Combinatorial synthesis and in vitro evaluation of a biaryl hydroxyketone library as antivirulence agents against mrsa

Article abstract of DOI:10.1021/co400142t

Antibiotic resistance coupled with decreased development of new antibiotics necessitates the search for novel antibacterial agents. Antivirulence agents offer an alternative to conventional antibiotics. In this work, we report on a family of small-molecule antivirulence agents against methicillin-resistant Staphylococcus aureus (MRSA), the most widespread bacterial pathogen. Structure-activity relationship studies led to the development of a concise synthesis of a 148-member biarylhydroxyketone library. An acylation bond-forming process afforded resorcinols (1) and aryloxy acetonitriles (2) as synthons. A Lewis-acid-activated Friedel-Crafts' acylation step involving a nitrile functionality of 2 by ZnCl₂, followed by nucleophilic attack by 1 was executed to obtain biaryl hydroxyketones in excellent yields. A large number of products crystallized. This strategy affords a range of biarylhydroxyketones in a single step. This is the first collective synthetic study documenting access to this class of compounds through a single synthetic operation. In vitro efficacy of compounds in this library was evaluated by a rabbit erythrocyte hemolysis assay. The most efficacious compound, 4f-12, inhibits hemolysis by 98.1 ± 0.1% compared to control in the absence of the compound.

Full text of DOI:10.1021/co400142t

Research Article
pubs.acs.org/acscombsci
Combinatorial Synthesis and in Vitro Evaluation of a Biaryl
Hydroxyketone Library as Antivirulence Agents against MRSA
Guanping Yu,†,§ David Kuo,† Menachem Shoham,† and Rajesh Viswanathan*,‡
‡
†Department of Biochemistry, and Department of Chemistry, Case Western Reserve University, Millis Science Center, Rm 216,
2074 Adelbert Road, Cleveland Ohio 44106, United States
S
* Supporting Information
ABSTRACT: Antibiotic resistance coupled with decreased development
of new antibiotics necessitates the search for novel antibacterial agents.
Antivirulence agents offer an alternative to conventional antibiotics. In this
work, we report on a family of small-molecule antivirulence agents against
methicillin-resistant Staphylococcus aureus (MRSA), the most widespread
bacterial pathogen. Structure−activity relationship studies led to the
development of a concise synthesis of a 148-member biarylhydroxyketone
library. An acylation bond-forming process afforded resorcinols (1) and
aryloxy acetonitriles (2) as synthons. A Lewis-acid-activated Friedel−
Crafts’ acylation step involving a nitrile functionality of 2 by ZnCl2,
followed by nucleophilic attack by 1 was executed to obtain biaryl
hydroxyketones in excellent yields. A large number of products crystallized.
This strategy affords a range of biarylhydroxyketones in a single step. This
is the first collective synthetic study documenting access to this class of
compounds through a single synthetic operation. In vitro efficacy of compounds in this library was evaluated by a rabbit
erythrocyte hemolysis assay. The most efficacious compound, 4f-12, inhibits hemolysis by 98.1 0.1% compared to control in
the absence of the compound.
KEYWORDS: biaryl hydroxyketone library, MRSA, antivirulence agents, antibiotic resistance
Antivirulence agents present alternatives to conventional
antibiotics.5 In contrast to antibiotics, antivirulence agents are
not bactericidal and, generally, are not even bacteriostatic. Since
the survival of the bacteria is not threatened there is less
pressure to develop resistance against an antivirulence agent.
The mechanism of action is based upon curtailing the
pathogen’s ability to elicit toxins against the host’s immune
system. An unimpaired immune system may be able to fight off
the infection on its own. Alternatively, a boost in the form of a
low-dose conventional antibiotic in combination with an
antivirulence agent may become a successful strategy against
more invasive infections. Antivirulence therapy offers the
attractive prospect of bringing back conventional and affordable
antibiotics into the clinic. In previous work, a series of simple
aromatic compounds were identified as effective antivirulence
candidates that included diflunisal, an FDA-approved anti-
inflammatory drug.6 In this study, we report a concise synthetic
route that affords a larger library of biaryl hydroxyketones and
show that the antivirulence activity displayed be these analogs is
much higher than diflunisal.
