-
micin. After overnight incubation at 37 °C under 5% CO2, the med-
ium from each well was removed and replenished with fresh
aliquots of the same medium containing various concentrations
of test agents dissolved in DMSO (final concentration, 0.1%). Con-
trol cells were treated with DMSO alone at a concentration equal
to that in drug-treated cells. After 24 h of drug exposure, the med-
We also thank Wei-Chun Hung, Hsiao-Jan Chen and the Micro-
array Core Facility of the National Research Program for Genomic
Medicine of the National Science Council in Taiwan for assistance
with experiments.
ium was removed and replaced by 100 ll of 0.5 mg/mL MTT in 10%
FBS-containing medium, and the cells were incubated in the CO2
incubator at 37 °C for 2 h. Subsequently, medium was removed
from each well, and the reduced MTT dye was dissolved with
100
ll of DMSO per well. Absorbance at 570 nm was measured
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Female C57BL/6 mice (8–10 weeks of age) were purchased from
the National Laboratory Animal Center (Taipei, Taiwan), and
housed as groups under conditions of constant photoperiod (12 h
light, 12 h dark) with ad libitum access to sterilized food and water
in the Laboratory Animal Center, College of Medicine, National
Taiwan University. All experimental procedures with these mice
were performed in accordance with protocols approved by the
Institutional Animal Care and Use Committee of the National
Taiwan University. Overnight-grown methicillin-resistant Staphy-
lococcus aureus (ATCC 33592) were diluted (1:100) in Luria Bertani
broth and grown at 37 °C to OD600 = 0.6, harvested by centrifuga-
tion, washed and resuspended in sterile PBS. Mice were infected by
intraperitoneal administration of MRSA (7 ꢀ 107 organisms in
0.3 ml PBS; a 100% lethal dose) through a 1 ml syringe with 25
gauge needle. The same bacterial culture was plated onto LB agar
to confirm the number of organisms inoculated. At 1 h post-infec-
tion, mice were assigned to treatment groups and then received a
single treatment of vehicle (40% PEG-400, 10% 2-hydroxyl-b-dex-
tran, 10% DMSO in normal saline), compound 36 or compound
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