INTRODUCTION
■
Antivirulence Agents against MRSA. Antibiotic resist-
ance coupled with the decline in the development of new
antibiotics necessitates the search for novel agents to prevent
and treat serious bacterial infections. Herein, we report a family
of antivirulence agents effective against methicillin-resistant
Staphylococcus aureus (MRSA), the most widespread bacterial
pathogen in the United States1 and in the developed world.2
MRSA causes a wide range of infections ranging from skin and
soft tissue to more invasive infections, such as pneumonia,
endocarditis, meningitis, bacteremia, and sepsis. The increase in
S. aureus infections has been associated with hospitalization,
affecting preferentially immunocompromised individuals. Re-
cently, such infections also increasingly occur in the community
in healthy individuals, such as athletes, students, prisoners, etc.
These community-associated infections (CA-MRSA) are
generally more virulent than hospital-associated infections
(HA-MRSA).2 Treatment of S. aureus infections is hampered
by the steady increase of resistance against conventional
antibiotics. Over two-thirds of S. aureus infections are now
resistant to methicillin, a second-generation β-lactam anti-
biotic.3 Vancomycin, linezolid, and daptomycin are the
antibiotics of last resort against MRSA. Alarmingly, strains
recently have emerged that are resistant to vancomycin.4
Therefore, the development of new therapeutic solutions
against MRSA represents an urgent medical need.
Biaryl Hydroxyketones as Antivirulence Compounds.
Recently, we reported the discovery of a small molecular library
Received: November 8, 2013
Revised: December 28, 2013
Published: December 30, 2013
© 2013 American Chemical Society
85
dx.doi.org/10.1021/co400142t | ACS Comb. Sci. 2014, 16, 85−91

ACS Combinatorial Science
Research Article
Scheme 1. (A) Ribbon Diagram of the Homology-Built Model of the N-Terminal Domain of AgrA (AgrA_N), (B) Structures of
a Few Representative Scaffolds Identified As Privileged Motifs for Inhibiting AgrA Activation and Thereby Toxin Synthesis by
MRSA, and (C) Library Design through Retrosynthetic Analysis of Biaryl Hydroxyketone Scaffold to Afford Resorcinols and
Aryloxyacetonitriles As Precursors
and showed that a privileged subset of chemical entities elicited
antivirulence activity against MRSA causing diminished
production of the staphylococcal toxins, α-hemolysin, also
known as α-toxin (Hla) and phenol-soluble modulin α (PSM-
α) in a dose-dependent manner.6 Virtual screening was
employed to discover small molecules that block the
phosphorylation site on the regulatory domain of AgrA, a
response regulatory protein integral to the toxin biosynthetic
pathway in MRSA (Scheme 1A,B). Initial results indicated that
the small molecules inhibited the transcription of Hla and PSM-
α.6 One of the compounds discovered as an initial candidate,
(referred to as compound VI),6 the parent compound (PC) in
this study, inhibited rabbit erythrocyte hemolysis by 98% and
9% at 10 and 1 μg/mL, respectively.6 On the basis of the biaryl
hydroxyketone structural scaffold of this candidate we
envisioned applying chemical synthesis to derivatize the
aromatic rings in order to discover compounds with more
potent antivirulence activity.
Herein, we describe a concise synthesis of 148 individual
chemical entities through a robust acylation method that affords
a range of biarylhydroxyketones in high yields and in a single
step. Furthermore, the in vitro efficacy of this library was
evaluated by a rabbit blood hemolysis assay affording a subset
of efficacious antivirulence agents for further in vivo studies.
The underlying bond disconnection, resulting in the generation
of a library of derivatives, is the acylation bond-forming process
affording resorcinols (1) as the nucleophilic synthon and
aryloxy acetonitriles (2) as the electrophilic synthon as shown
in Scheme 1C. This strategy led to the discovery of new
compounds with considerably higher quorum sensing inhib-
itory activity than the parent compound.
RESULTS AND DISCUSSION
■
Synthesis of Biarylhydroxyketone Library. The retro-
synthetic analysis of biaryl hydroxyketone moiety, as shown in
Scheme 1C allowed for resorcinol derivatives (represented by
1) and aryloxy acetonitriles (represented by 2) as convenient
starting points for the assembly of the targeted library. Scheme
2A shows the individual variations selected based on availability
of substituted resorcinols and aryloxyacetonitrile precursors. All
of the resorcinol derivatives were commercially available except
for 4-methyl and 4-propyl substitutions represented by 1d and
1e (necessary for synthesis of 4d and 4e series respectively).
These were synthesized using reduction reactions of corre-
sponding carbonyl compounds (see Supporting Information
(SI)). All the substitutions of aryloxyacetonitrile compounds
desired for the construction of targeted library needed to be
synthesized. Alkylation reaction of phenols with α-bromoace-
tonitriles reported by McManus et al.7 served as a template for
the synthesis of these precursors. Individual protocols were
adapted based on existing methods to provide precursors in
excellent yields (see SI).
As shown in Scheme 2B, a Lewis-acid catalyzed Friedel−
Crafts’ acylation step involving the activation of the nitrile
functionality of 2 by ZnCl2, followed by nucleophilic attack by
1 was executed in the presence of gaseous hydrogen chloride in
benzene-diethyl ether mixture to yield the iminium hydro-
chlorides 3 as the intermediate. Upon hydrolysis of 3 the biaryl
86
dx.doi.org/10.1021/co400142t | ACS Comb. Sci. 2014, 16, 85−91

ACS Combinatorial Science
Research Article
Scheme 2. (A) Synthesis of Biarylhydroxyketone Library through Friedel−Crafts Acylation between 1 and 2 and (B) Structural
Variations of Biarylhydroxyketone Library
hydroxy ketone library (represented by 4) was obtained in
moderate to excellent yields depending on individual
substitution pattern. Each derivatization reaction afforded
product 4 in moderate to high yields averaging about 70%.
Purification of the compounds was relatively simple as a large
number of products crystallized, thereby not requiring
chromatography. Though a few of these biaryl hydroxyketones
were reported in the literature, this is the first collective
synthetic study documenting this class of compounds directly
in a single operation. Structural characterization of each
magnitude of hemolysis inhibition (indicated by a lower % for
hemolysis) concomitantly with no or low inhibition of bacterial
growth is essential for a good antivirulence agent. Molecules
conferring such effect are desirable because they are good
candidates for eliminating virulence and low potential for
resistance development. The hemolysis data for all 148
compounds is shown as grouped sections in the SI (Tables
S5−1−S5−3) along with data on bacterial growth. By screening
this relatively large library for efficacy, we identified a subset of
24 analogs displaying the most efficacious hemolysis inhibition
at a concentration of 1 μg/mL. These results are plotted in
Figure 1. DMSO was used as a control, assigned 100%
hemolysis and 100% bacterial growth. The biaryl parent
compound (PC) is the control candidate having the same
chemical constituency as compound VI in prior work.6
Therefore PC served as a starting point for comparison of
hemolysis inhibitory effect of newer analogs. Diflunisal, an
FDA-approved anti-inflammatory was used as a comparative
standard. In general, among the 148 compounds tested,
compounds in the 4a and 4c series containing no substitution
on the resorcinol portion of the compound or containing a
hydroxyl functionality elicited much less hemolysis inhibition
when compared to compounds in the 4d, 4e, and 4f series. This
trend points to the beneficial effect of having sterically larger
substitutions on the resorcinol portion. In the presence of 1
μg/mL of the most efficacious compound, 4f-12, rabbit
1
member of the library was performed through H, 13C NMR
analyses (See SI). Compounds 4a-1 and 4a-11 yielded X-ray
diffraction data (See SI). Table 1 lists the individual
substitutions on each derivative that was synthesized using
this one-step method. Charts S1−S6 (SI) provide chemical
structures of each member of this family of molecules.
Efficacy of the Biarylhydroxyketone Library against
MRSA-Triggered Hemolysis. The antivirulence activity of
these compounds against MRSA strain USA 300 was measured
by the extent of hemolysis inhibition in rabbit erythrocytes in
vitro. MRSA secretes the cytotoxin Hla, which creates holes in
red blood cells of rabbits and other organisms, causing
hemolysis.8 The assay measures the level of hemoglobin
released from the erythrocytes through this rupture. In addition
to hemolysis, we measured the extent to which the family of
biaryl hydroxyketones 4 inhibited bacterial growth. A high
87
dx.doi.org/10.1021/co400142t | ACS Comb. Sci. 2014, 16, 85−91

ACS Combinatorial Science
Research Article
Table 1. Individual Structure of Biaryl Hydroxyketone Library Synthesized According to Scheme 2
4a R1= 5-H
compd
R2
4b R1= 5-Ethyl
compd
R2
4c R1= 6-OH
compd
R2
4d R1= 5-methyl
compd
R2
4e R1= 5-propyl
compd
R2
4f R1= 5-hexyl
compd
R2
a
a
f
4a-0
4a-1
4a-2
4a-3
4a-4
4a-5
4a-6
4a-7
4a-8
4a-9
H
4b-0
4b-1
4b-2
4b-3
4b-4
4b-5
4b-6
4b-7
4b-8
4b-9
H
4c-0
4c-1
4c-2
4c-3
4c-4
4c-5
4c-6
4c-7
4c-8
4c-9
H
4d-0
4d-1
4d-2
4d-3
4d-4
4d-5
4d-6
4d-7
4d-8
4d-9
H
4e-0
H
4f-0
H
m-F
o-Cl
m-Cl
p-Cl
o-F
m-F
o-Cl
m-Cl
p-Cl
o-F
m-F
o-Cl
m-Cl
p-Cl
o-F
m-F
o-Cl
m-Cl
p-Cl
o-F
4e-1
m-F
o-Cl
p-Cl
o-F
4f-1
m-F
g
4e-2
4f-12
4f-14
4f-19
4f-22
4f-26
4f-27
4f-28
4f-29
2,4-F2
4e-4
3,4-F2
b
b
b
a
g
g
e
g
f
4e-5
p-iPr
4e-6
p-F
p-NO2
f
f
f
g
p-F
p-F
p-F
p-F
4e-7
p-Br
p-I
p-OCH3
o-COOCH3
m-COOCH3
p-COOCH3
p-Br
p-I
p-Br
p-I
p-Br
p-I
p-Br
p-I
4e-8
g
4e-9
o-Me
b
o-Me
o-Me
o-Me
o-Me
4e-10
4e-11
4e-12
4e-13
4e-14
4e-15
4e-17
4e-18
4e-19
4e-20
4e-21
4e-22
4e-25
4e-26
4e-27
4e-28
4e-29
m-Me
b
c
4a-10
4a-11
4a-12
4a-13
4a-14
4a-15
4a-16
4a-17
4a-18
4a-19
4a-20
4a-21
4a-22
4a-23
4a-24
4a-26
4a-27
4a-28
4a-29
m-Me
4b-10
4b-11
4b-12
4b-13
4b-14
4b-15
4b-16
4b-17
4b-18
4b-19
4b-20
4b-21
4b-22
4b-24
4b-26
4b-27
m-Me
4c-10
4c-11
4c-12
4c-13
4c-14
4c-15
4c-16
4c-17
4c-18
4c-19
4c-20
4c-21
4c-22
4c-26
4c-27
4c-28
4c-29
m-Me
4d-10
4d-11
4d-12
4d-13
4d-14
4d-15
4d-16
4d-17
4d-18
4d-19
4d-20
4d-21
4d-22
4d-25
4d-26
4d-27
4d-28
4d-29
m-Me
p-Me
g
g
p-Me
p-Me
p-Me
p-Me
2,4-F2
2,4-F2
2,4-F2
2,4-F2
2,4-F2
2,6-F2
2,6-F2
2,6-F2
2,6-F2
2,6-F2
3,4-F2
3,4-F2
3,4-F2
3,4-F2
3,4-F2
3,5-F2
3,5-F2
3,5-F2
3,5-F2
3,5-F2
3,4,5-F3
Pentafluoro
p-iPr
2,4,5-F3
3,4,5-F3
pentafluoro
p-iPr
2,4,5-F3
3,4,5-F3
pentafluoro
p-iPr
2,4,5-F3
3,4,5-F3
pentafluoro
p-iPr
2,4,5-F3
3,4,5-F3
pentafluoro
p-iPr
p-tBu
g
g
g
b
d
g
g
p-Ph
p-tBu
p-tBu
p-tBu
p-tBu
p-NO2
g
g
p-Ph
p-Ph
p-Ph
p-Ph
p-COOH
p-OCH3
o-COOCH3
m-COOCH3
p-COOCH3
f
p-NO2
p-NO2
m-COOH
p-OCH3
o-COOCH3
p-NO2
p-NO2
f
o-COOH
m-COOH
p-OCH3
o-COOCH3
m-COOCH3
p-OCH3
o-COOCH3
m-COOCH3
p-COOCH3
p-COOH
p-OCH3
o-COOCH3
m-COOCH3
p-COOCH3
d
b
d
f
4b-28
m-COOCH3
p-COOCH3
b
p-COOCH3
4b-29
a
b
c
d
e
f
g
Ref 17. Ref 18. Ref 19. Ref 20. Ref 22. Ref 21. These compounds have a CAS Number, but no reference report.
Figure 1. Rabbit erythrocyte hemolysis (red bars) and bacterial growth (blue bars) of the most efficacious compounds at 1 μg/mL.
erythrocyte hemolysis was only 1.9 0.1% compared 100% in
the absence of the compound. However, this compound
inhibited bacterial growth by 26.7% at a concentration of 1 μg/
mL, as shown in Supporting Information (Table S5−3). Thus,
88
dx.doi.org/10.1021/co400142t | ACS Comb. Sci. 2014, 16, 85−91

ACS Combinatorial Science
Research Article
some decrease in hemolysis can be attributed to a lesser
amount of bacteria present. However, 4f-12 has been shown to
inhibit binding of AgrA to its cognate DNA by an electro-
phoretic mobility shift assay (data not shown), thereby
inhibiting the production of the α-toxin, the agent causing
hemolysis. Thus, 4f-12 has dual activity. It is a bacteriostatic
agent in addition to being an antivirulence compound.
therefore enable host defense factors to contain the infection.
If proven to be efficacious and nontoxic in animal models some
compounds discovered in this work could potentially be used as
adjuvants in conventional antibiotic therapy or perhaps in
monotherapy for topical applications. Further delineation of the
mechanism of action with the series of efficacious compounds
reported in Figure 1 is underway.
The presence of a larger hexyl side chain on the 5-position of
resorcinol ring of 4f-12 in addition to the presence of two
fluorine atoms on the aryloxy portion of this molecules are
noteworthy and seem to confer a significant hemolysis
inhibitory effect. This trend is across the rest of the 23
derivatives mapped in Figure 1 revealing the effects of
hydrophobic groups present on either one or both aromatic
rings. The exact nature of substitution seems only moderately
specific for rendering hemolysis inhibitory activity. For
example, the fluorine atoms (that adds favorable drug-like
properties)9−11 could be replaced by methyl groups or
isopropyl groups and the activity is maintained with minor
differences. The compounds described herein are novel anti-
MRSA agents. There are no other known efficacious anti-
MRSA agents except for antibiotics. Diflunisal and PC (the
parent compound of the combinatorial synthesis) are included
in the hemolysis graph to show that some of the synthesized
compounds are much better inhibitors than these two
previously discovered antivirulence agents in our laboratory.6
The nonsteroidal anti-inflammatory drug diflunisal contains
two aromatic fluorine atoms. Interestingly, diflunisal was
discovered in our preliminary screening to have antivirulence
activity against MRSA,6 demonstrating a new use for an old
drug.
Mechanism of Action of Antivirulence Compounds.
Toxins secreted by S. aureus cause damage to host cells and
impair the ability of host defense mechanisms to fight the
infection. The exact nature of the toxins varies between species.
α-hemolysin (Hla) and phenol-soluble modulin (PSMα) are
virulence factors transcriptionally regulated by the agr operon,
the most important S. aureus operon for the expression of toxin
biosynthesis and pathogenicity.6 The agr system is activated by
a quorum sensing mechanism. The autoinducing peptide serves
as the signaling molecule to activate the histidine kinase AgrC,
which in turn activates the response regulator AgrA to bind to
its cognate DNA to drive the expression of a series of toxins
and virulence factors.12 Multiple accounts of agr inhibition have
recently been reported. AgrC was the target in a synthetic
approach to inhibit agr activation by cyclic peptide mimics of
the autoinducing peptide (AIP).13 Sequestration of the
autoinducing peptide by designed inhibitory antibodies was
another approach to quench agr expression.14 Depsipeptides
isolated from a marine bacterium and ambuic acid isolated from
a fungus have been shown to inhibit expression of the agr
system, apparently by competition with the AIPs.15,16
CONCLUSION
■
In summary, we report the first collective synthetic study
documenting access to a biarylhydroxyketone library. Discovery
of a privileged subset led to the development of a concise
synthesis of a 148-member biarylhydroxyketone library. An
acylation bond-forming process afforded resorcinols (1) and
aryloxy acetonitriles (2) as synthons. A Lewis-acid activated
Friedel−Crafts’ acylation step involving a nitrile functionality of
2 by ZnCl2, followed by nucleophilic attack by 1 was executed
to obtain 4 in moderate to excellent yields. Each derivatization
reaction afforded 4 in yields averaging about 70%. A large
number of products crystallized. This strategy affords a range of
biarylhydroxyketones in a single step. The in vitro efficacy of
this library was evaluated by a hemolysis assay. Compounds
listed in this manuscript have advanced to in vivo efficacy
studies in an MRSA insect larvae model (data not shown). The
three most efficacious compounds from the insect larvae study
have been subjected to a murine MRSA wound infection model
with encouraging results. These in vivo results will be published
in a subsequent report.
EXPERIMENTAL PROCEDURE
■
General Methods. All commercially available reagents and
solvents were used as analytically pure substances as received.
Reactions were monitored by thin-layer chromatography
(TLC) on silica gel plates (60 F254) with a fluorescent
indicator, and independently visualized with UV light. Target
molecules 4a−4f were recrystallized from 2-propanol. Yields
refer to crystallized compounds. All separations of intermedi-
ates (1d, 1e, 2a-2aa) were carried out under flash
chromatography (Silica gel grade: 200−400 mesh, 40−63
μm) at medium pressure (20 psi). All new compounds gave
satisfactory spectroscopic analyses (1H NMR, 13C NMR).
NMR spectra were recorded at 400 MHz in CDCl3, acetone-d6
or DMSO-d6 and chemical shift values (δ) are given in parts per
1
million. H NMR spectra are reported in parts per million (δ)
1
relative to the residual (indicated) solvent peak. Data for H
NMR are reported as follows: chemical shift (δ ppm),
multiplicity (s = singlet, bs = broad singlet, d = doublet, t =
triplet, q = quartet, ddd = double double doublet, m =
multiplet, cm = complex multiplet), integration, and coupling
constants in hertz. 13C NMR spectra were obtained on 400
MHz spectrometers (100 MHz actual frequency) and are
reported in parts per million (δ) relative to the residual
(indicated) solvent peak. 19F NMR spectra were obtained on
400 MHz spectrometers (376 MHz actual frequency) and are
reported in parts per million (δ). High-resolution mass
spectrometry (HRMS) data were obtained on spectrometer
with a quadrupole analyzer. All melting points for solids are
reported uncorrected.
In a complementary study, on biaryl compounds sharing
structural features to those reported herein, we unambiguously
showed that antivirulence mechanism is effected through
disruption of AgrA binding to its cognate DNA counterpart.6
In a subsequent recent attempt, we have found that 4f-12
inhibits binding of AgrA to its cognate DNA by an
electrophoretic mobility shift assay (data not shown), thereby
inhibiting the production of the α-toxin agent that causes
hemolysis. Toxins secreted by S. aureus cause damage to host
cells, such as red blood cells and cells of the immune system.6
Blocking toxin expression by antivirulence agents might
General Procedure for Synthesis of α-Aryloxy-2,4-
dihydroxy-Substituted Acetophenones 4a−4f.18 Dry
hydrogen chloride was passed for l h into a solution of 2 (1
mmol, 1.0 equiv) in dry benzene (1.5 mL) at 0 °C. A solution
89
dx.doi.org/10.1021/co400142t | ACS Comb. Sci. 2014, 16, 85−91

ACS Combinatorial Science
Research Article
of the substituted-resorcinol 1 (1.2 mmol, 1.2 equiv.) and
ZnCl2 (0.136 g, 1 mmol, 1.0 equiv.) in 1.5 mL dry ether were
then added. Dry hydrogen chloride was bubbled for an
additional 2 h and the reaction mixture was left overnight.
The liquid was decanted from the solid, hot water (8 mL) was
added to the residue, and the mixture was boiled at 80−100 °C
for 2−3 h. After the mixture was cooled, the solid that formed
was filtered off, washed with water until pH reached 7, and
recrystallized from 2-propanol to yield 4.
ACKNOWLEDGMENTS
■
Dr. Robert Bonomo is thanked for supplying the MRSA USA
300, as well as for invaluable advice and thoughtful discussions.
The authors thank Case Western Reserve University, Depart-
ment of Chemistry, for providing instrumentation supporting
the chemical analyses of compounds reported herein. The
authors thank Dr. Dale Ray (CWRU, Chemistry) for assistance
with acquisition of NMR data and Dr. James Faulk (CWRU,
Chemistry) for assistance with acquisition of mass spectrometry
data. Dr. Jon Karty (Indiana University Mass Spectrometry
Facility) is thanked for assistance with mass spectrometry.
Growth and Hemolysis Assay. All reagents and culture
media were purchased from Fisher Scientific except rabbit
blood (HemoStat Lab., Dixon, CA, U.S.A.) and MRSA strain
USA300, which was obtained from Dr. Robert Bonomo at the
Louis Stokes Cleveland VA Medical Center. OD600 readings
were recorded on a BioPhotometer; OD541 readings were
recorded on a UV-1700 Pharmaspec instrument. MRSA strain
USA300 was cultured overnight at 37 °C in 1.5 mL of Tryptic
Soy (TS) broth. The overnight culture was diluted 1 to 100 and
2 mL was added to designated incubation tubes. DMSO
solutions of compound 4 were subsequently added to yield a
final concentration of either 1 or 10 μg/mL compound in 2%
DMSO. 100% DMSO was added to a control incubation tube.
The tubes were placed in a shaker and incubated at 37 °C for 6
h. OD600 was measured every hour to generate a growth curve.
After 6 h the bacterial samples were filtered through a 0.22 μm
syringe filter (Fisher Scientific). Bacterial filtrate (100 μL) was
added to 1 mL hemolysin buffer (0.145 M NaCl, 0.02 M
CaCl2). Twenty-five microliters of defibrinated rabbit blood
was added and incubated for 15 min at 37 °C. The unlysed
blood cells were pelleted by centrifugation (5500 × g, room
temperature, 2 min). The hemolytic activity of the supernatant
was determined by measuring the optical density of
hemoglobin at 541 nm. Defibrinated rabbit blood without
bacterial filtrate served as blank (Db), 2% DMSO supernatant
without antivirulence agent served as control culture (Dc). The
percent hemolysis was calculated by the formula H = (Dt −
Db)/(Dc − Db) × 100, where the Dt is the OD541 reading for
supernatant with antivirulence compound.
REFERENCES
■
(1) Gordon, R. J.; Lowy, F. D. Pathogenesis of Methicillin-Resistant
Staphylococcus aureus Infection. Clin. Infect. Dis. 2008, 46, S350−S359.
(2) Otto, M. Basis of Virulence in Community-Associated
Methicillin-Resistant Staphylococcus aureus. Annu. Rev. Microbiol.
2010, 64, 143−162.
(3) Lowy, F. D. Staphylococcus aureus Infections. N. Engl. J. Med.
1998, 339, 520−532.
(4) Pantosti, A.; Sanchini, A.; Monaco, M. Mechanisms of antibiotic
resistance in Staphylococcus aureus. Future Microbiol. 2007, 2, 323−334.
(5) Shoham, M. Antivirulence Agents against MRSA. Future Med.
Chem. 2011, 3, 775−777.
(6) Khodaverdian, V.; Pesho, M.; Truitt, B.; Bollinger, L.; Patel, P.;
Nithianantham, S.; Yu, G.; Shoham, M. Discovery of Anti-virulence
Agents against MRSA. Antimicrob. Agents Chemother. 2013, 57, 3645−
3652.
(7) McManus, J. M.; Herbst, R. M. Tetrazole Analogs of Plant
Auxins. J. Org. Chem. 1959, 24, 1464−1467.
(8) Inoshima, I.; Inoshima, N.; Wilke, G. A.; Powers, M. E.; Frank, K.
M.; Wang, Y.; Wardenburg, J. B. A Staphylococcus aureus Pore-Forming
Toxin Subverts the Activity of ADAM10 to Cause Lethal Infection in
Mice. Nat. Med. 2011, 17, 1310−1314.
(9) Muller, K.; Faeh, C.; Diederich, F. Fluorine in Pharmaceuticals:
̈
Looking Beyond Intuition. Science 2007, 317, 1881−1886.
(10) Bohm, H.-J.; Banner, D.; Bendels, S.; Kansy, M.; Kuhn, B.;
̈
Muller, K.; Obst-Sander, U.; Stahl, M. Fluorine in Medicinal
̈
Chemistry. ChemBioChem 2004, 5, 637−643.
(11) Hagmann, W. K. The Many Roles for Fluorine in Medicinal
Chemistry. J. Med. Chem. 2008, 51, 4359−4369.
(12) Koenig, R. L.; Ray, J. L.; Maleki, S. J.; Smeltzer, M. S.; Hurlburt,
B. K. Staphylococcus aureus AgrA Binding to the RNAIII-agr Regulatory
Region. J. Bacteriol. 2004, 186, 7549−7555.
ASSOCIATED CONTENT
■
S
* Supporting Information
(13) George, E. A.; Novick, R. P.; Muir, T. W. Cyclic Peptide
Inhibitors of Staphylococcal Virulence Prepared by Fmoc-Based
Thiolactone Peptide Synthesis. J. Am. Chem. Soc. 2008, 130, 4914−
4924.
(14) Park, J.; Jagasia, R.; Kaufmann, G. F.; Mathison, J. C.; Ruiz, D. I.;
Moss, J. A.; Meijler, M. M.; Ulevitch, R. J.; Janda, K. D. Infection
Control by Antibody Disruption of Bacterial Quorum Sensing
Signaling. Chem. Biol. 2007, 14, 1119−1127.
(15) Mansson, M.; Nielsen, A.; Kjærulff, L.; Gotfredsen, C. H.; Wietz,
M.; Ingmer, H.; Gram, L.; Larsen, T. O. Inhibition of Virulence Gene
Expression in Staphylococcus aureus by Novel Depsipeptides from a
Marine Photobacterium. Marine Drugs 2011, 9, 2537−2552.
(16) Nakayama, J.; Uemura, Y.; Nishiguchi, K.; Yoshimura, N.;
Igarashi, Y.; Sonomoto, K. Ambuic Acid Inhibits the Biosynthesis of
Cyclic Peptide Quormones in Gram-Positive Bacteria. Antimicrob.
Agents Chemother. 2009, 53, 580−586.
(17) Garazd, M. M.; Garazd, Y. L.; Ogorodniichuk, A. S.; Shilin, V.
V.; Turov, A. V.; Khilya, V. P. Mannich Reaction in the Series of 7-
Hydroxy-3-phenoxychromones and Their Derivatives. Chem. Nat.
Compd. 1998, 34, 442−447.
(18) Vasilyev, S. A.; Boyarchuk, V. L.; Lukyanchikov, M. S.; Khilya, V.
P. Elucidating Structure−Activity Relationships in the Series of 3-
Phenoxychromone Derivatives. Khim.-Farm. Zh. 1991, 25, 50−55.
Spectroscopic data and copies of H and 13C NMR spectra for
1
all new compounds and X-ray crystallography data for 4a-1 and
4a-11 (including the CIF files). This material is available free of
charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: rajesh.viswanathan@case.edu.
Present Address
§Guanping Yu: Department of Biomedical Engineering, School
of Engineering, Case Western Reserve University, Cleveland,
Ohio, United States.
Funding
This work was supported by grant-in-aid 09GRNT2380131
from the American Heart Association Great Rivers Affiliate and
by a grant from the Steris Foundation.
Notes
The authors declare no competing financial interests.
90
dx.doi.org/10.1021/co400142t | ACS Comb. Sci. 2014, 16, 85−91

ACS Combinatorial Science
Research Article
(19) Arkhipov, V. V.; Smirnov, M. N.; Khilya, V. P. Chemistry of
Modified Flavonoids. 19. Synthesis of Phenoxyl Analogs of Isoflavone.
Chem. Heterocycl. Compd. (N. Y.) 1997, 33, 515−519.
(20) Sonoda, N.; Tsutsumi, S. The Oxidation of β-Naphthol with
Hydrogen Peroxide and Selenium Dioxide. Technol. Rep. Osaka Univ.
1965, 14, 977−981.
(21) Garazd, M. M.; Ogorodniichuk, A. S.; Shilin, V. V.; Vasil’ev, S.
A.; Turov, A. V.; Khilya, V. P. 5,7-Dihydroxy-3-phenoxychromones:
Synthesis and Properties. Chem. Nat. Compd. 1999, 34, 435−441.
(22) Goldfarb, D. S. Method Using Lifespan-Altering Compounds
for Altering the Lifespan of Eukaryotic Organisms, and Screening for
Such Compounds. U.S. Patent 20090163545A1, 2009.
91
dx.doi.org/10.1021/co400142t | ACS Comb. Sci. 2014, 16, 85−91